Novel Formulations and Methods

Abstract

The present disclosure provides compositions comprising nanoparticles containing phosphocreatine and triiodothyronine (T3), and uses of the compositions in the treatment of cardiac conditions, particularly cardiac arrest and acute heart failure, and conditions generally associated with hypoxia, such as ischemia and stroke.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 933,504, filed November 10, 2019, the contents of which are incorporated herein by reference in their entirety. FIELD OF THEINVENTION The field relates to compositions comprising nanoparticles containing phosphocreatine and triiodothyronine (T3), and uses of the compositions in treating cardiac conditions, particularly cardiac arrest and acute heart failure, and conditions generally associated with hypoxia, such as ischemia and stroke, neurological disorders, and disorders characterized by low cellular energy (e.g., disorders characterized by mitochondrial dysfunction). [Background technology]

[0002] Cardiac arrest refers to a condition in which a patient's heart stops beating effectively and is no longer capable of pumping blood throughout the body. It is often due to myocardial infarction. If treated promptly, cardiac arrest can be reversed by cardiopulmonary resuscitation (CPR) and defibrillation. Medications to treat cardiac arrest include epinephrine, which stimulates the myocardium and increases aortic pressure, which promotes coronary perfusion. However, it is controversial whether epinephrine significantly improves overall survival, because while it increases the chances of resuscitation, it can also cause arrhythmias and strain on the heart, increasing the risk of late resuscitation problems.

[0003] Other forms of acute cardiac insufficiency include acute heart failure and cardiogenic shock. Acute heart failure is a common and serious condition in patients with chronic heart disease. During acute heart failure, the heart's ability to pump blood from the pulmonary circulation to the peripheral circulation is impaired. Cardiogenic shock is a form of shock resulting from insufficient blood circulation due to a primary inability of the ventricles of the heart to function effectively.

[0004] Triiodothyronine, also known as T3, is a thyroid hormone. Thyroid-stimulating hormone (TSH) activates the production of thyroxine (T4) and T3. T4 is converted to T3 by deiodination. T3 influences various body processes, including body temperature, growth, and heart rate. T3 has important effects on cardiac tissue. Thyroid hormones, especially T3, regulate ventricular function by genomic and non-genomic mechanisms. Cardiac stress events (cardiac arrest, myocardial infarction, etc.) are associated with a rapid drop in serum T3 levels. Post-resuscitation T3 levels are highly correlated with survival. T3 also has cardiac stimulating properties: it increases cardiac output by increasing heart rate and force of contraction. Overall, there is reason to believe that early bolus T3 injections may increase the chances of resuscitating a cardiac arrest victim, and that elevated T3 serum concentrations may increase the chances of survival to hospital discharge.

[0005] Cardioprotection is an important goal of therapeutic interventions in cardiology, aiming to reduce infarct size and therefore prevent progression to heart failure after acute ischemic and cardiac arrest events. Recent studies have highlighted the role of the thyroid system in cardioprotection, especially through the maintenance of mitochondrial function, its antifibrotic and proangiogenic effects, repolarization of cell membranes, and induction of cell regeneration. Triiodothyronine / thyroxine (T3) therapy has been used to reverse myocardial stunning. Hyperthyroidism is highly dependent on the mitochondrial sodium-calcium exchanger and mitochondrial K+ channels to prevent stunning.

[0006] The heart cannot store significant oxygen and is therefore dependent on the continuous delivery of blood flow to support its hypermetabolic state. After cardiac arrest, myocardial tissue oxygen tension rapidly declines and aerobic production of ATP ceases. Generally, return of spontaneous circulation (ROSC) cannot be achieved without reoxygenation of the ischemic myocardium. Epinephrine is currently used to induce ROSC recovery. However, the use of epinephrine has come under scrutiny as it causes various negative side effects, such as hypertension and pulmonary edema. It has also not been shown to improve long-term survival or mental function after recovery.

[0007] It is therefore desirable to create a procedure that can obtain ROSC without the potential side effects of epinephrine. Summary of the Invention

[0008] Phosphocreatine (hereafter also referred to as PCR) is a phosphorylated creatine molecule that functions in skeletal muscle and the brain as a rapidly mobilizable reserve of high-energy phosphate to recycle adenosine triphosphate, the energy currency of the cell. Phosphocreatine can anaerobically donate a phosphate group to ADP to form ATP. The use of phosphocreatine to rapidly regenerate ATP during strenuous activity can provide a spatial and temporal buffer of ATP concentration.

[0009] The inventors have surprisingly found that nanoparticles of T3 and phosphocreatine restore ATP levels in cardiomyocytes under hypoxic conditions. It is believed that this new combination can provide a treatment that can obtain ROSC without the potential side effects of epinephrine. This combination of nanoparticle forms of T3 and phosphocreatine represents a potentially new treatment for the control of tissue damage in cardiac ischemia and resuscitation. The inventors have also surprisingly found that the composition of the present disclosure can cross the blood-brain barrier, meaning that nanoparticles of T3 and phosphocreatine can also be used to treat various disorders associated with cerebral hypoxia. The results strongly suggest further applications in conditions characterized by low cellular energy, including conditions associated with hypoxia.

