BR112018005638B1 - PHARMACEUTICAL COMPOSITION COMPRISING COPPER CHELLATING TETRAMINE AND COPPER ION, USE OF THE PHARMACEUTICAL COMPOSITION TO INCREASE INTRACELLULAR COPPER LEVEL IN AN ISCHEMIC TISSUE AND KIT - Google Patents

Abstract

METHOD FOR INCREASING INTRACELLULAR COPPER LEVEL IN AN ISCHEMIC TISSUE, METHOD FOR SPECIFICALLY DELIVERING COPPER TO THE CELLS OF AN ISCHEMIC TISSUE, METHOD FOR INDUCING AT LEAST TWO TISSUE REPAIR EVENTS IN AN ISCHEMIC TISSUE, METHOD FOR INDUCING MIGRA STEM CELL ACTION FOR AN ISCHEMIC TISSUE, METHOD FOR PROMOTING COPPER-DEPENDENT HIF-1 TRANSCRIPTIONAL ACTIVITIES IN AN ISCHEMIC TISSUE, PHARMACEUTICAL COMPOSITION, USE OF A COMPOSITION, COMPOSITION AND KIT. These are methods of repair and regeneration of ischemic tissue by promoting tissue redistribution and copper reuse by administering a composition comprising a copper chelating tetramine, such as trientine. Methods and compositions for increasing the level of intracellular copper and/or inducing repair of an ischemic tissue in an individual. Increased copper level in an ischemic tissue can promote copper-dependent HIF-1 transcriptional activities and tissue repair.

Description

CROSS REFERENCE TO RELATED ORDERS

[001] This application claims the priority benefit of the International Patent Application no PCT/CN2015/090528, filed on September 24, 2015, the contents of which are incorporated into this document by reference in its entirety.

PRESENTATION OF SEQUENCE LISTING IN ASCII TEXT FILE

[002] The content of the following presentation in ASCII text file is incorporated herein by reference in its entirety: a machine readable form (CRF) of the Sequence Listing (file name: OP160744.160921.sequence listing.txt).

FIELD OF INVENTION TECHNIQUE

[003] The present invention relates to the repair and regeneration of ischemic tissue through the use of a composition comprising a tetramine, such as trientine.

BACKGROUND OF THE INVENTION

[004] Activation of hypoxia-inducible factors (HIFs) is the initial and primary molecular response of the human body to hypoxic or ischemic insult. The HIF-1 transcription factor belongs to the HIF family and controls the expression of several genes (such as VEGF) that are involved in multiple cellular adaptive responses to hypoxia and/or ischemia, including angiogenesis. HIF-1 comprises two subunits, namely HIF-1α and HIF-1β. Under hypoxic/ischemic conditions, HIF-1α accumulates in the cell nucleus to form a heterodimer with HIF-1β, which initiates transcription of downstream genes.

[005] However, under chronic myocardial ischemic conditions, the injured myocardium is commonly characterized by decreased capillary density and reduced angiogenesis. Defense mechanisms, such as those induced by accumulated HIF-1α under acute ischemic insults, do not function under chronic ischemic conditions due to mobilization of copper away from the myocardium triggered by prolonged ischemia. The transcriptional activity of HIF-1 has previously been shown to require the participation of the trace element copper. In patients with chronic ischemic cardiomyopathy, although HIF-1α levels persistently increase in ischemic myocardial tissue, the expression of genes regulated by HIF-1, such as VEGF, is reduced. Loss of cardiac copper blocks the activation of accumulated HIF-1α, and cardiac copper depletion correlates well with the degree of cardiac dysfunction in such patients. Additionally, decreased cardiac copper content is accompanied by a high blood copper level in patients with myocardial ischemic diseases. It is therefore believed that copper is released from the myocardium into the blood circulation in a form that cannot be reused by the ischemic myocardium. This drastic effusion of myocardial copper into the circulation in an unavailable form is believed to be a major cause for the reduced HIF-1α transcriptional activity accompanied by prolonged myocardial ischemia. Consequently, upregulation of HIF-1-controlled genes, an important step for tissue repair and regeneration, may fail to occur in patients with chronic ischemic myocardial diseases due to the loss of available copper. Therefore, promoting adequate tissue distribution of copper may serve as an effective strategy to treat various diseases and ischemic conditions.

[006] Trientine is a well-known copper chelating agent useful in copper detoxification. Trientine dihydrochloride is a pharmaceutically acceptable salt of trientine and has been widely employed to bind and remove excessive copper in the body to treat Wilson's disease, particularly in patients intolerant to penicillamine. Cooper et al. described the use of trientine and other copper antagonist compounds to treat various disorders, including diabetes mellitus and complications (e.g., diabetic cardiomyopathy), cardiovascular diseases, neurodegeneration, and mitochondria-related diseases. See, for example, U.S. Patent No. 7,459,446, U.S. Patent No. 7,928,094, International Application Publication WO2003077901 A1, International Application Publication WO2005058294 A1, and International Application Publication WO2007055598 A1.

[007] The disclosures of all publications, patents and patent applications mentioned in this document are incorporated into this document by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[008] The present application provides methods for increasing the level of intracellular copper or inducing tissue repair of an ischemic tissue in an individual by administering a composition comprising a copper chelating tetramine (such as trientine).

[009] In one aspect of the present application, there is provided a method for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue damage comprising administering to the individual an effective amount of a composition comprising a chelating tetramine. copper (such as trientine). Also provided is the use of a composition comprising a copper chelating tetramine (such as trientine) in the manufacture of a medicament for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue injury and a composition comprising a copper chelating tetramine (such as trientine) used to increase the intracellular copper level in ischemic tissue of an individual who has ischemic tissue injury.

[010] In one aspect of the present application, there is provided a method for specifically delivering copper to cells of an ischemic tissue in an individual having ischemic tissue injury which comprises administering to the individual an effective amount of a composition comprising a tetramine chelating tetramine. copper (such as trientine). Also provided is the use of a composition comprising a copper chelating tetramine (such as trientine) in the manufacture of a medicament for specifically delivering copper to the cells of an ischemic tissue in an individual having ischemic tissue injury and a composition comprising a copper chelating tetramine (such as trientine) used to specifically deliver copper to the cells of an ischemic tissue in an individual who has ischemic tissue injury.

[011] In one aspect of the present application, there is provided a method for inducing at least two tissue repair events in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a composition comprising a copper chelating tetramine. Also provided is the use of a composition comprising a copper chelating tetramine (such as trientine) in the manufacture of a medicament for inducing at least two tissue repair events in an ischemic tissue of an individual having ischemic tissue injury and a composition comprising a copper chelating tetramine (such as trientine) used to induce at least two tissue repair events in an ischemic tissue of an individual who has ischemic tissue injury.

[012] In one aspect of the present application, there is provided a method for inducing migration of stem cells into an ischemic tissue in an individual having ischemic tissue injury comprising administering to the individual an effective amount of a composition comprising a tetramine copper chelator. Also provided is the use of a composition comprising a copper chelating tetramine (such as trientine) in the manufacture of a medicament for inducing migration of stem cells into ischemic tissue in an individual having ischemic tissue injury and a composition that comprises a copper chelating tetramine (such as trientine) used to induce migration of stem cells into ischemic tissue in an individual who has ischemic tissue injury.

[013] In one aspect of the present application, there is provided a method for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury which comprises administering to the individual an effective amount of a composition that comprises a copper chelating tetramine. Also provided is the use of a composition comprising a copper chelating tetramine (such as trientine) in the manufacture of a medicament for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury and a composition comprising a copper chelating tetramine (such as trientine) used to promote copper-dependent HIF-1 transcriptional activities in ischemic tissue from an individual who has ischemic tissue injury.

[014] In some embodiments according to any of the methods described above, the individual has a compromised tissue repair system. In some embodiments, the individual does not have a compromised tissue repair system.

[015] In some embodiments according to any of the methods described above, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic tissue of the kidney, ischemic tissue of the skin, ischemic tissue of the digestive tract and tissue of the limbs.

[016] In some embodiments according to any of the methods described above, in which the copper chelating tetramine is trientine.

[017] In some embodiments according to any of the methods described above, the composition further comprises a copper ion. In some embodiments, the copper ion in the composition forms a complex with the copper chelating tetramine. In some embodiments, the complex of the copper chelating tetramine and the copper ion is crystalline. In some embodiments, the composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and a water molecule. In some embodiments, the copper ion in the composition does not form a complex with the copper chelating tetramine.

[018] In some embodiments according to any of the methods described above, the method further comprises administering to the individual an effective amount of a copper ion.

[019] In some embodiments according to any of the methods described above, the effective amount of the composition is insufficient to reduce the level of extracellular copper in the individual.

[020] In some embodiments according to any of the methods described above, the composition is administered orally. In some embodiments, the effective amount of the composition comprises about 80 mg to about 450 mg (including, for example, any of about 80 mg to about 150 mg, about 80 mg to about 200 mg, about 200 mg to about 300 mg, about 80 mg to about 300 mg, about 80 mg, about 100 mg, about 125 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, around 350 mg or around 400 mg) of the copper chelating tetramine per day. In some embodiments, the composition is administered at least twice daily (including, for example, about any one of twice, three times, or four times daily). In some embodiments, the composition is administered for at least about one month (including, for example, about any one of 1, 2, 3, 4, 5, 6, 8, 10, 12 or more months).

[021] In some embodiments according to any of the methods described above, administration of the composition leads to at least about 0.005 mg/l (including, for example, at least about any one of 0.01 mg/l, 0.05 mg/l, 0.1 mg/l, 0.5 mg/l, 1.0 mg/l, 2.0 mg/l, 3.0 mg/l, 4.0 mg/l or 5 mg/l) of the copper-chelating tetramine in the blood. In some embodiments, administration of the composition leads to at least about 0.005 mg/L of the copper chelating tetramine in the blood for at least about 1 week (including, for example, at least about any one of 2 weeks, 1 month , 2 months, 3 months, 4 months, 6 months, 12 months or more).

[022] In some embodiments according to any of the methods described above, the method further comprises monitoring the level of intracellular copper in the individual. In some embodiments, the method further comprises adjusting the dosage (including, for example, the effective amount, frequency of administration and combination thereof) of the composition based on the level of intracellular copper in the individual.

[023] In another aspect of the present application, a pharmaceutical composition is provided that comprises a copper chelating tetramine and a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and a water molecule.

[024] In some embodiments according to any of the pharmaceutical compositions described above, the pharmaceutical composition is formulated as a tablet, a capsule or a pill.

[025] Compositions, kits and articles of manufacture useful for the methods described herein are also provided.

[026] It should be understood that the aspects and embodiments of the invention described in this document include "consisting" and/or "essentially consisting" of the aspects and embodiments.

[027] Reference to “about” a value or parameter in this document includes (and describes) variations that are directed to that value or parameter itself. For example, the description referring to “about X” includes the description of “X”.

[028] The term “about X to Y” used in this document has the same meaning as “about X to about Y”.

[029] As used herein and in the attached claims, the singular forms "a" and "the" include plural references unless the context clearly indicates otherwise.

[030] As is evident to one skilled in the art, an individual evaluated, selected for and/or receiving treatment is an individual in need of such activities.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[031] Figure 1 shows a crystal structure of a trientine complex and a copper ion, which is also associated with two chlorine ions and a water molecule. The unidentified atoms are hydrogen atoms.

[032] Figure 2 lists an exemplary set of bond lengths, bond angles and torsional angles of the crystal structure of Figure 1.

[033] Figure 3 lists the structure and crystal refinement data of an exemplary crystal from Figure 1.

[034] Figures 4A to 4C list atomic coordinates and anisotropic parameters of atoms in the refined crystal structure of Figure 3. Figure 4A lists fractional atomic coordinates and equivalent isotropic displacement parameters of non-hydrogen atoms. Figure 4B lists anisotropic displacement parameters of non-hydrogen atoms. Figure 4C lists atomic coordinates and isotropic displacement parameters of hydrogen atoms.

[035] Figure 5 shows a flowchart of the experimental procedure of Example 2.

[036] Figure 6 shows intracellular copper concentrations of primary neonatal rat cardiomyocytes in different experimental groups of Example 2.

[037] Figure 7 shows a flowchart of the experimental procedure in Example 3.

[038] Figure 8A shows morphological changes detected by echocardiography in the interventricular septum depth (IVSD) of rats in the ascending aortic constriction (AAC) and sham operation groups with or without trientine treatment.

[039] Figure 8B shows morphological changes detected by echocardiography in the depth of the left ventricular posterior wall (IVPWD) of rats in the AAC and sham operation groups with or without trientine treatment.

[040] Figure 9A shows functional changes detected by echocardiography in the left ventricular ejection fraction (EF) of rats in the AAC and sham operation groups with or without trientine treatment.

[041] Figure 9B shows functional changes detected by echocardiography in the left ventricular shrinkage fraction (FF) of rats in the AAC and sham operation groups with or without trientine treatment.

[042] Figure 10A shows average copper concentrations in the cardiac tissues of rats in the sham control group and trientine-treated ACC groups.

[043] Figure 10B shows average copper concentrations in the plasma of rats in the sham control group and trientine-treated ACC groups. An initial high level of plasma copper concentration in the AAC rats was reduced by trientine treatment in both the high-dose trientine-treated group (ACC-Tr(H)) and the low-dose trientine-treated group (ACC-Tr(L )).

[044] Figure 11 shows a flowchart of the experimental procedure of Example 4.

[045] Figure 12 shows changes detected by echocardiography in the left ventricular ejection fraction (EF) of Rhesus monkeys with heart failure in the untreated and trientine-treated groups.

[046] Figure 13 shows copper concentrations in various samples from rhesus monkeys with heart failure in untreated and trientine-treated groups.

[047] Figure 14 shows a flowchart of the experimental procedure of Example 5.

[048] Figure 15 shows changes detected by echocardiography in the left ventricular ejection fraction (LVEF) of mice with myocardial infarction in the untreated and trientine-treated groups.

[049] Figure 16 shows copper concentrations in various samples from mice with myocardial infarction in untreated and trientine-treated groups.

DETAILED DESCRIPTION OF THE INVENTION

[050] The present application provides methods and compositions for repair and regeneration of ischemic tissue by promoting tissue redistribution and copper reuse. In particular, methods are described for increasing intracellular copper level in an ischemic tissue in an individual having ischemic tissue injury by administering a tetramine composition comprising a copper chelating tetramine (such as trientine) and, optionally, a composition copper promoter comprising a copper ion. The invention described herein is based on the surprising finding that a copper chelating tetramine (such as trientine), previously used to remove copper ions and reduce copper levels, can promote the redistribution of copper between circulating blood and myocardium. ischemic, when used in accordance with any of the methods of the present application. Trientine, for example, can bind specifically to ischemic tissue and serve to load copper into cells in the ischemic tissue. Therefore, trientine and other copper-chelating tetramines with similar properties are useful for increasing the intracellular copper level in the ischemic myocardium, thereby restoring copper-dependent HIF-1 transcriptional activities, promoting tissue repair, and reversing ischemic infarction. of the myocardium. Thus, the methods and compositions described herein are useful for treating various ischemic diseases and conditions.

METHODS FOR INCREASING INTRACELLULAR COPPER LEVELS

[051] The present application in one aspect provides a method for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a chelating tetramine. copper (hereinafter also referred to as “tetramine composition”). In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[052] In some embodiments, a method is provided for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper and administering to the subject an effective amount of a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the copper-promoting composition can alter the distribution of copper between cellular organelles. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[053] In some embodiments, a method is provided for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper and administering to the subject an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the effective amount of the copper-promoting composition increases the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[054] In some embodiments, a method is provided for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper, wherein the subject has previously received a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[055] In some embodiments, a method is provided for increasing the level of intracellular copper in an ischemic tissue of an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper, wherein the individual previously receives an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the effective amount of the copper-promoting composition increases the level of extracellular copper in the individual. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[056] In some embodiments, a method is provided for delivering copper ion to cells of an ischemic tissue in an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the method further comprises administering to the subject an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[057] In some embodiments, a method is provided for delivering copper ion to cells of an ischemic tissue in an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper and administering to the subject an effective amount of a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[058] In some embodiments, a method is provided for delivering copper ion to cells of an ischemic tissue in an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper and administering to the subject an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the effective amount of the copper-promoting composition increases the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[059] In some embodiments, a method is provided for delivering copper ion to cells of an ischemic tissue in an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper, wherein the subject has previously received a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[060] In some embodiments, a method is provided for delivering copper ion to cells of an ischemic tissue in an individual having ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper, wherein the subject previously receives an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the effective amount of the copper-promoting composition increases the level of extracellular copper in the individual. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[061] In some embodiments, a method is provided for promoting tissue redistribution and reuse of copper in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the method further comprises administering to the subject an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[062] In some embodiments, a method is provided for providing tissue redistribution and reuse of copper in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper and administering to the subject an effective amount of a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[063] In some embodiments, a method is provided for promoting tissue redistribution and reuse of copper in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper and administering to the subject an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the effective amount of the copper-promoting composition increases the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[064] In some embodiments, a method is provided for promoting tissue redistribution or reuse of copper in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper, wherein the subject has previously received a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[065] In some embodiments, a method is provided for promoting tissue redistribution and reuse of copper in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a tetramine chelating tetramine. copper, wherein the subject previously receives an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the effective amount of the copper-promoting composition increases the level of extracellular copper in the individual. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[066] In some embodiments, the intracellular copper level is increased by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 200%, 300%, 400%, 500% or more in the subject's ischemic tissue compared to the intracellular copper level of the subject's ischemic tissue before treatment. In some embodiments, the copper level (such as total copper level) of the subject's ischemic tissue is increased by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to the individual's ischemic tissue copper level before treatment. In some embodiments, the method does not reduce the level of extracellular copper (such as the level of copper in serum) in the individual. In some embodiments, the method does not reduce the extracellular copper level (such as the serum copper level) in the subject by more than about any of 5%, 10%, 20%, 30%, 40%, 50% or more compared to the individual's extracellular copper level before treatment. In some embodiments, the method does not reduce the total copper level in the individual. In some embodiments, the method does not reduce the total copper level in the individual by more than about any one of 5%, 10%, 20%, 30%, 40%, 50% or more compared to the total copper level. of the individual before treatment. In some embodiments, upon administration of the tetramine composition, the subject has at least about any one of 50%, 60%, 70%, 80%, 90% or more of the average total copper level in the serum of healthy subjects.

[067] Any of the methods described above may further comprise monitoring (including measuring and determining) the individual's copper level and adjusting the treatment plan based on the copper level. In some embodiments, the copper level is the extracellular copper level of the ischemic tissue. In some embodiments, the copper level is the individual's serum copper level. In some embodiments, the copper level is the intracellular copper level of the ischemic tissue. In some embodiments, the copper level is the total copper level, including both Cu1+ and Cu2+ levels and/or both intracellular and extracellular copper levels. In some embodiments, the copper level is the Cu2+ level. In some embodiments, the copper level is the Cu1+ level. In some embodiments, the copper level is the free (i.e., unbound) copper level. In some embodiments, the copper level comprises the level of both free and protein-bound copper. In some embodiments, the method further comprises monitoring the intracellular copper level in the individual. In some embodiments, the method further comprises adjusting the dosage (including, for example, the effective amount, frequency of administration and combination thereof) of the tetramine composition based on the level of intracellular copper in the individual.

[068] The various copper levels can be monitored singly or in combination before and/or after each administration step, and the corresponding copper levels before and after administration of the tetramine compositions can be compared to determine whether the copper level is increased or decreased by the current treatment plan. In some embodiments, the copper level measured after administration of the tetramine composition is compared to a predetermined copper level to determine whether the copper level needs to be further increased. The predetermined copper level may be a minimum copper level (such as an intracellular copper level or an extracellular copper level) that is necessary to promote copper-dependent HIF transcriptional activities and/or to induce one or more repair events. of ischemic tissue. The treatment plan can be adjusted (including, for example, whether to administer a copper-promoting composition, dose, frequency, or duration of the tetramine composition and, optionally, the copper-promoting composition, etc.) based on any of the extracellular copper level of the ischemic tissue, the individual's serum copper level, the intracellular copper level of the ischemic tissue, other individual copper levels, and combinations thereof. Additionally, the extent of repair of ischemic tissue injury can be monitored to evaluate the treatment plan. Methods for monitoring repair of ischemic tissue injury are described in the “Methods for Inducing Tissue Repair” section, which may include, but are not limited to, assessments of pathological, histological, or molecular markers associated with ischemic tissue injury.

[069] In some embodiments according to any of the methods described herein (including the methods in the section “Methods for inducing tissue repair), the method further comprises monitoring the level of intracellular copper in the individual. In some embodiments, the subject's intracellular copper level needs to be further increased after administration of the tetramine composition, if the intracellular copper level is at least about any one of 10%, 20%, 30%, 40%, 50 %, 60%, 70%, 80%, 90% or more below the predetermined intracellular copper level. In some embodiments, where the intracellular copper level needs to be further increased, the individual's treatment plan is adjusted by any one or a combination of the following: (a) continuing administration of the tetramine composition; (b) administering the tetramine composition at a higher dose; or (c) administering the tetramine composition at a higher dosing frequency. In some embodiments, the method further comprises adjusting the dosage (including, for example, the effective amount, frequency of administration and combination thereof) of the tetramine composition based on the level of intracellular copper in the individual. In some embodiments, the method further comprises monitoring the level of extracellular copper in the individual. In some embodiments, the subject's extracellular copper level needs to be increased, if the subject's extracellular copper level decreases by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more after administration of the tetramine composition or if the subject's extracellular copper level is at least about any one of 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90% or more below a predetermined extracellular copper level. In some embodiments, in which the subject's extracellular copper level needs to be increased, the subject's treatment plan is further adjusted by any one or a combination of the following: (a) administering a tetramine composition comprising a tetramine ion copper, wherein the tetramine composition of the current treatment plan does not comprise a copper ion; (b) administering a copper-promoting composition, wherein the current treatment plan does not comprise administering a copper-promoting composition; (c) increasing the dose of the copper-promoting composition; (d) increasing the dose frequency of the copper-promoting composition; (e) administering a different copper-promoting composition; or (f) stopping administration of the tetramine composition to the subject.

