Use of trientine to deliver copper to ischemic tissue

Abstract

The present invention relates to the field of medicine, and specifically to the repair and regeneration of ischemic tissue by using compositions comprising tetraamines, such as trientine. The present invention is a method for the repair and regeneration of ischemic tissue by promoting tissue redistribution and reuse of copper through the administration of compositions comprising tetraamines, such as trientine, that can chelate copper. Methods and compositions for increasing intracellular copper levels in ischemic tissue and / or inducing repair of ischemic tissue in a subject. Increased copper levels in ischemic tissue can promote copper-dependent HIF-1 transcriptional activity and tissue repair.

Description

[0001] This application claims priority to Patent Application No. PCT / CN2015 / 090528, filed on September 24, 2015, with the World Intellectual Property Organization, entitled "USE OF TRIENTINE TO DELIVER COPPER TO ISCHEMIC TISSUE", the entire contents of which are incorporated herein by reference.

[0002] This application is a divisional application of International Application No. PCT / CN2016 / 099852, filed on September 23, 2016, entered into the National Stage on March 26, 2018, with a National Application No. 201680056122.X, and entitled "USE OF TRIENTINE TO DELIVER COPPER TO ISCHEMIC TISSUE". TECHNICAL FIELD

[0003] The present application relates to the field of medicine, and in particular to the repair and regeneration of ischemic tissue by using compositions comprising a tetraamine, such as trientine. BACKGROUND

[0004] Activation of hypoxia-inducible factor (HIF) is the initial and primary molecular response of the human body to hypoxic or ischemic injury. HIF-1 transcription factor belongs to the HIF family and controls the expression of various genes (e.g., VEGF) involved in various cellular adaptive responses to hypoxia and / or ischemia, including angiogenesis. HIF-1 comprises two subunits, HIF-1a and HIF-1b. Under hypoxic / ischemic conditions, HIF-1a accumulates in the nucleus to form a heterodimer with HIF-1b, which initiates the transcription of downstream genes.

[0005] However, in chronic myocardial ischemic conditions, the characteristics of the damaged myocardium are generally a decrease in capillary density and reduced angiogenesis. The defense mechanisms, such as those induced by accumulated HIF-1a under acute ischemic injury, do not function under chronic ischemic conditions as the long-term ischemia triggers mobilization of copper away from the myocardium. It has been previously shown that HIF-1 transcriptional activity requires the involvement of the trace element copper. In patients with chronic ischemic cardiomyopathy, the expression of HIF-1 regulated genes, such as VEGF, is decreased despite the sustained elevation of HIF-1a levels in the ischemic myocardial tissue. The loss of cardiac copper blocks the activation of accumulated HIF-1a, and the depletion of cardiac copper is well correlated with the degree of cardiac dysfunction in such patients. Furthermore, in patients with myocardial ischemic disease, the decrease in cardiac copper content is accompanied by high blood copper levels. Thus, it is believed that copper is released from the myocardium into the blood circulation in a form that cannot be reused by the ischemic myocardium. This significant efflux of myocardial copper into the circulation in a non-usable form is believed to be the major cause of the decrease in HIF-1a transcriptional activity that accompanies long-term myocardial ischemia. Thus, upregulation of HIF-1 controlled genes, an important step for tissue repair and regeneration, can not occur in patients with chronic ischemic cardiomyopathy due to the loss of available copper. Therefore, facilitating the proper tissue distribution of copper can be used as an effective strategy for treating various ischemic diseases and conditions.

[0006] Trientine is a well-known copper chelator that can be used to detoxify copper. Trientine dihydrochloride is a pharmaceutically acceptable salt of trientine that has been widely used to bind and remove excess copper in the body to treat Wilson's disease, particularly in patients who are intolerant to penicillamine. Cooper et al. have described the use of trientine and other copper antagonist compounds for the treatment of various conditions, including diabetes and complications (e.g., diabetic cardiomyopathy), cardiovascular diseases, neurodegenerative and mitochondrial-related diseases. See, e.g., U.S. Patent No. 7,459,446, U.S. Patent No. 7,928,094, International Application Publication No. WO2003077901 Al, International Application Publication No. WO2005058294 Al, and International Application Publication No. WO2007055598 Al.

[0007] The disclosures of all publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. SUMMARY

[0008] The present application provides methods for increasing intracellular copper levels in ischemic tissue or inducing tissue repair in ischemic tissue in an individual by administering a composition comprising a tetraamine that can chelate copper, such as trientine.

[0009] In one aspect of the application, there is provided a method of increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a composition comprising a copper-chelatable tetramine (e.g., trientine). Also provided is the use of a composition comprising a copper-chelatable tetramine (e.g., trientine) in the manufacture of a medicament for increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage, and a composition comprising a copper-chelatable tetramine (e.g., trientine) for use in increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage.

[0010] In one aspect of the application, there is provided a method of specifically delivering copper to the intracellular space of ischemic tissue in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a composition comprising a copper-chelatable tetramine (e.g., trientine). Also provided is the use of a composition comprising a copper-chelatable tetramine (e.g., trientine) in the manufacture of a medicament for specifically delivering copper to the intracellular space of ischemic tissue in an individual having ischemic tissue damage, and a composition comprising a copper-chelatable tetramine (e.g., trientine) for use in specifically delivering copper to the intracellular space of ischemic tissue in an individual having ischemic tissue damage.

[0011] In one aspect of the application, there is provided a method of inducing at least two tissue repair events in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a composition comprising a copper-chelatable tetramine. Also provided is the use of a composition comprising a copper-chelatable tetramine (e.g., trientine) in the manufacture of a medicament for inducing at least two tissue repair events in ischemic tissue of an individual having ischemic tissue damage, and a composition comprising a copper-chelatable tetramine (e.g., trientine) for use in inducing at least two tissue repair events in ischemic tissue of an individual having ischemic tissue damage.

[0012] In one aspect of the application, there is provided a method of inducing stem cell migration to ischemic tissue in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a composition comprising a copper-chelatable tetramine. Also provided is the use of a composition comprising a copper-chelatable tetramine (e.g., trientine) in the manufacture of a medicament for inducing stem cell migration to ischemic tissue in an individual having ischemic tissue damage, and a composition comprising a copper-chelatable tetramine (e.g., trientine) for use in inducing stem cell migration to ischemic tissue in an individual having ischemic tissue damage.

[0013] In one aspect of the application, there is provided a method of promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a composition comprising a copper-chelatable tetramine. There is also provided the use of a composition comprising a copper-chelatable tetramine (e.g., trientine) in the manufacture of a medicament for promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage, and a composition comprising a copper-chelatable tetramine (e.g., trientine) for use in promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage.

[0014] In some embodiments according to any of the methods described above, the individual has an impaired tissue repair system. In some embodiments, the individual does not have an impaired tissue repair system.

[0015] In some embodiments according to any of the methods described above, the ischemic tissue is selected from the group consisting of ischemic cardiac tissue, ischemic hepatic tissue, ischemic brain tissue, ischemic pulmonary tissue, ischemic renal tissue, ischemic skin tissue, ischemic digestive tract tissue, and ischemic limb tissue.

[0016] In some embodiments according to any of the methods described above, wherein the copper-chelatable tetramine is trientine.

[0017] In some embodiments according to any of the methods described above, the composition further comprises copper ions. In some embodiments, the copper ions in the composition are complexed with the copper-chelatable tetramine. In some embodiments, the complex of the copper-chelatable tetramine with the copper ions is crystalline. In some embodiments, the composition comprises a crystalline complex of trientine with copper ions, wherein the copper ions are chelated by the four amine groups of trientine in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ions in the composition are not complexed with the copper-chelatable tetramine.

[0018] In some embodiments according to any of the methods described above, the method further comprises administering to the individual an effective amount of copper ions.

[0019] In some embodiments according to any of the methods described above, the effective amount of the composition is insufficient to reduce extracellular copper levels in the individual.

[0020] In some embodiments according to any of the above methods, 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, about 350 mg, or about 400 mg) of the tetramine that can chelate copper per day. In some embodiments, the composition is administered at least twice per day (including, for example, any of about twice, three times, or four times per day). In some embodiments, the composition is administered for at least about one month (including, for example, any of about 1, 2, 3, 4, 5, 6, 8, 10, 12, or more months).

[0021] In some embodiments according to any of the above methods, the administration of the composition results in at least about 0.005 mg / L (including, for example, at least about 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 tetramine that can chelate copper in the blood. In some embodiments, the administration of the composition results in at least about 0.005 mg / L of the tetramine that can chelate copper in the blood for at least about 1 week (including, for example, any of at least about 2 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 12 months, or more).

[0022] In some embodiments according to any of the above methods, the method further comprises monitoring intracellular copper levels in the individual. In some embodiments, the method further comprises adjusting the dosing (including, for example, effective amount, frequency of administration, and combinations thereof) of the composition based on the intracellular copper levels in the individual.

[0023] In another aspect of the application, a pharmaceutical composition is provided, comprising a tetramine that can chelate copper and a copper ion. In some embodiments, the tetramine that can chelate copper 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule.

[0024] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition is formulated as a tablet, capsule, or pill.

[0025] Also provided are compositions, kits, and articles of manufacture useful in the methods described herein.

[0026] It should be understood that the aspects and embodiments of the invention described herein include "consisting of these aspects and embodiments" and / or "consisting substantially of these aspects and embodiments".

[0027] When “about” or “approximately” is used, the numerical value or parameter in this text includes (and describes) variations with respect to that numerical value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0028] The term “about X to Y” used in this article has the same meaning as “about X to about Y”.

[0029] Unless the context clearly indicates otherwise, nouns without quantifiers used herein and in the appended claims mean one / type and / or more / types.

[0030] It will be apparent to those skilled in the art that such active individuals are needed for the treatment assessment, selection, and / or treatment of the individual. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0032] Figure 1 The crystal structure of the trientine complex with copper ions, which further associates with two chloride ions and water molecules, is shown. The unlabeled atoms are hydrogen atoms.

[0033] Figure 2 Listed Figure 1 An exemplary set of bond lengths, bond angles, and torsion angles in a medium crystal structure;

[0034] Figure 3 Listed Figure 1 Crystal and structural refinement data of the exemplary crystal in the image;

[0035] Figures 4A to 4C Listed Figure 3 Atomic coordinates and anisotropy parameters of atoms in refined crystal structures; Figure 4A The fractional atomic coordinates and equivalent isotropic displacement parameters of non-hydrogen atoms are listed; Figure 4B The anisotropic displacement parameters for non-hydrogen atoms are listed; Figure 4C The atomic coordinates and isotropic displacement parameters of the hydrogen atom are listed;

[0036] Figure 5 A flowchart of the experimental procedure in Example 2 is shown;

[0037] Figure 6 The intracellular copper concentrations of primary neonatal rat cardiomyocytes in different experimental groups of Example 2 are shown.

[0038] Figure 7 A flow chart showing the experimental procedure of Example 3 is shown;

[0039] Figure 8A A morphological change in the interventricular septum depth (IVSD) detected by echocardiography in rats with or without Trientine treatment of AAC and sham groups is shown;

[0040] Figure 8B A morphological change in the left ventricular posterior wall depth (IVPWD) detected by echocardiography in rats with or without Trientine treatment of AAC and sham groups is shown;

[0041] Figure 9A A functional change in the left ventricular ejection fraction (EF) detected by echocardiography in rats with or without Trientine treatment of AAC and sham groups is shown;

[0042] Figure 9B A functional change in the left ventricular shortening fraction (FS) detected by echocardiography in rats with or without Trientine treatment of AAC and sham groups is shown;

[0043] Figure 10A A mean copper concentration in heart tissue of rats in the sham control group and in the untreated and Trientine-treated ACC groups is shown;

[0044] Figure 10B A mean copper concentration in plasma of rats in the sham control group and in the untreated and Trientine-treated ACC groups is shown; Trientine treatment in both the high-dose Trientine-treated group (ACC-Tr(H)) and the low-dose Trientine-treated group (ACC-Tr(L)) reduced the initial high level of plasma copper concentration in AAC rats;

[0045] Figure 11 A flow chart showing the experimental procedure of Example 4 is shown;

[0046] Figure 12 A change in the left ventricular ejection fraction (EF) detected by echocardiography in Rhesus monkeys with heart failure in the untreated and Trientine-treated groups is shown;

[0047] Figure 13 Copper concentrations in different tissue samples of rhesus monkeys with heart failure in the untreated group and the trientine-treated group are shown;

[0048] Figure 14 A flow chart showing the experimental procedure of Example 5 is shown;

[0049] Figure 15 Changes in left ventricular ejection fraction (LVEF) detected by echocardiography in mice with myocardial infarction in the untreated group and the trientine-treated group are shown;

[0050] Figure 16 Copper concentrations in different tissue samples of mice with myocardial infarction in the untreated group and the trientine-treated group are shown. DETAILED DESCRIPTION

[0051] The use of trientine to deliver copper to ischemic tissue is disclosed, and those skilled in the art can make appropriate modifications to the process parameters to implement it with reference to the content herein. In particular, it is pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application, to implement and apply the present technology.

[0052] The present application provides methods and compositions for ischemic tissue repair and regeneration by promoting the tissue redistribution and reuse of copper. In particular, methods for increasing intracellular copper levels in ischemic tissue in an individual having ischemic tissue damage by administering a tetramine composition comprising a copper-chelatable tetramine (e.g., trientine) and optionally a copper ion-promoting composition are described. The inventions described herein are based on the surprising finding that copper-chelatable tetramines (e.g., trientine) previously used to remove copper ions and reduce copper levels can promote the redistribution of copper between ischemic myocardium and the blood circulation when used according to any of the methods of the present application. For example, trientine can specifically bind to ischemic tissue and be used to load copper into the cells of ischemic tissue. Thus, trientine and other copper-chelatable tetramines with similar properties can be used to increase intracellular copper levels in ischemic myocardium, thereby restoring copper-dependent HIF-1 transcriptional activity, promoting tissue repair, and reversing myocardial ischemic infarction. Thus, the methods and compositions described herein can be used to treat different ischemic diseases and conditions.

[0053] Methods for increasing intracellular copper levels

[0054] The present application provides, in one aspect, a method of increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine (also referred to hereinafter as "tetramine composition"). In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to decrease extracellular copper levels 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0055] In some embodiments, a method of increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage is provided, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine and administering to the individual an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the pro-copper composition can alter the distribution of copper in organelles. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0056] In some embodiments, there is provided a method of increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and administering to the individual an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before administering 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0057] In some embodiments, there is provided a method of increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and administering to the individual an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before administering 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0058] In some embodiments, there is provided a method of increasing intracellular copper levels in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the effective amount of the pro-copper composition increases extracellular copper levels in the individual. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before the 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 (e.g., 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 per day.

[0059] In some embodiments, there is provided a method of delivering copper ions into cells of ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the tetramine composition further comprises copper ions. In some embodiments, the copper ions in the tetramine composition are complexed with the tetramine that can chelate copper. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and copper ions, wherein the copper ions are chelated by the four amine groups of trientine in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ions in the tetramine composition are not complexed with the tetramine that can chelate copper. In some embodiments, the method further comprises administering to the individual an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the effective amount of the tetramine composition is insufficient to decrease extracellular copper levels 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 (e.g., 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 per day.

[0060] In some embodiments, there is provided a method of delivering copper ions into cells of ischemic tissue in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and administering to the individual an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper 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 (e.g., 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 per day.

[0061] In some embodiments, there is provided a method of delivering copper ions into cells of ischemic tissue in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and administering to the individual an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper composition are administered sequentially. In some embodiments, the effective amount of the pro-copper composition increases extracellular copper levels 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 (e.g., 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 per day.

[0062] In some embodiments, there is provided a method of delivering copper ions into cells of ischemic tissue in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has previously been administered a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0063] In some embodiments, there is provided a method of delivering copper ions into cells of ischemic tissue in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has previously been administered an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the effective amount of the pro-copper composition increases extracellular copper levels in the individual. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0064] In some embodiments, there is provided a method of promoting tissue redistribution and reuse of copper in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the method further comprises administering to the individual 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 extracellular copper levels in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0065] In some embodiments, there is provided a method of promoting tissue redistribution and reuse of copper in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine and administering to the individual an effective amount of a copper-promoting composition that can increase extracellular copper levels in the individual. In some embodiments, the copper-chelatable 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 uptake, 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 from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0066] In some embodiments, there is provided a method of promoting tissue redistribution and reuse of copper in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and administering to the individual an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper composition are administered sequentially. In some embodiments, the effective amount of the pro-copper composition increases extracellular copper levels in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0067] In some embodiments, there is provided a method of promoting tissue redistribution and reuse of copper in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has previously been administered a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more, prior to administering the tetramine composition. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0068] In some embodiments, there is provided a method of promoting tissue redistribution and reuse of copper in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the effective amount of the pro-copper composition increases extracellular copper levels in the individual. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before administering 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 (e.g., 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 per day.

[0069] In some embodiments, the intracellular copper levels in the ischemic tissue of the individual are increased by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more, compared to the intracellular copper levels of the individual's ischemic tissue prior to treatment. In some embodiments, the copper levels (e.g., total copper levels) of the individual's ischemic tissue are increased by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more, compared to the copper levels of the individual's ischemic tissue prior to treatment. In some embodiments, the method does not decrease extracellular copper levels (e.g., copper levels in serum) in the individual. In some embodiments, the method does not decrease extracellular copper levels (e.g., copper levels in serum) in the individual by more than about any of 5%, 10%, 20%, 30%, 40%, 50%, or more, compared to the extracellular copper levels of the individual prior to treatment. In some embodiments, the method does not decrease total copper levels in the individual. In some embodiments, the method does not decrease total copper levels in the individual by more than about any of 5%, 10%, 20%, 30%, 40%, 50%, or more, compared to the total copper levels of the individual prior to treatment. In some embodiments, after administration of the tetramine composition, the individual has at least about any of 50%, 60%, 70%, 80%, 90%, or more of the average total copper levels in serum of healthy individuals.

[0070] Any of the above methods can further comprise monitoring (including measuring and determining) the copper level of the individual, 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 serum copper level of the individual. 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 Cu 1+ and Cu 2+ levels, and / or both intracellular and extracellular copper levels. In some embodiments, the copper level is the Cu 2+ level. In some embodiments, the copper level is the Cu 1+ level. In some embodiments, the copper level is the free (i.e., unbound) copper level. In some embodiments, the copper level comprises both free copper level and protein-bound copper level. In some embodiments, the method further comprises monitoring the intracellular copper level in the individual. In some embodiments, the method further comprises adjusting the administration (including, for example, effective amount, frequency of administration, and combinations thereof) of the tetraamine composition based on the intracellular copper level in the individual.

[0071] Different copper levels can be monitored individually or in combination before and / or after each administration step, and the corresponding copper levels before and after administration of the tetraamine composition 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 tetraamine composition is compared to a predetermined copper level to determine whether further increase in copper level is needed. The predetermined copper level can be the minimum copper level (e.g., intracellular copper level or extracellular copper level) necessary to promote copper-dependent HIF transcriptional activity and / or induce one or more ischemic tissue repair events. The treatment plan can be adjusted (including, for example, whether to administer a copper-promoting composition; the dose, frequency, and duration of the tetraamine composition and optionally the copper-promoting composition, etc.) based on any one of the extracellular copper level of the ischemic tissue, the serum copper level of the individual, the intracellular copper level of the ischemic tissue, other copper levels of the individual, and combinations thereof. In addition, the extent of repair of the ischemic tissue damage can be monitored to evaluate the treatment plan. Methods for monitoring the repair of ischemic tissue damage are described in the section “Methods of inducing tissue repair,” which can include, but are not limited to, evaluation of pathologies, histological, or molecular markers associated with ischemic tissue damage.

[0072] In some embodiments according to any of the methods described herein, including the methods in the section "Methods of inducing tissue repair," the method further comprises monitoring intracellular copper levels in the individual. In some embodiments, if the intracellular copper levels are at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more lower than a predetermined intracellular copper level, further increasing the intracellular copper levels in the individual after administration of the tetramine composition is required. In some embodiments where further increasing the intracellular copper levels is required, the treatment plan for the individual 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 dosing of the tetramine composition (including, for example, the effective amount, the frequency of administration, and combinations thereof) based on the intracellular copper levels in the individual. In some embodiments, the method further comprises monitoring extracellular copper levels in the individual. In some embodiments, if the extracellular copper levels in the individual are decreased 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 extracellular copper levels in the individual are at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more lower than a predetermined extracellular copper level, increasing the extracellular copper levels in the individual is required. In some embodiments where increasing the extracellular copper levels in the individual is required, the treatment plan for the individual is also adjusted by any one or a combination of the following: (a) administering a tetramine composition comprising copper ions, where the tetramine composition of the current treatment plan does not comprise copper ions; (b) administering a pro-copper composition, where the current treatment plan does not include administration of a pro-copper composition; (c) increasing the dose of the pro-copper composition; (d) increasing the frequency of the dose of the pro-copper composition; (e) administering a different pro-copper composition; or (f) discontinuing administration of the tetramine composition to the individual.

[0073] Copper levels can be determined and / or monitored using any method known in the art. For example, copper levels can be quantitatively determined by atomic absorption spectrophotometry, by inductively coupled plasma mass spectrometry (ICPMS), or by protein induced x-ray emission microscopy (PIXE). See, e.g., Cooper G.J.S. et al. Diabetes (2004) 53:2501-2508; Lu J. et al. Drug Metabolism and Disposition (2007) 35(2):221-227; and U.S. Patent Publication No.