[070] Copper levels can be determined and/or monitored using any method known in the art. For example, the copper level can be quantitatively determined by atomic absorption spectrophotometry, by inductively coupled plasma mass spectrometry (ICPMS), or by protein-induced X-ray emission (PIXE) microscopy. See, for example, Cooper G.J.S. et al. Diabetes (2004) 53: 2501 to 2508; Lu J. et al. Drug Metabolism and Disposition (2007) 35(2): 221 to 227; and US Patent Publication No. 20100160428A1. For example, the level of total copper in a sample of ischemic tissue can be measured using a homogenized sample of ischemic tissue (such as ischemic tissue homogenized with nitric acid), which includes both the intracellular and extracellular contents of the tissue. ischemic. The level of intracellular copper in an ischemic tissue sample can be measured using isolated cells and an ischemic tissue sample, in which the cells are further lysed to release the intracellular contents before analysis. The level of extracellular copper in an individual can be measured using a sample of bodily fluid, including, but not limited to, blood serum, plasma, cerebrospinal fluid, lymph, and mucus. In some embodiments, blood serum is used to monitor the level of extracellular copper. In some embodiments, liver biopsy is used to determine the level of metabolized copper in an individual. Electron paramagnetic resonance spectroscopy, for example, can be used to detect the oxidation state (Cu1+ vs. Cu2+) of copper in the sample and provide a percentage of each oxidation state of copper in the sample. The Cu2+ level can thus be calculated using the percentage of Cu2+ in the sample and the total copper level, including both Cu1+ and Cu2+. Similarly, the Cu1+ level can be calculated using the percentage of Cu1+ in the sample and the total copper level, including both Cu1+ and Cu2+. In some embodiments, serum ceruloplasmin protein and/or serum albumin concentrations are measured, for example, using antibody-based methods (e.g., Western blot, ELISA, and the like) to monitor the level of copper that is available. for absorption and/or reuse by ischemic tissue. In some embodiments, a cross-sectional piece of an ischemic tissue sample can be used to measure both intracellular and extracellular copper levels using X-ray fluorescence (XRF) imaging methods.

[071] The methods described herein are generally applicable for redistributing copper (including, for example, increasing the level of intracellular copper and/or delivering copper to cells) in a variety of ischemic tissues. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue.

METHODS FOR INDUCING TISSUE REPAIR

[072] The present application in one aspect provides a method for inducing at least one (including, for example, at least any one of 2, 3, 4, 5, 6, 7 or more) tissue repair events in a tissue ischemic condition of an individual who has ischemic tissue damage comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[073] In some embodiments, a method is provided for inducing at least one (including, for example, at least any one of 2, 3, 4, 5, 6, 7 or more) tissue repair events in an ischemic tissue of an individual who has ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelating tetramine and an effective amount of a copper-promoting composition that can increase the individual's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[074] In some embodiments, a method is provided for inducing at least one (including, for example, at least any one of 2, 3, 4, 5, 6, 7 or more) tissue repair events in an ischemic tissue of a subject having ischemic tissue injury comprising administering to the subject an effective amount of a tetramine composition comprising a copper chelating tetramine and an effective amount of a copper promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[075] In some embodiments, a method is provided for inducing at least one (including, for example, at least any one of 2, 3, 4, 5, 6, 7 or more) tissue repair events in an ischemic tissue of an individual who has ischemic tissue damage comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the individual has previously received an effective amount of a copper promoting composition that can increase the level of extracellular copper in the individual. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[076] In some embodiments, a method is provided for inducing at least one (including, for example, at least any one of 2, 3, 4, 5, 6, 7 or more) tissue repair events in an ischemic tissue of a subject having ischemic tissue injury comprising administering to the subject an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the subject has previously received an effective amount of a copper promoting composition comprising a copper ion. copper. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[077] In some embodiments according to any of the methods for inducing tissue repair as described above, the at least one event (such as at least two events) of tissue repair comprises inducing migration of stem cells into the tissue ischemic, including, but not limited to, mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs), multipotent stem cells, induced pluripotent stem (iPS) cells, or various tissue-derived stem cells . In some embodiments, the at least one event (such as at least two events) of tissue repair comprises inducing differentiation of stem cells in the ischemic tissue. In some embodiments, the at least one event (such as at least two events) of tissue repair comprises inducing tissue regeneration in the ischemic tissue. In some embodiments, the at least one event (such as at least two events) of tissue repair comprises reversing damage to the ischemic tissue. In some embodiments, the at least one event (such as at least two events) of tissue repair comprises reconstructing the microenvironment of neurofibril cells and neurosecretory cells in the ischemic tissue. In some embodiments, the at least one event (such as at least two events) of tissue repair comprises inducing a signaling molecule that triggers tissue regeneration. In some embodiments, the at least one event (such as at least two events) of tissue repair comprises promoting copper-dependent HIF-1 transcriptional activities in ischemic tissue.

[078] The methods described herein can be used to induce tissue repair events (including, for example, promoting copper-dependent HIF-1 transcriptional activities and/or inducing stem cell migration into ischemic tissue) in various types of ischemic tissues. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue.

[079] In some embodiments, a method is provided for inducing the migration (i.e., addressing) of stem cells to an ischemic tissue of an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a composition of tetramine comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[080] In some embodiments, a method is provided for inducing the migration (i.e., addressing) of stem cells into an ischemic tissue of an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a composition of tetramine comprising a copper-chelating tetramine and an effective amount of a copper-promoting composition that can increase the individual's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[081] In some embodiments, a method is provided for inducing the migration (i.e., addressing) of stem cells into an ischemic tissue of an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a composition of tetramine comprising a copper chelating tetramine and an effective amount of a copper promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[082] In some embodiments, a method is provided for inducing the migration (i.e., addressing) of stem cells into an ischemic tissue of an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a composition of tetramine comprising a copper-chelating tetramine, wherein the subject has previously received an effective amount of a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[083] In some embodiments, a method is provided for inducing the migration (i.e., addressing) of stem cells into an ischemic tissue of an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a composition of tetramine comprising a copper-chelating tetramine, wherein the subject has previously received an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[084] In some embodiments, the stem cell is a mesenchymal stem cell (MSC), a bone marrow mesenchymal stem cell (BMSC), a multipotent stem cell, and an induced pluripotent stem cell (iPS) or a tissue-derived stem cell. In some embodiments, the stem cell is a stem cell derived from adipose tissue, a stem cell derived from cardiac tissue, or a stem cell derived from umbilical cord tissue. In other embodiments, the stem cell is an adult stem cell. In particular aspects, the adult stem cell is a hematopoietic stem cell, a mammalian stem cell, an intestinal stem cell, a mesenchymal stem cell in the placenta, adipose tissue, lung, bone marrow, blood, jelly Wharton from the umbilical cord or teeth (such as the perivascular niche of the dental pulp and periodontal ligament), an endothelial stem cell, a neural stem cell, an adult olfactory stem cell, a neural crest stem cell, or a -germline stem (e.g. a stem cell in the testis).

[085] In some embodiments, stem cells migrate in vivo from one organ or tissue compartment to a site of ischemic injury in another organ or tissue compartment of an individual who has ischemic tissue injury. For example, MSCs can migrate from bone marrow (BM), umbilical cord blood (UCB), umbilical cord stroma (Wharton's jelly), placenta, and adipose tissue (AT). In other embodiments, MSCs can be isolated from an organ or tissue compartment, enriched and/or treated in vitro, and then used in vivo for migration to the site of tissue or organ injury.

[086] Assays to measure cell migration that can be used herein include, but are not limited to, biomarkers, bioluminescence, fluorescence, positron emission tomography (PET)/CT and in vivo magnetic resonance imaging (MRI). In vivo assays can be validated and corroborated with other methods, for example, IHC on tissue sections.

[087] Non-invasive in vivo imaging techniques to assess stem cell migration include imaging gold-dextran coated particles that are loaded into MSCs, which can be visualized using X-ray modalities, Raman spectroscopy, computed tomography (CT) or ultrasound (US). In some embodiments, biocompatible nanoparticle constructs, beacons, or superparamagnetic particles are loaded into stem cells such as MSCs with properties to enable cellular visualization by X-ray, CT, US, PET, or MRI. In some embodiments, stem cell migration can be assessed using techniques such as cecal ligation and perforation (CLP). For example, performing CLP in a GFP chimeric mouse allows one to observe BMSC behavior in the establishment of abdominal sepsis. FACS, flow cytometry, and immunohistochemistry can be used to track BMSC migration to the peripheral blood, lung, liver, the skin wound, and the primary site of ischemic injury. BMSC behavior may be correlated to the timing of injury as well as local (using RT-PCR) and systemic levels of cytokines and chemokines. Tracking stem cell migration may help elucidate the contribution of BMSC to distant and local organ and tissue repair and regeneration after ischemic tissue injury.

[088] In some embodiments, stem cell migration can be monitored using identified cells administered to an individual. Approaches such as isotropic identification and staining are used to identify stem cells. In some embodiments, identification approaches include: injecting stem cells from male animals into the female, so the Y chromosome could be the tracer; inject stem cells from species A into species B, so the specific genes from species A could be the cell tracker; identify stem cells with pKH26, BrdU or other dyes, so the stem cells could be tracked by dyes or enzymatic reactions specific to the tracer.

[089] In some embodiments, isotropic identification is used to track stem cells in vivo. Stem cells could be tracked by the isotopes that identify the cells, but it is worth noting that safety issues and radioactive half-life have to be considered. Other in vivo stem cell tracking approaches include, but are not limited to: cellular staining by cellular dyes such as DID, live imaging of body surface cells by two-photon excited fluorescence microscopy, live imaging of stem cell cells specific body surface of transgenic animals by two-photon excited fluorescence microscopy, identify cells with SPIO and track the tracer by MRI, etc. Stem cells could be identified by multiple fluorescent dyes and then injected into animals. Just before the tracking experiment, target organs could be frozen, cut and observed directly under confocal laser scanning microscopy. This tracking approach does not use many identified cells (106 cell/rabbit), so autologous cells could be tracked in the natural context of organs and tissues.

[090] Stem cell identification can be achieved, for example, by a single tracer, such as pKH26. pKH26 is a lipid-soluble dye, and identification does not allow pKH26 to penetrate the cell membrane. Therefore, pKH26 is suitable for live imaging. The tracking process described in this document may involve multiple identification by 2 or 3 dyes. In some embodiments, the nucleus tracer (DAPI, Hoechst) plus the membrane tracer are used for multiple identification. The nucleus tracer confirms the cell nucleus and echoes the membrane tracer pKH26 at the same time. In some embodiments, 2 membrane tracers, e.g., Dio (3) & pKH26, are used for multiple identification. These tracers identify cells through similar mechanisms, but have different excitation and emission wavelengths, allowing simultaneous confirmation of migration (i.e., addressing) of stem cells (such as BMSCs) from 2 different fluorescent signals. In this tracking method, only overlapping signals of different wavelengths (such as red and green signals) are considered as the addressing signals.

[091] Many types of animal tissues are autofluorescent, and the most common autofluorescence in natural tissues is green fluorescence. Heart cells have relatively low fluorescence, but their fluorescence is strong enough to interfere with observations. The cut edge of the pieces is always the most strongly fluorescent. To deal with interference, only overlapping green and red signals could be recognized as the tracking signals. Red fluorescence is more suitable for statistical analysis with IOD value for its specificity (except for the obvious inaccuracy in red fluorescent signals).

[092] In some embodiments, a method for inducing stem cell differentiation in ischemic tissue is provided that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the stem cell has the ability to differentiate into a mesenchymal cell type, including osteoblasts, adipocytes, chondrocytes, endothelial cells, epithelial cells, enterocytes, osteocytes, neurocytes, hepatocytes, nephrocytes, myocytes (skeletal muscle and smooth muscle) and cardiomyocytes. In other embodiments, the stem cell has the capacity to differentiate into cells of non-mesodermal origin, including beta cells, hepatocytes and neurons. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[093] Assays known in the art can be used to elucidate the process of stem cell differentiation and the phenotypes of differentiated stem cells (such as MSCs, e.g., BMSC), including, but not limited to, alkaline phosphatase and staining. with alizarin red S for osteoblasts, oil red O staining for adipocytes and alcian blue staining for chondrogenesis. Differentiation of stem cells, such as MSCs, into various cell types can also be assessed by gene expression profiling. For example, transcriptional profiling identified specific genes implicated in osteogenic differentiation (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A), and tenogenesis (Smad8). In some embodiments, MSCs can generate high cell numbers by large-scale expansion.

[094] In some embodiments, a method for inducing tissue regeneration is provided that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the method induces cell proliferation in ischemic tissue. In some embodiments, the method induces angiogenesis in ischemic tissue. In some embodiments, the method induces blood vessel maturation in the ischemic tissue. In some embodiments, the method results in two or more of the effects described above. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[095] The tissue regeneration disclosed herein can be evaluated, for example, in an organism in which a portion of a tissue is damaged or removed. A tetramine composition as described herein is then administered to the organism and the rate of tissue regeneration is determined. The rate of tissue regeneration can be compared to the rate observed when an organism is given a control or left untreated. Other parameters that may be determined during a tissue regeneration assay include, but are not limited to, symptoms or outcomes, such as pain or pain producers, signs or symptoms of inflammation, final degree of regeneration, and quality of regeneration. In some embodiments, a tissue regeneration assay herein comprises evaluating one or more organ functional parameters, such as one or more cardiac functional markers, one or more renal functional markers, and one or more brain functional markers.

[096] In some embodiments, one or more of the following parameters in the analysis of cardiac regeneration and repair can be used to evaluate the methods described herein: (1) amount of reconstituted tissue or mass of myocardium and coronary vasculature; (2) number and size of restored myocytes and vessels; (3) integration of newly formed myocytes and vessels with the surrounding myocardium; and (4) origin of regenerated myocardial structures. In one aspect, magnetic resonance imaging (MRI) can be performed to study the scar area, global left ventricular function, regional function (motion and wall thickening), and regional ventricular perfusion. In another aspect, MRI is used to detect and/or confirm the presence of new vessels, tissue or cells that improve ventricular function. In yet another aspect, histopathology can be performed to determine the scar area and the identification and quantification of c-kit positive cardiac stem cells. Histopathology also provides data on the distribution, size, and density of new vessels and cardiomyocytes. Histopathology allows you to document the repair process at the cellular and tissue level. For example, tests are performed to evaluate, within infarct sections, microvessel density (vWF-positive vessels/mm2), BrdU-positive cells, and c-kit-positive cells. Quantifying microvessel density using von Willebrand factor (vWF) allows determining the number of new blood vessels created in the infarct zone. BrdU-positive cells represent proliferating cells, including cardiac cells. C-kit positive cell tests show the amount of stem cells within selected infarct sections.

[097] In some embodiments, a method for reversing damage to an ischemic tissue of an individual having ischemic tissue injury is provided that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[098] Reversal of tissue damage can be assessed by a suitable method, for example, detection of cellular markers of normal tissue homeostasis and/or of persistent tissue damage (for example, by immunohistochemistry or measurement of DNA levels and transcribed), measurement of the area of damage or volume of damage, or assessment of any clinically relevant indicators. For example, the reversal of cardiac tissue damage from infarcted tissue can be measured by quantifying cell numbers, such as the number of myocytes, fibroblasts, or amount of scar formation, or with functional assays for output or structural aspects of cardiac function. including LVEDP, LVDP, dp/dt max, dp/dt min, LV Weight, Chamber Volume and Diastolic Wall Voltage. In general, a method disclosed herein is said to reverse ischemic tissue damage if it results in a significant change (e.g., at least 2-fold) in any such clinical assessment or any combination thereof. In some embodiments, the method reverses fibrosis in ischemic tissue. Fibrosis is the abnormal accumulation of fibrous tissue that can occur as a part of the wound healing process in damaged tissue. Such tissue damage can result from physical injury, inflammation, infection, exposure to toxins, and other causes.

[099] Fibrotic tissues accumulate in the heart and blood vessels as a result of hypertension, hypertensive heart disease, atherosclerosis and myocardial infarction. High blood pressure, or hypertension, can be caused by a variety of factors and often leads to the development of Hypertensive Heart Disease (HHD) with progression to cardiac arrest and myocardial infarction. Similarly, atherosclerosis and other ischemic heart diseases often also result in cardiac arrest. These cardiovascular diseases also exhibit an accumulation of extracellular matrix or fibrotic deposition that results in stiffening of the vasculature and stiffening of the heart tissue itself. This deposition of fibrotic material is a response to damage induced by the hypertensive and/or sclerotic state, but the effects of this response also result in the negative effects of vascular and cardiac stiffening as well as ventricular enlargement. In some cases, increased cardiac fibrosis in cardiovascular disease disrupts or alters signals transmitted to cardiomyocytes through the heart's tissue scaffold, further leading to disruption of effective cardiac function and promoting cardiac arrest and myocardial infarction.

[0100] In accordance with the present disclosure, the expression profiles of genes differentially regulated during tissue damage can be used to evaluate the reversal of tissue damage and a method of treatment disclosed herein. For example, microarray-based analysis of gene expression can be based on the analysis of human cells (such as fibroblasts and cardiomyocytes) subjected to selected stimuli that result in changes in the accumulation and proliferation of extracellular collagen, the hallmarks of fibrosis. Stimuli can be selected to mimic those in the tissue-specific fibrosis process. Expression profiles of genes associated with fibrosis (e.g., liver fibrosis, lung fibrosis, cardiac tissue fibrosis, diabetic nephropathy, and kidney fibrosis) can then be used to assess fibrosis and the reversal of fibrotic tissue damage. . In other embodiments, expression profiles of genes associated with reversal of fibrosis (e.g., under a treatment known to at least partially reverse fibrosis) can be used to assess fibrosis and reversal of fibrotic tissue damage.

[0101] In some embodiments, there is provided a method for reconstructing the microenvironment of neurofibril cells and neurosecretory cells in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0102] The microenvironment is an intricate network of both structural and inflammatory cells, cytokines, proteins and growth factors. In the case of ischemia associated with diseases or fibrotic conditions of the heart, the heart comprises resident structural cells, such as cardiomyocytes, epithelial cells, resident fibroblasts and cardiomyocyte progenitors, and cytokine-secreting cells. These cells interact with fibrotic factors during the pathogenesis of fibrosis. In some respects, fibroblasts and myofibroblasts play an important role in creating a fibrotic environment as they secrete excess collagen and matrix materials that lead to irreversible scar formation. Cell-cell adhesion molecules and extracellular matrix ligands are important factors in the fibrotic microenvironment and promote fibrosis and fibroblast differentiation. In some embodiments, adhesion-mediated signaling is assessed in the tissue microenvironment. For example, cell differentiation and migration occur in response to microenvironmental mechanics, such as stiffening of the surrounding matrix. In one aspect, the elasticity of tissue or culture matrices of mesenchymal stem cells (MSCs) are assessed and modulated to promote stem cell targeting to ischemically injured tissue, stem cell differentiation at the site of ischemic injury, repair of tissue and/or reversal of tissue damage. In one embodiment, soft matrices result in differentiation of MSCs into neuron-like cells, wherein rigid matrices result in differentiation of MSCs into myogenic ones. In one aspect, the extracellular matrix and its components at the site of ischemic injury are evaluated to indicate whether the microenvironment promotes stem cell migration to the site, stem cell differentiation at the site of ischemic injury, tissue repair and/or reversal. of tissue damage.

[0103] In some embodiments, changes in cells in the context of their natural environment are measured to indicate the efficacy and/or toxicity of a therapeutic method disclosed herein. In some embodiments, the stem cell microenvironment of a donor tissue or organ (such as bone marrow) and a site of ischemic injury is assessed and/or modulated to promote stem cell migration to the site, differentiation of stem cells at the site of ischemic injury, tissue repair and/or reversal of tissue damage. The local tissue microenvironment can be assessed by protein staining (IHC and IF) and RNA staining with chromogenic or fluorescent ISH. For example, the hypoxic microenvironment can be indicated by hypoxic marker staining, endothelial marker staining, microvessel density analysis, and proximity analysis. The tissue microenvironment can also be studied using organ cultures or organotypic cultures as revealed in Benbrook, 2006, Drug Discovery Today: Disease Models, 3(2): 143 to 148.

[0104] In some embodiments, there is provided a method for inducing a signaling molecule that triggers tissue regeneration in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0105] Suitable signaling molecules described herein include, but are not limited to, HIF-1, VEGF, SDF-1, CXCR4, CXCL12 (also called SDF-1α), MMPs, HGF/c-met, TGF-β1 , IL-1β, TNF-α, CCR1, CCR4, CCR7, CCR10, CCR9, CXCR5, CXCR6, CD44, CD54, CD56, CD106, E-cadherin, P-selectin, integrins such as integrin-beta1 and CD49a, b , c, e, f (integrins a1, 2, 3, 4, 6) and integrin ligands such as VCAM and ICAM.

[0106] The SDF-1/CXCR4 geometric axis is one of the most important mechanisms for stem cell addressing. SDF-1 (stromal cell-derived factor 1 or CXCL12), belonging to the CXC-chemokine family, is a small molecule secreted protein. SDF-1 expression is regulated by HIF-1 (Hypoxia-inducible factor 1). HIF-1 is composed of HIF-1α and HIF-1β/ARNT (aryl hydrocarbon nuclear translocator, ARNT). HIF-1β is stable in the cytoplasm, so the expression and accumulation of HIF-1α determines HIF-1 activity. Under normoxia, HIF-1α protein is rapidly synthesized and degraded by the ubiquitin-proteasome system. Prolyl hydroxylases (PHDs) hydroxylate HIF-1α, and hydroxylated HIF-1α is recognized by the von Hippel-Lindau tumor suppressor protein (pVHL), which constitutes a ubiquitin-protein ligase that targets HIF-1α for protein degradation. Upon ischemic tissue injury, the damaged region is hypoxic, which inhibits the activity of PHDs, enabling the accumulation and translocation of HIF-1α to the nucleus, in which HIF-1α dimerizes with HIF-1β to form HIF-1, combines combines with other factors and initiates transcription of target genes. Injured tissues express a high level of SDF-1 and release SDF-1 into the circulation, building a concentration gradient from the injured region to the distant end of the circulation. The gradient thus attracts CXCR4-expressing stem cells, including BMSCs, to the injured tissues.

[0107] When the heart is under chronic hypoxia, the blood in the coronary arteries cannot meet the demand of the myocardium. Chronic ischemia can induce myocardial fibrosis, decrease microartery density, affect blood pumping, and ultimately result in ischemic heart infarction. Under chronic ischemia, HIF-1 activity is limited, resulting in inhibition of the expression of angiogenic factors that are regulated by HIF-1. The blood supply could therefore not be restored, and a heart attack could occur.

[0108] Usually, HIF-1 activity in ischemic injured tissues is temporally limited. Both animal experiments and clinical tests have shown that, under cardiac ischemia, HIF-1α in injured tissues accumulates instantly after injury, but gradually decreases thereafter. HIF-1 activity falls even faster than the HIF-1 level, resulting in decreased expression of HIF-1-regulated factors, such as VEGF and SDF-1, after a transient increase. Due to regulation by HIF-1, SDF-1 expression peaks on the first or second day after cardiac infarction. SDF-1 expression then gradually decreases and reduces to the baseline level in about a month. Due to the fact that SDF-1 is one of the mobilizers of stem cell addressing, the decrease in SDF-1 levels leads to the regression and even disappearance of stem cell addressing.

[0109] Importantly, HIF-1α-induced defense mechanisms as activated under acute ischemic conditions function differently than under prolonged ischemic conditions. Under long-term ischemic disease, HIF protein levels increase in the ischemic myocardium, while HIF-regulated genes (such as VEGF) are suppressed, which leads to decreased revascularization and impaired regeneration. Copper deprivation reduces HIF-1α binding to the HRE sequence of target genes and to P300, a component of the HIF-1 transcriptional complex. Furthermore, copper is substantially mobilized from the myocardium to the blood after prolonged ischemia. This mobilization of copper into the coronary flow sensitively follows prolonged, but not short, cardiac ischemia. The loss of copper from the myocardium correlates with the degree of loss of cardiac functions. Consequently, even under the condition of elevated HIF protein level, upregulation of HIF-controlled genes does not occur due to copper loss from the myocardium. Trace elements, such as copper, can lead to HIF-1 activation, including HIF-1α synthesis, stabilization, translocation from the cytosol to the nucleus, binding to the HRE sequence of target genes, and HIF-1 transcriptional complex formation. . Therefore, copper-dependent HIF-1 transcriptional activities, including copper-dependent induction of HIF-1 target genes or copper-dependent repression of HIF-1 target genes, may play important roles in the repair of ischemic tissues. The methods described herein are useful for inducing one or more signaling molecules, such as HIF-1α and HIF-1 copper-dependent target genes (such as HIF-1α).

[0110] In one aspect of the present application, there is provided a method for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a copper composition. tetramine comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the method induces expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the method represses the expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the at least one copper-dependent HIF-1 target gene is selected from the group consisting of VEGF, GAPDH, GLUT1, PGK1, and BNIP3. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0111] In some embodiments, there is provided a method for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelating tetramine and an effective amount of a copper-promoting composition that can increase the individual's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the method induces expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the method represses the expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the at least one copper-dependent HIF-1 target gene is selected from the group consisting of VEGF, GAPDH, GLUT1, PGK1, and BNIP3. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0112] In some embodiments, a method is provided for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine and an effective amount of a copper promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the method induces expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the method represses the expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the at least one copper-dependent HIF-1 target gene is selected from the group consisting of VEGF, GAPDH, GLUT1, PGK1, and BNIP3. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0113] In some embodiments, a method is provided for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the subject has previously received an effective amount of a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the method induces expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the method represses the expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the at least one copper-dependent HIF-1 target gene is selected from the group consisting of VEGF, GAPDH, GLUT1, PGK1, and BNIP3. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0114] In some embodiments, a method is provided for promoting copper-dependent HIF-1 transcriptional activities in an ischemic tissue of an individual having ischemic tissue injury comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the individual previously receives an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the method induces expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the method represses the expression of at least one copper-dependent HIF-1 target gene in an ischemic tissue of the subject. In some embodiments, the at least one copper-dependent HIF-1 target gene is selected from the group consisting of VEGF, GAPDH, GLUT1, PGK1, and BNIP3. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0115] HIF-1 target genes have been described in the art. See, for example, Benita Y. et al, (2009) Nucleic Acids Research, 37 (14): 4,587 to 4,602; Shen C. et al, (2008) J. Biol. Chem., 280: 20,580 to 20,588; Elvidge G.P. et al, (2006) J. Biol. Chem., 281: 15,215 to 15,266; Manalo DJ. et al, (2005) Blood, 105: 659 to 669; The HIF-1 target genes described in the references are incorporated herein by reference. HIF-1 transcriptional regulation of a subset of HIF-1 target genes is copper dependent, and the subset of HIF-1 target genes is referred to herein as copper-dependent HIF-1 target genes. . HIF-1 transcriptional regulation of some HIF-1 target genes by HIF-1 is copper independent. See, for example, Zhang Z. et al, (2014) Metallomics 6(10): 1889 to 1893. Exemplary copper-dependent HIF-1 target genes include, but are not limited to, vascular endothelial growth factor (VEGF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose transporter 1 (GLUT1), phosphoglycerate kinase 1 (PGK1 ) and 19 kDa BCL2/adenovirus E1B protein-interacting protein 3 (BNIP3).

[0116] The copper-dependent HIF-1 transcriptional activities contemplated by the present application include induction and repression (i.e., transcriptional regulation) of the expression of copper-dependent HIF-1 target genes in an ischemic tissue. In some embodiments, copper-dependent HIF-1 transcriptional activity (e.g., fold number of induction or repression of a copper-dependent HIF-1 target gene) is reduced by at least about any one of 10%. , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in an individual's ischemic tissue before receiving treatment, compared to a control level. In some embodiments, copper-dependent HIF-1 transcriptional activity (e.g., fold number of induction or repression of a copper-dependent HIF-1 target gene) in the subject's ischemic tissue is restored to at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the control level after the individual receives treatment. A control level of copper-dependent HIF-1 transcriptional activity can be based on the fold number of induction or repression of a HIF-1 target gene in a healthy tissue under ischemic conditions or under a comparable level of hypotoxic conditions, relative to normal conditions (e.g., undamaged or normoxic). Copper-dependent HIF-1 transcriptional activity can be determined by comparing the expression level (e.g., RNA level and/or protein level) of a copper-dependent HIF-1 target gene in an ischemic tissue. to the copper-dependent HIF-1 target gene expression level in healthy tissue. RNA expression levels can be measured using any of the methods known in the art, including, but not limited to, reverse transcription PCR (RT-PCR), quantitative RT-PCR, microarray and RNA sequencing methods. Protein expression levels can be measured using any of the methods known in the art, including, but not limited to, antibody-based methods (such as Western blot and ELISA) and quantitative proteomic methods (such as quantitative mass spectrometry). ).

[0117] In some embodiments, a method is provided for inducing at least two (including, for example, at least any one of 3, 4, 5, 6, 7 or more) tissue repair events in an individual who has injury of ischemic tissue comprising administering to the subject an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the at least two tissue repair events are selected from the group consisting of: inducing cell migration -stem cells, such as bone marrow mesenchyme stem cells, to ischemic tissue, induce stem cell differentiation in ischemic tissue, induce tissue regeneration in ischemic tissue, induce a signaling molecule that triggers tissue regeneration, reverse damage in ischemic tissue, reconstruct the microenvironment of neurofibril cells and neurosecretory cells in ischemic tissue, and promote copper-dependent HIF-1 transcriptional activities. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0118] In some embodiments, a method is provided for inducing migration of stem cells (such as MSC, e.g., BMSC) into ischemic tissue and inducing differentiation of stem cells in the ischemic tissue comprising administering to the subject an amount effective use of a tetramine composition comprising a copper chelating tetramine. In some embodiments, a method is provided for inducing migration of stem cells (such as MSC, e.g., BMSC) into ischemic tissue and inducing tissue regeneration in the ischemic tissue comprising administering to the subject an effective amount of a composition of tetramine comprising a copper chelating tetramine. In some embodiments, a method is provided for inducing migration of stem cells (such as MSC, e.g., BMSC) into ischemic tissue, inducing stem cell differentiation, and inducing tissue regeneration in the ischemic tissue comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0119] In some embodiments, there is provided a method for inducing ischemic tissue repair (or improving ischemic tissue function) in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0120] In some embodiments, there is provided a method for inducing ischemic tissue repair (or improving ischemic tissue function) in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a copper-chelating tetramine and an effective amount of a copper-promoting composition that can increase the individual's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0121] In some embodiments, there is provided a method for inducing ischemic tissue repair (or improving ischemic tissue function) in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine and an effective amount of a copper promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the tetramine composition and the copper promoting composition are administered simultaneously. In some embodiments, the tetramine composition and the copper promoting composition are administered sequentially. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0122] In some embodiments, there is provided a method for inducing ischemic tissue repair (or improving ischemic tissue function) in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the subject has previously received an effective amount of a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0123] In some embodiments, there is provided a method for inducing ischemic tissue repair (or improving ischemic tissue function) in an individual who has ischemic tissue injury that comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine, wherein the individual has previously received an effective amount of a copper-promoting composition comprising a copper ion. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the subject has received the copper-promoting composition about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more prior to injection. administration of the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0124] Methods for treating a disease or condition associated with ischemic tissue injury using any of the methods described herein are also provided.

[0125] In some embodiments, a method for preventing ischemic heart failure in an individual is provided which comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine (such as trientine). In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily. In some embodiments, the tetramine composition is administered for at least about 1 month (such as at least about 3 months or at least about 6 months). In some embodiments, the subject has left ventricular ejection function (LVEF) of at most about 35% at baseline. In some embodiments, the individual has class II or class III heart failure (based on the New York Heart Association or NYHA functional classification).

[0126] Heart failure of any class or stage that has ischemic origin (i.e., ischemic heart failure) can be treated with the methods described in this document. In some embodiments, the individual has NYHA Class I heart failure of ischemic origin. In some embodiments, the individual has NYHA Class II ischemic heart failure of ischemic origin. In some embodiments, the individual has NYHA Class III ischemic heart failure of ischemic origin. In some embodiments, the individual has NYHA Class IV ischemic heart failure of ischemic origin. In some embodiments, the individual has NYHA Class A ischemic heart failure of ischemic origin. In some embodiments, the individual has NYHA Class B ischemic heart failure of ischemic origin. In some embodiments, the individual has NYHA Class C ischemic heart failure of ischemic origin. In some embodiments, the individual has NYHA Class D ischemic heart failure of ischemic origin. In some embodiments, the individual has one or more symptoms of ischemic heart failure, such as fatigue, palpitation, dyspnea, or limitation of physical activity. In some embodiments, the individual has one or more symptoms of cardiovascular disease. In some embodiments, the individual has been hospitalized for at least about any one of 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30 or more days.

[0127] The effectiveness of any of the methods described herein can be further determined by evaluating the extent of ischemic tissue repair. Tissue repair can be assessed, for example, by area of damage or volume of damage. Repair of damaged tissue in a patient can be assessed using any clinically relevant standard. For example, cardiac tissue repair of infarcted tissue can be measured by quantifying cell numbers, such as the number of myocytes, fibroblasts, or amount of scar formation, or with functional assays for output or structural aspects of cardiac function, including LVEDP. , LVDP, dp/dt max, dp/dt min, LV Weight, Chamber Volume and Diastolic Wall Voltage. In general, a method disclosed herein is said to repair damaged tissue if it results in a significant change (e.g., at least 2-fold) in any such clinical assessment or any combination thereof.

[0128] Any appropriate method (or methods) can be performed to evaluate tissue repair. For example, methods can be performed to evaluate tissue healing, to evaluate the functionality of repaired tissue, and to evaluate cell growth in the tissue. To determine the extent of tissue healing, histology and cell staining can be performed to detect inoculated cell spreading and/or enhanced histological appearance. In some cases, portions of tissue can be collected and treated with a fixative such as, for example, neutral buffered formalin. Such tissue portions can be dehydrated, embedded in paraffin, and sectioned with a microtome for histological analysis. Sections can be stained with hematoxylin and eosin (H&E) and then mounted on glass slides for microscopic assessment of morphology and cellularity. In some cases, physiological tests may be performed to assess tissue movement and functionality following the methods and materials provided herein. For example, in vitro mechanical tests can be performed to measure the work of bending (WOF) or bending angle of a repaired tendon tissue or a repaired joint. In vivo assays may include organ functional assessment, symptom assessment, or imaging techniques.

[0129] In some embodiments, tissue and/or organ function before, during, or after administration of a therapeutic method disclosed herein may be assessed by any one or more of the following methods: biochemical analysis of at least one indicative biomarker of improved tissue function by methods such as flow cytometry, immunofluorescence, ELISA, phosphor identification, hybridization, nucleic acid amplification, or Western blot; cell function assays, such as cell apoptosis assays, necrosis assays, and cell viability assays, including Annexin V staining by immunofluorescence or flow cytometry, detection of caspase activity, hypoxia assays, TUNEL assay, cellular DNA fragmentation , number of rod-shaped cells in response to H2O2, qPCR assessment of gene expression and measurement of necrotic area by H&E staining; scar formation assays, including measuring the number of fibroblast cells in a damaged or infarcted area, measuring the level or deposition of collagen and other matrix proteins associated with scar formation; migration of stem cells or progenitor cells to the damaged area; and any other clinically relevant organ function tests.

[0130] In some embodiments, cardiac function can be assessed by any one or more of the following parameters: myocyte mechanics and cell fusion, e.g., frequency of myocyte size distribution, peak shortening, velocity of shortening and relengthening, and assessment of cell fusion (number of X chromosomes); output or structural aspects of cardiac function including LVEDP, LVDP, +dp/dT, LV Weight, Chamber Volume, Diastolic Wall Stress and comparison of MI-treated and non-MI-treated subjects; myocardial regeneration, such as composition of the regenerated myocardium, assessment of BrdU-positive cells in the infarcted area in treated versus untreated subjects and myosin-positive cells in the infarcted area in treated versus untreated subjects; cardiac structure, such as infarct size, amount of fibrosis and cardiomyocyte hypertrophy. In certain embodiments, a method disclosed herein further comprises measuring one or more indicia of cardiac function, wherein said indicia of cardiac function are thoracic cardiac output (CO), cardiac index (CI), pulmonary artery wedge pressure ( PAWP), cardiac index (CI), % fractional shortening (% FS), ejection fraction (EF), left ventricular ejection fraction (LVEF); left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), contractility (dP/dt), a decrease in atrial or ventricular functioning, an increase in pumping efficiency, a decrease in the rate of loss of effectiveness pumping, a decrease in loss of hemodynamic functioning, or a decrease in complications associated with cardiomyopathy, compared to a control.

[0131] In some embodiments, brain function before, during, or after administration of a therapeutic method disclosed herein may be assessed by a neurological test, or electrophysiologically, e.g., by a decreased signal-to-noise ratio, or biochemically. , for example, by analyzing at least one biomarker indicating organ function, tissue function and/or cellular function of the central or peripheral nervous system. Exemplary electrophysiological techniques include electroencephalography (EEG), electrocardiography (EKG), electromyography (EMG), event-related potentials (ERPs), evoked potentials (EPs), magnetoencephalography (MEG), and nerve conduction study (NCS). In other embodiments, brain function may be assessed by any one or more of the following methods or parameters: general intellectual function, such as the Wechsler Abbreviated Intelligence Scale and the Wechsler Adult Intelligence Scale III; basic attention, such as Digit Repetition (Digit Span), spatial location subtests (Spatial span) of the Wechsler Memory Scale III; complex attention (working memory), such as Spatial Location, Letter and Number Sequences and Arithmetic subtests of the Wechsler Adult Intelligence Scale III; executive functions, such as Wisconsin Card Sorting Test, Trail Making Test B, Stroop Test, Tower of London Test, Gambling Test, Frontal System Behavior Scale, and Iowa Scales of Lobe Function ; memory (visual and verbal), such as Wechsler Memory Scales III, Rey Auditory-Verbal Learning Test, California Verbal Learning Test II, Brief Visual Memory Test Revised; affect regulation, such as Minnesota Multiphasic Personality Inventory 2, Stroop Affective Test, Frontal System Behavior Scale, and Iowa Scales of Frontal Lobe Function; interpretation of emotion stimuli, such as DANVA (Diagnostic Analysis of Non-Verbal Behavior); processing speed, such as the Processing Speed (Symbol Search, Coding) index of the Wechsler Adult Intelligence Scale III, Trail Making Test and Digital Symbol Modalities Test; language, such as Boston Naming Test; Controlled Oral Word Association Test; Semantic Verbal Fluency Test; and Multilingual Aphasia Examination; visuoconstructive tests, such as the Rey-Osterrieth Complex Figure Test, Cubes and Object Assembly subtests of the Wechsler Adult Intelligence Scale III; and visuospatial tests, such as WAIS-III Logical Reasoning and Line Orientation Judgment Test.

[0132] In some embodiments, skeletal muscle health before, during and after administration of a therapeutic method disclosed herein is tested. In some embodiments, skeletal muscle health includes muscle pain, muscle damage, metabolic changes with exercise, and cytoskeletal reorganization. The function of skeletal muscle can be muscle resistibility, muscular endurance, training adaptation, a normal state of muscle that will allow joint movement, or standard physiological metabolism and function of skeletal muscle in a healthy mammal. Any functional variable of skeletal muscle can be measured, including muscle resistivity (maximum force generated in a specific movement), muscle endurance (the maximum number of contractions that can be performed at a defined frequency and force), and muscle power (force/time, the maximum effect generated by the muscle). Although not exhaustive, typical muscle-specific functions include myoblast differentiation, myoblast determination, muscle development, muscle contraction, sarcomeric changes, myoblast fusion, somatic muscle development, and myogenesis.

[0133] In some embodiments, a patient's skeletal muscle fibrosis is evaluated. Several methods are available to determine the status of skeletal muscle fibrosis, including obtaining a biopsy of muscle tissue from the patient and evaluating the biopsy with sensitive histochemical or immunohistochemical stains to detect the existence of fibrotic tissue. Examples of histochemical stains include, for example, hematoxylin and eosin (H&E), trichrome, and ATPase (e.g., at pH 4.3, 4.65, and 10.4). Representative antibodies that can be used to identify muscle fibers for immunohistochemical staining include, for example, myosin, type IV collagen, laminin, fibronectin, and dystrophin. Alternatively, a functional method for determining the extent to which fibrosis penetrates a patient's skeletal muscle can be employed. The functional method involves subjecting the patient to one or more of a battery of physical tests and measurements. Such tests and measurements typically include tests of neurological resistivity, muscular resistivity, balance, gait, posture, sensory coordination assessments, and tests of pulmonary function, e.g., vital capacity and forced expiratory capacity, all of which can be performed by methods known in the art. technique. In some embodiments, tissue repair can be assessed based on the level (or levels) of expression of one or more signaling molecules described herein. Biomarkers suitable as indicators of tissue repair include, but are not limited to, a DNA damage biomarker, an inflammatory response biomarker, a tissue damage biomarker, a tissue damage repair biomarker, or a hematology surrogate marker. such as p53, p21, GADD45a, ATM, phosphorylated histone H2AX, IL-6, CRP, SAA, IL-1, IL-5, IL-10, KC/GRO, IFN, IL-2, IL-4, TNF- alpha, IL-12, IL-3, IL-7, IL-6, salivary beta-amylase, citrullinated proteins, S100B, SP-D, BPI, TSP, CA15-3, CDBB, CKMB, CKMM, FABP2, GFAP, NSE, CD5, CD-16b, CD20, CD177, CD26, CD27, CD40, CD45, Flt-3L, G-CSF, KFG, EPO, TPO, GM-CSF or SDF-1α.

[0134] Copper (including copper ion) is a regulator of one or more factors (e.g., transcriptional factors) involved in tissue damage repair and/or tissue regeneration, and consequently, tissue repair can be assessed by evaluating any one or more of these factors. Copper-regulated factors include, but are not limited to: Cu homeostasis proteins such as Ctr 1, Ctr 3, DMT1, Atox 1, ATP7A/7B, Cox 17, CCS, Sco 1/2, Cox 11, D- glutamatergic N-methyl aspartate (NMDAR), Amyloid precursor protein (APP), MURR1 copper metabolism gene domain (COMMD1), X-linked inhibitor of apoptosis (XIAP), homocysteine (Hcy), subunit II of cytochrome c oxidase (COX II), cytochrome c oxidase subunit I (COX I), FGF-1, VEGF, angiopoietin (such as ANG1 or ANG2), fibronectin, collagenase, MMPs-TIMPs, elastin, PDGF and eNOS; intracellular Cu-binding proteins such as Cytochrome C oxidase (CCO), Superoxide dismutase (SOD), Metallothionein (MT), Glutathione (GSH), Dopamine-β-monooxygenase (DBH), Peptidylglycine-α-amidating mono- oxygenase (PAM), Tyrosinase, Phenylalanine hydroxylase, Diamine oxidase, Hephaestine cartilage matrix glycoprotein; Extracellular Cu-binding proteins such as Ceruloplasmin (CP), Lysyl oxidase (LOX), Albumin (ALB), Transcuprein, amine oxidase, Blood clotting factors V and VIII, Ferroxidase II, extracellular Superoxide dismutase and extracellular Metallothionein. Factors regulated by copper are disclosed in Zheng et al., Role of copper in regression of cardiac hypertrophy, Pharmacol. The R. doi:10.1016/j.pharmthera.2014.11.014 (2014), which is incorporated into this document by reference. In some embodiments, copper or copper ion regulates the transcriptional activity of one or more of HIF-1, SP1, MT, Atox 1, CCS and COMMD1 and the signaling networks regulated by these transcriptional factors.

[0135] In some embodiments, the level and/or activity of one or more copper-regulated factors disclosed herein are analyzed in an individual following treatment with a therapeutic or preventive composition disclosed herein. In some embodiments, the level and/or activity of one or more of HIF-1, SP1, MT, Atox 1, CCS, and COMMD1 are determined and then related to an individual's response to the preventive therapeutic composition. In some embodiments, the response is detected by measuring cellular markers of normal tissue homeostasis and/or persistent ischemic tissue damage (e.g., by immunohistochemistry or measuring DNA and transcript levels), measuring the area of damage or volume of harm or assessment of any clinically relevant indicators. Thus, in certain aspects, the level and/or activity of one or more copper-regulated factors (such as HIF-1, SP1, MT, Atox 1, CCS, and COMMD1) can be used as an endpoint biomarker of the response. of an individual to a therapeutic or preventive regimen disclosed herein.

[0136] In some embodiments, one or more copper-regulated factors disclosed herein may be used in a prognostic test to analyze and predict a response to a tetramine composition or treatment or preventive method disclosed herein. For example, the level and/or activity of one or more of HIF-1, SP1, MT, Atox 1, CCS and COMMD1 may indicate a probability that an individual will respond positively to a treatment or preventive composition disclosed herein, the treatment or preventive composition can be administered to the individual. Conversely, if the level and/or activity of one or more of HIF-1, SP1, MT, Atox 1, CCS, and COMMD1 indicates that an individual is unlikely to respond or will respond negatively to treatment or preventive composition, an alternative course of treatment can be prescribed. A negative response can be defined as either the absence of an effective response or the presence of negative side effects. The response to a therapeutic or preventive treatment can be predicted in a prior study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, treatment and a population that responds adversely to a treatment regimen (e.g., exhibits one or more side effects). These populations are provided as examples, and other populations and subpopulations can be analyzed. Based on the results of these analyses, an individual is genotyped to predict whether he or she will respond favorably to a treatment regimen, will not respond significantly to a treatment regimen, or will respond adversely to a treatment regimen. Thus, in some embodiments, the level and/or activity of one or more of HIF-1, SP1, MT, Atox 1, CCS, and COMMD1 can be used as indicators of an individual's response to a therapeutic or preventive regimen disclosed in the this document. Response indicators can be assessed before, during and/or after administration of the therapeutic or preventive regimen. For example, one or more indicators of response may be assessed during intervals between doses of continuous administration to assess whether the individual is likely to benefit from continued treatment or whether alternative treatment is necessary.