[0074] 20100160428A1. For example, total copper levels in an ischemic tissue sample can be measured using a homogenized sample of the ischemic tissue (e.g., ischemic tissue homogenized with nitric acid), which includes both intracellular and extracellular contents of the ischemic tissue. Intracellular copper levels in an ischemic tissue sample can be measured using cells isolated from the ischemic tissue sample, wherein the cells are further lysed to release intracellular contents prior to analysis. Extracellular copper levels in an individual can be measured using a body fluid sample, including but not limited to serum, plasma, cerebrospinal fluid, lymph, and mucus. In some embodiments, serum is used to monitor extracellular copper levels. In some embodiments, a liver biopsy is used to determine metabolic copper levels in an individual. For example, electron paramagnetic resonance spectroscopy can be used to detect the oxidation state of copper (Cu 1+ with respect to Cu 2 + ), and provide the percentage of each oxidation state of copper in the sample. Thus, Cu 2+ levels can be calculated using the percentage of Cu 1+ in the sample, as well as the total copper levels including both Cu 2+ and Cu 2+ . Similarly, Cu 1+ levels can be calculated using the percentage of Cu 1+ in the sample, as well as the total copper levels including both Cu 2+ and Cu 1+serum albumin concentration to monitor levels of copper available for uptake and / or reutilization by ischemic tissue. In some embodiments, cross-section slices of ischemic tissue samples can be used to measure both intracellular copper levels and extracellular copper levels using X-ray fluorescence imaging (XRF) methods.

[0075] The methods described herein are generally applicable to redistributing copper in various ischemic tissues (including, for example, increasing intracellular copper levels and / or delivering copper to cells). 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue.

[0076] Methods of inducing tissue repair

[0077] The present application provides, in one aspect, a method of inducing at least one (including, for example, any of at least 2, 3, 4, 5, 6, 7, or more) tissue repair event in an ischemic tissue of an individual having an ischemic tissue injury, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the tetramine composition is administered orally. In some embodiments, the effective amount of the tetramine composition is from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0078] In some embodiments, there is provided a method of inducing at least one (including, for example, any of at least 2, 3, 4, 5, 6, 7, or more) tissue repair event in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine and an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the tetramine composition and the pro-copper 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 (e.g., 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 per day.

[0079] In some embodiments, there is provided a method of inducing at least one (including, for example, any of at least 2, 3, 4, 5, 6, 7, or more) tissue repair event in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine and an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper 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 (e.g., 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 per day.

[0080] In some embodiments, there is provided a method of inducing at least one (including, for example, any of at least 2, 3, 4, 5, 6, 7, or more) tissue repair event in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before 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 (e.g., 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 per day.

[0081] In some embodiments, there is provided a method of inducing at least one (including, for example, any of at least 2, 3, 4, 5, 6, 7, or more) tissue repair event in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition that comprises copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before 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 (e.g., 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 per day.

[0082] In some embodiments according to any of the methods of inducing tissue repair described above, at least one tissue repair event (e.g., at least two tissue repair events) comprises inducing stem cells to migrate to the ischemic tissue, including but not limited to mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs), pluripotent stem cells, induced pluripotent stem cells (iPSs), or various tissue-derived stem cells. In some embodiments, at least one tissue repair event (e.g., at least two tissue repair events) comprises inducing stem cells to differentiate in the ischemic tissue. In some embodiments, at least one tissue repair event (e.g., at least two tissue repair events) comprises inducing tissue regeneration in the ischemic tissue. In some embodiments, at least one tissue repair event (e.g., at least two tissue repair events) comprises reversing damage in the ischemic tissue. In some embodiments, at least one tissue repair event (e.g., at least two tissue repair events) comprises reestablishing a microenvironment of neurofibrillary cells and neurosecretory cells in the ischemic tissue. In some embodiments, at least one tissue repair event (e.g., at least two tissue repair events) comprises inducing signaling molecules that trigger tissue regeneration. In some embodiments, at least one tissue repair event (e.g., at least two tissue repair events) comprises promoting copper-dependent HIF-1 transcriptional activity in the ischemic tissue.

[0083] The methods described herein can be used to induce tissue repair events (including, for example, promoting copper-dependent HIF-1 transcriptional activity and / or inducing stem cells to migrate to the ischemic tissue) in different types of ischemic tissue. 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic limb tissue). In some embodiments, the ischemic tissue is ischemic heart tissue. In some embodiments, the ischemic tissue is ischemic brain tissue.

[0084] In some embodiments, there is provided a method of inducing stem cells to migrate (i.e., home) to ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises copper ions. In some embodiments, the copper ions in the tetramine composition are complexed with the copper-chelatable tetramine. In some embodiments, the tetramine composition comprises a crystalline complex of trientine and copper ions, wherein the copper ions are chelated by the four amine groups of trientine in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ions in the tetramine composition are not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels 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 (e.g., 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 per day.

[0085] In some embodiments, there is provided a method of inducing stem cells to migrate (i.e., home) to ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine and an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the tetramine composition and the pro-copper 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 (e.g., 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 per day.

[0086] In some embodiments, there is provided a method of inducing stem cells to migrate (i.e., home) to ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper 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 (e.g., 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 per day.

[0087] In some embodiments, there is provided a method of inducing stem cells to migrate (i.e., home) to ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has previously been administered an effective amount of a pro-copper composition that can increase the individual’s extracellular copper levels. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before the 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 (e.g., 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 per day.

[0088] In some embodiments, methods of inducing stem cells to migrate (i.e., home) to ischemic tissue of an individual having an ischemic tissue injury are provided, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition comprising a copper ion. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more before 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 (e.g., 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 per day.

[0089] In some embodiments, the stem cells are mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs), pluripotent stem cells, induced pluripotent stem cells (iPS), or tissue-derived stem cells. In some embodiments, the tissue-derived stem cells are adipose tissue-derived stem cells, cardiac tissue-derived stem cells, or umbilical cord tissue-derived stem cells. In other embodiments, the stem cells are adult stem cells. In some particular aspects, the adult stem cells are hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells in the Wharton's jelly or dental (e.g., dental pulp and periodontal ligament perivascular niche) of the placenta, adipose tissue, lung, bone marrow, blood, umbilical cord, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or germ line stem cells (e.g., stem cells in the testes).

[0090] In some embodiments, the stem cells migrate in vivo from one organ or tissue compartment of an individual having an ischemic tissue injury to the site of ischemic injury of another organ or tissue compartment. For example, MSCs can migrate from bone marrow (BM), umbilical cord blood (UCB), umbilical cord mesenchyme (Wharton's jelly), placenta, and adipose tissue (AT). In other embodiments, MSCs can be isolated from an organ or tissue compartment, enriched and / or manipulated in vitro, and then used to migrate in vivo to a tissue or organ injury site.

[0091] Assays that can be used herein to measure cell migration include, but are not limited to, in vivo biomarkers, bioluminescence, fluorescence, positron emission tomography (PET) / CT, and magnetic resonance imaging (MRI). In vivo assays can be validated and confirmed with other methods, such as IHC on tissue sections.

[0092] In vivo non-invasive imaging techniques for assaying stem cell migration include imaging gold dextran-coated particles loaded into MSCs, which can be visualized using X-ray, Raman spectroscopy, computed tomography (CT), or ultrasound (US). In some embodiments, stem cells (e.g., MSCs) are loaded with biocompatible nanoparticle constructs, tracers, or superparamagnetic particles with properties that enable visualization of the cells by X-ray, CT, US, PET, or MRI. In some embodiments, stem cell migration can be assayed using techniques such as cecal ligation and puncture (CLP). For example, CLP in GFP chimeric mice allows for the observation of BMSC behavior in the context of abdominal sepsis. FACS, flow cytometry, and immunohistochemistry can be used to track the migration of BMSCs into the peripheral blood, lung, liver, skin wounds, and primary sites of ischemic injury. BMSC behavior can be correlated with the time of injury and local (using RT-PCR) and systemic levels of cytokines and chemokines. Tracking stem cell migration can help explain the contribution of BMSCs to local and distant organ and tissue repair and regeneration following ischemic tissue injury.

[0093] In some embodiments, stem cell migration can be monitored by administering labeled cells to an individual. Stem cells are labeled using methods such as isotope labeling and staining. In some embodiments, labeling methods include injecting female animals with male stem cells, so that Y chromosomes can be a tracer; injecting stem cells of species A into species B, so that specific genes of species A can be a cell tracer; labeling stem cells with pKH26, BrdU, or other dyes, so that stem cells can be tracked by the dye or specific enzymatic reactions against the tracer.

[0094] In some embodiments, isotopic labeling is used to track stem cells in vivo. Stem cells can be tracked by labeling the cells with an isotope, but it is important to consider safety issues and radioactive half-life. Other methods of tracking stem cells in vivo include, but are not limited to, cell staining by cell dyes such as DID; live imaging of surface cells by two-photon excitation fluorescence microscopy; live imaging of specific surface cells of transgenic animals by two-photon excitation fluorescence microscopy; labeling of cells with SPIO and tracking of the label by MRI, and the like. Stem cells can be labeled with a variety of fluorescent dyes and then injected into an animal. Shortly before the tracking experiment, the target organ can be frozen, sectioned, and directly observed under confocal laser scanning microscopy. This method of tracking does not require as many labeled cells (10^6 cells / rabbit) and thus autologous cells can be tracked in the natural background of organs and tissues.

[0095] Labeling of stem cells can be accomplished, for example, by a single tracker such as pKH26. pKH26 is a lipophilic dye and the label does not permeate the cell membrane. Thus, pKH26 is suitable for live imaging. The tracking methods described herein can involve multiple labeling by 2 or 3 dyes. In some embodiments, multiple labeling is performed using a nuclear tracker (DAPI, Hoechst) plus a membrane tracker. The nuclear tracker identifies the nucleus of the cell and simultaneously the membrane tracker pKH26 is attached. In some embodiments, multiple labeling is performed using 2 membrane trackers (e.g., Dio(3) & pKH26). These trackers label the cell by similar mechanisms but have different excitation and emission wavelengths, allowing simultaneous determination of the migration (i.e., homing) of stem cells (e.g., BMSCs) by 2 different fluorescent signals. In this tracking method, only the overlapping signal of different wavelengths (e.g., red and green signals) is considered as the homing signal.

[0096] Many animal tissues are autofluorescent and the most common autofluorescence in natural tissues is green fluorescence. Heart cells have a relatively low fluorescence, but it is strong enough to interfere with observation. The cut edge of a section is often the most fluorescent. To resolve the interference, only green and red overlapping signals can be identified as tracking signals. Red fluorescence is more suitable for statistical analysis with IOD values specific for it (except for obvious inaccuracy in red fluorescence signals).

[0097] In some embodiments, a method of inducing stem cells to differentiate in ischemic tissue is provided, comprising administering to an individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the tetramine that can chelate copper. In some embodiments, the tetramine composition comprises a crystalline complex of trientine with a copper ion, wherein the copper ion is chelated by the four amine groups of trientine in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the tetramine that can chelate copper. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior to the tetramine composition. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the tetramine composition is administered orally. In some embodiments, the stem cells are capable of differentiating into mesenchymal cell types, including osteoblasts, adipocytes, chondrocytes, endothelial cells, epithelial cells, enterocytes, osteocytes, neural cells, hepatocytes, renal cells, muscle cells (skeletal and smooth), and cardiomyocytes. In other embodiments, the stem cells are capable of differentiating into non-mesodermal-derived cells, including beta cells, hepatocytes, and neurons. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0098] The process of stem cell differentiation and the phenotype of differentiated stem cells (e.g., MSCs, e.g., BMSCs) can be elucidated using assays known in the art, including, but not limited to, alkaline phosphatase and alizarin red S staining for osteoblasts, oil red O staining for adipocytes, and Alcian blue staining for chondrogenesis. Differentiation of stem cells (e.g., MSCs) into multiple cell types can also be assayed by gene expression profiling. For example, transcriptional profiling has identified specific genes involved in osteogenic differentiation (FHL2, ITGA5, Fgf18), chondrogenesis (FOXOlA), and tenogenesis (Smad8). In some embodiments, MSCs can be produced in high cell numbers by large-scale expansion.

[0099] In some embodiments, a method of inducing tissue regeneration is provided, comprising administering to an individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior to the tetramine composition. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 vascular maturation in ischemic tissue. In some embodiments, the method produces two or more of the above effects. In some embodiments, the effective amount of the tetramine composition is about 80 mg to about 450 mg (e.g., 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 per day.

[0100] Tissue regeneration disclosed herein can be assayed, for example, in an organism in which a portion of the tissue is damaged or removed. A tetramine composition described herein is then administered to the organism, and the rate of tissue regeneration is determined. The rate of tissue regeneration is compared to the rate observed when a control or no treatment is administered to the organism. Other parameters that can be determined during the tissue regeneration assay include, but are not limited to, for example, symptoms or outcomes of pain or markers of pain, signs or symptoms of inflammation, extent of final regeneration, and quality of regeneration. In some embodiments, the tissue regeneration assay herein comprises assessing one or more organ functional parameters, such as one or more markers of cardiac function, one or more markers of renal function, and one or more markers of brain function.

[0101] In some embodiments, the methods described herein can be evaluated using one or more of the following parameters in the analysis of cardiac regeneration and repair: (1) amount of remodeled tissue or myocardial mass and coronary vascularization; (2) restoration of the number and size of myocytes and blood vessels; (3) integration of newly formed myocytes and blood vessels with surrounding myocardium; and (4) origin of the regenerated myocardial structure. In one aspect, magnetic resonance imaging (MRI) can be performed to study scar area, global left ventricular function, regional function (wall motion and thickening), and regional ventricular perfusion. In another aspect, MRI is used to detect and / or determine the presence of new blood vessels, tissue or cells that improve ventricular function. In yet another aspect, histopathology can be performed to determine scar area as well as identification and quantification of c-kit positive cardiac stem cells. Histopathology also provides data on the distribution, size and density of new blood vessels and myocardial cells. Histopathology allows documentation of the repair process at the tissue and cellular level. For example, tests are performed to evaluate microvessel density (vWF positive vessels / mm 2 ), BrdU positive cells and c-kit positive cells within the infarcted section. The use of von Willebrand factor (vWF) to quantify microvessel density allows determination of the amount of new blood vessels generated in the infarcted area. BrdU positive cells represent the proliferation of cells, including cardiac cells. The c-kit positive cell test shows the amount of stem cells within the selected infarcted section.

[0102] In some embodiments, a method of reversing injury in ischemic tissue of an individual having ischemic tissue injury is provided, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 (e.g., 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 per day.

[0103] Reversal of tissue damage can be measured by any suitable method, such as detecting cellular markers of normal tissue homeostasis and / or persistent tissue damage (e.g., by immunohistochemistry or measuring DNA and transcript levels), measuring lesion area or lesion volume, or assessing any clinically relevant indicator. For example, reversal of cardiac tissue damage in infarcted tissue can be measured by quantifying cell number (e.g., number of muscle cells, fibroblasts) or amount of scarring, or with functional assays for cardiac functional output or structural aspects including LVEDP, LVDP, max dp / dt, min dp / dt, LV Weight, Chamber Volume, and Diastolic Wall Stress. In general, the methods disclosed herein are considered to reverse damage in ischemic tissue if there is a significant change (e.g., at least a 2-fold change) in any such clinical assessment or any combination thereof. In some embodiments, the methods reverse fibrosis in ischemic tissue. Fibrosis is the abnormal accumulation of fibrous tissue, which can occur as part of the wound healing process in damaged tissue. Such tissue damage can be caused by physical injury, inflammation, infection, exposure to toxins, and other causes.

[0104] A consequence of hypertension, hypertensive heart disease, atherosclerosis, and myocardial infarction is the accumulation of fibrotic tissue in the heart and blood vessels. Hypertension, or high blood pressure, can be caused by a variety of factors and often leads to the development of hypertensive heart disease (HHD) and progression to cardiac arrest and myocardial infarction. Similarly, atherosclerosis and other ischemic heart diseases also often lead to cardiac arrest. These cardiovascular diseases all exhibit accumulation of extracellular matrix or fibrotic deposition, which leads to stiffening of blood vessels and the heart tissue itself. This deposition of fibrotic material is a response to damage caused by hypertension and / or hardening states, and the effects of this response also adversely affect the stiffening of blood vessels and the heart and the enlargement of the ventricles. In some cases, increased cardiac fibrosis in cardiovascular diseases interferes with or alters signals transmitted to cardiac muscle cells through the tissue scaffolding of the heart, further contributing to the disruption of effective cardiac function and promoting cardiac arrest and myocardial infarction.

[0105] According to the present disclosure, the expression profile of genes differentially regulated during tissue injury can be used to assess the reversal of tissue injury in the treatment methods disclosed herein. For example, microarray-based gene expression analysis can be based on analysis of human cells (e.g., fibroblasts and cardiomyocytes) subjected to selected stimuli that result in altered accumulation of extracellular collagen and proliferation, hallmarks of fibrosis. The stimuli can be selected to mimic those during tissue-specific fibrotic processes. Gene expression profiles associated with fibrosis (e.g., liver fibrosis, lung fibrosis, cardiac tissue fibrosis, diabetic nephropathy, and kidney fibrosis) can then be used to determine fibrosis and reversal of fibrotic injury to the tissue. In other embodiments, gene expression profiles associated with reversal of fibrosis (e.g., under treatment known to at least partially reverse fibrosis) can be used to determine fibrosis and reversal of fibrotic injury to the tissue.

[0106] In some embodiments, a method of reestablishing the microenvironment of neurofibrocytes and neurosecretory cells in ischemic tissue of an individual having ischemic tissue injury is provided, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior to the tetramine composition. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 (e.g., 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 per day.

[0107] The microenvironment is a complex network of both structural and inflammatory cells, cytokines, proteins, and growth factors. In the context of ischemia associated with a cardiac fibrotic disease or condition, the heart comprises resident structural cells such as cardiomyocytes, epithelial cells, fibroblasts, as well as resident cardiomyocyte progenitor and cytokine secreting cells. During fibrosis pathogenesis, these cells interact with fibrotic factors. In certain aspects, fibroblasts and myofibroblasts play an important role in creating a fibrotic environment as they secrete excess collagen and matrix material that leads to irreversible scarring. 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 assayed in the tissue microenvironment. For example, cell differentiation and migration occur in response to cues from the microenvironment (e.g., stiffness of the surrounding matrix). In one aspect, the elasticity of the tissue or culture matrix of mesenchymal stem cells (MSCs) is assayed and modulated to promote stem cell homing to an ischemically damaged tissue, stem cell differentiation at the site of ischemic injury, tissue repair, and / or tissue injury reversal. In one embodiment, a soft matrix leads to MSC differentiation into neuron-like cells, while a hard matrix leads to MSC differentiation into myogenic cells. In one aspect, the extracellular matrix and its components at the site of ischemic injury are assayed 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 tissue injury reversal.

[0108] In some embodiments, changes in cells in the context of their natural environment are measured to indicate efficacy and / or toxicity of the treatment methods disclosed herein. In some embodiments, the stem cell microenvironment of a donor tissue or organ (e.g., bone marrow) and the site of ischemic injury is assayed and / or modulated to promote stem cell migration to the site, stem cell differentiation at the site of ischemic injury, tissue repair, and / or tissue injury reversal. Local tissue microenvironments can be assayed by protein staining (IHC and IF) and RNA staining with chromogenic or fluorescent ISH. For example, hypoxic microenvironments can be indicated by hypoxic marker staining, endothelial cell marker staining, microvessel density analysis, and proximity analysis. Tissue microenvironments can also be studied using organ cultures or organotypic cultures as disclosed in Benbrook, 2006, Drug Discovery Today: Disease Models, 3(2): 143-148.

[0109] In some embodiments, there is provided a method of inducing a signaling molecule that triggers tissue regeneration in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 (e.g., 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 per day.

[0110] Suitable signaling molecules described herein include, but are not limited to, HIF-1, VEGF, SDF-1, CXCR4, CXCL12 (also known as SDF-1a), MMP, HGF / c-met, TGF-β1, IL-1β, TNF-a, CCR1, CCR4, CCR7, CCR10, CCR9, CXCR5, CXCR6, CD44, CD54, CD56, CD106, E-cadherin, P-selectin, integrins (e.g., integrin-β1 and CD49a, b, c, e, f (integrin al, 2, 3, 4, 6) and integrin ligands (e.g., VCAM and ICAM).

[0111] The SDF-1 / CXCR4 axis is one of the most important mechanisms for stem cell homing. SDF-1 (stromal cell-derived factor 1 or CXCL12) is a small secreted protein belonging to the CXC-chemokine family. SDF-1 expression is regulated by HIF-1 (hypoxia-inducible factor-1). HIF-1 is composed of HIF-1 alpha and HIF-1 beta / ARNT (aryl hydrocarbon nuclear translocator, ARNT). HIF-1 beta is stable in the cytoplasm, so the expression and accumulation of HIF-1 alpha determines the activity of HIF-1. Under normoxia, HIF-1 alpha protein is synthesized and rapidly degraded by the ubiquitin-proteasome system. Prolyl hydroxylases (PHDs) hydroxylate HIF-1 alpha, and the hydroxylated HIF-1 alpha is recognized by the von Hippel-Lindau tumor suppressor protein (pVHL), which constitutes a ubiquitin-protein ligase targeting HIF-1 alpha for protein degradation. After ischemic tissue injury, the injured area is hypoxic, which inhibits the activity of PHD, allowing HIF-1 alpha to accumulate and translocate into the nucleus, where HIF-1 alpha dimerizes with HIF-1 beta to form HIF-1, which combines with other factors and initiates target gene transcription. The injured tissue expresses high levels of SDF-1 and releases SDF-1 into the circulation, thereby establishing a concentration gradient from the injured area to the distal end of the circulation. This gradient thus attracts CXCR4-expressing stem cells, including BMSCs, to the injured tissue.

[0112] When the heart is under chronic hypoxia, the blood in the coronary arteries cannot meet the needs of the myocardium. Thus, chronic ischemia can induce myocardial fibrosis, reduce the density of arterioles, affect blood pumping, and ultimately lead to ischemic heart infarction. Under chronic ischemia, the activity of HIF-1 is limited, thereby causing the expression of angiogenic factors regulated by HIF-1 to be inhibited. Thus, the blood supply cannot be restored and infarction will occur.

[0113] In general, HIF-1 activity in ischemically injured tissue is temporarily limited. Both animal experiments and clinical trials have shown that under cardiac ischemia, HIF-1 alpha in the injured tissue accumulates immediately after injury, but then gradually decreases. The activity of HIF-1 even decreases faster than the level of HIF-1, thereby causing the expression of HIF-1-regulated factors, such as VEGF and SDF-1, to decrease after a short increase. Due to the regulation of HIF-1, SDF-1 expression peaks on the first or second day after heart infarction. Then, SDF-1 expression gradually decreases and drops to baseline levels within about a month. Since SDF-1 is one of the stem cell homing mobilizers, the decrease in SDF-1 levels leads to a decrease and even disappearance of stem cell homing.

[0114] Importantly, the role of HIF-1a-induced defense mechanisms activated as in acute ischemic conditions differs from that in chronic ischemic conditions. In chronic ischemic conditions, HIF protein levels are elevated in ischemic myocardium, and HIF-regulated genes (e.g., VEGF) are repressed, which results in decreased revascularization and impaired regeneration. Copper deficiency reduces HIF-1a binding to HRE sequences of target genes and to P300, a component of the HIF-1 transcription complex. Furthermore, after long-term ischemia, copper is significantly mobilized from myocardium to blood. This copper mobilization in coronary flow occurs sensitively after long-term but not short-term cardiac ischemia. Loss of myocardial copper correlates with the extent of loss of cardiac function. Thus, even in conditions of elevated HIF protein levels, upregulation of HIF-controlled genes does not occur due to loss of myocardial copper. Trace elements (e.g., copper) can contribute to the activation of HIF-1, including HIF-1a synthesis, stabilization, translocation from cytosol to nucleus, binding to HRE sequences of target genes, and HIF-1 transcription complex formation. Thus, copper-dependent HIF-1 transcriptional activity, including copper-dependent induction of HIF-1 target genes or copper-dependent repression of HIF-1 target genes, can play an important role in the repair of ischemic tissues. The methods described herein can be used to induce one or more signaling molecules, such as HIF-1a and copper-dependent HIF-1 (e.g., HIF-1a) target genes.

[0115] In one aspect of the application, a method of promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage is provided, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels 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 the ischemic tissue of the individual. In some embodiments, the method represses expression of at least one copper-dependent HIF-1 target gene in the ischemic tissue of the individual. 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 from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0116] In some embodiments, there is provided a method of promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and an effective amount of a copper-promoting composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the copper-promoting composition does not comprise copper ions. In some embodiments, the copper-promoting composition can increase copper uptake, 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 the ischemic tissue of the individual. In some embodiments, the method represses expression of at least one copper-dependent HIF-1 target gene in the ischemic tissue of the individual. 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 from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0117] In some embodiments, there is provided a method of promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and an effective amount of a copper-promoting composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the copper-promoting composition does not comprise copper ions. In some embodiments, the copper-promoting composition can increase copper uptake, 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 the ischemic tissue of the individual. In some embodiments, the method represses expression of at least one copper-dependent HIF-1 target gene in the ischemic tissue of the individual. 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 from about 80 mg to about 450 mg (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg) per day. In some embodiments, the tetramine composition is administered twice per day.

[0118] In some embodiments, there is provided a method of promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise copper ions. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more, prior to 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 the ischemic tissue of the individual. In some embodiments, the method represses expression of at least one copper-dependent HIF-1 target gene in the ischemic tissue of the individual. 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0119] In some embodiments, there is provided a method of promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is copper ions. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more, prior to 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 the ischemic tissue of the individual. In some embodiments, the method represses expression of at least one copper-dependent HIF-1 target gene in the ischemic tissue of the individual. 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 per day (e.g., about 80 mg to about 300 mg, or about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0120] HIF-1 target genes have been described in the art. See, e.g., Benita Y. et al., (2009) Nucleic Acids Research, 37(14):4587-4602; Shen C. et al., (2008) J. Biol. Chem., 280:20580-20588; Elvidge G.P. et al., (2006) J. Biol. Chem., 281 :15215-15266; Manalo DJ. et al., (2005) Blood, 105:659-669; HIF-1 target genes described in the references are incorporated herein by reference. A subset of HIF-1 target genes are dependent on copper for transcriptional regulation by HIF-1, and the subset of HIF-1 target genes are referred to herein as copper-dependent HIF-1 target genes. Some HIF-1 target genes by HIF-1 are independent of copper for transcriptional regulation by HIF-1. See, e.g., Zhang Z et al., (2014) Metallomics 6(10): 1889-93. 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 BCL2 / adenovirus E1B 19kDa protein-interacting protein 3 (BNIP3).

[0121] The copper-dependent HIF-1 transcriptional activity contemplated by the present application includes induction or repression (i.e., transcriptional modulation) of expression of a copper-dependent HIF-1 target gene in ischemic tissue. In some embodiments, the copper-dependent HIF-1 transcriptional activity (e.g., fold induction or repression of a copper-dependent HIF-1 target gene) in the ischemic tissue of an individual prior to receiving treatment is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, as compared to a control level. In some embodiments, the copper-dependent HIF-1 transcriptional activity (e.g., fold induction or repression of a copper-dependent HIF-1 target gene) in the ischemic tissue of an individual after receiving treatment is restored to at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, as compared to a control level. The control level of copper-dependent HIF-1 transcriptional activity can be based on the fold induction or repression of a HIF-1 target gene in healthy tissue under acute ischemic conditions or under comparable levels of hypoxic conditions relative to normal (e.g., unimpaired or normoxic) conditions. 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 ischemic tissue to the expression level of that copper-dependent HIF target gene in healthy tissue. RNA expression levels can be measured using any known method 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 known method in the art, including but not limited to antibody-based methods (e.g., Western blot and ELISA) and quantitative proteomic methods (e.g., quantitative mass spectrometry).

[0122] In some embodiments, there is provided a method of inducing at least two (including, for example, any of at least 3, 4, 5, 6, 7, or more) tissue repair events in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine, wherein the at least two tissue repair events are selected from the group consisting of: inducing stem cells (e.g., bone marrow mesenchymal stem cells) to migrate to the ischemic tissue, inducing stem cells to differentiate in the ischemic tissue, inducing tissue regeneration in the ischemic tissue, inducing signaling molecules that trigger tissue regeneration, reversing damage in the ischemic tissue, reestablishing the microenvironment of neurofibrillary cells and neurosecretory cells in the ischemic tissue, and promoting copper-dependent HIF-1 transcriptional activity. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior to the tetramine composition. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 (e.g., 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 per day.

[0123] In some embodiments, there is provided a method of inducing stem cells (e.g., MSCs, e.g., BMSCs) to migrate to an ischemic tissue and inducing the stem cells to differentiate in the ischemic tissue, comprising administering to an individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper. In some embodiments, there is provided a method of inducing stem cells (e.g., MSCs, e.g., BMSCs) to migrate to an ischemic tissue and inducing tissue regeneration in the ischemic tissue, comprising administering to an individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper. In some embodiments, there is provided a method of inducing stem cells (e.g., MSCs, e.g., BMSCs) to migrate to an ischemic tissue, inducing the stem cells to differentiate in the ischemic tissue, and inducing tissue regeneration in the ischemic tissue, comprising administering to an individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the tetramine that can chelate copper. 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the tetramine that can chelate copper. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior to the tetramine composition. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 (e.g., 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 per day.

[0124] In some embodiments, there is provided a method of inducing repair of ischemic tissue (or improving function of ischemic tissue) in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 from about 80 mg to about 450 mg (e.g., 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 per day.

[0125] In some embodiments, there is provided a method of inducing repair of ischemic tissue (or improving function of ischemic tissue) in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine and an effective amount of a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the tetramine composition and the pro-copper 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 from about 80 mg to about 450 mg (e.g., 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 per day.

[0126] In some embodiments, there is provided a method of inducing repair of ischemic tissue (or improving function of ischemic tissue) in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper and an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the tetramine composition and the pro-copper composition are administered simultaneously. In some embodiments, the tetramine composition and the pro-copper 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 from about 80 mg to about 450 mg (e.g., 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 per day.

[0127] In some embodiments, there is provided a method of inducing repair of ischemic tissue (or improving function of ischemic tissue) in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has previously been administered an effective amount of a pro-copper composition that can increase the individual’s extracellular copper levels. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more, prior to 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 from about 80 mg to about 450 mg (e.g., 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 per day.

[0128] In some embodiments, there is provided a method of inducing repair of ischemic tissue (or improving function of ischemic tissue) in an individual having ischemic tissue damage, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper, wherein the individual has been previously administered an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the tetramine that can chelate copper is trientine. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the individual has been administered the pro-copper composition about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more, prior to 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 (e.g., 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 per day.

[0129] Also provided is a method of treating a disease or condition associated with ischemic tissue damage using any of the methods described herein.

[0130] In some embodiments, there is provided a method of treating ischemic heart failure in an individual, comprising administering to the individual an effective amount of a tetramine composition comprising a tetramine that can chelate copper (e.g., trientine). In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels 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 (e.g., 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 per day. In some embodiments, the tetramine composition is administered for at least about 1 month (e.g., at least about 3 months or at least about 6 months). In some embodiments, the individual has a left ventricular ejection function (LVEF) of no more than about 35% at baseline. In some embodiments, the individual has class II or class III heart failure (based on New York Heart Association (NYHA) Functional classification).

[0131] Any classification or stage of heart failure of ischemic origin (i.e., ischemic heart failure) can be treated with the methods described herein. 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, palpitations, shortness of breath, or limited 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 of 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30 days or more.

[0132] The efficacy of any of the methods described herein can additionally be determined by assessing the degree of repair of ischemic tissue. Tissue repair can be assessed by, for example, lesion area or lesion volume. Any clinically relevant criteria can be used to assess repair of damaged tissue in a patient. For example, repair of infarcted tissue can be measured by quantifying cell number (e.g., number of muscle cells, fibroblasts), or amount of scar formation, or with functional assays for cardiac functional output or structural aspects including LVEDP, LVDP, max dp / dt, min dp / dt, LV weight, chamber volume, and diastolic wall stress. Generally, a method disclosed herein is considered to repair damaged tissue if it produces a significant change (e.g., at least a 2-fold change) in any such clinical assessment or any combination thereof.

[0133] Any suitable method can be performed to determine tissue repair. For example, methods to assess tissue healing, assess functionality of the repaired tissue, and assess cell growth in the tissue can be performed. To determine the extent of tissue healing, histology and cell staining can be performed to detect proliferation of seeded cells and / or improved histological appearance. In some cases, tissue portions can be collected and treated with a fixative, e.g., such as neutral buffered formalin. These 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 evaluation of morphology and cellular composition. In some cases, physiological tests can be performed to assess tissue movement and functionality following treatment according to the methods and materials provided herein. For example, in vitro mechanical assays can be performed to measure work of flexion (WOF) or flexion angle of repaired tendon tissue or repaired joints. In vivo assays can include functional evaluation of an organ, symptom assessment, or imaging techniques.

[0134] In some embodiments, tissue and / or organ functionality before, during, or after administration of the treatment methods disclosed herein can be assessed by any one or more of the following: biochemical analysis of at least one biomarker indicative of improved tissue functionality by methods such as flow cytometry, immunofluorescence, ELISA, phosphorimetry, hybridization, nucleic acid amplification, or Western blotting; cell function assays such as apoptosis assays, necrosis assays, and cell viability assays including annexin V staining by immunofluorescence or flow cytometry, detection of caspase activity, hypoxia assays, TUNEL assays, cell DNA laddering, number of rod-shaped cells in response to H2O2, qPCR evaluation of gene expression, and measurement of necrotic area by H&E staining; scar formation assays including measurement of the number of fibroblasts in the damaged or infarcted area, measurement of collagen deposition and levels of other matrix proteins associated with scar formation; migration of stem or progenitor cells into the damaged area; and any other clinically relevant test of organ functionality.

[0135] In some embodiments, cardiac function can be assessed by any one or more of the following parameters: myocyte mechanics and cell fusion, such as distribution frequency of myocyte size, peak shortening, velocity of shortening and re-lengthening, 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 MI untreated subjects; myocardial regeneration, such as composition of regenerated myocardium, assessment of BrdU positive cells in infarct zone in treated versus untreated subjects, and myosin positive cells in infarct zone in treated versus untreated subjects; cardiac structure, such as infarct size, amount of fibrosis, and myocyte hypertrophy. In certain embodiments, the methods disclosed herein further comprise measuring one or more markers of cardiac function, wherein the markers of cardiac function are 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), reduction in atrial or ventricular function, increase in pumping efficiency, decrease in rate of loss of pumping efficiency, decrease in loss of hemodynamic function, or reduction in complications associated with cardiomyopathy, compared to a control.

[0136] In some embodiments, brain function can be assessed before, during, or after administration of the treatment methods disclosed herein by neurological testing, or in electrophysiological terms (e.g., by reduced signal-to-noise ratio), or in biochemical terms (e.g., by analysis of at least one biomarker indicative of 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 potential (ERP), evoked potential (EP), magnetoencephalography (MEG), and nerve conduction study (NCS). In other embodiments, brain function can be assessed by any one or more of the following methods or parameters: general intellectual functioning, such as the Wechsler Abbreviated Scale of Intelligence and the Wechsler Adult Intelligent Scale-III; basic attention, such as the Digit Span, Spatial Span Subtests from the Wechsler Memory Scale-III; complex attention (working memory), such as the Digit Span, Digit Letter Order, and Arithmetic Subtests from the Wechsler Adult Intelligent Scale-III; executive function, such as the Wisconsin Card Sorting Test, Trail Making Test B, Stroop Test, Tower of London Test, Gambling Test, Frontal System Behavior Scale, and Iowa Scale of Frontal Lobe Function;Memory (visual and verbal), such as the Wechsler Memory Scale-III, Rey Auditory, Verbal Learning Test, California Verbal Learning Test-II, Brief Visual Memory Test Revised; affect regulation, such as the Minnesota Multiphasic Personality Inventory-2, Affective Stroop Test, Frontal Systems Behavior Scale, and Iowa Gambling Task; interpretation of emotional stimuli, such as the DANVA (Diagnostic Analysis of Nonverbal Behavior); processing speed, such as the Processing Speed Index (Symbol Search, Coding) from the Wechsler Adult Intelligence Scale-III, Trail Making Test B, and Symbol Digit Modalities Test; language, such as the Boston Naming Test; Controlled Oral Word Association Test; Semantic Word Fluency Test; and Multilingual Aphasia Examination; visuo-constructional tests, such as the Rey-Osterrieth Complex Figure Test from the Wechsler Adult Intelligence Scale-III, Block Design, and Object Assembly subtest; and visual-spatial tests, such as Matrix Reasoning from the WAIS-III, and Judgment of Line Orientation Test.

[0137] In some embodiments, skeletal muscle health is tested before, during, or after administration of the therapeutic methods disclosed herein. In some embodiments, skeletal muscle health includes muscle pain, muscle injury, metabolic changes to exercise, and cytoskeletal reorganization. Skeletal muscle function can be muscle strength, muscle endurance, training adaptation, normal muscle state 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 strength (the maximum force generated in a particular movement), muscle endurance (the maximum number of contractions that can be performed at a set frequency and force), and muscle power (force / time, the maximum effect produced by a muscle). While not exhaustive, typical muscle-specific functions include myoblast differentiation, myoblast determination, muscle development, muscle contraction, sarcomere changes, myoblast fusion, somatic muscle development, and myogenesis.

[0138] In some embodiments, the patient is assessed for skeletal muscle fibrosis. A number of methods can be used to determine the status of skeletal muscle fibrosis, including obtaining a biopsy of muscle tissue from the patient and evaluating the biopsy with histochemical or immunohistochemical stains that are sensitive to the presence 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 label muscle fibers for immunohistochemical staining include, for example, myosin, collagen type IV, laminin, fibronectin, and dystrophin. Alternatively, a functional method to determine the extent to which fibrosis has infiltrated the patient's skeletal muscle can be employed. This functional method involves subjecting the patient to one or more of a battery of tests and physical measurements. Such tests and measurements typically include nerve strength testing, muscle strength, balance, gait, posture, sensory coordination evaluation, and pulmonary function tests (e.g., vital capacity and forced expiratory volume), all of which can be performed by methods known in the art. In some embodiments, tissue repair can be assessed based on the expression level of one or more signaling molecules described herein. Suitable biomarkers that are indicators of tissue repair include, but are not limited to, DNA damage biomarkers, inflammatory response biomarkers, tissue damage biomarkers, tissue damage repair biomarkers, or hematological surrogate markers, such as p53, p21, GADD45a, ATM, phosphorylated H2AX histone, 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-amyloid, citrulinated 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-1 alpha.

[0139] Copper (including copper ions) is a regulator of one or more factors (e.g., transcription factors) involved in tissue damage repair and / or tissue regeneration, and thus can be assessed by evaluating any one or more of these factors for tissue repair. Copper regulatory 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, glutamatergic N-methyl D-aspartate receptor (NMDAR), amyloid precursor protein (APP), copper metabolism gene MURR1 domain (COMMD1), X-linked inhibitor of apoptosis (XIAP), homocysteine (Hcy), subunit II of cytochrome c oxidase (COX II), subunit I of cytochrome c oxidase (COX I), FGF-1, VEGF, angiogenin (e.g., ANG1 or ANG2), fibronectin, collagenase, MMP-TIMP, 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 monooxygenase (PAM), tyrosinase, phenylalanine hydroxylase, diamine oxidase, Hephaestin, and cartilage matrix glycoprotein; extracellular Cu-binding proteins, such as ceruloplasmin (CP), lysyloxidase (LOX), albumin (ALB), transcuprein, amine oxidase, coagulation factors V and VIII, ferroxidase II, extracellular superoxide dismutase, and extracellular metallothionein. Copper regulatory factors are described in Zheng et al., Role of copper in regression of cardiac hypertrophy, Pharmacol. Ther.

[0140] doi: 10.1016 / j.pharmthera.2014.11.014 (2014), which is incorporated herein by reference. In some embodiments, copper or copper ions modulate the transcriptional activity of one or more of HIF-1, SP1, MT, Atox 1, CCS, and COMMD1, and the signaling networks modulated by these transcription factors.

[0141] In some embodiments, the levels and / or activities of one or more of the factors modulated by copper disclosed herein are analyzed in an individual after treatment with a therapeutic or prophylactic composition disclosed herein. In some embodiments, the levels and / or activities of one or more of HIF-1, SP1, MT, Atox 1, CCS, and COMMD1 are determined and then correlated with the individual's response to the therapeutic or prophylactic 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 damage, or evaluating any clinically relevant indicators. Thus, in certain aspects, the levels and / or activities of one or more copper modulated factors (e.g., HIF-1, SP1, MT, Atox 1, CCS, and COMMD1) can be used as an endpoint biomarker of an individual's response to a therapeutic or prophylactic regimen disclosed herein.

[0142] In some embodiments, one or more of the factors modulated by copper disclosed herein can be used in a prognostic test to analyze and predict response to a tetramine composition or treatment or prevention method disclosed herein. For example, the level and / or activity of one or more of HIF-1, SP1, MT, Atox 1, CCS, and COMMD1 can indicate the likelihood that an individual will respond positively to a therapeutic or prophylactic composition disclosed herein, and can be administered the therapeutic or prophylactic composition. 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 likely to not respond or to respond negatively to a therapeutic or prophylactic composition, an alternative course of treatment can be prescribed. A negative response can be defined as the absence of an effective response or the presence of toxic side effects. Response to a therapeutic or prophylactic treatment can be predicted in the context of a background 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, and a population that has an adverse response 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 it will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or have an adverse response 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 an indicator of response of an individual to a treatment or prevention regimen disclosed herein. The response indicator can be assessed prior to, during, and / or after administration of a treatment or prevention regimen. For example, one or more response indicators can be assessed during intervals between successive doses of administration to evaluate whether an individual can benefit from continued treatment or requires an alternative treatment.

[0143] The prognostic tests described above are also applicable to clinical trials. One or more response indicators (e.g., HIF-1, SP1, MT, Atox 1, CCS, and COMMD1) can be identified using the methods described herein. Thereafter, potential participants in a clinical trial comprising a tetramine composition of a tetramine that can chelate copper and optionally a pro-copper composition comprising copper ions can be screened to identify those individuals most likely to respond favorably to the tetramine composition and exclude those individuals likely to experience side effects. In this way, the effectiveness of a treatment can be measured in individuals that respond positively to the tetramine composition without diluting the measurements and without risking undesirable safety issues due to the inclusion of individuals in the study that are unlikely to respond positively.