[0137] The prognostic tests described above are applicable to clinical tests. One or more response indicators (such as HIF-1, SP1, MT, Atox 1, CCS and COMMD1) can be identified using the methods described herein. Thereafter, potential participants in clinical trials of a tetramine composition comprising a copper-chelating tetramine and, optionally, a copper-promoting composition comprising a copper ion can be screened to identify those individuals who are most likely to respond to the tetramine composition. and exclude those likely to experience side effects. In this way, treatment efficacy can be measured in individuals who respond positively to the tetramine composition, without reducing the measurement as a result of including individuals who are not likely to respond positively in the study and without risking undesirable safety issues.

[0138] In some embodiments, a method is provided for inducing tissue repair in an individual who has ischemic tissue injury without increasing VEGF expression at the injection site comprising administering to the individual an effective amount of a tetramine composition that comprises a copper chelating tetramine. In some embodiments, a method is provided for inducing blood vessel growth toward the site of ischemic injury in a subject comprising administering to the subject an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, a method is provided for inducing blood vessel growth toward the site of ischemic injury in a subject without increasing VEGF expression at the injection site comprising administering to the subject an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0139] The formation and growth of blood vessels within a tissue can occur by angiogenesis and/or vasculogenesis. In some embodiments, the blood vessels include capillary-like structures that are fully functional to support blood transport. In some embodiments, angiogenesis includes a process involving the growth of new blood vessels from pre-existing vessels, sprouting angiogenesis, the formation of new blood vessels by sprouting from existing ones, or splitting angiogenesis (intussusception), the formation of new blood vessels by dividing from existing ones. In some embodiments, vasculogenesis includes a process that involves the de novo production of new blood vessels by proliferation of endothelial stem cells, such as the formation of new blood vessels when there are no pre-existing ones.

[0140] In some embodiments, the formation and growth of blood vessels requires signals from growth factors and other proteins that directly control the process, such as angiopoietins (such as Ang-1 and Ang-2), ephrin (Eph), vascular endothelial growth factors (such as VEGF-A and VEGF-C), platelet-derived growth factor (PDGF), fibroblast growth factors (such as FGF-1 and FGF-2), tumor necrosis factor-α (TNF-α ), interleukin (IL), monocyte chemoattractant protein 1 (MCP-1) (also known as CCL-2), transforming growth factor α (TGF-α), transforming growth factor βs (such as TGF-β1, TGF-β2, TGF-β3 and TGF-β4), endostatin, vasoibin, chemokines, thrombospondin, angiostatin, vascular cell adhesion molecule (such as VCAM-1), matrix metalloproteinases (such as MMP-2 and MPP-9), integrins , cadherins, plasminogen activators and plasminogen activator inhibitors.

[0141] In some embodiments, blood vessel growth is assessed by measuring endothelial cell proliferation, which is necessary to develop capillaries in an intact animal. In some embodiments, the action of administering a tetramine composition comprising a copper-chelating tetramine on endothelial proliferation can be assessed by direct cell counts, DNA synthesis and/or metabolic activity. For example, endothelial cells can be isolated from the site of ischemic injury and assessed for their rate of proliferation after treatment with a tetramine composition comprising a copper chelating tetramine. In other embodiments, endothelial cell proliferation at the site of ischemic injury can be monitored by identifying cells and measuring cell counts, DNA synthesis and/or metabolic activity in situ. In other embodiments, the identified endothelial cells can be administered to an individual, and the proliferation of identified endothelial cells at the site of ischemic injury can be monitored in situ. In some embodiments, endothelial cells are identified with a radioisotope, a fluorescent chemical moiety, or a marker that can be specifically detected, for example, by an antibody. In specific embodiments, cells are identified with [3H]thymidine or bromodeoxyuridine (BrdU).

[0142] In some embodiments, blood vessel growth is assessed by measuring the migration of endothelial cells, which degrade the basement membrane and migrate along chemical gradients established by proangiogenic growth factors, for example, during sprouting angiogenesis. In certain embodiments, endothelial cells at the site of ischemic injury are identified and cell migration is monitored in vivo. In other aspects, the identified endothelial cells are administered to a subject, and their migration toward the site of ischemic injury is monitored in vivo. In other aspects, endothelial cells at the site of ischemic injury can be isolated and their migratory properties can be assessed by a variety of in vitro assays, including the Boyden chamber assay, agarose assay, wound healing assay, circ. Teflon, phagokinetic screening assay and similar assays.

[0143] In some embodiments, blood vessel growth is assessed by measuring endothelial cell formation tubes with lumens to conduct blood flow, e.g., tubulogenesis. In some embodiments, blood vessel growth is assessed by an aortic ring assay. An aortic ring assay to evaluate blood vessel growth is disclosed in Li et al., “Copper promotion of angiogenesis in isolated rat aortic ring: role of vascular endothelial growth factor”, Journal of Nutritional Biochemistry 25(2014) 44 to 49 , the disclosure of which is incorporated into this document by reference in its entirety. Budding microvessels from the aortic ring interact closely with resident macrophages, pericytes, and fibroblasts in an ordered sequence that emulates angiogenesis in the intact animal. In some embodiments, the endothelial cells have been preselected by passaging and are thus in a quiescent state similar to that of the intact animal. Other angiogenesis assays that incorporate angiogenic functions (such as matrix degradation, migration, proliferation, tube formation) include the embryoid assay, mouse metatarsal assay, and similar assays.

[0144] In some embodiments, an in vivo assay is used to measure blood vessel growth after administration of a tetramine composition comprising a copper chelating tetramine. These assays include and are not limited to the corneal angiogenesis assay, chick chorioallantoic membrane assay, and Matrigel plug assay. For example, the cornea is the only tissue in the body that is both avascular and transparent, making it ideal for observing angiogenesis. In some embodiments, pellets or sponges containing pro-angiogenic molecules (e.g., a tetramine composition comprising a copper-chelating tetramine as disclosed herein) can be implanted into surgically created stromal pockets. The ingrowth of new vessels from the peripheral limb vasculature can be monitored daily, allowing rates of angiogenesis to be determined. In a Matrigel plug assay, a Matrigel containing the tetramine composition (with or without copper ion) as disclosed herein may be implanted in an individual at or near the site of ischemic injury, and the Matrigel plug is subsequently removed to visualize blood vessels. In some embodiments, endothelial cells are identified with one or more markers, and their proliferation, migration, tubulogenesis, blood vessel formation, and/or blood vessel growth at the site of ischemic injury are assessed in vivo, e.g., using of an appropriate imaging technique.

COMBINATION THERAPY

[0145] The tetramine composition and, optionally, in combination with the copper-promoting composition described above can be used as a single agent or as part of a combination therapy with stem cells or stem cell inducers to induce repair of an ischemic tissue. In some embodiments, there is provided a method for inducing tissue repair (or improving tissue function) in a subject who has ischemic tissue injury comprising: a) administering to the subject an effective amount of a tetramine composition comprising a copper chelating tetramine; and b) administering to the subject an effective quantity of stem cells (such as mesenchymal stem cells (MSC), e.g., bone marrow mesenchymal stem cells (BMSC)) or a stem cell inducer. In some embodiments, the method comprises administering to the individual an effective quantity of stem cells (such as MSC, e.g., BMSC). In some embodiments, the method comprises administering to the subject an effective amount of stem cell inducer. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0146] In some embodiments, the stem cell disclosed herein is a mesenchymal stem cell (MSC), a bone marrow mesenchymal stem cell (BMSC), a multipotent stem cell, and a pluripotent stem cell induced (iPS) or a tissue-derived stem cell. In some embodiments, the stem cell is a stem cell derived from adipose tissue, a stem cell derived from cardiac tissue, or a stem cell derived from umbilical cord tissue. In some embodiments, the stem cell is an inducer of an adult stem cell. In some embodiments, the adult stem cell is a hematopoietic stem cell, a mammalian stem cell, an intestinal stem cell, a mesenchymal stem cell in the placenta, adipose tissue, lung, bone marrow, blood, marrow jelly, Wharton from the umbilical cord or teeth (such as the perivascular niche of the dental pulp and periodontal ligament), an endothelial stem cell, a neural stem cell, an adult olfactory stem cell, a neural crest stem cell, or a -germline stem (e.g. a stem cell in the testis).

[0147] In some embodiments, the stem cell inducer disclosed herein is an inducer of a mesenchymal stem cell (MSC), a bone marrow mesenchymal stem cell (BMSC), a multipotent stem cell, an induced pluripotent stem (iPS) cell or a tissue-derived stem cell, such as a stem cell derived from adipose tissue, a stem cell derived from cardiac tissue, or a stem cell derived from umbilical cord tissue. In some embodiments, the stem cell inducer is an inducer of an adult stem cell, such as a hematopoietic stem cell, a mammalian stem cell, an intestinal stem cell, a mesenchymal stem cell in the placenta, adipose tissue, lung, bone marrow, blood, Wharton's jelly from the umbilical cord or teeth (such as the perivascular niche of the dental pulp and periodontal ligament), an endothelial stem cell, a neural stem cell, an adult stem cell olfactory cell, a neural crest stem cell, or a germline stem cell (e.g., a stem cell in the testis).

[0148] In some embodiments, stem cells or stem cell inducer are administered systemically. In some embodiments, the stem cells or stem cell inducer are administered locally to the ischemic tissue. In some embodiments, the stem cells or stem cell inducer are administered locally to a site other than the site of ischemic injury.

[0149] In some embodiments, the stem cells (or the stem cell inducer), the tetramine composition (with or without copper ion) and, optionally, the copper promoting composition are administered simultaneously. In some embodiments, a stem cell disclosed herein (or the stem cell inducer) and the tetramine composition (with or without copper ion) and, optionally, the copper promoting composition are administered sequentially in any suitable order. .

[0150] Since the stem cells (or stem cell inducer), the tetramine composition (with or without copper ion) and, optionally, the copper promoting composition described herein are administered to a mammal (e.g., a human), the presence and/or biological activity of cells, in some embodiments, is monitored by any of several known methods. In some embodiments, cells migrate in vivo from an ischemic tissue of an individual, and the presence and/or biological activity of the cells en route to a site of tissue damage is monitored and/or regulated.

[0151] Although the methods described herein are generally applicable to all aspects of tissue repair, it should be understood that combination therapy methods can be used for the purpose of any one or more of the following : induce stem cell migration from bone marrow mesenchyme to ischemic tissue, induce stem cell differentiation in ischemic tissue, induce tissue regeneration in ischemic tissue, induce a signaling molecule that triggers tissue regeneration, promote copper-dependent HIF-1 transcriptional activities, reverse damage at the site of ischemic injury, and reconstruct the microenvironment of neurofibril cells and neurosecretory cells at the site of ischemic injury.

METHODS FOR PREVENTION AND PROPHYLACTIC USE

[0152] Also provided herein are methods for preventing damage to ischemic tissue in an individual comprising administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine. In some embodiments, the ischemic tissue is selected from the group consisting of ischemic heart tissue, ischemic liver tissue, ischemic brain tissue, ischemic lung tissue, ischemic kidney tissue, ischemic skin tissue, ischemic tract tissue. digestive and ischemic skeletal muscle tissue (such as limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition forms a complex with the copper chelating tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises , two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition does not form a complex with the copper chelating tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the method further comprises administering to the subject a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the subject previously receives a copper-promoting composition that can increase the subject's extracellular copper level. In some embodiments, the copper promoting composition is a copper ion. In some embodiments, the copper promoting composition does not comprise a copper ion. In some embodiments, the copper-promoting composition can increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily.

[0153] In some embodiments, a method for preventing ischemic heart failure in an individual is provided which comprises administering to the individual an effective amount of a tetramine composition comprising a copper chelating tetramine (such as trientine). In some embodiments, the effective amount of the tetramine composition is insufficient to reduce the level of extracellular copper in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (such as about 80 mg to about 300 mg or about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice daily. In some embodiments, the tetramine composition is administered for at least about 1 month (such as at least about 3 months or at least about 6 months). In some embodiments, the subject has left ventricular ejection function (LVEF) of at most about 35% at baseline. In some embodiments, the individual has class II or class III heart failure (based on the New York Heart Association or NYHA functional classification). In some embodiments, the individual has at least about 150 pg/ml of plasma B-type natriuretic peptide (BNP). In some embodiments, the individual has a maximum of about 600 pg/ml of NT-proBNP (N-terminal pro-BNP).

[0154] “Prevent”, as used herein, includes providing prophylaxis in relation to the occurrence or recurrence of a disease in an individual who may be predisposed to the disease, but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the development of a disease or to slow the progression of a disease, such as tissue damage.

[0155] For the prevention or treatment of disease, the appropriate dosage or route of administration depends on the type of disease being treated, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, the previous therapy , the individual's clinical history and response to tetramine compositions and/or cells and the discretion of the treating physician. Tetramine compositions, copper-promoting compositions, stem cells, and stem cell inducers are, in some embodiments, suitably administered to the individual at one time or over a series of treatments.

[0156] In some embodiments, the present disclosure provides compositions and methods for treating and preventing damage to ischemic tissue. In some embodiments, the tetramine compositions, the copper-promoting compositions and/or the cells disclosed herein are administered before, during and/or after a treatment that will cause or is likely to cause tissue damage in an individual, and the administration prevents or reduces damage to ischemic tissue associated with cancer treatment, such as radiotherapy and chemotherapy.

[0157] In some embodiments, the tetramine composition or the method disclosed herein prevents ischemic tissue damage or reduces the area, volume, or duration of ischemic tissue damage by inducing migration (e.g., addressing) of a stem cell to the tissue, even after the tissue in the individual has otherwise lost the inherent ability to spontaneously recruit stem cells. In embodiments, administration of the tetramine composition and/or cells of the present disclosure triggers a series of other events that lead to increased resistance to ischemic tissue damage, including, for example, inducing stem cell differentiation at the tissue site. , induce tissue regeneration at the tissue site, induce a signaling molecule that triggers tissue regeneration, promote tissue-dependent HIF-1 transcriptional activities, reverse damage at the site of an initial ischemic injury before further damage occurs, and/or reconstruct the microenvironment of neurofibril cells and neurosecretory cells at the tissue site.

[0158] For example, myocardial ischemia or infarction can lead to irreversible loss of functional heart tissue with possible deterioration of pump function and death. Occlusion of a coronary vessel leads to interruption of the blood supply to the dependent capillary system. If nutrition and oxygen, cardiomyocytes die and undergo necrosis. Inflammation of the surrounding tissue occurs with invasion of inflammatory cells and phagocytosis of cell debris. Fibrotic scar formation occurs, and the afflicted region of the heart loses its contractive strength. Without intervention, the only way for the heart muscle to compensate for tissue loss is hypertrophy of the remaining cardiomyocytes (accumulation of cellular protein and contractive elements within the cell). Endocrine, metabolic (alcohol) or infectious agents (viral myocarditis) and cancer treatment agents also lead to cell death, with consequently reduced myocardial function. In some embodiments, the tetramine composition or method disclosed herein prevents damage to ischemic heart tissue or reduces the area, volume, or duration of damage to ischemic heart tissue. In some embodiments, the tetramine composition disclosed herein induces migration (e.g., targeting) and/or retention of mesenchymal stem cells (e.g., BMSCs) to ischemic cardiac tissue. In some modalities, in cases of ischemia or myocardial infarction, the cardiac muscle can compensate for tissue loss by differentiating stem cells into cardiomyocytes, thus preventing or reducing cardiac hypertrophy and damage to cardiac tissue. .

TETRAMINE COMPOSITIONS

[0159] Also provided herein are tetramine compositions (including pharmaceutical compositions) comprising a copper-chelating tetramine (such as trientine) to increase the level of intracellular copper, deliver copper to cells, induce at least one (such as at least any of 2, 3, 4, 5, 6, 7 or more) tissue repair events, induce stem cell migration, or promote copper-dependent HIF-1 transcriptional activities in an ischemic tissue of a individual who has ischemic tissue damage. Any of the tetramine compositions, optionally in combination with copper and/or stem cell promoting (or stem cell inducer) compositions, can be used in the methods described above.

[0160] In some embodiments, a tetramine composition is provided that comprises a copper chelating tetramine or a pharmaceutically acceptable salt thereof. The copper chelating tetramine (such as trientine) contemplated herein includes, but is not limited to, the copper chelating tetramine compound itself, pharmaceutically acceptable salts thereof, active metabolites thereof, prodrugs thereof and derivatives thereof. In some embodiments, the copper chelating tetramine is trientine. In some embodiments, the copper-chelating tetramine is a trientine analog, such as a tetramine of Formula (II), as described in the “Copper and copper-chelating tetramines” section. In some embodiments, the tetramine composition does not comprise a trace element, such as copper. In some embodiments, the composition can chelate copper ion in the blood and deliver the copper ion to cells in an ischemic tissue.

[0161] In some embodiments, a tetramine composition is provided that comprises a mixture of a copper chelating tetramine and a copper ion. In some embodiments, the tetramine composition comprises a mixture of trientine and a copper ion. In some embodiments, the relative ratio (mol for mole) between the copper chelating tetramine (such as trientine) and the copper ion is about any one of 100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:50 or 1: 100. In some embodiments, the relative molar ratio between the copper chelating tetramine (such as trientine) and the copper ion is about 1:1. In some embodiments, the relative ratio (mol for mole) between the copper chelating tetramine (such as trientine) and the copper ion is about any one of 50:1 to 100:1, 20:1 to 50:1, 10:1 to 20:1, 5:1 to 10:1, 4:1 to 5:1, 3:1 to 4:1, 2:1 to 3:1, 1:1 to 2:1, 1: 2 to 1:1, 1:3 to 1:2, 1:4 to 1:3, 1:5 to 1:4, 1:10 to 1:5, 1:20 to 1:10, 1:50 to 1:20, 1:100 to 1:50, 1:100 to 1:10, 1:10 to 1:1, 1:1 to 10:1, 10:1 to 100:1 or 1:10 to 10: 1. In some embodiments, at least a fraction of the copper ion is in a complex with the copper chelating tetramine. In some embodiments, the copper ion is not in a complex with the copper chelating tetramine.

[0162] In some embodiments, a tetramine composition is provided that comprises a complex of a copper-chelating tetramine and a copper ion. In some embodiments, the tetramine composition comprises a trientine complex and a copper ion. In some embodiments, the stoichiometry between the copper chelating tetramine (such as trientine) and the copper ion is about 1:1. In some embodiments, the complex of the copper chelating tetramine (such as trientine) and the copper ion is crystalline. In some embodiments, the crystalline complex of the copper chelating tetramine (such as trientine) and the copper ion is a thermodynamic polymorph. Different thermodynamic polymorphs of the crystal complex can be described by a specific set of geometric structures, unit cell dimensions, space groups, and structural coordinates, which can be determined using skills known in the art, such as X-ray crystallography. embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises two chloride ions and a water molecule. In some embodiments, the tetramine composition comprises a crystalline complex of Formula (I), as shown below, where Cu is a copper ion and the dotted lines denote hydrogen bonds. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and a copper ion, wherein the crystalline complex has the crystal structure as shown in Figure 1. In some embodiments, the crystal structure has bond lengths, angles, bond angles and torsional angles as listed in Figure 2. In some embodiments, the crystal complex comprises crystals with the space group and parameters as listed in Figure 3. In some embodiments, the tetramine composition comprises a trientine crystal complex and an ion copper, where the crystal structure of the crystal complex is defined by the atomic coordinates as listed in Figures 4A to 4C.

[0163] In some embodiments, the tetramine composition comprises a complex of the copper chelating tetramine (such as trientine) and the copper ion, wherein the complex is not crystalline. In some embodiments, the tetramine composition further comprises the copper ion is not in a complex with the copper chelating tetramine. In some embodiments, the relative ratio (mol for mole) between the copper ion that is not in complex with the copper chelating tetramine and the complex is about any one of 1:1, 1:2, 1:3, 1 :4, 1:5, 1:10, 1:20, 1:50 or 1:100. In some embodiments, the relative ratio (mol for mole) between the copper ion that is not in complex with the copper chelating tetramine and the complex is about any one of 1:2 to 1:1, 1:3 to 1 :2, 1:4 to 1:3, 1:5 to 1:4, 1:10 to 1:5, 1:20 to 1:10, 1:50 to 1:20, 1:100 to 1:50 , 1:100 to 1:10 or 1:10 to 1:1. In some embodiments, the percentage of total copper in the tetramine composition that is in complex with the copper chelating tetramine (such as trientine) is about any one of 1%, 5%, 10%, 20%, 30%, 40 %, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, the percentage of total copper in the tetramine composition that is in complex with the copper chelating tetramine (such as trientine) is about any one of 1% to 5%, 5% to 10%, 10% to 20%. %, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 1% to 10%, 10% to 50%, 50% to 80% or 80% to 100%. In some embodiments, the percentage of total copper chelating tetramine (such as trientine) in the tetramine composition that is in complex with the copper ion is at least about any one of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, the percentage of total copper chelating tetramine (such as trientine) in the tetramine composition that is in complex with the copper ion is about any one of 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100 %, 1% to 10%, 10% to 50%, 50% to 80% or 80% to 100%. In some embodiments, the copper ion is not in complex with the copper chelating tetramine (such as trientine) is present as a salt, such as copper sulfate, chloride chloride, copper oxide, copper nitrate, copper acetate , copper formate, copper gluconate, copper amino acid chelates and the like.

[0164] In some embodiments, a tetramine composition is provided that comprises a copper-chelating tetramine and a copper ion, wherein the copper ion does not form a complex with the copper-chelating tetramine. In some embodiments, the tetramine composition comprises trientine and a copper ion, wherein the copper ion does not form a complex with trientine. In some embodiments, the copper ion is present as a salt, such as copper sulfate, copper chloride, copper oxide, copper gluconate, copper amino acid chelates, and the like. In some embodiments, the relative ratio (mol for mole) between the copper chelating tetramine (such as trientine) and the copper ion is about any one of 100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:50 or 1: 100. In some embodiments, the relative ratio (mol for mole) between the copper chelating tetramine (such as trientine) and the copper ion is about any one of 50:1 to 100:1, 20:1 to 50:1, 10:1 to 20:1, 5:1 to 10:1, 4:1 to 5:1, 3:1 to 4:1, 2:1 to 3:1, 1:1 to 2:1, 1: 2 to 1:1, 1:3 to 1:2, 1:4 to 1:3, 1:5 to 1:4, 1:10 to 1:5, 1:20 to 1:10, 1:50 to 1:20, 1:100 to 1:50, 1:100 to 1:10, 1:10 to 1:1, 1:1 to 10:1, 10:1 to 100:1 or 1:10 to 10: 1.

[0165] Many factors of the tetramine composition, including, but not limited to, chemical structure of the copper chelating tetramine, the ratio of the copper chelating tetramine to the copper in the tetramine composition, interactions of the copper ion with the copper chelating tetramine copper (e.g., if it is in a complex, if the complex is crystalline, etc.), may affect the ability of the tetramine composition to deliver (such as discharge) copper into cells in an ischemic tissue. For example, the copper chelating tetramine may have a conformation (including chelate denticity, donor linkage groups, and cavity size) that promotes reversible binding of the copper ion. In some embodiments, the tetramine composition comprises a copper-chelating tetramine with sufficiently low affinity for the copper ion within cells in ischemic tissue, wherein the tetramine composition dissociates and discharges the copper ion within the cells. In some embodiments, the tetramine composition comprises additional compounds and/or agents that increase copper ion unloading within cells in ischemic tissue.

[0166] There are also provided pharmaceutical compositions comprising any of the tetramine compositions described herein and one or more carriers, excipients, stabilizing agents, diluents and/or other pharmaceutically acceptable agents, which are known in the art, for use in the methods described in this document.

[0167] Accordingly, in one aspect of the present application, there is provided a pharmaceutical composition comprising a copper chelating tetramine and a copper ion. In some embodiments, a pharmaceutical composition comprising trientine and a copper ion is provided.

[0168] In some embodiments, a pharmaceutical composition is provided that comprises a complex of a copper chelating tetramine and a copper ion. In some embodiments, a pharmaceutical composition comprising a trientine complex and a copper ion is provided. In some embodiments, the complex of the copper chelating tetramine (such as trientine) and the copper ion is crystalline. In some embodiments, the crystalline complex of the copper chelating tetramine (such as trientine) and the copper ion is a thermodynamic polymorph. In some embodiments, the pharmaceutical composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by the four amine groups of trientine to adopt a planar square geometry, and wherein the crystalline complex further comprises two chloride ions and a water molecule. In some embodiments, the complex of the copper chelating tetramine (such as trientine) and the copper ion is not crystalline.

[0169] In some embodiments, there is provided a pharmaceutical composition comprising a copper chelating tetramine and a copper ion, wherein at least a fraction (such as at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) of the copper ion is not in complex with the copper chelating tetramine. In some embodiments, there is provided a pharmaceutical composition comprising trientine and a copper ion, wherein at least a fraction (such as at least about any one of 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90% or more) of the copper ion is not in complex with trientine. In some embodiments, a pharmaceutical composition is provided that comprises a copper chelating tetramine and a copper ion, wherein the copper ion does not form a complex with the copper chelating tetramine. In some embodiments, a pharmaceutical composition comprising trientine and a copper ion is provided, wherein the copper ion does not form a complex with the copper chelating tetramine. In some embodiments, the copper ion is not in complex with the copper chelating tetramine is present as a salt, such as copper sulfate, chloride chloride, copper oxide, copper nitrate, copper acetate, copper formate, copper gluconate, copper amino acid chelates and the like.

[0170] Any of the pharmaceutical compositions described herein can be used to increase the level of intracellular copper in an ischemic tissue in an individual who has ischemic tissue injury, inducing at least two (such as any one of at least 2, 3, 4, 5, 6, 7 or more) tissue repair events, promote copper-dependent HIF-1 transcriptional activities and/or treat (including prevent) any disease or condition associated with ischemic tissue injury.

[0171] The pharmaceutical compositions described herein can be formulated as solutions, emulsions, suspensions, dispersions or inclusion complexes, such as cyclodextrins in suitable solvents or pharmaceutical carriers or as pills, tablets, lozenges, suppositories, sachets, dragees, granules , powders, powders for reconstitution or capsules together with solid carriers in accordance with conventional methods known in the art for preparing various dosage forms. The pharmaceutical compositions of the embodiments may be administered by a suitable route of delivery, such as oral, parenteral, rectal, nasal, topical or ocular routes or by inhalation. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for parental administration (such as intravenous administration).

[0172] For oral administration, the pharmaceutical composition may be provided in a solid form, such as a tablet or a capsule, or as a solution, an emulsion or a suspension. In some embodiments, the pharmaceutical composition is formulated as a tablet, capsule, or pill. Oral tablets may include the active ingredient (or ingredients) mixed with compatible pharmaceutically acceptable excipients, such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preserving agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Exemplary liquid oral excipients include ethanol, glycerol, water and the like. Starch, polyvinyl pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose and alginic acid are exemplary disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract or may be coated with an enteric coating. Oral formulations may be presented as distinct units, such as capsules, tablets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as a liquid oil-in-water emulsion or a liquid water-in-oil emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

[0173] A tablet can be produced by compression or shaping, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient into a free-flowing form, such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, diluent inert, preservative, disintegrator (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Shaped tablets can be produced by shaping, in a suitable machine, a mixture of the moistened powder compound with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated to provide slow and controlled release of the active ingredient therein with the use, for example, of hydroxypropylmethylcellulose in varying proportions to provide the desired profile and release.

[0174] Capsules for oral administration include hard or soft gelatin capsules. To prepare hard gelatin capsules, the active ingredient (or ingredients) can be mixed with a solid, semisolid, or liquid diluent. Soft gelatin capsules can be prepared by mixing the active ingredient with water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of short-chain fatty acid mono- and diglycerides, polyethylene glycol 400 or propylene glycol. Capsules may also contain gelatin, iron oxides, stearic acid and titanium dioxide as inactive ingredients.

[0175] Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups or may be lyophilized or presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically acceptable excipients, such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g. oil (e.g. almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol or water; preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents, such as lecithin; and, if desired, flavoring or coloring agents.

[0176] For parenteral use, including intravenous, intramuscular, intraperitoneal, intranasal or subcutaneous routes, tetramine compositions can be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms may be presented in unit dose form, such as ampoules or disposable injection devices, in multiple dose forms, such as vials from which the appropriate dose can be withdrawn, or in a solid or pre-concentrate form which can be used to prepare an injectable formulation. Formulations suitable for parenteral, including intravenous, administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Formulations may be represented in single-dose or multi-dose containers, e.g., ampoules and sealed vials, and may be stored in a freeze-dried (lyophilized) condition that requires only the addition of sterile liquid carrier, e.g., water. for injections, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above.

DOSAGE AND ADMINISTRATION METHODS

[0177] When used in vivo for any of the treatment methods described herein, the tetramine composition (including the pharmaceutical composition) and, optionally, the copper and/or stem cell promoting composition (or stem cell inducer) trunk) are administered to the individual in effective amounts. An “effective amount” is at least the minimum concentration required to effect a measurable improvement or prevention of the disease or condition associated with ischemic tissue injury. An effective amount herein may vary depending on factors such as the degree of ischemic injury in the individual, the characteristics of the particular tetramine composition, the copper ion and/or stem cells (or stem cell inducers) used. , for example, its therapeutic index, the individual (such as age, gender, weight and medical history). An effective amount is also one in which any toxic or harmful effects of the treatment are offset by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, decrease in severity or delay of onset of the disease or condition, including biochemical, histological and/or behavioral symptoms of the disease or condition, its complications and phenotypes. intermediate pathological conditions that appear during the development of the disease or condition. For therapeutic use, beneficial or desired results include clinical results, such as decreasing one or more symptoms resulting from the disease or condition, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications necessary to treat the disease, intensifying the effect of another medication, such as directing, slowing the progression of the disease and/or prolonging survival.

[0178] In some embodiments, the effective amount of the tetramine composition (such as pharmaceutical composition) and, optionally, in combination with the effective amount of the copper promoting composition are effective to result in an increase of more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more tissue intracellular copper level ischemic status of the subject compared to the intracellular copper level of the subject's ischemic tissue prior to treatment. In some embodiments, the effective amount of the tetramine composition (such as pharmaceutical composition) and, optionally, in combination with the effective amount of the copper promoting composition are effective to result in an increase of more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more in the total copper level of an ischemic tissue in the individual compared to the total ischemic tissue copper level in the individual before treatment. In some embodiments, the effective amount of the tetramine composition (such as a pharmaceutical composition) and, optionally, in combination with the effective amount of the copper-promoting composition do not decrease the extracellular copper level (such as the blood copper level) in the individual. In some embodiments, the effective amount of the tetramine composition (such as a pharmaceutical composition) and, optionally, in combination with the effective amount of the copper-promoting composition do not reduce the level of extracellular copper (such as the level of copper in the blood) in the individual by more than about any one of 5%, 10%, 20%, 30%, 40%, 50% or more compared to the individual's extracellular copper level before treatment. In some embodiments, the effective amount of the tetramine composition (such as pharmaceutical composition) and, optionally, in combination with the effective amount of the copper-promoting composition do not decrease the total copper level in the individual. In some embodiments, the effective amount of the tetramine composition (such as pharmaceutical composition) and, optionally, in combination with the effective amount of the copper-promoting composition do not reduce the total copper level in the individual by more than about any one of 5 %, 10%, 20%, 30%, 40%, 50% or more compared to the total copper level in the individual before treatment.

[0179] An effective amount can be administered in one or more administrations. As understood in the clinical context, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be achieved. or is hit.

[0180] The effective amount, doses and dosage regimen of the tetramine composition alone or in combination with the copper (such as copper ion) and/or stem cell (or stem cell inducer) promoting composition can be determined during preclinical testing and clinical trials by methods familiar to physicians and clinicians. In some embodiments, the effective amount of the copper chelating tetramine (such as trientine) in the tetramine composition is less than about any one of 0.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 80 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg or 1,200 mg. In some embodiments, the effective amount of the copper chelating tetramine (such as trientine) in the tetramine composition is any one of about 0.5 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 75 mg, about 75 mg to about 100 mg, about 100 mg to about 125 mg, about 125 mg to about 150 mg, about 150 mg to about 175 mg, about 175 mg to about 200 mg, about 200 mg to about 225 mg, about 225 mg to about 250 mg, about 250 mg to about 275 mg, about 275 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg to about 400 mg, about 400 mg to about 500 mg, about 500 mg to about 600 mg, about 600 mg to about 1,200 mg, about 1,200 mg to about 2,400 mg, about 0.5 mg to about 50 mg, about 50 mg to about 100 mg, about 10 mg to about 125 mg, about 80 mg to about 200 mg, about 150 mg to about 300 mg, about 200 mg to about 300 mg, about 300 mg to about 600 mg, about 0.5 mg to about 200 mg, about 80 mg to about 300 mg, or about 80 mg to about 400 mg, or about 80 mg to about 450 mg. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg of the copper-chelating tetramine (such as in dichloride salt form) per day for a human patient.

[0181] In some embodiments, the effective amount of copper-chelating tetramine (such as trientine) in the tetramine composition (such as pharmaceutical composition) includes at least about any one of 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg. In some embodiments, the effective amount of copper-chelating tetramine (such as trientine) in the tetramine composition (such as pharmaceutical composition) includes less than about any one of 35 mg/kg, 30 mg/kg, 25 mg/kg, 20 mg/kg, 18 mg/kg, 15 mg/kg, 10 mg/kg, 5 mg/kg, 2.5 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg or 0 .1 mg/kg. In some embodiments, the effective amount of copper-chelating tetramine in the tetramine composition is any one of about 1 mg/kg to about 10 mg/kg, about 10 mg/kg to about 15 mg/kg, about 15 mg/kg to about 20 mg/kg, about 20 mg/kg to about 25 mg/kg, about 25 mg/kg to about 30 mg/kg, about 30 mg/kg to about 40 mg/kg, about 1 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50 mg/kg, about 15 mg/kg to about 50 mg/kg, about 15 mg /kg to about 40 mg/kg or about 20 mg/kg to about 35 mg/kg per day. In some embodiments, the effective amount of the tetramine composition is at most about 20 mg/kg per day, such as about 18 mg/kg per day for a Rhesus monkey. In some embodiments, the effective amount of the tetramine composition is at most about 15 mg/kg to about 35 mg/kg per day for a mouse.

[0182] In some embodiments, the effective amount of the copper chelating tetramine (such as trientine) in the pharmaceutical composition (e.g., a unit dosage form) is in the range of about 5 mg to about 300 mg, such as about from 80 mg to about 150 mg, about 80 mg to about 200 mg, about 200 mg to about 300 mg, or about 80 mg to about 300 mg. In some embodiments, the concentration of the copper chelating tetramine (such as trientine) in the pharmaceutical composition is diluted (about 0.1 mg/ml) or concentrated (about 100 mg/ml), including, for example, any of about 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml or about 1 to about 10 mg/ml.

[0183] Exemplary dosage frequencies include, but are not limited to, any of four times a day, three times a day, twice a day, once for two days, once for three days, once for four days or weekly. In some embodiments, the tetramine composition (such as pharmaceutical composition) is administered at least about any one of 1x, 2x, 3x, 4x, 5x, 6x or 7x (i.e., daily) per week. In some embodiments, the intervals between each administration are less than about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 4 hours, or 3 hours. In some embodiments, the tetramine composition is administered daily. In some embodiments, the tetramine composition is administered at least twice daily. In some embodiments, the tetramine composition is administered at least once, including, for example, at least any one of 2x, 3x, or 4x daily.

[0184] In some embodiments, the effective amount of the tetramine composition combined with the dosing frequency are sufficient to maintain a high concentration of the tetramine (such as trientine) in the individual, such as in the blood or ischemic tissue. In some embodiments, a high concentration of the tetramine is a concentration that promotes copper-dependent HIF-1 transcriptional activities or induces at least one (including at least 2, 3, 4, 5, 6, 7 or more) copper repair events. tissue. In some embodiments, administration of the tetramine composition (such as a pharmaceutical composition) leads to at least about 0.005 mg/l (including, for example, at least about any one of 0.01 mg/l, 0.05 mg /l, 0.1 mg/l, 0.5 mg/ml,1.0 mg/l, 2.0 mg/l, 3.0 mg/l, 4.0 mg/l or 5.0 mg/ l) copper chelating tetramine in the blood. The concentration of tetramine in a biological sample (such as blood or a biopsy of ischemic tissue) can be determined using methods known in the art, such as fluorescence spectroscopy, mass spectroscopy or chromatography methods, or by measuring the level of an identified tetramine.

[0185] In some embodiments, the effective amount of the tetramine composition combined with the dosing frequency are sufficient to maintain a high concentration of the tetramine (such as trientine) in the individual for at least about 4, 5, 6, 7, 8 , 9, 10 or more hours. In some embodiments, the effective amount of the tetramine composition combined with the dosing frequency are sufficient to maintain a high concentration of the tetramine (such as trientine) in the subject for at least about 8 or more hours.

[0186] In some embodiments, the effective amount of the tetramine composition is about at least any one of 1 mg/kg/day, 2 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 12 mg/kg/day, 15 mg/kg/day, 18 mg/kg/day, 20 mg/kg/day, 30 mg/kg/day, 40 mg/kg/day, 50 mg/kg/day or more of trientine in the tetramine composition. In some embodiments, the effective amount of the tetramine composition is at most any one of 1 mg/kg/day, 2 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 12 mg/kg/day. day, 15 mg/kg/day, 18 mg/kg/day, 30 mg/kg/day, 40 mg/kg/day or 50 mg/kg/day of trientine in tetramine composition. In some embodiments, the effective amount of the tetramine composition is any one of about 1 mg/kg/day to about 2 mg/kg/day, about 2 mg/kg/day to about 5 mg/kg/day. , about 5 mg/kg/day to about 10 mg/kg/day, about 10 mg/kg/day to about 15 mg/kg/day, about 15 mg/kg/day to about 20 mg /kg/day, about 20 mg/kg/day to about 30 mg/kg/day, about 30 mg/kg/day to about 50 mg/kg/day, about 1 mg/kg/day to about 5 mg/kg/day, about 5 mg/kg/day to about 15 mg/kg/day, about 1 mg/kg/day to about 10 mg/kg/day or about 1 mg/day kg/day to about 18 mg/kg/day of trientine in the tetramine composition. In some embodiments, the effective amount of the tetramine composition is about 18 mg/kg/day of trientine in the tetramine composition. In some embodiments, the effective amount of the tetramine composition is about 1 mg/kg/day to about 10 mg/kg/day of trientine in the tetramine composition.

[0187] Administration of the tetramine composition (such as pharmaceutical composition) can be over an extended period of time, such as from about any one week to about several years. In some embodiments, the tetramine composition (such as a pharmaceutical composition) is administered over a period of at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 18, 24, 30, 36, 48, 60, 72 or 84 months or more. In some embodiments, the tetramine composition (such as pharmaceutical composition) is administered for at least about one month, including, for example, about any one of 1, 2, 3, 4, 5, 6, 8, 10, 12 or more months. In some embodiments, the tetramine composition (such as a pharmaceutical composition) is administered for at least about one month, and wherein the tetramine composition is administered at least once (such as twice, three times, or four times) per month. day. In some embodiments, administration of the tetramine composition (including pharmaceutical composition) leads to at least about 0.005 mg/l (including, for example, at least about any one of 0.01 mg/l, 0.05 mg/l). l, 0.1 mg/l, 0.5 mg/ml,1.0 mg/l, 2.0 mg/l, 3.0 mg/l, 4.0 mg/l or 5.0 mg/l ) of the copper chelating tetramine in the blood for at least about 1 week (including, for example, at least about any one of 2 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 12 months or more ).

[0188] Any of the tetramine compositions (such as pharmaceutical compositions) described herein can be administered to an individual (such as a human being) via various routes, including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, transdermal, intratumoral, direct injection into the blood vessel wall, intracranial or intracavity. In some embodiments, sustained continuous release formulation of the tetramine composition (such as pharmaceutical composition) may be used. In some embodiments, the tetramine composition is administered parenterally. In some embodiments, the tetramine composition is administered orally. In some embodiments, the tetramine composition is administered directly to the ischemic tissue (e.g., using direct delivery methods described below). In some embodiments, the tetramine composition is administered via an interventional method, such as angioplasty.

[0189] In some embodiments, the tetramine composition (such as a pharmaceutical composition) can be administered with a second therapeutic compound and/or a second therapy. The dose and dosage frequency of the tetramine composition (such as pharmaceutical composition) and the second compound may be adjusted over the course of the treatment based on the judgment of the administering physician. In some embodiments, the first and second therapies are administered simultaneously, sequentially or concomitantly. “Concurrent administration,” as used herein, may indicate that a first therapy and a second therapy in a combination therapy are administered at a time separation of at most about 15 minutes, such as at most about any one of 10, 5 or 1 minute. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both a first and a second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition). “Sequential administration” may indicate that the first therapy and the second therapy in a combination therapy are administered at a time separation of at most about 15 minutes, such as at most about any one of 20, 30, 40, 50 minutes. , 60 or more minutes. The first therapy or the second therapy may be administered first. The first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits. Concomitant administration may indicate that the administration of the first therapy and the second therapy in a combination therapy overlap each other. When administered separately, the pharmaceutical composition and the second compound may be administered at different frequency or dosage intervals. For example, the tetramine composition (such as a pharmaceutical composition) may be administered daily, while a second compound may be administered more or less frequently. In some embodiments, the sustained continuous release formulation of the tetramine composition and/or the second compound may be used. Various formulations and devices for achieving sustained release are known in the art. A combination of the administration settings described in this document can be used. In some embodiments, the second compound is a copper ion (such as a copper salt or chelated copper). In some embodiments, the second therapy comprises stem cells or a stem cell inducer.

[0190] In some embodiments, an effective amount of a copper-promoting composition comprising a copper ion is further administered to the individual. In some embodiments, the effective amount of the copper-promoting composition administered to the subject is sufficient to increase the subject's extracellular copper level by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more compared to the individual's extracellular copper level before treatment. In some embodiments, the effective amount of the copper-promoting composition administered to the subject is sufficient to increase the total copper level of the subject's ischemic tissue by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more compared to the total copper level of the subject's ischemic tissue before treatment.

[0191] In some embodiments, an effective amount of the copper-promoting composition administered to the subject is sufficient to increase the subject's extracellular copper level by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more compared to the individual's extracellular copper level before treatment. In some embodiments, the effective amount of the copper-promoting composition administered to the subject is sufficient to increase the total copper level of the subject's ischemic tissue by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more compared to the total copper level of the subject's ischemic tissue before treatment. In some embodiments, a copper-promoting composition that does not comprise a copper ion can be used to increase the level of extracellular copper. For example, the copper-promoting composition may increase copper absorption, decrease copper excretion, and/or decrease zinc toxicity.

[0192] In some embodiments, the effective amount of the copper ion in a copper-promoting composition comprising a copper ion is included in any of the following ranges: about 0.01 mg to about 0.1 mg, about from 0.1 mg to about 0.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 8 mg, about 8 mg to about 10 mg, about 0.01 mg to about 1 mg or about 0 .1 mg to about 2.5 mg. In some embodiments, the effective amount of the copper ion in the copper-promoting composition administered to the subject includes at least about any one of 5 mcg/kg, 10 mcg/kg, 20 mcg/kg, 30 mcg/kg, 50 mcg/kg, kg, 100 mcg/kg, 200 mcg/kg, 300 mcg/kg, 400 mcg/kg, 500 mcg/kg, 600 mcg/kg, 700 mcg/kg, 800 mcg/kg, 900 mcg/kg or 1,000 mcg/kg kg. In some embodiments, the effective amount of the copper ion in the copper-promoting composition administered to the subject includes less than about any of 1,000 mcg/kg, 900 mcg/kg, 800 mcg/kg, 700 mcg/kg, 600 mcg/kg, kg, 500 mcg/kg, 400 mcg/kg, 300 mcg/kg, 200 mcg/kg, 100 mcg/kg, 50 mcg/kg, 30 mcg/kg, 20 mcg/kg, 10 mcg/kg or 5 mcg/kg kg.

[0193] In some embodiments, the copper-promoting composition is administered daily or twice daily. In some embodiments, the copper-promoting composition is administered with the same dosage frequency and duration as the tetramine composition. In some embodiments, the copper-promoting composition is administered with a different dosage frequency and/or duration than the tetramine composition. In some embodiments, the copper-promoting composition is administered orally. The effective amount and dosage frequency of the copper-promoting composition can be determined during preclinical testing and clinical testing by methods familiar to physicians and clinicians.

[0194] Once improvement in the patient's disease has occurred, the dose can be adjusted for preventive or maintenance treatment. For example, the dosage or frequency of administration, or both, may be reduced as a function of symptoms to a level at which the desired therapeutic or prophylactic effect is maintained. Obviously, if symptoms have been alleviated to an adequate level, treatment can cease. Patients, however, may require intermittent treatment on a long-term basis upon any recurrence of symptoms. Patients may also require chronic treatment on a long-term basis.