[0144] In some embodiments, there is provided a method of inducing tissue repair in an individual having ischemic tissue damage without increasing VEGF expression at the injection site, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, there is provided a method of inducing blood vessel growth toward an ischemic damage site in an individual, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, there is provided a method of inducing blood vessel growth toward an ischemic damage site in an individual without increasing VEGF expression at the injection site, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to decrease extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is administered to the individual prior to the tetramine composition. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 from about 80 mg to about 450 mg per day (e.g., from about 80 mg to about 300 mg, or from about 150 mg to about 350 mg). In some embodiments, the tetramine composition is administered twice per day.

[0145] Formation and growth of blood vessels within tissue can be performed by angiogenesis and / or vasculogenesis. In some embodiments, blood vessels include capillary-like structures that are fully functional to support transport of blood. In some embodiments, angiogenesis includes a process that involves generation of new blood vessels from previously existing blood vessels: sprouting angiogenesis (sprouting from pre-existing vessels), intussusceptive angiogenesis (invagination of existing vessels), and vasculogenesis (de novo formation of blood vessels from endothelial cells). In some embodiments, vasculogenesis includes a process that involves de novo formation of blood vessels from endothelial cells.

[0146] angiogenesis) or splitting angiogenesis (formation of new blood vessels by splitting of existing blood vessels). In some embodiments, angiogenesis includes processes involving the de novo production of new blood vessels by proliferation of endothelial stem cells, for example, formation of new blood vessels in the absence of pre-existing blood vessels.

[0147] angiogenesis) or splitting angiogenesis (formation of new blood vessels by splitting of existing blood vessels). In some embodiments, angiogenesis includes processes involving the de novo production of new blood vessels by proliferation of endothelial stem cells, for example, formation of new blood vessels in the absence of pre-existing blood vessels.

[0148] In some embodiments, blood vessel formation and growth requires signals from growth factors and other proteins that directly control the process, such as angiopoietins (e.g., Ang-1 and Ang-2), ephrins (Eph), vascular endothelial growth factors (e.g., VEGF-A and VEGF-C), platelet-derived growth factor (PDGF), fibroblast growth factors (e.g., FGF-1 and FGF-2), tumor necrosis factor-α (TNF-α), interleukins (IL), monocyte chemotactic protein-1 (MCP-1) (also known as CCL-2), transforming growth factor-α (TGF-α), transforming growth factors-β (e.g., TGF-β1, TGF-β2, TGF-β3, and TGF-β4), endostatin, vasostatin, chemotactic factors, thrombospondin, angiostatin, vascular cell adhesion molecules (e.g., VCAM-1), matrix metalloproteinases (e.g., MMP-2 and MPP-9), integrins, cadherins, plasminogen activator, and plasminogen activator inhibitors.

[0149] In some embodiments, vascular growth is determined by measuring endothelial cell proliferation, which is required for capillary formation in intact animals. In some embodiments, the effect of administering a tetramine composition comprising a copper-chelatable tetramine on endothelial cell proliferation can be assessed by direct cell counting, DNA synthesis, and / or metabolic activity. For example, endothelial cells can be isolated from an ischemic injury site following treatment with a tetramine composition comprising a copper-chelatable tetramine and the rate of proliferation determined. In other embodiments, endothelial cell proliferation at an ischemic injury site can be monitored by labeling the cells and measuring cell counts, DNA synthesis, and / or metabolic activity in situ. In other embodiments, labeled endothelial cells can be administered to a subject and the proliferation of the labeled endothelial cells at an ischemic injury site monitored in situ. In some embodiments, the endothelial cells are labeled with a radioisotope, a fluorescent moiety, or a marker that can be specifically detected by, for example, an antibody. In some particular embodiments, the cells are labeled with 3 H] thymidine or bromodeoxyuridine (BrdU).

[0150] In some embodiments, vascular growth is determined by measuring endothelial cell migration, which involves degradation of the basement membrane and migration along a chemotactic gradient established by proangiogenic growth factors, for example, during sprouting angiogenesis. In certain embodiments, endothelial cells at an ischemic injury site are labeled and cell migration is monitored in vivo. In other aspects, labeled endothelial cells are administered to a subject and their migration toward an ischemic injury site is monitored in vivo. In other aspects, endothelial cells at an ischemic injury site can be isolated and their migratory properties can be determined by a variety of in vitro assays, including Boyden chamber assays, under-agarose assays, wound healing assays, Teflon fence assays, phagokinetic track assays, and the like.

[0151] In some embodiments, vascular growth is determined by measuring endothelial cells forming tubes with lumens that direct blood flow (i.e., tube formation). In some embodiments, vascular growth is determined by an aortic ring assay. An aortic ring assay for determining vascular 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) 44-49, the disclosure of which is incorporated by reference herein in its entirety. Sprouting microvessels from aortic rings closely interact with resident macrophages, pericytes, and fibroblasts in a sequential series that mimics the sequence of angiogenesis in intact animals. In some embodiments, the endothelial cells have not been pre-selected by passaging and are thus in a quiescent state similar to that of intact animals. Other angiogenic assays that incorporate vascular growth functions (e.g., matrix degradation, migration, proliferation, tube formation) include spheroid assays, mouse metatarsal assays, and similar assays.

[0152] In some embodiments, in vivo assays are used to measure vascular growth following administration of a tetramine composition comprising a copper-chelatable tetramine. These assays include, but are not limited to, a corneal angiogenesis assay, a chick chorioallantoic membrane assay, and a 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, a pellet or sponge comprising a pro-angiogenic molecule (e.g., a tetramine composition comprising a copper-chelatable tetramine disclosed herein) can be implanted into a stromal pocket created by surgical procedure. The ingrowth of new blood vessels from the peripheral limbal vasculature can be monitored daily, allowing the rate of angiogenesis to be determined. In a Matrigel plug assay, Matrigel comprising a tetramine composition disclosed herein (with or without copper ions) is implanted at or near the site of an ischemic injury in an individual, and the Matrigel plug is later removed to visualize the blood vessels. In some embodiments, endothelial cells are labeled with one or more markers, and their proliferation, migration, tube formation, vascularization, and / or vascular growth at the site of ischemic injury are determined in vivo, e.g., using suitable imaging techniques.

[0153] Combination therapy

[0154] The tetramine composition described above, and optionally in combination with a pro-copper composition, 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 ischemic tissue. In some embodiments, there is provided a method of inducing tissue repair (or improving tissue function) in an individual having ischemic tissue damage, comprising: a) administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine; and b) administering to the individual an effective amount of a stem cell (e.g., a mesenchymal stem cell (MSC), such as a bone marrow mesenchymal stem cell (BMSC)) or a stem cell inducer. In some embodiments, the method comprises administering to the individual an effective amount of a stem cell (e.g., a MSC, such as a BMSC). In some embodiments, the method comprises administering to the individual an effective amount of a stem cell inducer. In some embodiments, the copper-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the individual is pre-administered a pro-copper composition that can increase extracellular copper levels in the individual. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition can increase copper uptake, 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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 (e.g., 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 per day.

[0155] In some embodiments, the stem cells disclosed herein are mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs), pluripotent stem cells, induced pluripotent stem cells (iPS), or tissue-derived stem cells. In some embodiments, the tissue-derived stem cells are adipose tissue-derived stem cells, cardiac tissue-derived stem cells, or umbilical cord tissue-derived stem cells. In some embodiments, the stem cells are inducers of adult stem cells. In some embodiments, the adult stem cells are hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells in the placenta, adipose tissue, lung, bone marrow, blood, Wharton's jelly of the umbilical cord, or teeth (e.g., the perivascular niche of dental pulp and periodontal ligament), endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or germ line stem cells (e.g., stem cells in the testes).

[0156] In some embodiments, the stem cell inducers disclosed herein are inducers of mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs), pluripotent stem cells, induced pluripotent stem cells (iPS), or tissue-derived stem cells such as adipose tissue-derived stem cells, cardiac tissue-derived stem cells, or umbilical cord tissue-derived stem cells. In some embodiments, the stem cell inducers are inducers of adult stem cells, such as hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells in the placenta, adipose tissue, lung, bone marrow, blood, Wharton's jelly of the umbilical cord, or teeth (e.g., the perivascular niche of dental pulp and periodontal ligament), endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or germ line stem cells (e.g., stem cells in the testes).

[0157] In some embodiments, the stem cells or stem cell inducers are administered systemically. In some embodiments, the stem cells or stem cell inducers are administered locally to the ischemic tissue. In some embodiments, the stem cells or stem cell inducers are administered locally to a site other than the site of ischemic injury.

[0158] In some embodiments, the stem cells (or stem cell inducers), tetraamine compositions (with or without copper ions), and optional pro-copper compositions are administered simultaneously. In some embodiments, the stem cells (or stem cell inducers) and tetraamine compositions (with or without copper ions), and optional pro-copper compositions disclosed herein are administered sequentially in any suitable order.

[0159] Once the stem cells (or stem cell inducers), tetraamine compositions (with or without copper ions), and optional pro-copper compositions described herein have been administered to a mammal (e.g., a human), the presence and / or biological activity of the cells is monitored in some embodiments by any of a number of known methods. In some embodiments, the cells migrate from the individual's ischemic tissue in vivo, and the presence and / or biological activity of the cells en route to the site of tissue injury is monitored and / or modulated.

[0160] While the methods described herein are generally applicable to all aspects of tissue repair, it is understood that the combination therapy methods can be used for any one or more of the following purposes: inducing migration of bone marrow mesenchymal stem cells to an ischemic tissue, inducing differentiation of stem cells in an ischemic tissue, inducing tissue regeneration in an ischemic tissue, inducing signaling molecules that trigger tissue regeneration, promoting copper-dependent HIF-1 transcriptional activity, reversing damage at an ischemic injury site, reestablishing the microenvironment of neurofibrillary cells and neurosecretory cells at an ischemic injury site.

[0161] Preventative methods and uses

[0162] Also provided herein are methods of preventing ischemic tissue damage in an individual comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable 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 digestive tract tissue, and ischemic skeletal muscle tissue (e.g., ischemic 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-chelatable tetramine is trientine. In some embodiments, the tetramine composition further comprises a copper ion. In some embodiments, the copper ion in the tetramine composition is complexed with the copper-chelatable 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule, and wherein the crystalline complex further comprises two chloride ions and a water molecule. In some embodiments, the copper ion in the tetramine composition is not complexed with the copper-chelatable tetramine. In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels in the individual. In some embodiments, the method further comprises administering to the individual a pro-copper composition that increases extracellular copper levels in the individual. In some embodiments, the individual is previously administered a pro-copper composition that increases extracellular copper levels in the individual. In some embodiments, the pro-copper composition is a copper ion. In some embodiments, the pro-copper composition does not comprise a copper ion. In some embodiments, the pro-copper composition increases copper absorption, decreases copper excretion, and / or decreases 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 (e.g., 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 per day.

[0163] In some embodiments, methods of preventing ischemic heart failure in an individual are provided, comprising administering to the individual an effective amount of a tetramine composition comprising a copper-chelatable tetramine (e.g., trientine). In some embodiments, the effective amount of the tetramine composition is insufficient to reduce extracellular copper levels 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 (e.g., 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 per day. In some embodiments, the tetramine composition is administered for at least about 1 month (e.g., at least about 3 months or at least about 6 months). In some embodiments, the individual has no more than about 35% left ventricular ejection function (LVEF) at baseline. In some embodiments, the individual has class II or class III heart failure (based on New York Heart Association (NYHA) functional classification). In some embodiments, the individual has a plasma B-type natriuretic peptide (BNP) of at least about 150 pg / mL. In some embodiments, the individual has an NT-proBNP (N-terminal pro-BNP) of no less than about 600 pg / mL.

[0164] As used herein, “preventing” includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual who can be predisposed to the disease, but has not yet been diagnosed with the disease. In some embodiments, the cells and compositions provided are used to delay development of a disease or slow development of a disease (e.g., tissue damage).

[0165] For the prevention or treatment of disease, the appropriate dosage or route of administration depends on the type of disease to be treated, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, the previous treatment status, the individual’s clinical history and response to the tetramine composition and / or cells, and the judgment of the treating physician. In some embodiments, the tetramine composition, pro-copper composition, stem cells, and stem cell inducers are suitably administered to the individual once or in a series of treatments.

[0166] In some embodiments, the present disclosure provides compositions and methods for treating and preventing ischemic tissue damage. In some embodiments, the tetramine compositions, pro-copper compositions, and / or cells disclosed herein are administered prior to, during, or after a treatment that will or can cause tissue damage in an individual, and prevent or reduce ischemic tissue damage associated with the treatment (e.g., cancer radiation or chemotherapy).

[0167] In some embodiments, the tetramine compositions or methods disclosed herein prevent or reduce the area, volume, or duration of ischemic tissue injury by inducing stem cell migration (e.g., homing) to the tissue, even after the tissue of the individual has lost its inherent ability to spontaneously recruit stem cells. In some embodiments, administration of the tetramine compositions and / or cells of the present disclosure elicit a cascade of other events that result in enhanced resistance to ischemic tissue injury, including, for example, inducing stem cell differentiation at the tissue site, inducing tissue regeneration at the tissue site, inducing signal molecules that trigger tissue regeneration, promoting copper-dependent HIF-1 transcriptional activity, reversing injury at the site of initial ischemic injury before additional injury occurs, and / or reestablishing the microenvironment of neural fibrocytes and neurosecretory cells at the ischemic site.

[0168] For example, myocardial ischemia or infarction can result in irreversible loss of functional heart tissue and can reduce pump function and death. Occlusion of the coronary blood vessels results in disruption of blood supply to the dependent capillary system. Without nutrients and oxygen, the myocardial cells die and necrosis occurs. Inflammation of the surrounding tissue occurs with invasion of inflammatory cells and phagocytosis of cell debris. A fibrotic scar occurs and the affected heart region loses contractility. Without intervention, the only way the myocardium can compensate for tissue loss is hypertrophy of the remaining myocardial cells (accumulation of intracellular cellular proteins and contractile elements). Cell death resulting from endocrine, metabolic (alcohol) or infectious (viral myocarditis) agents and cancer therapeutics also results in decreased myocardial function. In some embodiments, the tetramine compositions or methods disclosed herein prevent or reduce the area, volume, or duration of ischemic heart tissue injury. In some embodiments, the tetramine compositions disclosed herein induce mesenchymal stem cell (e.g., BMSC) migration (e.g., homing) and / or retention to ischemic heart tissue. In some embodiments, in the case of myocardial ischemia or infarction, the myocardium can compensate for tissue loss by differentiating stem cells into cardiomyocytes, thereby avoiding or reducing cardiac hypertrophy and further cardiac tissue injury.

[0169] Tetramine compositions

[0170] Also provided herein are tetramine compositions (including pharmaceutical compositions) comprising a tetramine that can chelate copper (e.g., trientine) for use in increasing intracellular copper levels, delivering copper to cells, inducing at least one (e.g., any of at least 2, 3, 4, 5, 6, 7, or more) tissue repair event, inducing stem cell migration, or promoting copper-dependent HIF-1 transcriptional activity in ischemic tissue of an individual having ischemic tissue injury. Any tetramine composition, optionally in combination with a pro-copper composition and / or stem cells (or stem cell inducers), can be used in the methods described above.

[0171] In some embodiments, a tetramine composition comprising a copper-chelating tetramine or a pharmaceutically acceptable salt thereof is provided. The copper-chelating tetramines contemplated herein (e.g., trientine) include, but are 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 an analog of trientine, such as the tetramine of Formula (II) described in the “Copper and Copper-Chelating Tetramines” section. In some embodiments, the tetramine composition does not comprise a trace element, e.g., copper. In some embodiments, the composition can chelate copper ions in the blood and deliver the copper ions to cells of ischemic tissue.

[0172] In some embodiments, a tetramine composition comprising a mixture of a copper-chelating tetramine and copper ions is provided. In some embodiments, the tetramine composition comprises a mixture of trientine and copper ions. In some embodiments, the relative ratio (molar ratio) of the copper-chelating tetramine (e.g., trientine) to copper ions is any of about 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 of the copper-chelating tetramine (e.g., trientine) to copper ions is about 1:1. In some embodiments, the relative ratio (molar ratio) of the copper-chelating tetramine (e.g., trientine) to copper ions is any of about 50:1-100:1, 20:1-50:1, 10:1-20:1, 5:1-10:1, 4:1-5:1, 3:1-4:1, 2:1-3:1, 1:1-2:1, 1:2-1:1, 1:3-1:2, 1:4-1:3, 1:5-1:4, 1:10-1:5, 1:20-1:10, 1:50-1:20, 1:100-1:50, 1:100-1:10, 1:10-1:1, 1:1-10:1, 10:1-100:1, or 1:10-10:1. In some embodiments, at least a portion of the copper ions are complexed with the copper-chelating tetramine. In some embodiments, the copper ions are not complexed with the copper-chelating tetramine.

[0173] In some embodiments, a tetraamine composition comprising a complex of a copper-chelating tetraamine and a copper ion is provided. In some embodiments, the tetraamine composition comprises a complex of trientine and a copper ion. In some embodiments, the stoichiometric ratio of the copper-chelating tetraamine (e.g., trientine) to the copper ion is about 1 : 1. In some embodiments, the complex of the copper-chelating tetraamine (e.g., trientine) and the copper ion is crystalline. In some embodiments, the crystalline complex of the copper-chelating tetraamine (e.g., trientine) and the copper ion is a thermodynamic polymorph. Different thermodynamic polymorphs of the crystalline complex can be described by a particular set of geometric structure, unit cell dimensions, space group, and structure coordinates, which can be determined using techniques known in the art, such as X-ray crystallography. In some embodiments, the tetraamine composition comprises a crystalline complex of trientine and a copper ion, wherein the copper ion is chelated by four amine groups of trientine in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the tetraamine composition comprises a crystalline complex of Formula (I) as shown below,

[0174]

[0175] wherein Cu is a copper ion, and the dashed lines represent hydrogen bonds. In some embodiments, the tetraamine composition comprises a crystalline complex of trientine and a copper ion, wherein the crystalline complex has a crystal structure as shown in Figure 1 In some embodiments, the crystal structure has bond lengths, bond angles, and torsion angles as listed in Figure 2 In some embodiments, the crystalline complex comprises a crystal having a space group and parameters as listed in Figure 3 In some embodiments, the tetraamine composition comprises a crystalline complex of trientine and a copper ion, wherein the crystal structure of the crystalline complex is defined by atomic coordinates listed in Figures 4A-4C

[0176] ​In some embodiments, the tetramine composition comprises a complex of a copper- chelatable tetramine (e.g., trientine) and copper ions, wherein the complex is not crystalline. In some embodiments, the tetramine composition further comprises copper ions that are not complexed with the copper-chelatable tetramine. In some embodiments, the relative ratio (molar ratio) of copper ions that are not complexed with the copper-chelatable tetramine to the complex is any of about 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 (molar ratio) of copper ions that are not complexed with the copper-chelatable tetramine to the complex is any of about 1 :2-1 : 1, 1 :3-1 :2, 1 :4-1 :3, 1 :5-1 :4, 1 : 10-1 :5, 1 :20-1 : 10, 1 :50-1 :20, 1 : 100-1 :50, 1 : 100-1 : 10, or 1 : 10-1 : 1. In some embodiments, the percentage of total copper complexed with the copper-chelatable tetramine (e.g., trientine) in the tetramine composition is any of about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the percentage of total copper complexed with the copper-chelatable tetramine (e.g., trientine) in the tetramine composition is any of about 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 1-10%, 10-50%, 50-80%, or 80-100%. In some embodiments, the percentage of total copper-chelatable tetramine (e.g., trientine) complexed with copper ions in the tetramine composition is at least any of about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the percentage of total copper-chelatable tetramine (e.g., trientine) complexed with copper ions in the tetramine composition is any of about 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 1-10%, 10-50%, 50-80%, or 80-100%. In some embodiments, the copper ions that are not complexed with the copper-chelatable tetramine (e.g., trientine) are present in the form of a salt, such as copper sulfate, copper chloride, copper oxide, copper nitrate, copper acetate, copper formate, copper gluconate, copper amino acid chelates, and the like.

[0177] In some embodiments, there is provided a tetraamine composition comprising a tetraamine that can chelate copper and copper ions, wherein the copper ions are not complexed with the tetraamine that can chelate copper. In some embodiments, the tetraamine composition comprises trientine and copper ions, wherein the copper ions are not complexed with the trientine. In some embodiments, the copper ions are present as a salt, such as copper sulfate, copper chloride, copper oxide, copper gluconate, copper amino acid chelate, and the like. In some embodiments, the relative ratio (molar ratio) of the tetraamine that can chelate copper (e.g., trientine) to the copper ions is any of about 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 (molar) of the tetraamine that can chelate copper (e.g., trientine) to the copper ions is any of about 50: 1-100: 1, 20: 1-50: 1, 10: 1-20: 1, 5: 1-10: 1, 4: 1-5: 1, 3: 1-4: 1, 2: 1-3: 1, 1: 1-2: 1, 1:2-1: 1, 1:3-1:2, 1:4-1:3, 1:5-1:4, 1: 10-1:5, 1:20-1: 10, 1:50-1:20, 1: 100-1:50, 1: 100-1: 10, 1: 10-1: 1, 1: 1-10: 1, 10: 1-100: 1, or 1: 10-10: 1.

[0178] Many factors of the tetraamine composition, including but not limited to the chemical structure of the tetraamine that can chelate copper, the ratio of the tetraamine that can chelate copper to copper in the tetraamine composition, the interaction of the copper ions with the tetraamine that can chelate copper (e.g., whether a complex, whether the complex is crystalline, etc.) can affect the ability of the tetraamine composition to deliver (e.g., unload) copper intracellularly in ischemic tissue. For example, the tetraamine that can chelate copper can have a conformation (including chelating bite, donor binding groups, and cavity size) that facilitates reversible binding of copper ions. In some embodiments, the tetraamine composition comprises a tetraamine that can chelate copper that has a sufficiently low affinity for copper ions intracellularly in ischemic tissue, wherein the tetraamine composition dissociates and unloads copper ions intracellularly. In some embodiments, the tetraamine composition comprises an additional compound and / or agent that enhances unloading of copper ions intracellularly in ischemic tissue.

[0179] Also provided are pharmaceutical compositions for use in the methods described herein, comprising any of the tetraamine compositions described herein and one or more pharmaceutically acceptable carriers, excipients, stabilizers, diluents, and / or other agents known in the art.

[0180] Thus, in one aspect of the application, there is provided a pharmaceutical composition comprising a tetraamine that can chelate copper and copper ions. In some embodiments, there is provided a pharmaceutical composition comprising trientine and copper ions.

[0181] In some embodiments, provided are pharmaceutical compositions comprising a complex of a copper-chelatable tetramine and a copper ion. In some embodiments, provided are pharmaceutical compositions comprising a complex of trientine and a copper ion. In some embodiments, the complex of a copper-chelatable tetramine (e.g., trientine) and a copper ion is crystalline. In some embodiments, the crystalline complex of a copper-chelatable tetramine (e.g., trientine) and a 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 in a square planar geometry, and wherein the crystalline complex further comprises two chloride ions and one water molecule. In some embodiments, the complex of a copper-chelatable tetramine (e.g., trientine) and a copper ion is not crystalline.

[0182] In some embodiments, provided are pharmaceutical compositions comprising a copper-chelatable tetramine and a copper ion, wherein at least a portion (e.g., at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of the copper ions are not complexed with the copper-chelatable tetramine. In some embodiments, provided are pharmaceutical compositions comprising trientine and a copper ion, wherein at least a portion (e.g., at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of the copper ions are not complexed with trientine. In some embodiments, provided are pharmaceutical compositions comprising a copper-chelatable tetramine and a copper ion, wherein the copper ions are not complexed with the copper-chelatable tetramine. In some embodiments, provided are pharmaceutical compositions comprising trientine and a copper ion, wherein the copper ions are not complexed with the copper-chelatable tetramine. In some embodiments, the copper ions that are not complexed with the copper-chelatable tetramine are present as a salt, e.g., copper sulfate, copper chloride, copper oxide, copper nitrate, copper acetate, copper formate, copper gluconate, copper amino acid chelates, etc.

[0183] Any of the pharmaceutical compositions described herein can be used to increase intracellular copper levels, induce at least two (e.g., at least any of 2, 3, 4, 5, 6, 7, or more) tissue repair events, promote copper-dependent HIF-1 transcriptional activity, and / or treat (including prevent) any disease or disorder associated with ischemic tissue damage in an individual having ischemic tissue damage.

[0184] The pharmaceutical compositions described herein can be formulated into solutions, emulsions, suspensions, dispersions or inclusion complexes (e.g., cyclodextrins) in a suitable pharmaceutical solvent or carrier according to conventional methods known in the art for preparing various dosage forms, or with solid carriers into pills, tablets, troches, suppositories, sachets, lozenges, granules, powders, reconstitutable powders, or capsules. The pharmaceutical compositions of the embodiments can be administered by a suitable delivery route, e.g., oral, parenteral, rectal, nasal, topical, or ocular route, or by inhalation. In some embodiments, the pharmaceutical compositions are formulated for oral administration. In some embodiments, the pharmaceutical compositions are formulated for parenteral administration (e.g., intravenous administration).

[0185] For oral administration, the pharmaceutical compositions can be provided in solid form, as tablets or capsules, or in solution, suspension, or emulsion. In some embodiments, the pharmaceutical compositions are formulated as tablets, capsules, or pills. Oral tablets can include the active ingredient in admixture with a compatible pharmaceutically acceptable excipient such as diluents, disintegrating agents, binding agents, lubricants, sweetening agents, flavoring agents, coloring agents, and preservatives. Suitable inert fillers include sodium and calcium carbonates, sodium and calcium phosphates, lactose, starches, sugars, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrating agents. Binding agents can include starch and gelatin. Lubricants, if present, can be magnesium stearate, stearic acid, or talc. If desired, tablets can be coated with a material such as a glycerol monostearate or glycerol distearate to delay absorption in the gastrointestinal tract, or can be coated with an enteric coating. Oral formulations can be presented in discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be presented as a bolus, electuary, or paste.

[0186] Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., polyvinylpyrrolidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, primogel, corn starch, potato starch), surface-active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets can optionally be coated or scored and can be formulated to provide a sustained or controlled release of the active ingredient, using, for example, different proportions of hydroxypropylmethyl cellulose.

[0187] Capsules for oral administration include hard and soft gelatin capsules. For making hard gelatin capsules, the active ingredient can be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules can be made by mixing the active ingredient with water, an oil such as peanut or olive oil, a mixture of the liquid paraffin with water, a mixture of mono- and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol. The capsules can also contain gelatin, iron oxide, stearic acid, and titanium dioxide as inactive ingredients.

[0188] Liquids for oral administration can be in the form of suspensions, solutions, emulsions, or syrups, or can be lyophilized or provided as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions can optionally contain: pharmaceutically acceptable excipients such as suspending agents (for example sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and the like); non-aqueous vehicles, for example oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents, for example, lecithin; and, if desired, flavoring or coloring agents.

[0189] For parenteral use, including intravenous, intramuscular, intraperitoneal, intranasal or subcutaneous routes, the tetramine composition can be provided in sterile aqueous solution or suspension buffered to an appropriate pH and isotonicity, or in a parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms can be presented in unit-dose form, such as ampules or disposable injection devices, in multi-dose forms, such as vials from which the appropriate dose can be withdrawn, or in solid form or pre-concentrate useful in the preparation of injectable formulations. Formulations suitable for parenteral, including intravenous, administration include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit- dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above.

[0190] Dosage and method of administration

[0191] When used in vivo for any one of the therapeutic methods described herein, the tetramine composition, including the pharmaceutical composition, and optionally the copper-promoting composition and / or stem cells (or stem cell inducers) are administered to the individual in an effective amount. An "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a disease or condition associated with ischemic tissue damage. The effective amount herein can vary according to factors such as the extent of ischemic damage in the individual, the particular tetramine composition, copper ion, and / or stem cell (or stem cell inducer) used (e.g., its therapeutic index), the individual (e.g., age, sex, weight, and medical history). An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include, for example, a reduction and / or alleviation of a symptom associated with the disease or condition, including biochemical, histological and / or behavioral symptoms, complications, and intermediate pathological phenomena developing during the course of the disease or condition, and the elimination, reduction, or delay of the onset of the disease or condition. For therapeutic use, beneficial or desired results include, for example, clinical results such as alleviation of one or more symptoms resulting from the disease or condition, improvement in quality of life, reduction in the dose of other medications required to treat the disease, enhancement of effects of another medication, e.g., by targeting, delay in the progression of the disease, and / or an extension of survival.

[0192] In some embodiments, an effective amount of a tetramine composition (e.g., a pharmaceutical composition), and optionally in combination with an effective amount of a pro-copper composition, is effective to increase intracellular copper levels in ischemic tissue in a subject by more than any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more, compared to intracellular copper levels in the subject’s ischemic tissue prior to treatment. In some embodiments, an effective amount of a tetramine composition (e.g., a pharmaceutical composition), and optionally in combination with an effective amount of a pro-copper composition, is effective to increase total copper levels in ischemic tissue in a subject by more than any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more, compared to total copper levels in the subject’s ischemic tissue prior to treatment. In some embodiments, an effective amount of a tetramine composition (e.g., a pharmaceutical composition), and optionally in combination with an effective amount of a pro-copper composition, does not decrease extracellular copper levels (e.g., copper levels in the blood) in a subject. In some embodiments, an effective amount of a tetramine composition (e.g., a pharmaceutical composition), and optionally in combination with an effective amount of a pro-copper composition, does not decrease extracellular copper levels (e.g., copper levels in the blood) in a subject by more than any of about 5%, 10%, 20%, 30%, 40%, 50%, or more, compared to extracellular copper levels in the subject prior to treatment. In some embodiments, an effective amount of a tetramine composition (e.g., a pharmaceutical composition), and optionally in combination with an effective amount of a pro-copper composition, does not decrease total copper levels in a subject. In some embodiments, an effective amount of a tetramine composition (e.g., a pharmaceutical composition), and optionally in combination with an effective amount of a pro-copper composition, does not decrease total copper levels (e.g., copper levels in the blood) in a subject by more than any of about 5%, 10%, 20%, 30%, 40%, 50%, or more, compared to total copper levels in the subject prior to treatment.

[0193] 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 can or can not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” can be considered in the context of administration of one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if the desired result can or is achieved if combined with one or more other agents.

[0194] The effective amount, dosage, and dosing regimen of the tetramine composition alone or in combination with a pro-copper composition (e.g., copper ions) and / or stem cells (or stem cell inducers) can be determined during preclinical and clinical trials by methods familiar to physicians and clinicians. In some embodiments, the effective amount of the tetramine (e.g., trientine) that can chelate copper in the tetramine composition is less than about any 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 1200 mg. In some embodiments, the effective amount of the tetramine (e.g., trientine) that can chelate copper in the tetramine composition is about any of 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 1200 mg, about 1200 mg to about 2400 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 for a human patient is about 80 mg to about 450 mg of the tetramine (e.g., in the form of a dichloride salt) that can chelate copper per day.

[0195] In some embodiments, an effective amount of a copper-chelatable tetraamine (e.g., trientine) in a tetraamine composition (e.g., a pharmaceutical composition) includes at least about any 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, an effective amount of a copper-chelatable tetraamine (e.g., trientine) in a tetraamine composition (e.g., a pharmaceutical composition) includes less than about any 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, an effective amount of a copper-chelatable tetraamine in a tetraamine composition is about any of 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, an effective amount of a tetraamine composition is no more than about 20 mg / kg per day, e.g., about 18 mg / kg per day, for a rhesus monkey. In some embodiments, an effective amount of a tetraamine composition is no more than about 15 mg / kg to about 35 mg / kg per day for a mouse.

[0196] In some embodiments, an effective amount of a copper-chelatable tetraamine (e.g., trientine) in a pharmaceutical composition (e.g., a unit dosage form) is about 5 mg to about 300 mg, e.g., about 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, a concentration of a copper-chelatable tetraamine (e.g., trientine) in a pharmaceutical composition is diluted (about 0.1 mg / ml) or concentrated (about 100 mg / ml), including, e.g., about any of 0.1 to about 50 mg / ml, 0.1 to about 20 mg / ml, or 1 to about 10 mg / ml.

[0197] Exemplary dosing frequencies include, but are not limited to, any of four times per day, three times per day, twice per day, once per day, once every two days, once every three days, once every four days, or once per week. In some embodiments, the tetraamine composition (e.g., pharmaceutical composition) is administered at least about any of lx, 2x, 3x, 4x, 5x, 6x, or 7x per week (i.e., daily). In some embodiments, the interval between each administration is less than about any 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 tetraamine composition is administered daily. In some embodiments, the tetraamine composition is administered at least twice daily. In some embodiments, the tetraamine composition is administered at least once daily, including, for example, at least any of 2x, 3x, or 4x.

[0198] In some embodiments, the effective amount of the tetraamine composition in combination with the dosing frequency is sufficient to maintain a high concentration of tetraamine (e.g., trientine) in the individual (e.g., blood or ischemic tissue). In some embodiments, the high concentration of tetraamine is a concentration that promotes copper-dependent HIF-1 transcriptional activity or induces at least one (including at least 2, 3, 4, 5, 6, 7, or more) tissue repair event. In some embodiments, administration of the tetraamine composition (e.g., pharmaceutical composition) results in at least about 0.005 mg / L (including, for example, at least any 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) of copper-chelatable tetraamine in the blood. The concentration of tetraamine in a biological sample (e.g., blood or biopsy of ischemic tissue) can be determined using methods known in the art, such as fluorescence spectroscopy, mass spectrometry, or chromatography, or by measuring the level of labeled tetraamine.

[0199] In some embodiments, the effective amount of the tetraamine composition in combination with the dosing frequency is sufficient to maintain a high concentration of tetraamine (e.g., trientine) in the individual for at least any of about 4, 5, 6, 7, 8, 9, 10, or more hours. In some embodiments, the effective amount of the tetraamine composition in combination with the dosing frequency is sufficient to maintain a high concentration of tetraamine (e.g., trientine) in the individual for at least about 8 hours.

[0200] 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 no more than 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, 30 mg / kg / day, 40 mg / kg / day, or 50 mg / kg / day of trientine in the tetramine composition. In some embodiments, the effective amount of the tetramine composition is about any one of 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 / 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.

[0201] Administration of a tetraamine composition (e.g., a pharmaceutical composition) can be over an extended period of time, e.g., from about one week to about any of several years. In some embodiments, administration of a tetraamine composition (e.g., a pharmaceutical composition) is for at least about any 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, administration of a tetraamine composition (e.g., a pharmaceutical composition) is for at least about one month, including, e.g., any of 1, 2, 3, 4, 5, 6, 8, 10, 12, or more months. In some embodiments, administration of a tetraamine composition (e.g., a pharmaceutical composition) is for at least about one month, and wherein the tetraamine composition is administered at least once (e.g., twice, three times, or four times) per day. In some embodiments, administration of a tetraamine composition (including a pharmaceutical composition) results in at least about 0.005 mg / L (including, e.g., at least about any 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) of a copper-chelatable tetraamine in the blood for at least about one week (including, e.g., at least about any of 2 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 12 months, or more).

[0202] Any of the tetraamine compositions (e.g., pharmaceutical compositions) described herein can be administered to an individual (e.g., a human) by a variety of routes, including, e.g., intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intracapsular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, transdermal, intratumoral, direct injection into a blood vessel wall, intracranial, or intracavitary. In some embodiments, a sustained continuous release formulation of the tetraamine composition (e.g., a pharmaceutical composition) can be used. In some embodiments, the tetraamine composition is administered parenterally. In some embodiments, the tetraamine composition is administered orally. In some embodiments, the tetraamine composition is administered directly to ischemic tissue (e.g., using a direct delivery method described below). In some embodiments, the tetraamine composition is administered by an interventional procedure, such as angioplasty.

[0203] In some embodiments, the tetramine composition (e.g., pharmaceutical composition) can be administered with a second therapeutic compound and / or a second treatment. The dosage and frequency of administration of the tetramine composition (e.g., pharmaceutical composition) and the second compound can be adjusted over the course of therapy according to the judgment of the administering physician. In some embodiments, the first treatment and the second treatment are administered simultaneously, sequentially, or concurrently. As used herein, “simultaneous administration” can refer to the administration of the first treatment and the second treatment in the combination therapy with a time interval of no more than about 15 minutes, e.g., no more than about any of 10, 5, or 1 minutes. When the first treatment and the second treatment are administered simultaneously, the first treatment and the second treatment can be contained in the same composition (e.g., a composition containing both the first treatment and the second treatment) or in separate compositions (e.g., the first treatment in one composition and the second treatment contained in another composition). “Sequential administration” can refer to the administration of the first treatment and the second treatment in the combination therapy with a time interval of greater than about 15 minutes, e.g., greater than about any of 20, 30, 40, 50, 60, or more minutes. The first treatment or the second treatment can be administered first. The first treatment and the second treatment are contained in separate compositions, which can be contained in the same or different packaging or kit. Concurrent administration can refer to the administration of the first treatment and the administration of the second treatment in the combination therapy overlapping each other. When administered separately, the pharmaceutical composition and the second compound can be administered at different frequencies or intervals. For example, the tetramine composition (e.g., pharmaceutical composition) can be administered daily, while the second compound can be administered more or less frequently. In some embodiments, a sustained continuous release formulation of the tetramine composition and / or the second compound can be used. Various formulations and devices for achieving sustained release are known in the art. Combinations of the administration configurations described herein can be used. In some embodiments, the second compound is a copper ion (e.g., a copper salt or chelated copper). In some embodiments, the second treatment comprises a stem cell or a stem cell inducer.

[0204] In some embodiments, the individual is also administered an effective amount of a pro-copper composition comprising copper ions. In some embodiments, the effective amount of the pro-copper composition administered to the individual is sufficient to increase the extracellular copper level of the individual by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more, compared to the extracellular copper level of the individual prior to treatment. In some embodiments, the effective amount of the pro-copper composition administered to the individual is sufficient to increase the total copper level of the ischemic tissue of the individual by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more, compared to the total copper level of the ischemic tissue of the individual prior to treatment.

[0205] In some embodiments, the effective amount of the pro-copper composition administered to the individual is sufficient to increase the extracellular copper level of the individual by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more, compared to the extracellular copper level of the individual prior to treatment. In some embodiments, the effective amount of the pro-copper composition administered to the individual is sufficient to increase the total copper level of the ischemic tissue of the individual by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more, compared to the total copper level of the ischemic tissue of the individual prior to treatment. In some embodiments, a pro-copper composition that does not comprise copper ions can be used to increase extracellular copper levels. For example, the pro-copper composition can increase copper uptake, decrease copper excretion, and / or decrease zinc toxicity.

[0206] In some embodiments, the effective amount of copper ions in the pro-copper composition comprising copper ions includes any range from about 0.01 mg to about 0.1 mg, about 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 copper ions in the pro-copper composition administered to an individual includes at least any of about 5 mcg / kg, 10 mcg / kg, 20 mcg / kg, 30 mcg / kg, 50 mcg / 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 1000 mcg / kg. In some embodiments, the effective amount of copper ions in the pro-copper composition administered to an individual includes less than any of about 1000 mcg / kg, 900 mcg / kg, 800 mcg / kg, 700 mcg / kg, 600 mcg / 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.

[0207] In some embodiments, the pro-copper composition is administered daily or twice daily. In some embodiments, the pro-copper composition is administered at the same dosing frequency and duration as the tetramine composition. In some embodiments, the pro-copper composition is administered at a different dosing frequency and / or duration than the tetramine composition. In some embodiments, the pro-copper composition is administered orally. The effective amount and dosing frequency of the pro-copper composition can be determined during preclinical and clinical trials by methods familiar to physicians and clinicians.

[0208] Once improvement of the patient's disease has occurred, the dose can be adjusted to a maintenance dose for prevention or maintenance therapy. For example, the dosage or the frequency of administration, or both, can be reduced to a level at which the desired therapeutic or prophylactic effect is maintained. Of course, if an adverse effect occurs, a suspension of the treatment can be warranted. Upon reoccurrence of any symptoms, however, intermittent treatment of the patient can be desired. Long-term intermittent treatment of the patient can also be desired.

[0209] A variety of direct delivery methods are known in the art and can be used to administer the tetramine composition (e.g., pharmaceutical composition) and / or the pro-copper composition.

[0210] In some embodiments, the tetramine composition and / or the pro-copper composition is delivered by microbubbles. In some embodiments, the copper ion is delivered by a peptide-based nanoparticle comprising copper. In some embodiments, the tetramine composition and / or the pro-copper composition is passively delivered or targeted to the ischemic tissue by the physical action of the nanoparticle (or microsphere) to induce release or delivery to the ischemic tissue.

[0211] In some embodiments, the tetramine composition and / or the pro-copper composition is delivered by direct administration of the tetramine composition and / or the pro-copper composition to the ischemic tissue. In some embodiments, the tetramine composition and / or the pro-copper composition disclosed herein is orally administered to the site of ischemic tissue injury. In some embodiments, the tetramine composition and / or the pro-copper composition is absorbed through the digestive tract. In one aspect, the absorbed composition and / or copper ion is targeted (by active targeting or passive targeting) to the site of ischemic injury and released locally at the site of ischemic injury to provide an effective local concentration of the tetramine composition and / or the pro-copper 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 a monosaccharide, a disaccharide, or a 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.

[0212] In some embodiments, the sustained delivery compositions disclosed herein include long-acting injectables (e.g., oil-based injectables, injectable suspensions, injectable microspheres, and injectable in-situ systems) containing the tetramine composition and / or the copper composition, polymers for depot injection, commercially available depot injectables, and injectable sustained release delivery systems. In certain embodiments, the sustained delivery compositions disclosed herein include a polymeric matrix from which the agent is released by diffusion and / or degradation of the polymeric matrix. Thus, the release pattern of the agent is determined primarily by the polymeric matrix, as well as the percentage loading and the method of manufacture. In some embodiments, the sustained release formulation uses a biodegradable polymer. In this case, the sustained release formulation does not require surgical removal of the formulation from the subject. Typically, such formulations degrade slowly and are absorbed by the patient’s body and eventually disposed of with other soluble metabolic waste.