[0195] Various direct delivery methods are known in the art and can be used to administer the tetramine composition (such as a pharmaceutical composition) and/or the copper promoting composition.

[0196] In some embodiments, the tetramine composition and/or the copper promoting composition are delivered via a microbubble. In some embodiments, the copper ion is delivered via peptide-based nanoparticles comprising copper. In some embodiments, the tetramine composition and/or the copper-promoting composition are delivered or targeted to the ischemic tissue passively through a physical effect that induces release or delivery to the ischemic tissue via a nanoparticle (or microsphere).

[0197] In some embodiments, the tetramine composition and/or the copper-promoting composition are delivered by directly administering the tetramine composition and/or the copper-promoting composition to the ischemic tissue. In some embodiments, the tetramine composition and/or the copper-promoting composition disclosed herein are administered orally to the site of ischemic tissue injury. In some embodiments, the tetramine composition and/or the copper-promoting composition are absorbed through the digestive tract. In one aspect, the absorbed copper composition and/or ion is directed (by active targeting or passive targeting) to a site of ischemic injury and is released locally at the site of ischemic injury to provide an effective local concentration of the tetramine composition and /or the copper-promoting composition for tissue repair. In some embodiments, the orally delivered copper ion forms a compound or complex with a protein, a peptide, an amino acid, or mono-, di-, or polysaccharide. In some embodiments, the copper ion forms a compound or complex with one or more polymers. In other embodiments, the copper ion is in an organometallic compound, such as a small molecule organometallic compound.

[0198] In some embodiments, a sustained delivery composition disclosed herein includes long-acting injectables (e.g., base injections, injectable suspensions, injectable microspheres, and injectable in situ systems) containing the tetramine composition and/or the composition of copper, agents and polymers for depot injections, commercially available depot injections and injectable sustained release delivery systems. In certain embodiments, a sustained release composition disclosed herein comprises a polymeric matrix from which an agent is released by diffusion and/or degradation of the polymeric matrix. Therefore, the agent release pattern is mainly determined by the polymer matrix, as well as the percentage loading and manufacturing method. In some embodiments, sustained release preparations use a biodegradable polymer. In this case, sustained-release preparations do not require surgical removal of the preparations from the subject. Typically, such preparations are slowly degraded and absorbed by the patient's body and ultimately disposed of along with other soluble metabolic waste products.

[0199] In some embodiments, a polymeric injectable depot system is used to deliver an in situ forming implant containing the tetramine composition and/or the copper composition to the site of ischemic injury. In situ formation implant systems are typically produced from biodegradable products, which can be injected via a syringe into the body, and once injected, freeze to form a solid biodegradable implant. In some embodiments, the implant is formed from thermoplastic pastes, in situ cross-linked polymers, in situ polymeric precipitation, thermally induced gelation, or in situ solidifying organogels. The deposition formation mechanism of thermoplastic pastes must form a semisolid upon cooling to body temperature after injection into the body in molten form. Cross-linked polymer networks can be achieved in situ in various ways, forming solid polymer systems or gels. Methods for in situ cross-linked systems include free radical reactions, usually initiated by heat or photon absorption, or ionic interactions between small polymeric cations and anions. In situ formations can be produced causing polymer precipitation from solution. A biodegradable, water-insoluble polymer is solubilized in a biocompatible organic solvent to which a drug is added that forms a solution or suspension after mixing. When this formulation is injected into the body, the water-miscible organic solvent dissipates and water penetrates the organic phase. This leads to phase separation and precipitation of the polymer that forms a deposit at the injection site. Thermally induced systems show thermoreversible solid/gel transitions and are characterized by a lower critical solution temperature. They are at room temperature and produce a gel at and above the critical solution temperature. In situ solidifying organogels comprise water-insoluble amphiphilic lipids, which swell in water and form various types of lyotropic liquid crystals.

[0200] In some embodiments, the tetramine composition and/or the copper-promoting composition is injected into the site of ischemic injury, for example, by percutaneous perforation, by an interventional catheter, or by intravertebral injection. In some embodiments, the tetramine composition and/or the copper-promoting composition is delivered directly to a site of ischemic injury with the use of a coated implant, stent or plate or an implant impregnated with the tetramine composition and/or the copper-promoting composition. copper promoter. In some embodiments, the tetramine composition and/or the copper-promoting composition is delivered directly to a site of ischemic injury by slowly releasing the tetramine composition and/or the copper-promoting composition from an intravascular stent secured with the tetramine composition. and/or the copper promoting composition. In some embodiments, the tetramine composition and/or the copper-promoting composition is delivered to the site of ischemic injury by a positive targeting liposome or an acceptor-donor complex. In some embodiments, the tetramine composition and/or the copper-promoting composition is delivered to the site of ischemic injury using physical therapy, ultrasound, iontophoresis, ultrasound penetration enhancement, electroporation and/or sponge application. Application of the tetramine composition and/or cells to the site of ischemic injury may be topical (e.g., through the skin), may be to some location in the injured tissue that is internal to the surface of the body, or both. For example, the tetramine composition and/or the copper promoting composition can be delivered via iontophoresis through the blood vessel, an endothelial cell layer, or other tissues internal to the site of ischemic injury to provide an effective local concentration of the copper composition. tetramine and/or the copper-promoting composition for tissue repair.

[0201] In some embodiments, a sustained release composition disclosed herein comprises a biodegradable polymer for controlled delivery of the tetramine composition and/or the copper promoting composition. Suitable biodegradable polymers typically include polylactides (PLA), polyglycolides (PGA), poly(lactide-co-glycolide) (PLGA), poly(ε-caprolactone) (PCL), polyglyconate, polyanhydrides, polyorthoesters, poly(dioxanone) and polyalkylcyanoacrylates. In some embodiments, the sustained release composition comprises injectable biodegradable microspheres such as PLGA microspheres, PCL microspheres, polyanhydride microspheres, polyorthoester microspheres and polyalkylcyanoacrylate.

[0202] In particular embodiments, a range of types of copper-containing compounds can be used for localized delivery of the copper-promoting composition directly to a site of ischemic injury. Examples of suitable copper ion-containing solutions are solutions of copper (I) chloride, copper (II) chloride, copper acetate and copper sulfate. In some embodiments, copper forms a compound or complex with a protein, a peptide, an amino acid, a mono-, di-, or polysaccharide, one or more polymers, or a small molecule and the compound or complex is used to direct localized delivery to the site of ischemic injury. In some embodiments, an organometallic compound containing copper ion is used to direct localized delivery to the site of injury.

[0203] In some embodiments, the concentration of copper ions in the copper-promoting composition used for localized delivery directly to a lesion site is from about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 40 μM, about 40 μM to about 60 μM, about 60 μM to about 80 μM, about 80 μM to about 100 μM, about 100 μM to about 200 μM, about 200 μM to about 400 μM, about 400 μM to about 600 μM, about 600 μM to about 800 μM, about 800 μM to about 1 mM, about 1 mM to about 5 mM, about 5mM to about 10mM, about 10mM to about 20mM, about 20mM to about 40mM or about 40mM to about 60mM. Copper ion concentration can be determined during preclinical testing and clinical testing by methods familiar to physicians and clinicians.

COPPER AND COPPER CHELATING TETRAMINES

[0204] The terms “copper”, “copper ion” and “copper element” are used interchangeably herein. In biological systems, copper ions usually exist in two oxidation states, cuprous state (Cu1+, copper (I) or reduced) and cupric state (Cu2+, copper (II) or oxidized). In some embodiments, copper includes both cuprous and copper states. In some embodiments, the copper is in the cupric (Cu2+) state. In some embodiments, the copper is in the cuprous state (Cu1+). In some embodiments, the copper is a free ion, that is, unbound or in a complex with another molecule, such as a protein or a small organic molecule. In some embodiments, the copper is in a salt form. In some embodiments, copper is present as a salt selected from copper sulfate, copper chloride, copper oxide, copper gluconate and copper amino acid chelates. In some embodiments, copper is present as a complex-forming ion. In some embodiments, the copper is in an organometallic compound, such as a small molecule organometallic compound. In some embodiments, the copper is in a complex with a copper chelating tetramine. In some embodiments, copper is a complex ion, including the various species of ions resulting from the introduction of copper into a cell, a tissue or an organism in accordance with the present disclosure. In some embodiments, copper forms a compound or complex with a protein, a peptide, an amino acid, or mono, di, or polysaccharide. Important copper-binding proteins found in biological systems include, but are not limited to, cytochrome c oxidase (CcO), copper-zinc superoxide dismutase (Cu, Zn-SOD), dopamine D-hydroxylase (DBH), prion protein ( PrP), tyrosinase, X-linked inhibitor of apoptosis proteins (XIAP), lysyl oxidase, metallothionein (MT) and ceruloplasmin. In some embodiments, the copper is in a form not available for absorption or use by an ischemic tissue, such as in a complex with ceruloplasmin. In some embodiments, the copper is in a form available for absorption or use by an ischemic tissue. In some embodiments, the copper disclosed herein is an inducer of HIF-1 transcriptional activity.

[0205] The copper promoting composition comprising any species of copper ion described above can be used in the methods described herein. In some embodiments, the copper promoting composition comprises a Cu2+. In some embodiments, the copper promoting composition comprises a Cu1+. In some embodiments, the copper-promoting composition comprises a copper ion in a salt form (such as any one or a combination selected from copper sulfate, copper chloride, copper oxide, copper gluconate, and copper amino acid chelates). ). In some embodiments, the copper-promoting composition comprises an organometallic compound. In some embodiments, the copper-promoting composition does not comprise a copper ion or a copper compound.

[0206] “Copper level” in reference to any of the methods described herein refers to the concentration of any of the copper species described above, such as Cu2+, Cu1+, or the concentration of total copper (e.g., Cu1+ and Cu2+ and/or free or alloyed copper). In some embodiments, the copper level refers to the copper state level. In some embodiments, the copper level refers to the level of copper that is in a form available for absorption or use by an ischemic tissue.

[0207] “Copper chelating tetramine” refers to a copper-binding or chelating tetramine compound. In some embodiments, the copper chelating tetramine binds to Cu2+. In some embodiments, the copper chelating tetramine binds to Cu1+. In some embodiments, the copper chelating tetramine is specific for (i.e., with higher affinity to) Cu2+ relative to Cu1+. In some embodiments, the copper chelating tetramine forms a complex with a copper ion in a distorted square-flat, distorted square-flat, trigonal-pyramidal, square-pyramidal, or distorted octahedral conformation. In some embodiments, the copper chelating tetramine alters the balance between Cu1+ and Cu2+ in cells or organisms. In some embodiments, the copper chelating tetramine may alter (such as decrease) the copper level (such as total copper level) in the individual. In some embodiments, the copper chelating tetramine may alter (such as decrease) the copper level (such as total copper level) in the individual's blood. In some embodiments, the copper chelating tetramine can increase the intracellular copper level (such as total or cupric copper level). In some embodiments, the copper chelating tetramine can increase the concentration of copper in a form available to an ischemic tissue in the individual. In some embodiments, the copper chelating tetramine can redirect intracellular trafficking and/or inter-tissue or inter-organ transport of copper. In some embodiments, the copper chelating tetramine specifically binds to and/or is absorbed by an ischemic tissue, such as ischemic cardiac tissue. In some embodiments, the copper chelating tetramine (including its complex with copper) is permeable through the membrane. In some embodiments, the copper chelating tetramine (including its complex with copper) is lipid soluble. In some embodiments, the stoichiometry between the copper chelating tetramine and copper in a complex thereof is about 1:1. In some embodiments, the copper chelating tetramine binds to the copper ion reversibly. In some embodiments, the copper chelating tetramine binds to the copper ion with a sufficiently low affinity within cells in ischemic tissue that allows unloading or dissociation of the copper ion.

[0208] Any copper-chelating tetramine that can increase the level of intracellular copper can be used in the methods described herein. Copper chelating tetramine may refer to the compounds themselves, pharmaceutically acceptable salts, active metabolites, derivatives and prodrugs thereof, as well as stereoisomers, enantiomers, racemic mixtures and the like, when applicable. In some embodiments, the copper chelating tetramine is linear. In some embodiments, the copper chelating tetramine is branched. In some embodiments, the copper chelating tetramine is cyclic. In some embodiments, the copper chelating tetramine is selected from triethylenetetramine (2,2,2-tetramine), 2,3,2-tetramine and 3,3,3-tetramine.

[0209] In some embodiments, the copper chelating tetramine is trientine. “Trientine” is also called triethylenetetramine, 2,2,2-tetramine, N,N'-Bis(2-aminoethyl)-1,2-ethanediamine, 1,8-diamino-3,6-diazaoctane, 3,6- diazaoctane-1,8-diamine, 1,4,7,10-tetraazadecane, triene, TETA, TECZA, N,N'- Bis(aminoethyl)ethylenediamine, N,N'-Bis(2-aminoethyl)ethanediamine and N,N'-Bis(2-aminoethyl)-ethylenediamine. In some embodiments, trientine is a compound of the formula: NH2(CH2)2NH(CH2)2NH(CH2)2NH2 or a pharmaceutically acceptable salt thereof.

[0210] Other copper-chelating tetramines with similar copper-chelating properties may include, but are not limited to, compounds of Formula (II) and their pharmaceutically acceptable salts thereof:

[0211] In some embodiments, the copper chelating tetramine is an acyclic compound of Formula (II), in which R1, R2, R3, R4, R5 and R6 are independently chosen from H, CH3, C2-C10 alkyl of straight or branched chain, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono-, di-, tri-, tetra- and penta-substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 mono alkyl aryl , di, tri, tetra and penta substituted, C1-C5 alkyl heteroaryl, C1-C6 fused alkyl aryl, CH2COOH, CH2SO3H, CH2PO(OH)2, CH2P(CH3)O(OH); n1, n2, and n3 are independently chosen to be 2 or 3; and R7, R8, R9, R10, R11 and R12 are independently chosen from H, CH3, C2-C10 straight or branched chain alkyl, C3-C10 cycloalkyl, C1-C6 alkyl, C3-C10 cycloalkyl, aryl, mono aryl , di, tri, tetra and penta substituted, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl aryl mono, di, tri, tetra and penta substituted, C1-C5 alkyl heteroaryl, C1C6 fused alkyl aryl. Additionally, one or more of R1, R2, R3, R4, R5 or R6 may be functionalized for binding, for example, to peptides, proteins, polyethylene glycols and other chemical entities in order to modify the overall pharmacokinetics, delivery capacity and/or or the half-lives of the constructs. Examples of such functionalization include, but are not limited to, C1-C10 alkyl-CO-peptide, C1C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1C10 alkyl-NH- protein, C1-C10 alkyl-NH—CO-PEG, C1-C10 alkyl-S-peptide, C1C10 alkyl-S-protein. Furthermore, one or more of R7, R8, R9, R10, R11, or R12 may be functionalized for binding, for example, to peptides, proteins, polyethylene glycols, and other chemical entities to modify the overall pharmacokinetics, deliverability, and /or the half-lives of the constructs. Examples of such functionalization include, but are not limited to, C1-C10 alkyl-CO-peptide, C1C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1C10 alkyl-NH- protein, C1-C10 alkyl-NH—CO-PEG, C1-C10 alkyl-S-peptide and C1-C10 alkyl-S-protein.

[0212] In some embodiments, the copper chelating tetramine is a cyclic compound of Formula (II), in which R1 and R6 are joined to form the bridging group (CR13R14)n4, and in which R2, R3, R4 and R5 are independently chosen from H, CH3, C2-C10 straight or branched chain alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl aryl mono, di, tri, tetra and penta substituted, C1-C5 alkyl heteroaryl, C1-C6 fused alkyl aryl, CH2COOH, CH2SO3H, CH2PO(OH)2, CH2P(CH3)O(OH); n1, n2 and n3 are independently chosen to be 2 or 3; and R7, R8, R9, R10, R11, R12, R13 and R14 are independently chosen from H, CH3, C2-C10 straight or branched chain alkyl, C3-C10 cycloalkyl, C1-C6 alkyl, C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1 -C6 alkyl aryl, C1 -C6 mono, di, tri, tetra and penta substituted alkyl aryl, C1 - C5 alkyl heteroaryl, C1 - C6 fused alkyl aryl. Additionally, one or more of R2, R3, R4 or R5 may be functionalized for binding, for example, to peptides, proteins, polyethylene glycols and other chemical entities in order to modify the general pharmacokinetics, delivery capacity and/or half- lives of the constructs. Examples of such functionalization include, but are not limited to, C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH—CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein. Furthermore, one or more of R7, R8, R9, R10, R11, R12, R13 and R14 can be functionalized for binding, for example, to peptides, proteins, polyethylene glycols and other chemical entities to modify the general pharmacokinetics, delivery capacity and/or the half-lives of the constructs. Examples of such functionalization include, but are not limited to, C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl- NH-protein, C1-C10 alkyl-NH—CO-PEG, C1-C10 alkyl-S-peptide and C1-C10 alkyl-S-protein.

[0213] “Pharmaceutically acceptable salts” refer to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases or inorganic or organic acids and the like. When a compound is basic, for example, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such salts include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic acid, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic and similar. In some embodiments, pharmaceutically acceptable salts of the copper chelating tetramine are selected from hydrochloride salt (e.g., triethylenetetramine dihydrochloride), succinate salt (e.g., triethylenetetramine disuccinate), maleate salt (e.g., triethylenetetramine tetramaleate), and fumarate (e.g. triethylenetetramine tetrafumarate). The copper chelating tetramine, such as trientine, may also be in the form of quaternary ammonium salts in which the nitrogen atom bears a suitable organic group, such as an alkyl, alkenyl, alkynyl or aralkyl moiety. Copper chelating tetramine metabolites may include, but are not limited to, acetylated metabolites, such as N-acetyl triethylenetetramine (e.g., monoacetyl-triethylenetetramine). Copper chelating tetramine derivatives may include, but are not limited to, PEG-modified tetramines (such as trientine-PEG).

[0214] Copper chelating tetramine, including trientine, can be prepared using any of a variety of chemical synthesis, isolation and purification methods known in the art. For example, see U.S. Patent No. 4,806,517, U.S. Patent No. 4,550,209, U.S. Patent No. 5,225,599, U.S. Patent No. 4,766,247, Patent No. EP262562, U.S. Patent No. 8,394,992, and Patent Publication No. US20130108709 A1.

INDIVIDUAL WHO HAS ISCHEMIC TISSUE INJURY

[0215] “Ischemic tissue injury” described herein refers to an injury to a tissue, including, for example, cardiovascular, liver, brain, skeletal muscle and the like, that results in a restriction in the blood supply to the tissue , causing a shortage of oxygen and glucose necessary for cellular metabolism in the tissue. The injury may involve any of a number of pathological conditions or trauma by an external force, resulting in disturbance of blood flow. In some embodiments, the ischemic tissue injury is cardiovascular ischemia. In some embodiments, the ischemic tissue injury is cerebral ischemia or ischemic stroke. In some embodiments, the ischemic tissue injury is limb ischemia, such as lower limb ischemia. In some embodiments, the ischemic tissue injury is intestinal ischemia, such as ischemic colitis or mesenteric ischemia. In some embodiments, the ischemic tissue injury is cutaneous ischemia. In some embodiments, ischemic tissue injury is associated with embolism, thrombosis, aneurysm, trauma, myocardial infarction, mitral valve disease, chronic atrial fibrillation, cardiomyopathy, prosthesis, exit from thoracic outlet syndrome, atherosclerosis, hypoglycemia, tachycardia, hypotension, include tumor of a blood vessel, sickle cell disease, cold ulcer, arteriovenous malformation, peripheral artery occlusive disease, rupture of significant blood vessels, anemia, diabetes, diabetic foot ulcers, necrotizing enterocolitis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, restenosis (after angioplasty or stent implantation) or pancreatitis. In some embodiments, ischemic tissue damage is associated with cardiomyopathy. In some embodiments, ischemic tissue injury is associated with myocardial infarction. In some embodiments, ischemic tissue damage is associated with diabetes.

[0216] The methods described herein are therefore generally applicable to many diseases involving ischemic tissue injury. These diseases include, but are not limited to, myocardial infarction, cardiomyopathy, aneurysm, angina, aortic stenosis, aortitis, arrhythmias, arteriosclerosis, arteritis, asymmetric septic hypertrophy (ASH), atherosclerosis, atrial fibrillation and ripple, bacterial endocarditis, Barlow Syndrome ( mitral valve prolapse), bradycardia, Buerger's disease (thromboangiitis obliterans), cardiomegaly, carditis, carotid artery disease, coarctation of the aorta, congenital heart defects, congestive heart failure, coronary artery disease, Eisenmenger Syndrome, embolism, endocarditis, erythromelalgia, fibrillation, fibromuscular dysplasia, bundle branch block, heart murmur, hypertension, hypotension, idiopathic infantile arterial calcification, Kawasaki disease (mucocutaneous lymph node syndrome, mucocutaneous lymph node disease, infantile polyarteritis), metabolic syndrome, microvascular angina , myocarditis, paroxysmal atrial tachycardia (PAT), periarteritis nodosa (polyarteritis, polyarteritis nodosa), pericarditis, peripheral vascular disease, critical limb ischemia, phlebitis, pulmonary valve stenosis (pulmonary stenosis), Raynaud's disease, renal artery stenosis, renovascular hypertension, rheumatic heart disease, diabetic vasculopathy, septic defects, silent ischemia, syndrome varicose veins, vasculitis, ventricular septic defect, Wolff-Parkinson-White Syndrome, endocardial cushion defect, acute rheumatic fever, acute rheumatic pericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis, chronic rheumatic heart disease, mitral valve disease, mitral stenosis, rheumatic mitral insufficiency, aortic valve diseases, diseases of other endocardiac structures, ischemic heart disease (acute and subacute), angina pectoris, acute pulmonary heart disease, pulmonary embolism, chronic pulmonary heart disease, kyphoscoliotic heart disease, myocarditis, endocarditis, fibrosis endomyocardial disease, endocardial fibroelastosis, atrioventricular block, cardiac dysrhythmia, myocardial degeneration, cerebrovascular disease, a disease of arteries, arterioles and capillaries or a disease of veins and lymphatic vessels; an acquired brain injury, traumatic brain injury, infarction (which includes ischemic, intracerebral hemorrhagic, subarachnoid hemorrhagic), anoxic injuries, metabolic disorders, encephalitis, and brain injuries due to infection. In certain embodiments, diseases involving ischemic tissue damage include systemic sarcoidosis, a skin disease or condition, Lofgren syndrome, a lung disease or condition, a heart disease or condition, an eye disease or condition, a liver disease or condition, a musculoskeletal disease or condition and a kidney disease or condition. The present application, therefore, also comprises treating any of the diseases using methods described herein.