[0213] In some embodiments, a polymeric injectable depot system is used to deliver an in situ forming implant comprising a tetraamine composition and / or a copper composition at the site of ischemic injury. In situ forming implant systems are typically made from biodegradable products that can be injected into the body through a syringe and once injected, solidify to form a solid biodegradable implant. In some embodiments, the implant is formed from a thermoplastic paste, an in situ cross-linked polymer, in situ polymer precipitation, thermal induced gelation, or in situ solidifying organic gel. The depot formation mechanism for a thermoplastic paste is to form a semi-solid upon cooling to body temperature after being injected into the body in a molten form. Cross-linked polymer networks can be achieved in situ in a variety of ways, forming a solid polymer system or a gel. Methods for in situ cross-linking systems include free radical reactions, typically initiated by heat or absorption of photons or ionic interactions between small cations and polymeric anions. In situ formation can be produced by causing a polymer to precipitate from solution. A polymer that is insoluble in water and biodegradable is dissolved in a biocompatible organic solvent with a drug added, which after mixing forms a solution or suspension. When this formulation is injected into the body, the water-miscible organic solvent dissipates and water penetrates into the organic phase. This results in phase separation and polymer precipitation, forming a depot at the injection site. Thermal induced gelation systems exhibit a thermoreversible sol / gel transition and have a lower critical solution temperature. It is a liquid at room temperature and produces a gel at or above the lower critical solution temperature. In situ solidifying organic gels contain water-insoluble amphiphilic lipids that swell in water and form multiple types of lyotropic liquid crystals.

[0214] In some embodiments, the tetramine composition and / or the pro-copper composition is injected into the site of ischemic injury, for example, by direct percutaneous puncture, through an interventional catheter, or by intraosseous injection. In some embodiments, the tetramine composition and / or the pro-copper composition is delivered directly to the site of ischemic injury by using a coated implant, stent, or plate or an implant impregnated with the tetramine composition and / or the pro-copper composition. In some embodiments, the tetramine composition and / or the pro-copper composition is delivered directly to the site of ischemic injury by slow release from an intravascular stent with the tetramine composition and / or the pro-copper composition attached. In some embodiments, the tetramine composition and / or the pro-copper composition is delivered to the site of ischemic injury by positive targeting liposomes or receptor-donor complexes. In some embodiments, the tetramine composition and / or the pro-copper composition is delivered to the site of ischemic injury using physical therapy, ultrasound, iontophoresis, ultrasound penetration enhancement, electroporation, and / or sponge application. The tetramine composition and / or the cells can be administered to the site of ischemic injury can be superficial (e.g., through the skin), can be at some location at an injured tissue inside the body surface, or both. For example, the tetramine composition and / or the pro-copper composition can be delivered to the site of ischemic injury with iontophoresis through a blood vessel, endothelial cell layer, or other internal tissue to provide an effective local concentration of the tetramine composition and / or the pro-copper composition for tissue repair.

[0215] In some embodiments, the sustained release compositions disclosed herein comprise a biodegradable polymer for controlled release delivery of the tetramine composition and / or the pro-copper composition. Suitable biodegradable polymers generally include polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-epsilon-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 microspheres.

[0216] In some particular embodiments, a variety of types of copper-containing compounds can be used to deliver the pro-copper composition locally to the site of ischemic injury. Examples of suitable solutions comprising copper ions are copper (I) chloride, copper (II) chloride, copper acetate, and copper sulfate solutions. In some embodiments, copper forms a compound or complex with a protein, a peptide, an amino acid, a monosaccharide, a disaccharide, or a polysaccharide, one or more polymers, or a small molecule, and the compound or complex is used for direct local delivery at the site of ischemic injury. In some embodiments, an organometallic compound containing copper ions is used for direct local delivery at the site of ischemic injury.

[0217] In some embodiments, the concentration of copper ions in a pro-copper composition for direct local delivery to a site of injury is about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 20 mM to about 40 mM, about 40 mM to about 60 mM, about 60 mM to about 80 mM, about 80 mM to about 100 mM, about 100 mM to about 200 mM, about 200 mM to about 400 mM, about 400 mM to about 600 mM, about 600 mM to about 800 mM, about 800 mM to about 1 mM, about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 20 mM to about 40 mM, or about 40 mM to about 60 mM. The concentration of copper ions can be determined in preclinical and clinical trials by methods familiar to physicians and clinicians.

[0218] Copper and tetraamines that can chelate copper

[0219] The terms "copper," "copper ions," and "copper element" are used interchangeably herein. In biological systems, copper ions exist in two oxidation states, cuprous copper (Cu 1+ , Cu(I) or reduced) and cupric copper (Cu 2+ , Cu(II) or oxidized). In some embodiments, copper includes both cuprous and cupric states. In some embodiments, copper is cupric state (Cu 2+ ). In some embodiments, copper is cuprous state (Cu 1+). In some embodiments, copper is a free ion, i.e., not bound or complexed with another molecule such as a protein or small organic molecule. In some embodiments, copper is in the form of a salt. In some embodiments, copper is in the form of a salt selected from the group consisting of copper sulfate, copper chloride, copper oxide, copper gluconate, and copper amino acid chelate. In some embodiments, copper is present as a complex ion. In some embodiments, copper is in an organometallic compound, such as a small molecule organometallic compound. In some embodiments, copper is in a complex with a tetramine that can chelate copper. In some embodiments, copper is a complex ion, which includes various ion species that result from introducing copper into a cell, tissue, or organism according to the present disclosure. In some embodiments, copper forms a compound or complex with a protein, peptide, amino acid, or monosaccharide, disaccharide, 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-β-hydroxylase (DBH), prion protein (PrP), tyrosinase, X-linked inhibitor of apoptosis protein (XIAP), lysyl oxidase, metallothionein (MT), and ceruloplasmin. In some embodiments, copper is in a form that cannot be taken up or used by ischemic tissue, for example, in a complex with ceruloplasmin. In some embodiments, copper is in a form that can be taken up or used by ischemic tissue. In some embodiments, the copper disclosed herein is an inducer of HIF-1 transcriptional activity.

[0220] A pro-copper composition comprising any of the copper ion species described above can be used in the methods described herein. In some embodiments, the pro-copper composition comprises Cu 2+ . In some embodiments, the pro-copper composition comprises Cu 1+ . In some embodiments, the pro-copper composition comprises copper ions in the form of a salt (e.g., any one or combination selected from the group consisting of copper sulfate, copper chloride, copper oxide, copper gluconate, and copper amino acid chelate). In some embodiments, the pro-copper composition comprises an organometallic compound. In some embodiments, the pro-copper composition does not comprise copper ions or copper compounds.

[0221] A “copper level” referred to in any of the methods described herein can refer to the concentration of any of the copper species described above (e.g., Cu 2+ , Cu 1+ ), or the concentration of total copper (e.g., Cu 1+ and Cu 2+ , and / or free or bound copper). In some embodiments, the copper level refers to the level of copper in the divalent state. In some embodiments, the copper level refers to the level of copper in a form that can be taken up or used by ischemic tissue.

[0222] “Tetramine that can chelate copper” refers to a copper binding or chelating tetramine compound. In some embodiments, the tetramine that can chelate copper binds to Cu2+ binds. In some embodiments, the copper-chelatable tetraamine is more specific (i.e., has a higher affinity) for Cu 1+ binds. In some embodiments, the copper-chelatable tetraamine is more specific (i.e., has a higher affinity) for Cu 1+ In some embodiments, the copper-chelatable tetraamine is Cu 2+ specific (i.e., has a higher affinity). In some embodiments, the copper-chelatable tetraamine forms a complex with copper ions in square planar, distorted square planar, trigonal pyramidal, square pyramidal, or distorted octahedral conformations. In some embodiments, the copper-chelatable tetraamine alters the balance between Cu 1+ and Cu 2+ In some embodiments, the copper-chelatable tetraamine can alter (e.g., decrease) copper levels (e.g., total copper levels) in an individual. In some embodiments, the copper-chelatable tetraamine can alter (e.g., decrease) copper levels (e.g., total copper levels) in the blood of an individual. In some embodiments, the copper-chelatable tetraamine can increase intracellular copper levels (e.g., total copper or divalent copper levels). In some embodiments, the copper-chelatable tetraamine can increase the concentration of available forms of copper in ischemic tissue in an individual. In some embodiments, the copper-chelatable tetraamine can redirect intracellular transport and / or inter-tissue or inter-organ transport of copper. In some embodiments, the copper-chelatable tetraamine specifically binds and / or is taken up by ischemic tissue, such as ischemic cardiac tissue. In some embodiments, the copper-chelatable tetraamine (including its complex with copper) can permeate membranes. In some embodiments, the copper-chelatable tetraamine (including its complex with copper) is lipophilic. In some embodiments, the stoichiometric ratio of copper-chelatable tetraamine to copper in the complex is about 1 : 1. In some embodiments, the copper-chelatable tetraamine reversibly binds copper ions. In some embodiments, the copper-chelatable tetraamine binds copper ions intracellularly in ischemic tissue with sufficiently low affinity to allow for unloading or dissociation of the copper ions.

[0223] Any copper-chelatable tetraamine that can increase intracellular copper levels can be used in the methods described herein. Copper-chelatable tetraamine can refer to the compound itself, pharmaceutically acceptable salts, active metabolites, derivatives, and prodrugs thereof, as well as applicable stereoisomers, enantiomers, racemic mixtures, and the like. In some embodiments, the copper-chelatable tetraamine is linear. In some embodiments, the copper-chelatable tetraamine is branched. In some embodiments, the copper-chelatable tetraamine is cyclic. In some embodiments, the copper-chelatable tetraamine is selected from the group consisting of triethylenetetramine (2,2,2-tetraamine), 2,3,2-tetraamine, and 3,3,3-tetraamine.

[0224] In some embodiments, the copper-chelatable tetraamine is trientine. "Trientine" is also known as triethylenetetramine, 2,2,2-tetramine, N,N'-bis(2-aminoethyl)-l,2-ethanediamine, 1,8-diamino-3,6-diazaoctane, 3,6-diazaoctane-l,8-diamine, 1,4,7,10-tetraazodecane, triethylenetetramine, TETA, TECZA, N,N'-bis(aminoethyl)ethylenediamine, N,N'-bis(2-aminoethyl)ethylenediamine, and N,N'-bis(2-aminoethyl)-ethylenediamine. In some embodiments, trientine is a compound of the formula NH2(CH2)2NH(CH2)2NH(CH2)2NH2or a pharmaceutically acceptable salt thereof.

[0225] Other copper-chelatable tetraamines having similar copper-chelating properties can include, but are not limited to, compounds of formula (II) and pharmaceutically acceptable salts thereof:

[0226]

[0227] In some embodiments, the copper-chelatable tetraamine is an acyclic compound of formula (II), wherein R1, R2, R3, R4, R5, and R6are independently selected from H, CH3, C2-C10straight or branched alkyl, C3-C10cycloalkyl, C1-C6alkyl C3-C10cycloalkyl, aryl, mono-, di-, tri-, tetra-, and penta-substituted aryl, heteroaryl, fused aryl, C1-C6alkyl aryl, C1-C6alkyl mono-, di-, tri-, tetra-, and penta-substituted aryl, C1-C5alkyl heteroaryl, C1-C6alkyl fused aryl, CH2COOH, CH2SO3H, CH2PO(OH)2, CH2P(CH3)O(OH); n1, n2, and n3are independently selected to be 2 or 3; and R7, R8, R9, R10, R11, and R12are independently selected from H, CH3, C2-C10straight or branched alkyl, C3-C10cycloalkyl, C1-C6alkyl C3-C10cycloalkyl, aryl, mono-, di-, tri-, tetra-, and penta-substituted aryl, heteroaryl, fused aryl, C1-C6alkyl aryl, C1-C6alkyl mono-, di-, tri-, tetra-, and penta-substituted aryl, C1-C5alkyl heteroaryl, C1-C6alkyl fused aryl. Additionally, one or more of R1, R2, R3, R4, R5, or R6may be functionalized to link, for example, a peptide, a protein, a polyethylene glycol, and other such chemical entities to alter the overall pharmacokinetics, delivery ability, and / or half-life of the construct. Examples of such functionalization include, but are not limited to, C1-C10alkyl-CO-peptide, C1-C10alkyl-CO-protein, C1-C10alkyl-CO-PEG, C1-C10alkyl-NH-peptide, C1-C10alkyl-NH-protein, C1-C10alkyl-NH-CO-PEG, C1-C10alkyl-S-peptide, C1-C10alkyl-S-protein. Furthermore, one or more of R7, R8, R9, R10, R11, or R12may be functionalized to link, for example, a peptide, a protein, a polyethylene glycol, and other such chemical entities to alter the overall pharmacokinetics, delivery ability, and / or half-life of the construct. Examples of such functionalization include, but are not limited to, C1-C10alkyl-CO-peptide, C1-C10alkyl-CO-protein, C1-C10alkyl-CO-PEG, C1-C10alkyl-NH-peptide, C1-C10alkyl-NH-protein, C1-C10alkyl-NH-CO-PEG, C1-C10alkyl-S-peptide, C1-C10alkyl-S-protein.

[0228] In some embodiments, the copper-chelatable tetraamine is a cyclic compound of formula (II), wherein R1and R6are linked together to form a bridging group ((CR13R14) n4and wherein R2, R3, R4, and R5 are independently selected 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 mono-, di-, tri-, tetra-, and penta-substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH2COOH, CH2SO3H, CH2PO(OH)2, CH2P(CH3)O(OH); n1, n2, and n3 are independently selected to be 2 or 3; and R7, R8, R9, R10, R11, and R12 are independently selected 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 mono-, di-, tri-, tetra-, and penta-substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl. Additionally, one or more of R2, R3, R4, or R5 can be functionalized to link, for example, a peptide, a protein, a polyethylene glycol, and other such chemical entities to alter the overall pharmacokinetics, delivery ability, and / or half-life of the construct. 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, or R12 can be functionalized to link, for example, a peptide, a protein, a polyethylene glycol, and other such chemical entities to alter the overall pharmacokinetics, delivery ability, and / or half-life of the construct. 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.

[0229] "Pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid, including inorganic or organic bases and inorganic or organic acids, and the like. When the compound is basic, for example, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids and organic acids. Such acids include, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. In some embodiments, the pharmaceutically acceptable salt of the copper-chelatable tetramine is selected from the group consisting of a hydrochloride salt (e.g., triethylenetetramine dihydrochloride), a succinate salt (e.g., triethylenetetramine disuccinate), a maleate salt (e.g., triethylenetetramine tetramaleate), and a fumarate salt (e.g., triethylenetetramine tetra-fumarate). The copper-chelatable tetramine such as trientine can also be in the form of a quaternary ammonium salt, wherein the nitrogen atom bears a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety. Metabolites of the copper-chelatable tetramine can include, but are not limited to, acetylated metabolites such as N-acetyl triethylenetetramine (e.g., monoacetyl-triethylenetetramine). Derivatives of the copper-chelatable tetramine can include, but are not limited to, PEG-modified tetramines (e.g., trientine-PEG).

[0230] The copper-chelatable tetramine including trientine can be prepared using any of a variety of chemical synthesis, isolation, and purification methods known in the art. See, e.g., 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, European Patent No. EP 262 562, U.S. Patent No. 8,394,992, and U.S. Patent Publication No. US20130108709 Al.

[0231] Individuals having ischemic tissue injury

[0232] "Ischemic tissue injury" as described herein refers to injury to tissue (including, for example, cardiovascular, liver, brain, skeletal muscle, etc.) that results in restricted blood supply to the tissue, causing a lack of oxygen and glucose needed for cellular metabolism in the tissue. The injury can involve any of several pathologies that result in interference with blood flow or trauma caused by external forces. 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 extremity 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 skin ischemia. In some embodiments, the ischemic tissue injury is associated with embolism, thrombosis, aneurysm, trauma, myocardial infarction, mitral valve disease, chronic atrial fibrillation, cardiomyopathy, prosthesis, thoracic outlet syndrome, atherosclerosis, hypoglycemia, tachycardia, hypotension, tumor compression of blood vessels, Sickel cell disease, frostbite, arteriovenous malformation, peripheral arterial occlusive disease, rupture of vital blood vessels, anemia, diabetes, diabetic foot ulcer, necrotizing enterocolitis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, restenosis (after angioplasty or stent implantation), or pancreatitis. In some embodiments, the ischemic tissue injury is associated with cardiomyopathy. In some embodiments, the ischemic tissue injury is associated with myocardial infarction. In some embodiments, the ischemic tissue injury is associated with diabetes.

[0233] Accordingly, the methods described herein are generally applicable to a number of diseases involving ischemic tissue damage. These include, but are not limited to: myocardial infarction, cardiomyopathy, aneurysm, angina, aortic valve stenosis, aortitis, arrhythmia, arteriosclerosis, arteritis, asymmetric septal hypertrophy (ASH), atherosclerosis, atrial fibrillation and flutter, bacterial endocarditis, Barlow’s Syndrome (mitral valve prolapse), bradycardia, Buerger’s Disease (thromboangiitis obliterans), cardiac hypertrophy, carditis, carotid artery disease, coarctation of the aorta, congenital heart defects, congestive heart failure, coronary artery disease, Eisenmenger’s Syndrome, embolism, endocarditis, erythromelalgia, fibrillation, fibromuscular dysplasia, heart block, heart murmur, hypertension, hypotension, idiopathic infantile arterial calcification, Kawasaki Disease (cutaneous mucocutaneous lymph node syndrome, cutaneous mucocutaneous lymph node disease, infantile polyarteritis), metabolic syndrome, microvascular angina, myocarditis, paroxysmal atrial tachycardia (PAT), polyarteritis nodosa (polyarteritis, nodosa polyarteritis), pericarditis, peripheral vascular disease, severe limb ischemia, phlebitis, pulmonary artery stenosis (pulmonary stenosis), Raynaud’s Disease, renal artery stenosis, renal vascular hypertension, rheumatic heart disease, diabetic vasculopathy, septal defect, silent ischemia, syndrome X, tachycardia, Takayasu’s Arteritis, Tetralogy of Fallot, transposition of the great vessels, tricuspid atresia, truncus arteriosus, valvular heart disease, varicose ulcer, varicose veins, vasculitis, ventricular septal defect, Wolff-Parkinson-White Syndrome, endocardial cushion defects, acute rheumatic fever, acute rheumatic pericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis, chronic rheumatic heart disease, mitral valve disease, mitral valve stenosis, rheumatic mitral insufficiency, aortic valve disease, other endocardial structure disease, ischemic heart disease (acute and subacute), angina pectoris, acute pulmonary heart disease, pulmonary embolism, chronic pulmonary heart disease, scoliotic heart disease, myocarditis, endocarditis, endomyocardial fibrosis, endocardial fibroelastosis, atrioventricular block, cardiac dysrhythmias, myocardial degeneration, cerebrovascular disease, arterial, arteriolar, and capillary disease, or venous and lymphatic disease; acquired brain injury, traumatic brain injury, stroke (including ischemic, hemorrhagic, subarachnoid hemorrhage), hypoxic injury, metabolic disease, encephalitis, and brain injury caused by infection. In certain embodiments, the disease involving ischemic tissue damage includes systemic sarcoidosis, a skin disease or disorder, syndrome, a pulmonary disease or disorder, a cardiac disease or disorder, an ocular disease or disorder, a hepatic disease or disorder, a musculoskeletal disease or disorder, and a renal disease or disorder. Accordingly, the present application also includes the treatment of any disease using the methods described herein.

[0234] An "individual," "subject," or "patient" described herein refers to a mammal, such as a mouse, rat, rabbit, cat, dog, pig, cow, bull, sheep, goat, horse, monkey, and other non-human primate, as well as a human, a vertebrate such as a fish, and a bird such as a chicken. Mammals can include farm animals, sport animals, rodents, and pets. In some embodiments, the individual is a human.

[0235] The methods described herein are applicable to individuals having one or more ischemic tissue injuries, including but not limited to: ischemic myocardial injury, ischemic brain injury, ischemic spinal cord injury, ischemic muscle injury, ischemic skeletal injury, acute renal tubular necrosis, ischemic intestinal injury, ischemic lung injury, ischemic liver injury, ischemic kidney injury, ischemic skin injury, hernia, vascular anastomosis, atherosclerotic plaque, hemangioma, and blunt or penetrating traumatic injury.

[0236] In some embodiments according to any of the methods described herein, the individual does not have a limited tissue repair system. In some embodiments, the individual has a limited tissue repair system. An individual having a limited tissue repair system can have one or more of the following characteristics: (a) old age (e.g., at least about 60 years old, including, for example, at least about 65, 70, 75, 80, 85, 90 years old or older); (b) chronic tissue injury (e.g., an individual having a tissue injury for at least about any of 6, 7, 8, 9, 10, 11, 12, 18, or 24 months or more); (c) stem cell deficiency; (d) stem cell migration (i.e., homing) deficiency; (e) a deficient tissue repair system; (f) one or more of the following symptoms or disorders: decreased memory, low or decreased motor ability (including, but not limited to, strength ability, speed endurance, flexibility, and joint mobility), decreased sensation, muscle weakness, hearing loss, and chronic strain.

[0237] In some embodiments, the individual is at least about any one of 20, 30, 40, 50, 60, 70, 80 years old or older. In some embodiments, the individual is less than about any one of 20, 30, 40, 50, 60, 70, 80 years old. In some embodiments, the individual has chronic ischemia. In some embodiments, the individual has copper efflux from ischemic tissue (e.g., ischemic myocardium) into the blood circulation. In some embodiments, the individual has reduced copper levels (less than about any one of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) in the ischemic tissue. In some embodiments, the reduced copper levels in the ischemic tissue are caused by a chronic ischemic condition. In some embodiments, the individual has suppressed HIF-1 transcriptional activity.

[0238] In some embodiments, the individual is selected for treatment based on his or her copper levels, such as intracellular copper levels, extracellular copper levels, total copper levels, and copper levels in serum (i.e., blood). Individuals under chronic ischemic conditions typically have low intracellular copper levels in the ischemic tissue, such as less than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or lower of the average intracellular copper levels of the corresponding tissue in healthy individuals. For individuals under chronic ischemic conditions, the copper levels in serum, such as total copper levels in serum, levels of protein (e.g., ceruloplasmin) bound copper in serum, or levels of free (i.e., unbound) copper in serum are typically higher than the average copper levels in serum of healthy individuals (e.g., about any of 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more). In some embodiments, the total copper levels in serum of the individual are at least about any of 60 pg / dL, 70 pg / dL, 80 pg / dL, 90 pg / dL, 100 pg / dL, 110 pg / dL, 120 pg / dL, 130 pg / dL, 140 pg / dL, 150 pg / dL, 175 pg / dL, 200 pg / dL, 250 pg / dL, 300 pg / dL, or more prior to administration of the tetraamine composition. In some embodiments, the individual has no more than about any of 1-fold, 1.2-fold, 1.5-fold, 1.75-fold, or 2-fold of the average total copper levels in serum of healthy individuals prior to administration of the tetraamine composition. In some embodiments, the total copper levels in serum of the individual are no more than about any of 60 pg / dL, 70 pg / dL, 80 pg / dL, 90 pg / dL, 100 pg / dL, 110 pg / dL, 120 pg / dL, 130 pg / dL, 140 pg / dL, 150 pg / dL, 175 pg / dL, 200 pg / dL, 250 pg / dL, 300 pg / dL prior to administration of the tetraamine composition. In some embodiments, the individual has at least about any of 50%, 60%, 70%, 80%, 90%, or more of the average total copper levels in serum of healthy individuals after administration of the tetraamine composition. In some embodiments, the total copper levels in serum of the individual are at least about any of 60 pg / dL, 70 pg / dL, 80 pg / dL, 90 pg / dL, 100 pg / dL, 110 pg / dL, 120 pg / dL, 130 pg / dL, 140 pg / dL, or 150 pg / dL after administration of the tetraamine composition.

[0239] In some embodiments, an individual is selected for treatment based on his or her level of HIF-1 activity. In some embodiments, the individual has suppressed HIF-1 target gene transcriptional activity in ischemic tissue. In some embodiments, the individual has high levels of HIF-1 alpha (e.g., protein or RNA levels) in ischemic tissue, but suppressed HIF-1 target gene transcriptional activity in ischemic tissue. In some embodiments, the individual has chronic ischemia that results in suppressed HIF-1 activity.

[0240] Kits and articles of manufacture

[0241] The present application also provides kits, pharmaceuticals, compositions, and unit dosage forms for use in any of the methods described herein.

[0242] Kits provided herein include one or more containers comprising any of the tetramine compositions (including pharmaceutical compositions) described herein and / or other reagents, and in some embodiments, instructions for use according to any of the methods described herein. The kits can further include a description of the selection of an individual suitable for treatment. Instructions provided in kits of the application are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0243] For example, in some embodiments, the kit comprises a) a tetramine composition comprising a tetramine that can chelate copper (e.g., trientine) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the tetramine composition to treat a disease or disorder associated with ischemic tissue damage.

[0244] In some embodiments, the kit comprises a) a tetramine composition comprising a tetramine that can chelate copper (e.g., trientine) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; b) a copper- promoting composition comprising copper ions (e.g., CuSO4or CuCl2) and a pharmaceutically acceptable carrier; and optionally c) instructions for administering the tetramine composition to treat a disease or disorder associated with ischemic tissue damage.

[0245] Kits of the application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kits can optionally provide additional components, such as buffers and explanatory information. Accordingly, the present application also provides articles of manufacture, which include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like.

[0246] In some embodiments, the kit comprises one or more components that facilitate delivery of the tetramine composition, the pro-copper composition, and / or the additional therapeutic agent to the individual. For example, in some embodiments, the kit comprises a component that facilitates intralesional delivery of the tetramine composition and / or the pro-copper composition to the individual. In some embodiments, the kit comprises, e.g., a syringe and needle suitable for delivery of the cells to the individual. In such embodiments, the tetramine composition and / or the pro-copper composition can be contained in the kit in a bag or one or more vials. In some embodiments, the kit comprises a component that facilitates intravenous or intraarterial delivery of the tetramine composition and / or the pro-copper composition to the individual. In some embodiments, the tetramine composition and / or the pro-copper composition can be contained within, e.g., a bottle or bag (e.g., a blood bag or similar bag capable of holding up to 1.5 L of a solution containing cells), and the kit further includes a tube and needle suitable for delivery of the tetramine composition and / or the pro-copper composition to the individual.

[0247] Instructions relating to the use of the compositions generally include information as to dosage, dosing regimen, and route of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. For example, a kit comprising a sufficient amount of a tetramine disclosed herein that can chelate copper to provide effective treatment of an individual for an extended period of time, e.g., any of 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 can be provided. The kit can also include multiple unit doses of the pharmaceutical composition and instructions for use, and the amount packaged is sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy and a compounding pharmacy.

[0248] Also provided are medicaments, compositions, and unit dosage forms useful in the methods described herein.

[0249] The following non-limiting examples further illustrate the compositions and methods of the present application. Those skilled in the art will recognize that there can be numerous embodiments within the scope and spirit of the application. The present application will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the present application, but of course should not be construed as in any way limiting its scope.

[0250] Examples

[0251] Example 1: Crystal structure of a complex of trientine with copper ions

[0252] A complex comprising trientine dichloride, copper ions and water was crystallized. A suitable single crystal (150116_s2_lzh_m) was selected and used to collect X-ray diffraction data on an Xcalibur Eos diffractometer. The crystal was kept at 143.00-143.10 K during data collection. The structure was solved using Charge Flipping with Superflip (Palatinus et al., (2008) J. Appl. Cryst. 41 : 975-984; Palatinus et al., (2012) J. Appl. Cryst. 45: 575-580) structure solution program using Olex2 (Dolomanov et al., (2009) J. Appl. Cryst. 42: 339-341) and refined using ShelXL (Sheldrick G. M. (2008) Acta Cryst. A64: 112-122) refinement package using Least Squares minimization algorithm. The empirical formula of the complex in each unit cell was determined to be C6H 20 Cl2CuN4O. The refined crystal structure and its parameters are shown in Figures 1-3 and 4A-4C.

[0253] Embodiment 2: Intracellular delivery of copper to cardiomyocytes by trientine and trientine-copper complexes

[0254] This example describes an in vitro copper delivery assay to cardiomyocytes by trientine and trientine-copper complexes. A flowchart of the experimental procedure is shown in Figure 5

[0255] ​Primary cultures of neonatal rat cardiomyocytes were incubated in serum-free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for 48 h at 37 °C, 10% CO2. Cells were then transferred to serum-free DMEM and incubated for 12 h at 37 °C, 10% CO2, after which the cells were divided into 5 experimental groups (one control group and four treatment groups). In the control group, cells were incubated for a further 6 h in serum-free DMEM at 37 °C, 10% CO2. In the four treatment groups, cells were incubated with CuCl2 alone, trien alone, trien-copper complex, and a mixture of trien and CuCl2 for 6 h at 37 °C, 10% CO2, each at a final concentration of 10 mM trien and / or 10 mM copper. The trien-copper complex was synthesized in-house and characterized by mass spectrometry and X-ray diffraction (XRD). The trien-copper complex had the composition and structure as described in Example 1. The mixture of trien and copper was prepared by adding equimolar amounts of trien and CuCl2 to serum-free DMEM at a final concentration of 10 mM at 37 °C for 24 h, after which the mixture was used to treat neonatal rat cardiomyocytes.

[0256] After treatment, cells were collected by cell scraper, washed three times with ice-cold PBS containing 10 mM EDTA (Sigma, USA) to ensure complete removal of extracellular copper, and centrifuged at 3000 rpm for 5 min. Cell pellets were lysed using 1% SDS solution (Beyotime, CN). The lysate was divided into two parts. One part was digested with concentrated nitric acid at 50 °C for 72 h and analyzed using a graphite furnace atomic absorption spectrophotometer to assess intracellular copper concentration. The other part was used to determine total protein concentration by bicinchoninic acid (BCA) protein assay (Bio-Rad, USA). Intracellular copper concentration was normalized to total protein concentration for each treatment group.

[0257] Figure 6 Normalized intracellular copper concentrations for the five experimental groups are shown. All data are expressed as mean ± standard deviation (SD). One-way ANOVA was used for initial analysis, with Student-Newman-Keuls for comparison between multiple groups. Differences between experimental groups were considered significant at P < 0.05. As Figure 6As shown, the intracellular copper concentration of the trientine-copper complex (i.e., Cu-trientine) treatment group and the treatment group with a mixture of trientine and CuCl2(i.e., Cu+ trientine) was significantly increased compared to the control group, and the increase of intracellular copper concentration of these two groups was more significant compared to CuCl2treatment alone. Notably, the mixture of trientine and CuCl2(i.e., Cu+ trientine) resulted in the greatest increase in intracellular copper concentration under all test conditions, indicating that trientine can transport copper from the cellular environment with high copper levels into cardiomyocytes.

[0258] Example 3. Trientine treatment in a rat model of pathological cardiac hypertrophy

[0259] This example describes an in vivo experiment for evaluating the efficacy of trientine treatment in Sprague-Dawley rats with pathological cardiac hypertrophy. The rat model of pathological cardiac hypertrophy was established by ascending aortic constriction surgery. Figure 7 A flowchart showing the experimental procedure is shown.

[0260] 1.1 Establishment of pathological cardiac hypertrophy in rats

[0261] Prior to the surgical procedure, all subjects received an intraperitoneal injection of 10% chloral hydrate (0.35 mg / kg) to induce sedation. The left side of the chest was completely shaved for surgery. A tracheal cannula was introduced for ventilation. Assist breathing was performed to achieve a tidal volume of 1.2 to 1.5 mL. The breathing rate was about 80 times / min, and the inspiration / expiration ratio was 1:1.

[0262] During the surgery, the rat was adjusted to a right lateral position and placed under a stereomicroscope. The surgical area was isolated in a sterile manner. Isolation was performed with a piece of disposable sterile drape.

[0263] The surgical area was gently incised in the middle of the left second intercostal space and a transverse incision of 1-1.5 cm was made outwards from the left side of the presternum. The subcutaneous tissue and muscle plane was incised down to the pleura, into the pleural cavity. A cotton swab was inserted to clean the pleural cavity, and the lung was pushed away from the surgical area to avoid lung injury, then the intercostal incision was enlarged with a retractor to open the chest cavity and expose the thymus and fat.

[0264] After pulling apart the thymus and fat, the main blood vessels of the upper left atrial appendage were exposed. The ascending part of the aorta was cut off from the aortic trunk on the right side. The constriction site was located between the aortic valve and the innominate artery on the ascending aorta.

[0265] The ascending aorta was constricted with a 20-gauge needle (O.D. 0.9 mm). The ascending aorta and the needle were tied together with a 6-0 surgical suture. The needle was then immediately removed to provide an inner lumen with a stenotic aorta. After constriction, the left ventricle and left auricle swelled.

[0266] Before closing the chest, the chest retractor is removed and the thymus and fat are moved back to their normal position. The chest cavity is closed by suturing the second and third ribs together with two 3-0 silk sutures. Care is taken to avoid puncturing the dilated heart and damaging the lung during the rib suturing process to avoid massive bleeding and pneumothorax. When closing the intercostal incision, the lung is reinflated by turning off the ventilator for 1-2 seconds using the finger, so that air can be expelled from the pleural cavity. After closing the intercostal incision, the muscle and skin incision is closed layer by layer with 5-0 silk suture and cleaned in a sterile manner. After spontaneous respiration is restored, the endotracheal tube is withdrawn. To alleviate postoperative pain, analgesic buprenorphine (0.8 mg / kg) is given intramuscularly once a day for 2 days.

[0267] 1.2 Echocardiography

[0268] Rats were sedated for echocardiography measurements by intraperitoneal injection of 10% chloral hydrate (0.35 mg / kg). A series of echocardiography was performed using an 11.5-MHz transducer (Vivid 7 Dimension, GE) 4 months after the aortic constriction surgery and 1, 3, and 5 weeks after the treatment with curcumin. The interventricular septum depth (IVSD) and left ventricular posterior wall depth (LVPWD) were obtained by measuring the short-axis cross-sectional area and left ventricular length using a two-dimensional model.

[0269] Left ventricular ejection fraction (EF) and fractional shortening (FS) were assessed with the Simpson's single-plane method. Left ventricular end-diastolic volume (LVEDV), end-systolic volume (LVESV), end-diastolic internal diameter (LVID d ), and end-systolic internal diameter (LVID s ) were recorded directly. EF and FS were calculated according to the following equations: EF = (LVEDV - LVESV) / LVEDV x 100%, FS = (LVID d - LVID s ) / LVID d x 100%.

[0270] 1.3 Curcumin treatment

[0271] At 4 months after surgery, left ventricular concentric hypertrophy and myocardial interstitial fibrosis were observed. The establishment of the pathological cardiac hypertrophy model was confirmed by echocardiographic evaluation of cardiac morphology and function. The treatment with trientine was started after confirmation of the pathological cardiac hypertrophy status. The aortic constriction group (AAC) was divided into three groups: control group (NS group) and two trientine treatment groups (Tr(H) group and Tr(L) group). The sham-operated group rats were subjected to the same surgery except for the step of aortic constriction. The sham-operated group rats were also divided into three groups: control group (NS group) and two trientine treatment groups (Tr(H) group and Tr(L) group). The rats in the control group were treated with saline solution. In the trientine treatment groups, trientine was administered orally twice a day. Two doses of trientine (calculated based on trientine dihydrochloride) were administered, 45 mg / kg / day (Tr(H) group) and 90 mg / kg / day (Tr(L) group). The treatment lasted for 6 weeks.

[0272] The experimental procedures and results in the following sections of this example focus on the treatment with a trientine composition consisting essentially of trientine dihydrochloride. The same experimental protocol was used to evaluate the efficacy of other trientine compositions in treating cardiac hypertrophy in the rat model. For example, in one experiment, in addition to the trientine treatment, the ACC rats in the trientine treatment group were also treated with an oral copper supplement (e.g., copper chloride) at a dose of 54 mg / kg per day for 6 weeks. In another experiment, the ACC rats in the trientine treatment group were treated with the trienolate-copper complex of Example 1 at a dose of 120 mg / kg / day orally for 6 weeks.

[0273] 1.4 Cardiac morphology and function evaluation

[0274] Cardiac morphology and function were evaluated by echocardiography and according to the protocol shown in Figure 7 Plasma copper concentration (i.e., blood test) was measured according to the protocol shown in

[0275] In addition, heart tissue sections were obtained after the rats were sacrificed at the end of the experiment. Immunohistochemistry experiments were performed on the tissue sections. Capillary density of the heart tissue sections was determined and changes in collagen content were detected. mRNA and protein levels of HIF-1a and its targets such as VEGF and VEGFR-1 were measured in infarct tissue, border zone of infarct tissue, and heart tissue distal to the infarct area.

[0276] 1.5 Copper concentration in heart tissue

[0277] Tissue samples were freshly frozen and stored at -80°C prior to lyophilization. After lyophilization of the tissues and digestion with nitric acid, the digest was colorless or light yellow and clear with no visible precipitates or residues. Ultrapure water was added to each container to dilute the HNO3 to 2% for subsequent analysis of copper concentration. Copper concentration was determined by graphite furnace atomic absorption spectrophotometry (ICE 3500, Thermo) according to the procedure shown in Table 1 below.

[0278] Table 1 Graphite furnace atomic absorption spectrophotometry procedure

[0279] Temperature (°C) Time (s) Argon flow rate (L / min) 90 20 0.2 120 20 0.2 850 20 0.2 2100 3 0 2500 3 0.2

[0280] 1.6 Statistical analysis

[0281] All data are expressed as mean ± SD. Variations in each parameter were compared between experimental groups using Levene's test of homogeneity and coefficient of variance (CV). Statistical software package SPSS 14.0 (SPSS, Chicago, IL) was used and significant differences were considered when P value < 0.05.

[0282] 2. Results

[0283] 2.1 Heart morphology and function

[0284] Echocardiography examination showed that after treatment with trientine, a reversal of the pathological cardiac hypertrophy occurred at the morphological level. The interventricular septum depth (IVSD) and left ventricular posterior wall depth (LVPWD) of rats significantly decreased after 3 weeks of treatment. The efficacy of trientine was more pronounced as the treatment time was prolonged. When AAC rats were treated for 5 weeks, IVSD and LVPWD were almost normal compared to the sham group. As shown in Figures Figure 8A and Figure 8B , according to the results of continuous monitoring, the treatment group showed a significant decreasing trend in IVSD and LVPWD values over time. In contrast, in the control group, IVSD and LVPWD increased steadily over time.

[0285] Although the cardiac function parameters, ejection fraction (EF) and fractional shortening (FS) were within the normal range in all experimental groups due to compensatory effects, Figure 9A and 9B showed no very significant fluctuations in EF and FS in the trientine-treated group. In contrast, a significant decreasing trend in EF and FS was observed in the untreated group.

[0286] The copper concentration in the plasma and myocardium of rats in each treatment group was determined by atomic absorption spectroscopy. As shown in Figure 10AAs shown, the copper concentration in the hypertrophic myocardium of rats was reduced when AAC surgery was performed. The copper concentration in the heart tissue of treated rats was increased after 6 weeks of treatment with trientine. Figure 10A The bar corresponding to the AAC-Tr group shows the average copper concentration in the heart tissue of AAC rats treated with both high and low doses of trientine (i.e., the AAC-Tr(H) and AAC-Tr(L) groups combined).

[0287] Due to the efflux of copper from the heart tissue of AAC rats, the copper concentration in the plasma of AAC rats was higher than that of the sham-operated group rats. However, the high copper concentration in the plasma of AAC rats was significantly reduced over time according to the measurements at different time points (i.e., every two weeks) during the treatment process. In contrast, the plasma copper concentration of the sham-operated group rats remained fairly stable Figure 10B ) during the treatment process.

[0288] 3. Discussion

[0289] This study used trientine to increase the copper concentration in the hypertrophic heart tissue of rats. The results showed that trientine was able to promote the tissue redistribution and reutilization of copper and restore the morphology and function of the hypertrophic heart. Due to the trientine treatment in the heart hypertrophy rats, the transcriptional activity of HIF-1 and the capillary density in the infarct heart tissue were also increased. In addition, echocardiography examination showed that normal cardiac function was maintained throughout the trientine treatment period. The results of this experiment provide strong evidence that the trientine treatment of the present invention can effectively deliver copper to ischemic heart tissue to treat cardiac hypertrophy.

[0290] Example 4. Trientine treatment of a rhesus monkey heart failure model after myocardial ischemic infarction

[0291] This example describes an in vivo experiment for evaluating the efficacy of trientine treatment in a rhesus monkey model of heart failure. Rhesus monkeys have a more advanced heart with similar internal structure, electrical activity, coronary artery distribution, coronary collateral circulation, and its location and attachment in the thoracic cavity as humans. Therefore, the rhesus monkey model of heart failure provides a good surrogate for evaluating the efficacy for heart failure conditions in humans. In this experiment, myocardial ischemic infarction was established by a coronary ligation surgery. After the surgery, the ischemic myocardial tissue was gradually replaced by collagen fibers and became infarct tissue. After 1 year of surgery, the non-infarct heart tissue in the animals could not compensate for the functional loss of the infarct heart tissue, thus developing a heart failure model. The monkeys were then provided with trientine treatment to treat heart failure.

[0292] 1.1 Establishment of heart failure in rhesus monkeys

[0293] Prior to the surgical procedure, all subjects received an intramuscular injection of 5 mg / kg ketamine and 0.2 mg / kg midazolam to induce sedation. The electrode attachment sites over the chest and limbs were completely shaved for the surgery and to improve electrocardiogram (ECG) recording results. Standard bipolar and unipolar limb leads were recorded. Animals showing abnormal ECGs such as tachycardia (more than 200 beats per minute), arrhythmia, and significant deviation of the ST segment from baseline were excluded from the study.

[0294] Standard non-invasive measurements, including electrocardiogram, cuff blood pressure, pulse oximetry, and capnography, were continuously monitored (Dash 3000, GE, USA), and intravenous catheters were established in the subjects. After induction of anesthesia by intravenous infusion of fentanyl (10 μg / kg), midazolam (0.2 mg / kg), propofol (1 mg / kg), and vecuronium (0.1 mg / kg), all monkeys undergoing the surgical procedure were first intubated. Assisted respiration was performed under pressure-controlled ventilation to achieve an end-tidal CO2 of 35-40 mmHg. The inspiratory pressure was set in the range of 12-20 cmH2O. The respiratory rate was 40 breaths per minute, and the inspiration / expiration ratio was 1 :2.

[0295] To maintain anesthetic conditions during the surgical procedure, 2 mL of fentanyl (0.1 mg) and 10 mL of propofol (100 mg) were diluted to 20 mL with a saline solution. The mixture was continuously infused at a rate of 5-10 mL / hour by a syringe pump. The pump rate was adjusted according to the anesthetic state and the duration of the surgery. An arterial cannula was inserted into the femoral artery with an indwelling needle and connected to a pressure monitoring tube for invasive blood pressure monitoring during the surgery. Generally, the femoral artery pulse was palpated midway between the anterior superior iliac spine and the pubic symphysis. The surgical area was isolated in a sterile manner. Isolation was performed with four disposable sterile sheets.

[0296] The surgical area was gently incised midway at the left fourth intercostal space and a 4-5 cm transverse incision was made out from the left side of the manubrium. Monopolar diathermy was used for tissue cutting and coagulation purposes. The subcutaneous tissue and muscle planes were incised down to the pleura, into the pleural cavity, and then the incision was enlarged by opening forceps. A cotton swab was inserted to clear the pleural cavity, the lung was pushed away from the hole, and then the intercostal incision was enlarged to open the thoracic cavity and expose the pericardium.