[0217] An “individual”, “subject” or “patient” described herein refers to a mammal, such as mice, rats, rabbits, cats, dogs, pigs, cows, cattle, sheep, goats, horses, monkeys and other non-human primates and humans, a vertebrate such as fish, and a bird such as chicken. Mammals can include farm animals, sporting animals, rodents, and pets. In some embodiments, the individual is a human being.

[0218] The methods described in the present invention are applicable to an individual who has one or more ischemic tissue injuries, including, but not limited to: ischemic myocardial injury, ischemic brain injury, ischemic spinal cord injury, ischemic muscle injury, skeletal ischemic injury, acute tubular necrosis, ischemic injury of the intestines, ischemic lung injury, ischemic liver injury, ischemic kidney injury, ischemic skin injury, hernia, vascular anastomoses, atherosclerotic plaque, hemangioma and after penetrating or blind traumatic injury.

[0219] In some embodiments according to any of the methods described herein, the individual does not have a compromised tissue repair system. In some embodiments, the individual has a compromised tissue repair system. Individuals with a compromised tissue repair system may have one or more of the following characteristics: (a) advanced age (such as at least about 60 years of age, including, for example, at least about any one of 65, 70, 75, 80, 85, 90 or more years of age); (b) chronic tissue injury (such as an individual who has had tissue injury for at least about any one of 6, 7, 8, 9, 10, 11, 12, 18, or 24 months); (c) stem cell deficiency; (d) deficiency in migration (i.e., addressing) of stem cells; (e) a defective tissue repair system; and (f) one or more of the following symptoms or conditions: memory loss, low or reduced mobility capacity (including, but not limited to, strength capacity, speed resistance, flexibility and joint mobility), hypoesthesia, muscle weakness, hearing loss and chronic stressors.

[0220] In some embodiments, the individual is at least about any one of 20, 30, 40, 50, 60, 70, 80 or more years of age. In some embodiments, the individual is younger than about anyone 20, 30, 40, 50, 60, 70, 80 years of age. In some embodiments, the individual has chronic ischemia. In some embodiments, the individual has copper efflux from ischemic tissue (such as ischemic myocardium) into the blood circulation. In some embodiments, the individual has a decreased level (less than about any one of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) copper in ischemic tissue. In some embodiments, the decreased level of copper in ischemic tissue is caused by chronic ischemic conditions. In some embodiments, the individual has repressed HIF-1 transcriptional activity.

[0221] In some embodiments, the individual is selected for treatment based on their copper level, such as intracellular copper level, extracellular copper level, total copper level, and serum (i.e., blood) copper level. Individuals with chronic ischemic conditions usually have an individually low copper level in ischemic tissue, such as less than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the average intracellular copper level of the corresponding tissue in healthy individuals. Serum copper level, such as serum total copper level, serum protein-bound (e.g., ceruplasmin) copper level, or serum free (i.e., unbound) copper, for individuals under chronic ischemic conditions is usually higher (such as about any one of 1.2 times, 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times or more) than the average serum copper level of healthy individuals. In some embodiments, prior to administration of the tetramine composition, the subject has at least about any one of 60 μg/dl, 70 μg/dl, 80 μg/dl, 90 μg/dl, 100 μg/dl, 110 μg/dl, dl, 120 μg/dl, 130 μg/dl, 140 μg/dl, 150 μg/dl, 175 μg/dl, 200 μg/dl, 250 μg/dl, 300 μg/dl or more serum total copper level . In some embodiments, prior to administration of the tetramine composition, the subject has at most about any of 1 times, 1.2 times, 1.5 times, 1.75 times, or 2 times the average serum total copper level. of healthy individuals. In some embodiments, prior to administration of the tetramine composition, the subject has a maximum of about any one of 60 μg/dl, 70 μg/dl, 80 μg/dl, 90 μg/dl, 100 μg/dl, 110 μg/dl, dl, 120 μg/dl, 130 μg/dl, 140 μg/dl, 150 μg/dl, 175 μg/dl, 200 μg/dl, 250 μg/dl, 300 μg/dl serum total copper level. In some embodiments, upon administration of the tetramine composition, the subject has at least about any one of 50%, 60%, 70%, 80%, 90% or more of the average total copper level in the serum of healthy subjects. In some embodiments, upon administration of the tetramine composition, the individual has at least about any of 60 μg/dl, 70 μg/dl, 80 μg/dl, 90 μg/dl, 100 μg/dl, 110 μg/dl , 120 μg/dl, 130 μg/dl, 140 μg/dl, or 150 μg/dl serum total copper level.

[0222] In some embodiments, the individual is selected for treatment based on their level of HIF-1 activity. In some embodiments, the individual has repressed transcriptional activity of HIF-1 target genes in ischemic tissue. In some embodiments, the individual has a high level (such as protein or RNA level) of HIF-1α in the ischemic tissue, but repressed transcriptional activity of HIF-1 target genes in the ischemic tissue. In some embodiments, the individual has chronic ischemia that results in repressed HIF-1 activity.

MANUFACTURING KITS AND ITEMS

[0223] The present application also provides kits, medicaments, compositions and unit dosage forms for use in any of the methods described herein.

[0224] The kits provided herein include one or more containers comprising any of the tetramine compositions (including pharmaceutical compositions) described herein and/or other agent (or agents) and, in some embodiments, further comprise instructions for use in accordance with any of the methods described herein. The kit may also include a description of the selection of a suitable individual for treatment. Instructions provided in kits of the invention are typically instructions written on an identification or leaflet (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions loaded onto a magnetic or optical storage disk ) are also acceptable.

[0225] For example, in some embodiments, the kit comprises a) a tetramine composition comprising a copper chelating tetramine (such as trientine) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and, optionally, b) instructions for administering the tetramine composition for treating a disease or condition associated with ischemic tissue injury.

[0226] In some embodiments, the kit comprises a) a tetramine composition comprising a copper chelating tetramine (such as trientine) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; b) a copper-promoting composition comprising a copper ion (such as CuSO4 or CuCl2) and a pharmaceutically acceptable carrier; and, optionally, c) instructions for administering the tetramine composition and the copper-promoting composition for treating a disease or condition associated with ischemic tissue injury.

[0227] The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed plastic or Mylar pouches) and the like. Kits can optionally provide components such as buffers and interpretive information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging and the like.

[0228] In some embodiments, the kits comprise one or more components that facilitate delivery of the tetramine composition, the copper-promoting composition and/or additional therapeutic agents to the individual. For example, in some embodiments, the kit comprises components that facilitate intralesional delivery of the tetramine composition and/or the copper-promoting composition to the individual. In some embodiments, the kit may comprise, for example, syringes and needles suitable for delivering cells to the individual and the like. In such embodiments, the tetramine composition and/or the copper promoting composition may be contained in the kit in a pouch or in one or more vials. In some embodiments, the kit comprises components that facilitate intravenous or intra-arterial delivery of the tetramine composition and/or the copper-promoting composition to the individual. In some embodiments, the tetramine composition and/or the copper-promoting composition may be contained, for example, within a bottle or bag (e.g., a blood bag or similar bag that may contain up to about 1.5 l of solution comprising the cells), and the kit further comprises tubing and needles suitable for delivering the tetramine composition and/or the copper-promoting composition to the individual.

[0229] Instructions related to the use of the compositions, in general, include information regarding the dosage, the dosage schedule and the route of administration for the intended treatment. Containers may be unit doses, batch packages (e.g., multi-dose packages), or subunit doses. For example, kits may be provided that contain sufficient dosages of the copper chelating tetramine as disclosed herein to provide effective treatment of an individual for an extended period, such as any one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months or more. The kits may also include multiple unit doses of the pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

[0230] Medicaments, compositions and unit dosage forms useful for the methods described herein are also provided.

[0231] The following non-limiting examples further illustrate the compositions and methods of the present invention. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the invention, but, of course, should not be considered in any way as limiting it.

EXAMPLES EXAMPLE 1: CRYSTAL STRUCTURE OF A TRIENTINE COMPLEX AND A COPPER ION

[0232] A complex comprising trientine dichloride, a copper ion and water was crystallized. A suitable single crystal (150116_s2_lzh_m) was selected and X-ray diffraction data was collected using the single crystal on an Xcalibur Eos diffractometer. The crystal was maintained at -130.15 to -130.05 °C (143.00 to 143.10 K) during data collection. Using Olex2 (Dolomanov et al., (2009) J. Appl. Cryst. 42: 339 to 341), the structure was solved with the Superflip structure solution program (Palatinus et al., (2008) J. Appl. Cryst. 41: 975 to 984; Palatinus et al., (2012) J. Cryst. 45: 575 to 580) using Charge Flipping and refined with the ShelXL refinement package (Sheldrick G.M. (2008) Acta Cryst. A64: 112 to 122) using the least squares minimization algorithm. The empirical formula of the complex in each unit cell was determined to be C6H20Cl2CuN4O. The refined crystal structure and its parameters are shown in Figures 1 to 3 and 4A to 4C.

EXAMPLE 2: INTRACELLULAR DELIVERY OF COPPER TO CARDIOMYOCYTES BY TRIENTINE AND TRIENTINE-COPPER COMPLEX

[0233] This example describes an in vitro copper delivery assay for cardiomyocytes by trientine and trientine-copper complex. A flowchart of the experimental procedure is shown in Figure 5.

[0234] Primary cultures of neonatal rat cardiomyocytes were grown in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) at 37 ° C, 10% CO2 for 48 hours. The cells were then transferred to serum-free DMEM and cultured at 37°C, 10% CO2 for 12 hours, before the cells were divided into five experimental groups (one control group and four treatment groups). In the control group, cells were cultured with serum-free DMEM for an additional 6 hours at 37 °C, 10% CO2. In the four treatment groups, cells were incubated for 6 hours at 37 °C, 10% CO2 with CuCl2 alone, trientine alone, a trientine-copper complex, and a mixture of trientine and CuCl2, respectively, each at a concentration final dose of 10 μM trientine and/or 10 μM copper. The trientine-copper complex was locally synthesized and characterized by mass spectrometry and X-ray diffraction (XRD). The trientine-copper complex had a composition and structure as described in Example 1. The trientine-copper mixture was prepared by adding equal moles of trientine and CuCl2 to serum-free DMEM at a final concentration of 10 μM at 37 °C. for 24 hours before the mixture was used to treat neonatal rat cardiomyocytes.

[0235] After treatment, cells were collected by a cell scraper, washed three times with ice-cold PBS containing 10 mM EDTA (Sigma, USA) to ensure that extracellular copper was completely removed, and centrifuged at 3,000 rpm for 5 minutes. Cell pellets were lysed using 1% SDS solution (Beyotime, CN). The lysates were divided into two parts. A portion was digested with concentrated nitric acid at 50°C for 72 hours and analyzed using a graphite furnace atomic mass absorption spectrophotometer to assess intracellular copper concentration. Another part was used to determine total protein concentration by Bicinchoninic Acid (BCA) Protein Assay (Bio-Rad, USA). The intracellular copper concentration of each treatment group was normalized to the total protein concentration.

[0236] Figure 6 shows the normalized intracellular copper concentrations of the five experimental groups. All data were expressed as mean ± standard deviation (SD). One-way analysis of variance was used for initial analysis, and Student-Newman-Keuls was employed for comparison between multiple groups. Differences between experimental groups were considered significant at P < 0.05. As shown in Figure 6, the intracellular copper concentration of the trientine-copper complex treatment group (i.e., Cu-trientine) and the trientine and CuCl2 mixture treatment group (i.e., Cu+trientine) increased. significantly compared to the control group, and the increase in intracellular copper concentrations in these two groups was more pronounced than the CuCl2 treatment alone. Notably, the mixture of trientine and CuCl2 (i.e., Cu+trientine) led to the greatest increase in intracellular copper concentration among all conditions tested, suggesting that trientine could release copper from a high copper cellular environment into cardiomyocytes.

EXAMPLE 3. TRIENTINE THERAPY IN A RAT MODEL OF PATHOLOGICAL CARDIAC HYPERTROPHY.

[0237] This example describes an in vivo experiment to evaluate the efficacy of trientine therapy in Sprague-Dawley rats with pathological cardiac hypertrophy. The rat model of pathological cardiac hypertrophy was established by an ascending aortic constriction operation. Figure 7 shows a flowchart of the experimental procedure.

1.1 ESTABLISHMENT OF PATHOLOGICAL CARDIAC HYPERTROPHY IN RATS

[0238] Before the surgical procedure, all subjects received an intraperitoneal injection of 10% chloral hydrate (0.35 mg/kg) to induce sedation. The hair covering the left part of the chest was shaved throughout the operation. Endotracheal intubation was introduced by ventilation. Assisted breathing was conducted to achieve periodic volume between 1.2 ml to 1.5 ml. The respiratory rate was almost 80/min, and the inspiratory/expiratory ratio was 1:1.

[0239] During the operation, the position of the rat was adjusted to the right lateral decubitus position, and the rat was placed under a stereomicroscope. The operational area was aseptically isolated. Isolation was performed with a piece of sterile disposable tape.

[0240] The surgical area was cut slightly medial to the line of the left second intercostal space, and a 1 to 1.5 cm transverse incision was made outward from the left side of the presternum. The subcutaneous tissue and muscular planes were dissected up to the pleura, entering the pleural space. A cotton swab was inserted to sweep the pleural space and push the lung away from the operating field to avoid injury to the lung, and then the intercostal incision was enlarged with a retractor to open the chest and expose the thymus and fat.

[0241] After the thymus and fat were removed, the larger vessels were exposed in the upper part of the left atrial appendage. The ascending portion of the aorta was dissected from the pulmonary trunk on the right. The construction site was located in the ascending aorta between the aortic valve and the innominate artery.

[0242] The ascending aorta was constricted with a 20 gauge needle (O.D. 0.9 mm). The ascending aorta and needle were ligated using a single piece of 6-0 surgical suture. The needle was then immediately removed to provide a lumen with a stenotic aorta. After constriction, the left ventricle and left atrium swelled.

[0243] Before closing the chest, the chest retractor was removed, and the thymus and fat were moved back to their normal position. The chest cavity was closed by joining the second and third ribs with two 3-0 nylon sutures. To avoid hemorrhage and pneumothorax, care was taken to avoid puncturing the reported heart and damaging the lung while suturing the ribs. The lungs were reinflated by turning off the outflow on the ventilator for 1 to 2 seconds using a finger while closing the intercostal incision so that air could be expelled from the pleural cavity. After the intercostal incision was closed, the muscle and skin incisions were closed in layers with 5-0 silk sutures and cleaned in a sterile manner. The endotracheal tube was retracted after spontaneous breathing was reestablished. To alleviate pain after surgery, analgesic dezocine (0.8 mg/kg) was given intramuscularly and once daily for the next 2 days.

1.2 ECHOCARDIOGRAPHY

[0244] Rats were sedated by intraperitoneal injection of 10% chloral hydrate (0.35 mg/kg) for echocardiography measurements. At 4 months after the aortic construction operation and at 1, 3, and 5 weeks after trientine treatment, a series of echocardiograms were performed using an 11.5 MHz transducer (Vivid 7 Dimension, GE). Interventricular septal depth (IVSD) and left ventricular posterior wall depth (LVPWD) were obtained using two-dimensional mode by taking measurements of the short axis cross-sectional areas and the length of the left ventricle.

[0245] Left ventricular ejection fraction (EF) and shrinkage fraction (FS) were evaluated with Simpson's single-plane method. Left ventricular end-diastolic volume (LVEDV), end-systolic volume (LVESV), end-diastolic internal diameter (LVIDd), and end-systolic internal diameter (LVIDs) were directly recorded. EF and FS were calculated according to the following formulas: EF = (LVEDV-LVESV)/LVEDVx100%, FS = (LVIDd-LVIDs)/LVIDdXl00%

1.3 TREATMENT WITH TRIENTINE

[0246] Four months after the operations, concentric hypertrophy of the left ventricle and interstitial fibrosis of the myocardium were observed. The establishment of the pathological cardiac hypertrophy model was confirmed by ultrasonic assessments of cardiac morphology and functions. Treatment with trientine was initiated after confirmation of the state of pathological cardiac hypertrophy. The ascending aortic constriction (AAC) group was divided into three groups: a control group (NS group) and two trientine treatment groups (Tr(H) group and Tr(L) group). Rats in the sham group underwent the same surgical procedure except for the ascending aortic constriction steps. The rats in the sham group were also divided into three groups: a control group (NS group) and two trientine treatment groups (Tr(H) group and Tr(L) group). Rats in the control groups were treated with a saline solution. Trientine was administered orally twice daily in the trientine treatment groups. Two doses of trientine (dose calculated based on trientine dihydrochloride) were administered, 45 mg/kg/day (Tr(H) group) and 90 mg/kg/day (Tr(L) group). Treatment continued for 6 weeks.

[0247] The experimental procedure and results in the following sections of the present example are focused on treatment with a trientine composition consisting essentially of trientine dihydrochloride. The same experimental protocol is used to evaluate the therapeutic efficacy of other trientine compositions to treat cardiac hypertrophy in the rat model. For example, in one experiment, in addition to trientine treatment, rats with ACC in the trientine treatment groups are further treated with an oral copper supplement (such as copper chloride) at a dose of 54 mg/kg at the same time. day for 6 weeks. In another experiment, the trientine-copper complex of Example 1 is used to treat rats with ACC in the trientine treatment group at a dosage of 120 mg/kg/day by oral administration for 6 weeks.

1.4 EVALUATIONS OF CARDIAC MORPHOLOGY AND FUNCTION

[0248] Cardiac functions and morphology are assessed by echocardiography, and plasma copper concentrations were measured (i.e., blood detection) to assess the therapeutic efficacy of trientine treatments according to the schedule shown in Figure 7.

[0249] Additionally, heart tissue sections are obtained from the mice after they are sacrificed at the end of the experiments. Immunohistochemical experiments are performed on the tissue sections. The capillary density of heart tissue sections is determined, and changes in collagen contents are detected. The mRNA and protein levels of HIF-1α and its targets, such as VEGF and VEGFR-1, in the infarcted tissue, the border zone of the infarcted tissue, and cardiac tissue remote from the healthy infarcted area are measured.

1.5 COPPER CONCENTRATION IN HEART TISSUE

[0250] The tissue samples were frozen again and stored at -80 °C before freeze-drying. After lyophilization and digestion of the tissues with nitric acid, the sample was colorless or soft yellow and clear with no visible precipitate or residue. Ultrapure water was added to each vessel to dilute HNO3 to 2% for subsequent analysis of copper concentrations. Copper concentrations were determined by graphite furnace atomic absorption spectrophotometry (ICE3500, Thermo) according to the program shown in Table 1 below. TABLE 1 GRAPHITE FURNACE ATOMIC ABSORPTION SPECTROPHOTOMETRY PROGRAM

1.6 STATISTICAL ANALYSIS

[0251] All data were expressed as mean ± standard deviation. The variation of each parameter was compared between the various experimental groups using Levene's homogeneity test and coefficient variance (CV). An SPSS 14.0 statistical package (SPSS, Chicago, IL) was used and significant difference was assumed when P values were <0.05.

2 RESULTS 2.1 CARDIAC MORPHOLOGY AND FUNCTIONS

[0252] Echocardiography exams showed that, after treatment with trientine, the reversal of pathological cardiac hypertrophy occurred at the morphological level. After 3 weeks of treatment, the interventricular septal depth (IVSD) and left ventricular posterior wall depth (LVPWD) of rats decreased significantly. With the extension of treatment time, the treatment effect of trientine became even more obvious. When AAC mice were treated for 5 weeks, IVSD and LVPWD were almost normal compared with those in sham groups. As shown in Figures 8A and Figures 8B, the treated groups showed a significantly downward trend in IVSD and LVPWD values over time according to the continuous monitoring results. In contrast, in the control group, IVSD and LVPWD increased stably over time.

[0253] Although the cardiac function parameters, ejection fraction (EF) and shrinkage fraction (FS) were within the normal range for all experimental groups due to compensatory effects, Figures 9A and 9B show that EF of FS of the groups treated with trientine did not fluctuate very significantly. In contrast, a significant downward trend of EF and FS was observed in the untreated groups.

[0254] Copper concentrations in the plasma and myocardium of rats from the various treatment groups were determined by atomic absorption spectroscopy. As shown in Figure 10A, copper concentrations decreased in the hypertrophic myocardium when rats underwent AAC operations. After treatment with trientine for 6 weeks, copper concentrations increased in the heart tissues of treated rats. The bar in Figure 10A corresponding to the AAC-Tr group shows the mean copper concentration in the heart tissue of rats with AAC treated with either high-dose trientine or low-dose trientine (i.e., AAC-Tr(H) groups and AAC-Tr(L) combined).

[0255] As a consequence of copper efflux from the heart tissues of rats with AAC, copper concentrations in the plasma of rats with AAC were higher than those of rats in the sham group. However, after a 6-week treatment of trientine, according to measurements at different time points during the course of treatment (i.e., every two weeks), the high concentration of copper in the plasma of AAC rats decreased significantly as over time. In contrast, the plasma copper concentration of rats in the sham group remained quite stable throughout the course of treatment (Figure 10B).

3 . DISCUSSION

[0256] The present study used trientine to increase the concentration of copper and hypertrophic heart tissues in rats. The results showed that trientine had the ability to promote tissue redistribution and copper reuse and reverse the morphology and functions of hypertrophic hearts. HIF-1 transcriptional activities and capillary density may also be increased in infarcted cardiac tissues as a result of trientine treatment in rats with hypertrophic hearts. Furthermore, porechocardiography exams showed that normal cardiac functions were maintained throughout the treatment with trientine. The results of this experiment provide strong evidence that the trientine treatment of the present invention can effectively deliver copper to ischemic cardiac tissues in vivo for treating cardiac hypertrophy.

EXAMPLE 4. TRIENTINE THERAPY FOR RHESUS MONKEY HEART FAILURE MODEL AFTER MYOCARDIAL ISCHEMIC INFARCTION.

[0257] This example describes an in vivo experiment to evaluate the efficacy of trientine therapy in a Rhesus monkey model of heart failure. The Rhesus monkey has a higher-order heart that resembles that of humans in terms of internal structure, electrical activities, distribution of coronary arteries, coronary collateral circulation, and their placement and fixation in the thoracic cavity. Thus, the Rhesus monkey model of heart failure provides a good surrogate for evaluating the efficacy of therapies for heart failure conditions in humans. In this experiment, myocardial ischemic infarction was established by a coronary artery ligation operation. After the operation, the ischemic heart tissue was gradually replaced by collagen fiber and became infarcted tissue. One year after the operation, the non-infarcted heart tissue in the animal became unable to compensate for the lost functions of the infarcted heart tissue, and a model of heart failure was thus developed. Trientine treatment was subsequently given to the monkeys to treat heart failure.

1.1 ESTABLISHMENT OF HEART FAILURE IN RHESUS MONKEYS

[0258] Before the surgical procedure, all subjects received an intramuscular injection of 5 mg/kg ketamine and 0.2 mg/kg midazolam for induced sedation. The hair covering the chest and limbs at the electrode attachment sites was completely shaved for the operation and to improve electrocardiography (ECG) recording results. Standard bipolar and unipolar limb connectors were recorded. Animals exhibiting abnormal ECG such as tachycardia (more than 200 beats per minute), arrhythmia, and obvious ST segment deviations from baseline were excluded from the study.

[0259] Standard non-invasive measurements including electrocardiogram, blood pressure meter, pulse oximetry and capnography were constantly monitored (Dash3000, GE, USA.) and vein catheters were established in the subjects. All monkeys undergoing the surgical procedure were first intubated after anesthesia induced by intravenous infusion of fentanyl (10 μg/kg), midazolam (0.2 mg/kg), propofol (1 mg/kg) and vecuronium (0.1 mg /kg). Assisted breathing was conducted with pressure-controlled ventilation to achieve end-tidal CO2 between 35 mmHg and 40 mmHg. Inspiratory pressure was adjusted within a range of 12 to 20 cm H2O. The respiratory rate was 40/min, and the inspiratory/expiratory ratio was 1:2.

[0260] In order to maintain anesthetic conditions during the surgical procedure, 2 ml of fentanyl (0.1 mg) and 10 ml of propofol (100 mg) were diluted in 20 ml of saline solution. The mixture was infused continuously by a syringe pump at a speed of 5 to 10 ml/h. The pump speed was adjusted according to the anesthetic state and duration of the operation. The arterial cannulation was pierced into the femoral artery with a permanent needle and was connected to the pressure monitoring tubing for invasive blood pressure monitoring during the operation. Typically, the femoral arterial pulsation was palpated halfway between the anterior superior iliac spine and symphysis pubis. The operational area was aseptically isolated. Isolation was performed with four pieces of sterile disposable sheets.

[0261] The surgical area was cut slightly medial to the line of the left fourth intercostal space, and a 4 to 5 cm transverse incision was made outward from the left side of the presternum. Monopolar diathermy was applied for both cutting and tissue coagulation purposes. The subcutaneous tissue and muscular planes were dissected up to the pleura, entering the pleural space, and then the incision was enlarged by opening the forceps. A cotton swab was inserted to sweep the pleural space and push the lung away from the hole, and then the intercostal incision was enlarged to open the chest and expose the pericardium.

[0262] The heart was exposed through the left fourth thoracotomy incision (4 to 5 cm) and the apex and left atrium were identified. The epicardial end of the left anterior descending artery (LAD) was defined as level zero; the origin of the LAD below the left atrium was defined as level 100. Ligation was performed at 60% of the LAD. Additionally, the greater diagonal branch was also ligated parallel to the local LAD artery ligation in some monkeys if the diagonal artery branch site was above the ligation site.

[0263] The artery was occluded for 1 minute followed by a 5-minute reperfusion, and this occlusion-reperfusion was repeated 3 times before final ligation. After the final ligation, the difference in left ventricular wall motion, color changes of the anterior ventricular wall, and changes in the electrocardiogram and blood pressure were monitored to ensure that the ligation was successful. Methylene blue (1 ml) was injected as a bolus into the left atrium with a 1.0 ml syringe after the final ligation. The methylene blue filling defect indicated completion of binding as well as helped predict the ischemic area.

[0264] Before closing the chest, the condition of the heart was intensively monitored for 45 minutes. Dobutamine (3 to 5 μg^kg-^min-1) was infused to support cardiac functions and the defibrillator (HEARTSTART XL, Philips) was used if necessary. Care was taken to avoid damage to the heart during pericardial closure. Sodium hyaluronate was infused into the pericardial chamber for antiadhesion treatment. The pericardium and pleura were closed with 4-0 polyethylene sutures. The intercostal incision was closed with silk sutures. To avoid pneumothorax, care was taken to avoid lung damage during intercostal closure. The lungs were reinflated while the intercostal incision was closed so that air could be expelled from the pleural cavity. After the intercostal incision was closed, saline was poured into the subcutaneous space, and the lung was inflated again to verify that the chest incision was firmly closed. The muscle and skin incisions were closed in layers with 2-0 silk sutures and cleaned in a sterile manner. The endotracheal tube was retracted after spontaneous breathing was reestablished. The incision was covered with sterile gauze and bandage. Tramadol (2 mg/kg) was injected intramuscularly to relieve pain. The bandage was changed every other day, and the sutures were removed one week after the operation.

1.2 ELECTROCARDIOGRAM (ECG) MONITORING

[0265] A 12-lead ECG (MAC8000, GE, USA.) was recorded in the supine position of each monkey at the time before the operation, immediately after the operation (about 2 hours for the entire surgical procedure) and at four weeks and eight weeks after the operation using pediatric electrodes at 25 mm/s paper speed and 10 mm/mV amplitude. The chest wall of a monkey was not wide enough to allow 6 precordial connectors at the same time even with the pediatric electrodes. Therefore, the 6 precordial leads were divided into two groups: V1, V3 and V5 were recorded in one group, and V2, V4 and V6 were recorded in another group.

1.3 ECHOCARDIOGRAPHY

[0266] Two-dimensional echocardiographic measurements were performed in standard apical 2- or 4-chamber views with three consecutive cardiac cycles. The frame rate was maintained between 70 fps and 100 fps. All monkeys underwent transthoracic echocardiographic evaluation with the 10.3 MHz transducer (P10-4, Siemens ACUSON Antares system, German) in the left lateral position before the operation and four weeks and eight weeks after the operation.

[0267] Left ventricular ejection fraction (EF) was assessed with Simpson's single-plane method. Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were directly recorded, and EF was calculated using the following: EF = (LVEDV-LVESV)/LVEDVx100%. Left ventricular stroke volume (SV) was calculated as follows: SV=LVEDV-LVESV.

1.4 TREATMENT WITH TRIENTINE

[0268] One year after the operation, the ischemic heart tissue was completely replaced by collagen fiber and became an infarcted tissue. A model of heart failure was established as confirmed by ultrasound assessments of cardiac functions. Trientine treatment was subsequently conducted. In the trientine-treated group, trientine was administered to each monkey orally twice a day. The dose of trientine was 18 mg/kg/day. This treatment continued for eight weeks. Monkeys in the untreated group (i.e., control) did not receive any treatment. Cardiac functions and morphology were assessed according to the schedule shown in Figure 11 to evaluate the therapeutic efficacy of trientine.

[0269] The experimental procedure and results in the following sections of the present example are focused on treatment with a trientine composition consisting essentially of trientine dihydrochloride. The same experimental protocol is used to evaluate the therapeutic efficacy of other trientine compositions to treat heart failure in the Rhesus monkey model. For example, in one experiment, in addition to trientine treatment, rhesus monkeys with heart failure in the treatment group are also treated with an oral copper supplement (such as copper chloride) at a dose of 16.5 mg/kg. kg per day for 6 weeks. In another experiment, the trientine-copper complex of Example 1 is used to treat rhesus monkeys with heart failure in the treatment group at a dosage of 36.7 mg/kg/day by oral administration for 6 weeks.

1.5 HISTOPATHOLOGICAL EXAMINATIONS

[0270] Monkeys are sacrificed by intravenous injection of potassium chloride (10%, 10 ml) and a complete autopsy of each monkey is performed. The harvested hearts are washed and grossly inspected for visible lesions and fixed in 10% formaldehyde solution. Then, the heart is cut into six blocks from the apex to the base along the geometric axis. The thickness of each block is 0.5 cm. The surface of each section is smooth and uniform during incision, and the sections are marked by ligature with identification. Thin sections were cut and stained with Masson and H/E for microscopic examination.

IMMUNOHISTOCHEMISTRY

[0271] Tissue sections are examined using immunohistochemical methods to detect HIF-1α, VEGFA and VEGFR1. The following antibodies are used respectively: mouse anti-human HIF-1α monoclonal antibody (ab16066, Abcam); mouse anti-human VEGFA monoclonal antibody (sc-57496, Santa Cruz); rabbit anti-human VEGFR1 monoclonal antibody (1303-12, Epitomics); mouse anti-human CD31 monoclonal antibody (Maixin bio-tech company, Fuzhou). HIF-1α is recovered by a high-pressure heat-induced antigen retrieval method using EDTA (pH 9.0), VEGF and VEGFR1 are recovered by microwave heat-induced antigen retrieval methods using the use of a citrate buffer solution (pH 6.0), and CD31 is recovered by a microwave heat-induced antigen retrieval method using EDTA. The working concentrations of antibodies are as follows: 1:800 for anti-HIF-1α, 1:100 for anti-VEGF and 1:100 for anti-VEGFR1. Negative control samples are incubated with PBS upon binding of the first antibody in immunohistochemical experiments. CD31 is an endothelial cell marker. Ki-67 identification is examined by immunofluorescence using confocal microscopy.

HAIR DENSITY

[0272] Capillary densities of tissue sections are evaluated as follows. First, a visual field of maximum capillary distribution is determined under a light microscope at 100x magnification, and then 5 randomized visual fields are collected under the light microscope at 200x magnification to determine capillary density. Capillary is defined as a lumen with a diameter less than the sum of 8 times the diameter of red blood cells. The measurement is carried out by two independent technicians.

SEMIQUANTITATIVE PROTEIN EXPRESSION ANALYSIS

[0273] Immunohistochemical slides are observed under a light microscope, and images are taken and used to determine protein expression levels in a semiquantitative manner using Image-Pro Plus 6.0 image analysis software (Media Cybemetics). . Slides from different groups are evaluated by two independent technicians. Images of 5 randomized visual fields of the border area and remote areas relative to the infarct on each slide are taken under the light microscope at 400x magnification.

1.6 WESTERN BLOT TISSUE PREPARATION

[0274] The heart is removed from the chest. The left ventricular wall is carefully examined, and tissue samples from the infarcted area, the border area, and the remote area are isolated. The infarcted area can be distinguished from the non-infarcted area based on its pale appearance. The border area is defined as an area from 1 mm inward from the infarcted area to 3 mm outside the infarcted area. Remote areas are defined as more than 3 mm outside the infarcted area. Samples are preserved in liquid nitrogen for Western blot analysis.

WESTERN BLOT

[0275] Protein extracts are obtained after grinding each tissue in liquid nitrogen and lysing the ground tissue in RIPA lysis buffer (Beyotime, CN) containing 1% complete EDTA-free protease inhibitor cocktail (Roche, DE) for 40 minutes on ice. Protein concentrations are determined by Pierce BCA Protein Assay Kit (Thermo SCIENTIFIC, 23227, USA). Equal amounts of protein (30 μg) from each sample are solubilized in 5x SDS sample buffer and separated on 10% SDS and 8% polyacrylamide gels. The proteins are then electrophoretically transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). Membranes are blocked for 1 hour in Tris-buffered saline/Tween 20 (TBST) (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) containing nonfat dry milk at 5% (blocking solution) and incubated overnight at 4 °C against primary antibodies, such as anti-HIF-1α (Abcam, ab113642, USA), anti-VEGF (Santa Cruz, sc57496, USA) and anti-VEFGR-1 (Abcam, ab32152, USA), diluted in the blocking solution according to the supplier's recommendations. After washing with TBST, membranes are incubated for 1 hour at 37°C with appropriate secondary antibodies. Target proteins are visualized using an HRP chemiluminescence substrate (Millipore, USA) and analyzed by densitometry using QUANTITY ONETM Software.

1.7 mRNA levels of HIF-1 target genes

[0276] In order to define the transcriptional activities of HIF-1 in the ischemic myocardium, the mRNA levels of HIF-1α, HIF-1 target genes such as VEGF and VEGFR-1 (also known as Flt-1 ), are determined by real-time PCR (RT-PCR).

[0277] Total RNA from each sample is isolated using TRIZOL® (Invitrogen, 15596-026, USA) according to the manufacturer's instructions. 1 μg of total RNA is reverse transcribed using the PRIMESCRIPTTM RT Reagent Kit (TaKaRa, RR037A, Japan) at 37°C for 15 minutes followed by 85°C for 5 seconds and 4°C for 5 minutes. Real-time RT-PCR reactions were performed using the SYBR® Premix Ex TaqTM II kit (TaKaRa, RR820A, Japan). To amplify HIF-1α, VEGF and VEGFR1 cDNA fragments, samples are processed using a BIO-RAD CFX96 Real-Time System with the following program: denaturation at 95 °C for 30 seconds, followed by 35 cycles 95°C for 5 seconds and 60°C for 30 seconds. The log linear phase results of the growth curve are analyzed, and relative qualification is performed using the 2-ΔCT method. The gene expression levels of HIF-1α, VEGF, and VEGFR1 are each normalized to the level of Actin expression in each sample. At least 3 replicates are made for each sample. Primer sequences are shown in Table 2 below. TABLE 2. RT-PCR PRIMER SEQUENCES

1.8 COPPER CONCENTRATION IN THE HEART

[0278] The tissue samples were frozen again and stored at -80 °C before freeze-drying. After lyophilization and digestion of the tissues with nitric acid, the digests were colorless or light yellow and clear with no visible precipitate or residue. Ultrapure water was added to each vessel to dilute HNO3 to 2% for subsequent analysis of copper concentrations. Copper concentrations were determined by graphite furnace atomic absorption spectrophotometry (ICE3500, Thermo) using the program shown in Table 1 of Example 3.

1.9 STATISTICAL ANALYSIS

[0279] All data were expressed as mean ± standard deviation. The variation of each parameter was compared between the various experimental groups using Levene's homogeneity test and coefficient variance (CV). An SPSS 14.0 statistical package (SPSS, Chicago, IL) was used and significant difference was assumed when P values were <0.05.

2. RESULTS 2.1 CARDIAC FUNCTIONS

[0280] Echocardiography exams showed that, after treatment with trientine, the left ventricular ejection fraction increased significantly over time. However, in the untreated group, the left ventricular ejection fraction decreased over time. See Figure 12

2.2 COPPER CONCENTRATIONS IN THE INFARCTED HEART

[0281] The concentration of copper in the myocardium was determined by atomic absorption spectroscopy. As shown in Figure 13, copper concentrations increased significantly after trientine treatment in tissue samples from the infarcted area and border zone of the treated group compared with those of the untreated group. In contrast, copper concentrations of tissue samples in the remote areas are comparable in the trientine-treated group and the untreated group.

3. DISCUSSION

[0282] Myocardial ischemia leads to HIF-1α accumulation and deprivation. Under ischemic conditions, accumulated HIFα cannot activate HIF transcription due to the fact that copper is required for HIF transcriptional complex formation and for the interaction of HIF with HIF response element (HRE) sequences in target genes. Therefore, although HIF accumulation occurs in the ischemic myocardium, copper deficiency blocks HIF-regulated expression of genes involved in angiogenesis, which leads to suppression of myocardial angiogenesis. This effect results in myocardial infarction, which further progresses to heart failure.

[0283] The present study used trientine to increase copper concentrations in local ischemic tissues for the treatment of myocardial infarction. The results showed that trientine promoted tissue redistribution and copper reuse. Furthermore, porechocardiography exams showed that cardiac functions were improved after treatment with trientine. The dose of trientine in this experiment was 18 mg/kg per day for the Rhesus monkeys, which is equivalent to about 420 mg per day for a human subject. Compared to typical doses of trientine used to lower the serum copper level in patients with Wilson's disease (500 to 700 mg/day to a maximum of 1,500 mg/day for pediatric patients and 750 to 1,250 mg/day to a maximum of 2,000 mg/day for adult patients), the dose used in this experiment is much lower. The results of this experiment provide strong evidence that the low-dose trientine treatment described herein is an effective strategy for delivering copper in vivo for the treatment of myocardial infarction.

EXAMPLE 5. TRIENTINE THERAPY IN THE MOUSE MODEL OF ISCHEMIC MYOCARDIAL INFARCTION

[0284] In this experiment, a mouse model of myocardial ischemic infarction was established by a permanent coronary artery ligation operation. Within four weeks after the operation, the ischemic heart tissue was replaced by collagen fiber and became infarcted tissue. Trientine treatments were conducted as described in the protocol shown in Figure 14.

[0285] Trientine treatment was administered to the four groups of modeled mice via the intragastric route twice daily at a dose of 16.75, 33.49, 55.94 or 78.25 mg/kg per day. This treatment continued for 4 weeks. The untreated group did not receive any trientine treatment.

ECHOCARDIOGRAPHY

[0286] Cardiac functions were evaluated by echocardiography to evaluate the therapeutic efficacy of trientine. All mice underwent transthoracic echocardiographic evaluation with a 12 MHz transducer (i13L, Vivid7, GE Ultrasound). Left ventricular ejection fraction (EF) was assessed with the Simpson single-plane method. Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were directly recorded, and EF was calculated using the following: EF = (LVEDV-LVESV)/LVEDVx1QQ%.

COPPER CONCENTRATION IN THE HEART

[0287] Tissue samples were frozen again and stored at -8°C before freeze-drying. After lyophilization and digestion of the tissues with nitric acid, the digests were colorless or light yellow and clear with no visible precipitate or residue. Ultrapure water was added to each vessel to dilute HNO3 to 2% for subsequent analysis of copper concentrations. Copper concentrations were determined by graphite furnace atomic absorption spectrophotometry (ICE35QQ, Thermo) using the program shown in Table 1 of Example 3.

RESULTS

[0288] Cardiac performance detected by echocardiography showed that cardiac function as measured by the left ventricular ejection fraction of mice treated with trientine improved at lower doses of trientine treatment, and such improvement diminished at higher doses of trientine treatment (see Figure 15). The rate of ejection fraction improvement peaked at a dose of 33.49 mg/kg per day and then decreased even at a higher dose. This experiment suggested that trientine therapy for myocardial infarction is effective within a narrow low-dose range. As shown in Figure 16, the copper concentration in the infarcted area increased significantly in response to trientine treatment. Notably, in the treatment group with the dose of 33.49 mg/kg per day, the copper content in the infarct area was the highest.

DISCUSSION

[0289] The present study used a series of increasing doses of trientine to treat myocardial ischemic infarction in mice. The results showed that in the group treated with trientine at a dose of 33.49 mg/kg per day, the copper content in the infarcted area was the highest among all experimental groups, which corresponds to the highest improvement ratio of copper fraction. ejection observed in this group compared to other experimental groups. At the highest trientine doses tested, no further improvement in the copper content of the infarcted area or ejection fraction was observed.

[0290] The doses, 16.75, 33.49, 55.94 and 78.25 mg/kg per day, tested in this experiment are equivalent to about 150, 300, 500 and 700 mg per day, respectively, for a human patient. In contrast, the dose of trientine used to treat Wilson's disease by lowering the serum copper level in these patients is about 500 to 700 mg per day to a maximum of 1,500 mg per day for pediatric patients and about 750 mg to 1,250 mg per day. mg per day up to a maximum of 2,000 mg per day for adult patients. Thus, the dose of trientine with the highest efficacy of restoring copper level in ischemic heart tissue and restoring cardiac functions observed in this experiment is much lower than the doses used to treat patients with Wilson's disease. The results of this trial provide strong evidence that trientine therapy for myocardial infarction is effective within a narrow low-dose range.

[0291] Without being bound by any theory or hypothesis, trientine may serve as a copper delivery shuttle to transfer copper from a high concentration tissue or environment (such as serum after ischemia) to a copper-deprived ischemic tissue in the heart, thus alleviating copper depletion in ischemic tissue and improving cardiovascular conditions. Several publications describe the elevated level of copper in the serum of patients who have cardiovascular diseases, specifically myocardial infarction. See, for example, ES Ford. Am. J. Epidem. 151 (12): 1,182 (2000); E. Gomez et al. J. Trace Elements Med. Biol. 14: 65 to 70 (2000); and Singh MM et al. Angiology—Journal of Vascular Diseases, 504 to 506 (1985)

EXAMPLE 6. A CLINICAL STUDY OF TRIENTINE THERAPY IN PATIENTS WITH HEART FAILURE

[0292] A clinical study is conducted to evaluate the clinical effects of treatment with low dose trientine and patients who have heart failure. The main objective of the study is to evaluate the effectiveness of trientine compared to placebo in treating patients with heart failure before and after treatment.

[0293] The study is a randomized, double-blind, placebo-controlled clinical study in patients with heart failure (e.g., NYHA functional class II and III) with reduced ejection fraction (e.g., LVEF < 35%). Patients in the control group receive standard of care (SOC) plus placebo twice daily. Patients in the treatment group receive SOC plus oral administration of trientine twice daily at 150 mg/dose. Patients are evaluated at screening, at baseline (week 0), throughout the course of treatment, and after treatment.

[0294] The primary endpoint of the study may be survival, hospitalization related to heart failure, or change in a biomarker related to heart failure. For example, circulating natriuretic peptide levels over time have been used to stratify heart failure risk and thus may serve as biomarkers for heart failure severity.

[0295] Secondary endpoints of the study may include change in cardiac structure and function from baseline to the end of treatment. Cardiac structure and function can be determined by echocardiography. Exemplary metrics that can serve as secondary endpoints include left ventricular end-diastolic volume, left ventricular ejection fraction, and E/E' ratio. Secondary study endpoints may also include functional status based on the six-minute walk distance test, changes in symptoms (NYHA class), and Quality of Life scores.

[0296] Serum copper levels and other biomarkers can be monitored as tertiary end points of the study.

[0297] Safety is assessed by analyzing subject-reported spontaneous adverse events (AEs) and other appropriate medical and safety assessments, such as vital signs, ECG, laboratory tests, etc.

Claims (7)

1. Use of a composition comprising copper chelating tetramine and a copper ion characterized in that it is for the manufacture of a medicament to treat a disease associated with ischemic tissue injury in an individual, in which the copper chelating tetramine is trientine, in that the disease associated with ischemic tissue damage is selected from the group consisting of cardiomegaly and heart failure. 2. Use according to claim 1, characterized in that the ischemic tissue is ischemic heart tissue. 3. Use according to any one of claims 1 to 2, characterized in that the copper ion in the medicament forms a complex with the copper chelating tetramine. 4. Use, according to claim 3, characterized in that the complex of the copper chelating tetramine and the copper ion is crystalline. 5. Use, according to claim 4, characterized in that the copper ion is chelated by the four trientine amine groups to adopt a flat square geometry, and in which the complex further comprises two chloride ions and a water molecule . 6. Use according to any one of claims 1 to 2, characterized in that the copper ion in the medicament does not form a complex with the copper chelating tetramine. 7. Use according to any one of claims 1 to 2, characterized in that the medicine is an oral composition.