[0297] The heart was exposed through a left fourth intercostal thoracotomy (4-5 cm) and the apex and left atrial appendage were identified. The epicardial end of the left anterior descending artery (LAD) was defined as zero grade; the origin of the LAD at the lower end of the left atrial appendage was defined as 100 grade. Ligation was performed at 60% of the LAD. Additionally, the main diagonal branch was also ligated in parallel to the ligation site on the LAD artery in some monkeys if the branching point of the diagonal branch artery was above the ligation site.

[0298] The artery was occluded for 1 minute, followed by 5 minutes of reperfusion, and the occlusion-reperfusion was repeated 3 times before the final ligation. After the final ligation, the left ventricular wall motion was monitored for differences, color changes in the anterior wall of the ventricle, and changes in the electrocardiogram and blood pressure to ensure the success of the ligation. After the final ligation, methylene blue (1 mL) was injected into the left atrial appendage through a 1.0 mL syringe. The opacification of the methylene blue indicated the completion of the ligation and helped to predict the ischemic area.

[0299] The cardiac status was monitored intensively for 45 minutes before closing the chest. Dobutamine (3 to 5 μg·kg -1 ·min -1 ) was infused to support cardiac function, and a defibrillator (HEARTSTART XL, Philips) was used if necessary. Care was taken to avoid damage to the heart during the closure of the pericardium. Sodium hyaluronate was infused into the pericardial cavity for an anti-adhesion treatment. The pericardium and the pleura were closed with 4-0 polyethylene sutures. The intercostal incision was closed with silk sutures. To avoid pneumothorax, care was taken to avoid damage to the lung during the intercostal closure. The lung was re-inflated while closing the intercostal incision so that air could be expelled from the pleural cavity. After closing the intercostal incision, the lung was re-inflated by dripping a saline solution into the subcutaneous space to ensure a tight closure of the chest incision. The muscle and skin incisions were closed layer by layer with #2-0 silk sutures and cleaned in a sterile manner. The endotracheal tube was withdrawn after the spontaneous breathing was restored. The incision was covered with sterile gauze and bandages. Tramadol (2 mg / kg) was injected intramuscularly to reduce pain. The bandages were changed every other day and the sutures were removed one week after surgery.

[0300] 1.2 Electrocardiogram (ECG) monitoring

[0301] A 12-lead ECG was recorded in the supine position of each monkey before surgery, immediately after surgery (the entire surgical procedure was about 2 hours), 4 weeks and 8 weeks after surgery using pediatric electrodes at a paper velocity of 25 mm / s and an amplitude of 10 mm / mV (MAC8000, GE, USA.). Even with pediatric electrodes, the width of the monkey’s chest wall was not sufficient to allow 6 precordial leads at the same time. Therefore, the 6 precordial leads were divided into two groups: V1, V3 and V5 were recorded in one group and V2, V4 and V6 in the other group.

[0302] 1.3 Echocardiography

[0303] Two-dimensional echocardiographic measurements were performed in standard apical 2- and 4-chamber views and for three consecutive cardiac cycles. Frame rate was kept between 70 fps to 100 fps. Transthoracic echocardiographic evaluation was performed in all monkeys with a 10.3 MHz transducer (P10-4, Siemens ACUSON Antares System, German) in left lateral position before surgery, 4 weeks and 8 weeks after surgery.

[0304] 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 recorded directly and EF was calculated using the following: EF = (LVEDV - LVESV) / LVEDV x 100%. Stroke volume (SV) of the left ventricle was calculated as follows: SV = LVEDV - LVESV.

[0305] 1.4 Trientine treatment

[0306] At one year after surgery, the ischemic heart tissue was completely replaced by collagenous fibers and became infarcted tissue. The establishment of the heart failure model was confirmed by echocardiographic assessment of cardiac function. Subsequently, trientine treatment was performed. In the trientine treatment group, trientine was administered orally to each monkey twice a day. The dose of trientine was 18 mg / kg / day. The treatment lasted for eight weeks. The monkeys in the untreated group (i.e., control group) did not receive any treatment. The monkeys in the trientine treatment group were monitored for adverse effects of trientine treatment. Figure 11 Cardiac function and morphology were evaluated according to the protocol shown to assess the efficacy of trientine.

[0307] The experimental procedures and results in the following sections of this example focus on treatment with trientine compositions consisting essentially of trientine dihydrochloride. The same experimental protocol was used to assess the efficacy of other trientine compositions in treating heart failure in the rhesus monkey model. For example, in one experiment, in addition to trientine treatment, the rhesus monkeys with heart failure in the trientine treatment group were also treated with an oral copper supplement (e.g., copper chloride) at a dose of 16.5 mg / kg per day for 6 weeks. In another experiment, the trientine treatment group of rhesus monkeys with heart failure were treated with the trienolate-copper complex of Example 1 at a dose of 36.7 mg / kg / day orally for 6 weeks.

[0308] 1.5 Histopathological examination

[0309] The monkeys were sacrificed by intravenous injection of potassium chloride (10%, 10 mL) and complete necropsy was performed on each monkey. The harvested heart was washed, grossly examined for visible lesions and fixed in 10% formaldehyde solution. Then, the heart was cut into 6 pieces along the long axis from the apex to the base. The thickness of each piece was fixed at 0.5 cm. It was ensured that the surface of each section was smooth and uniform during the cutting and the sections were marked by a marked ligature. Thin sections were cut and stained with Masson and H / E for microscopic examination.

[0310] Immunohistochemistry

[0311] Tissue sections were examined for HIF-1a, VEGFA and VEGFR1 using immunohistochemistry method. The following antibodies were used respectively: mouse anti-human HIF-1a monoclonal antibody (ab16066, Abeam); 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 (Maixinbio-tech company, Fuzhou). HIF-1a was recovered by high pressure heat-induced antigen recovery using EDTA (pH 9.0), VEGF and VEGFR1 were recovered by microwave heat-induced antigen recovery using citrate buffer solution (pH 6.0), and CD31 was recovered by microwave heat-induced antigen recovery using EDTA. The working concentrations of the antibodies were as follows: anti-HIF-1a was 1:800, anti-VEGF was 1:100, and anti-VEGFR1 was 1:100. In the immunohistochemistry experiment, the negative control was incubated with PBS instead of the first antibody. CD31 is a marker of endothelial cells. Ki-67 labeling was examined by immunofluorescence using a confocal microscope.

[0312] Capillary density

[0313] The capillary density of the tissue sections was evaluated as follows. First, the maximum capillary distribution field was determined under a light microscope at 100x magnification, and then 5 random fields were collected under a light microscope at 200x magnification to determine the capillary density. Capillary was defined as the sum of the lumen diameter less than 8 times the diameter of red blood cells. The measurements were made by two independent technicians.

[0314] Semi-quantitative protein expression analysis

[0315] Immunohistochemical sections were observed under light microscope, images were taken, and protein expression levels were determined in a semi-quantitative manner using Image-Pro Plus 6.0 image analysis software (MediaCybemetics). Slides of different groups were evaluated by two independent technicians. Five random fields of the edge and distal regions of infarction of each slide were photographed under light microscope at 400x magnification.

[0316] 1.6 Western blot

[0317] Tissue preparation

[0318] The heart was removed from the thorax. The left ventricular wall was carefully examined and tissue samples from the infarcted, border and distal regions were isolated. The infarcted region can be distinguished from the non-infarcted region according to its pale appearance. The border zone was defined as the region from 1 mm inside the infarcted region to 3 mm outside the infarcted region. The distal region was defined as the region more than 3 mm outside the infarcted region. The samples were stored in liquid nitrogen for Western blot analysis.

[0319] Western blot

[0320] Each tissue was ground in liquid nitrogen and the ground tissue was lysed in RIPA lysis buffer (Beyotime, CN) containing 1% complete protease inhibitor cocktail without EDTA (Roche, DE) on ice for 40 min, after which the protein extract was obtained. The protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo SCIENTIFIC, 23227, USA). An equal amount of protein (30 pg) from each sample was dissolved in 5x SDS sample buffer and separated on 10%-SDS and 8% polyacrylamide gel. Then, the protein electrophoresis was transferred to a polyvinylidene difluoride membrane (Bio-Rad, USA). The membrane was blocked in Tris-buffered saline / Tween 20 (TBST) (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% Tween 20) containing 5% skim milk (blocking solution) for 1 h and incubated with the corresponding primary antibody, such as anti-HIF-la (Abeam, ab113642, USA), anti-VEGF (Santa Cruz, sc57496, USA) and anti-VEFGR-1 (Abeam, ab32152, USA), diluted with blocking buffer at 4°C overnight according to the supplier's recommendations. After washing with TBST, the membrane was incubated with the appropriate secondary antibody at 37°C for 1 h. The target protein was visualized using the chemiluminescent HRP substrate (Millipore, USA) and QUANTITY ONETM Analysis was performed using density measurement techniques.

[0321] 1.7 mRNA levels of HIF-1 target genes

[0322] To determine the transcriptional activity of HIF-1 in ischemic myocardium, the mRNA levels of HIF-1α and HIF-1 target genes such as VEGF and VEGFR-1 (also known as Flt-1) were determined by real-time PCR (RT-PCR).

[0323] According to the manufacturer's instructions, use Total RNA was isolated from each sample (Invitrogen, 15596-026, USA). PRIMESCRIPT was used. TM The RT kit (TaKaRa, RR037A, Japan) was used to reverse transcribe 1 μg of total RNA 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 Premix Ex Taq™ II kit (TaKaRa, RR820A, Japan). To amplify HIF-1α, VEGF, and VEGFR1 cDNA fragments, samples were processed using a BIO-RAD CFX96 real-time system with the following procedure: denaturation at 95°C for 30 seconds, followed by 35 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Logarithmic phase results of growth curves were analyzed and relative quantification was performed using the 2-ΔCT method. The gene expression levels of HIF-1α, VEGF, and VEGFR1 were each normalized relative to the actin expression level in each sample. Each sample was tested in at least three replicates. Primer sequences are shown in Table 2.

[0324] Table 2. Primer sequences for RT-PCR

[0325]

[0326]

[0327] 1.8 Copper concentration in the heart

[0328] Tissue samples were freshly frozen and stored at -80°C before lyophilization. After lyophilization and digestion with nitric acid, the digestate was colorless or pale yellow and transparent, with no visible precipitate or residue. Ultrapure water was added to each container to dilute HNO3 to 2% for subsequent analysis of copper concentration. Copper concentration was determined by graphite furnace atomic absorption spectrophotometry (ICE3500, Thermo) according to the procedure shown in Table 1 of Example 3.

[0329] 1.9 Statistical Analysis

[0330] All data are expressed as mean ± SD. Changes in individual parameters were compared between experimental groups using Levene's test for equality of variances and coefficient of variation (CV). Statistical software package SPSS 14.0 (SPSS, Chicago, IL) was used and a P value < 0.05 was considered significant.

[0331] 2. Results

[0332] 2.1 Cardiac function

[0333] Echocardiography examination showed that left ventricular ejection fraction significantly increased over time after trientine treatment. However, in the untreated group, left ventricular ejection fraction decreased over time. See Figure 12 .

[0334] 2.2 Copper concentration in infarcted heart

[0335] Copper concentration in myocardium was determined by atomic absorption spectroscopy. As shown in Figure 13 , copper concentration was significantly increased in tissue samples from infarcted and border zones from the treated group after trientine treatment compared to the untreated group. In contrast, copper concentration in tissue samples from the remote zone was comparable in the trientine treated and untreated groups.

[0336] 3. Discussion

[0337] Myocardial ischemia leads to HIF-1a accumulation and copper depletion. Under ischemic conditions, accumulated HIFa cannot activate HIF transcription because copper is required for HIF transcription complex formation and interaction of HIF with HIF response elements in target genes. Thus, despite HIF accumulation in ischemic myocardium, copper deficiency blocks HIF-regulated expression of genes involved in angiogenesis, leading to suppression of myocardial angiogenesis. This effect leads to myocardial infarction, further development into heart failure.

[0338] This study used trientine to increase copper concentration in ischemic tissue for the treatment of myocardial infarction. The results showed that trientine promoted tissue redistribution and reutilization of copper. In addition, echocardiography showed that cardiac function was improved after trientine treatment. In this experiment, the dose of trientine was 18 mg / kg per day for rhesus monkeys, which is equivalent to about 420 mg per day for a human individual. Compared to the usual dose of trientine for reducing serum copper levels in Wilson's disease patients (500-700 mg / day to a maximum of 1500 mg / day for pediatric patients, and 750-1250 mg / day to a maximum of 2000 mg / day for adult patients), the dose used in this experiment is much lower. The results of this experiment provide strong evidence that low-dose trientine treatment described herein is an effective strategy for delivering copper in vivo to treat myocardial infarction.

[0339] Example 5. Treatment with trientine in a mouse model of myocardial ischemic infarction

[0340] In this experiment, a mouse model of myocardial ischemic infarction was established by a permanent coronary artery ligation procedure. Four weeks after the procedure, the ischemic heart tissue was replaced by collagen fibers and became infarcted tissue. The mice were treated with trientine as described in the protocol below. Figure 14

[0341] Four groups of model mice were administered trientine treatment by intragastric route twice daily at doses of 16.75, 33.49, 55.94, or 78.25 mg / kg per day. This treatment lasted for 4 weeks. The untreated group did not receive any trientine treatment.

[0342] Echocardiography

[0343] Cardiac function was assessed by echocardiography to evaluate the efficacy of trientine. All mice were evaluated by transthoracic echocardiography with a 1 MHz transducer (i13L, Vivid 7, GE Ultrasound). Left ventricular ejection fraction (EF) was assessed with the Simpson monoplane method. Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were recorded directly, and EF was calculated as follows: EF = (LVEDV - LVESV) / LVEDV x 100%.

[0344] Copper concentration in the heart

[0345] Tissue samples were freshly frozen and stored at -80°C before lyophilization. After lyophilization of the tissue and digestion with nitric acid, the digest was colorless or light yellow and transparent, with no visible precipitates or residues. Ultrapure water was added to each container to dilute the HNO3 to 2% for subsequent analysis of copper concentration. Copper concentration was determined by graphite furnace atomic absorption spectrophotometry (ICE 3500, Thermo) according to the procedure shown in Table 1 of Example 3.

[0346] Results

[0347] Cardiac performance detected by echocardiography showed that cardiac function, measured by left ventricular ejection fraction, was improved in mice treated with trientine at lower doses of trientine treatment, and this improvement decreased at higher doses of trientine treatment (see Figure 15 ). A dose of 33.49 mg / kg per day resulted in an improvement in ejection fraction that peaked and then decreased even with higher doses. This experiment showed that trientine treatment for myocardial infarction was effective in a narrow range of low doses. As Figure 16 ​As shown, copper concentration in the infarct region was significantly elevated in response to trientine treatment. Notably, the copper content in the infarct region was highest in the treatment group at a dose of 33.49 mg / kg per day.

[0348] Discussion

[0349] The present study used a series of increasing doses of trientine to treat myocardial ischemic infarction in mice. The results showed that the copper content in the infarct region was highest in the treatment group at a dose of 33.49 mg / kg per day of trientine, which corresponded to the highest improvement in ejection fraction observed in that group compared to the other experimental groups. No further improvement in copper content in the infarct region or ejection fraction was observed at the higher trientine doses tested.

[0350] The doses of 16.75, 33.49, 55.94, and 78.25 mg / kg per day tested in this experiment correspond to approximately 150, 300, 500, and 700 mg per day, respectively, in a human patient. In comparison, the dose of trientine used to treat Wilson's disease by lowering serum copper levels in patients ranges from 500-700 mg / day to a maximum of 1500 mg / day for pediatric patients and 750-1250 mg / day to a maximum of 2000 mg / day for adult patients. Thus, the trientine dose observed in this experiment to have the highest efficacy in supplementing copper content in ischemic heart tissue and restoring cardiac function is far lower than the dose used to treat Wilson's disease patients. The results of this experiment provide strong evidence that trientine treatment for myocardial infarction is effective within a narrow, low-dose range.

[0351] Without being bound by any theory or hypothesis, trientine can act as a copper delivery shuttle to transfer copper from high concentration tissues or environments, such as post-ischemia serum, to ischemic tissue in the heart that is lacking copper, thereby alleviating copper depletion in the ischemic tissue and improving cardiovascular disease. Multiple publications describe elevated copper levels in serum of patients with cardiovascular disease, especially myocardial infarction. See, e.g., ES Ford. Am. J. Epidem. 151(12): 1182 (2000); E. Gomez et al. J. Trace Elements Med. Biol. 14:65-70 (2000); and Singh MM et al. Angiology— Journal of Vascular Diseases, 504-506 (1985).

[0352] Example 6. Clinical study of trientine treatment in heart failure patients

[0353] A clinical study is conducted to assess the clinical effects of low-dose trientine treatment in patients with heart failure. The primary objective of the study is to assess the efficacy of trientine compared to placebo in treating heart failure patients before and after treatment.

[0354] The study is a randomized, double-blind, placebo-controlled clinical study in heart failure patients with reduced ejection fraction (e.g., LVEF < 35%) (e.g., NYHA functional class II and III). Patients in the control group are given standard of care (SOC) plus placebo twice daily. Patients in the treatment group are given SOC plus trientine orally at 150 mg / dose twice daily. Patients are assessed at screening, baseline (week 0), during treatment, and post-treatment.

[0355] The primary endpoint of the study can be survival, hospitalization related to heart failure, or changes in biomarkers related to heart failure. For example, circulating levels of natriuretic peptides over time can be used to stratify risk of heart failure and thus can serve as a biomarker of heart failure severity.

[0356] Secondary endpoints of the study can include changes in cardiac structure and function compared to baseline at the end of treatment. Cardiac structure and function can be determined by echocardiography. Exemplary measures that can be used as secondary endpoints include left ventricular end-diastolic volume, left ventricular ejection fraction, and E / E' ratio. Secondary endpoints of the study can also include functional status based on six-minute walk distance test, changes in symptoms (NYHA classification), and quality of life scores.

[0357] Serum copper levels and other biomarkers can be monitored as tertiary endpoints of the study.

[0358] Safety is assessed by review of subject-reported spontaneous adverse events (AEs) and other appropriate medical and safety assessments, such as vital signs, ECGs, laboratory tests, etc.

[0359] The above only is the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can also make several improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of a copper-chelating tetramine or a pharmaceutically acceptable salt thereof and a copper-promoting composition comprising copper ions in the manufacture of a medicament for treating a disease or condition associated with ischemic tissue injury; the copper-chelating tetramine is trientine; the disease or condition associated with ischemic tissue injury is cardiac hypertrophy, myocardial ischemic infarction, or ischemic heart failure; both the trientine and the copper-promoting composition comprising copper ions are administered orally; the effective amount of copper-chelating tetramine in a unit dosage form of the medicament is from 5 mg to 300 mg.

2. Use of a combination in the manufacture of a medicament for treating a disease or condition associated with ischemic tissue injury; the combination comprises a copper-chelating tetramine or a pharmaceutically acceptable salt thereof and a copper-promoting composition comprising copper ions; the copper-chelating tetramine is trientine; the disease or condition associated with ischemic tissue injury is cardiac hypertrophy, myocardial ischemic infarction, or ischemic heart failure; both the trientine and the copper-promoting composition comprising copper ions are administered orally; the effective amount of copper-chelating tetramine in a unit dosage form of the medicament is from 5 mg to 300 mg.

3. A kit comprising a combination of a copper-chelating tetramine or a pharmaceutically acceptable salt thereof and a copper-promoting composition comprising copper ions for use in treating a disease or condition associated with ischemic tissue injury; the copper-chelating tetramine is trientine; the disease or condition associated with ischemic tissue injury is cardiac hypertrophy, myocardial ischemic infarction, or ischemic heart failure; both the trientine and the copper-promoting composition comprising copper ions are administered orally; the effective amount of copper-chelating tetramine in a unit dosage form of the combination is from 5 mg to 300 mg.

4. The use according to claim 1 or 2 or the kit according to claim 3, wherein the effective amount of copper-chelating tetramine in combination with the effective amount of copper-promoting composition comprising copper ions results in an increase in intracellular copper levels or total copper levels in the ischemic tissue of the individual of greater than about 10% as compared to the intracellular copper levels or total copper levels in the ischemic tissue of the individual prior to treatment.

5. The use according to claim 1 or 2 or the kit according to claim 3, wherein the effective amount of copper-chelating tetramine in combination with the effective amount of copper-promoting composition comprising copper ions does not decrease extracellular copper levels or total copper levels in the individual.

6. The use according to claim 1 or 2 or the kit according to claim 3, wherein the effective amount of copper-chelating tetramine in combination with the effective amount of copper-promoting composition comprising copper ions does not decrease extracellular copper levels or total copper levels in the individual by greater than about 5% as compared to the extracellular copper levels or total copper levels in the individual prior to treatment.

7. The use or kit according to any one of claims 4 to 6, wherein the ischemic heart failure is NYHA functional class II or III heart failure.

8. The use or kit according to any one of claims 4 to 6, wherein the ischemic heart failure is a reduced ejection fraction heart failure.

9. The use or kit according to any one of claims 4 to 6, wherein the ischemic heart failure is a reduced ejection fraction heart failure with LVEF < 35%.

10. The use or kit of any one of claims 4 to 6, wherein the pharmaceutically acceptable salt of a copper-chelatable tetraamine is trientine dihydrochloride.

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