[0010] In one embodiment, the present invention provides nanoparticles that encapsulate both T3 and PCR, the nanoparticles comprise chitosan and PLGA, and the relative ratio of chitosan to PLGA can be changed to adjust the release of active ingredient, such as T3 and / or PCR.Without being bound by theory, it is believed that chitosan is hydrophilic.Therefore, when the active ingredient may possibly be hydrophobic (such as T3 and PCR), the addition of more chitosan compared to PLGA, for example, in a relative amount of chitosan to PLGA of 80 / 20 (e.g., %w / w 80 / 20, chitosan to PLGA) or in a relative amount of chitosan to PLGA of 90 / 10 (e.g., %w / w 90 / 10, chitosan to PLGA), can result in nanoparticles that release active ingredient quickly when applied or administered. Without being bound by theory, if the active ingredient is more hydrophobic, the addition of more PLGA compared to the amount of chitosan, for example in a relative ratio of chitosan to PLGA of 20 / 80 (e.g., % w / w 20 / 80, chitosan to PLGA), or chitosan to PLGA of 10 / 90 (e.g., % w / w 10 / 90, chitosan to PLGA), may result in nanoparticles that release the active ingredient more slowly.

[0011] In a further aspect, the present disclosure provides a method for preventing or treating a disease, disorder or condition characterized by a deficiency of adenosine triphosphate (ATP), comprising administering a therapeutically effective amount of triiodothyronine (T3) and phosphocreatine (PCR) to a subject in need of such prevention or treatment.In various embodiments, the disease, disorder or condition is selected from a cardiovascular disorder, a hypoxia-related disorder, or a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction or a disorder characterized by ATP synthase dysfunction), a neurodegenerative disorder, a respiratory disorder, obesity, a metabolic disorder, or diabetes.

[0012] In one embodiment, the disclosure provides a method for treating a cardiac condition, e.g., cardiac arrest, cardiac arrhythmia, cardiac dysfunction, myocardial infarction, myocardial ischemia / reperfusion injury, myocardial infarction, myocardial hypoxia, or congestive heart failure, comprising administering to a patient in need of treatment a composition comprising an effective amount of nanoparticles of T3 and phosphocreatine (PCR), wherein the composition comprises a bioabsorbable polymer, e.g., as described above.

[0013] In another embodiment, the present disclosure also provides a method for treating a disease or condition associated with hypoxia, ischemia or ischemia-reperfusion injury, e.g., myocardial hypoxia, stroke (e.g., ischemic stroke or hemorrhagic stroke), traumatic brain injury (e.g., concussion), ischemia (e.g., myocardial ischemia or retinal ischemia), hemorrhagic shock, or edema (e.g., cerebral edema), comprising administering to a subject in need of treatment a composition comprising an effective amount of T3 nanoparticles and phosphocreatine (PCR), e.g., having the characteristics of any of compositions 1 or 1.1-1.16 above, wherein the composition comprises a bioabsorbable polymer, e.g., as described above.

[0014] In certain examples of the methods disclosed herein, the nanoparticles administered include chitosan-PLGA nanoparticles that encapsulate T3 and PCR.

[0015] In yet another example, the administered nanoparticles include chitosan-PLGA nanoparticles that immobilize both T3 and PCR. Alternatively, the administered nanoparticles include chitosan-PLGA nanoparticles that immobilize T3 and PCR, and chitosan-PLGA nanoparticles that encapsulate T3 and PCR. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the effect that the combination of T3 nanoparticles and PCR has on ATP levels in neonatal cardiomyocytes compared to controls.

[0017] [Diagram 2]FIG. 2 shows the particle size distribution of PLGA nanoparticles.

[0018] [Diagram 3] FIG. 3 shows the particle size distribution of PLGA-PEG nanoparticles encapsulating phosphocreatine.

[0019] [Figure 4] FIG. 4 shows the particle size distribution of T3-PLGA nanoparticles loaded with phosphocreatine.

[0020] [Diagram 5] FIG. 5 shows the particle size distribution of T3-PLGA nanoparticles.

[0021] [Figure 6] FIG. 6 shows the cardioprotective effect of Nano-T3 under normal and hypoxic conditions on ATP levels in neonatal cardiomyocytes (bioluminescence assay).

[0022] [Figure 7] FIG. 7 shows the cardioprotective effect of Nano-T3 under normal and hypoxic conditions on troponin T release levels in neonatal cardiomyocytes (bioluminescence assay).

[0023] [Figure 8] FIG. 8 shows the effect of T3, Nano-T3, and Nano-T3 and phosphocreatine on neuronal (PC12) cells under hypoxia on ATP levels.

[0024] [Figure 9] FIG. 9 shows the effect of T3, Nano-T3, and Nano-T3 and phosphocreatine on neuronal (PC12) cells under hypoxia on troponin T release levels.

[0025] [Figure 10]FIG. 10 shows an embodiment of a PLGA nanoparticle morphology of the present disclosure in which T3 is bound to the outer surface of the PLGA nanoparticle and PCR is encapsulated within the nanoparticle.

[0026] [Figure 11A] 11A shows heart rates in BPM of porcine subjects observed at regular intervals after cardiac arrest. Results are shown for porcine subjects treated with T3-nanoparticles, T3-nanoparticles with phosphocreatine, and epinephrine compared to controls.

[0027] [Figure 11B] Figure 11B shows the left ventricular dP / dtmax of porcine subjects observed at regular intervals after cardiac arrest. Results are shown for porcine subjects treated with T3-nanoparticles, T3-nanoparticles containing phosphocreatine, and epinephrine compared to controls.

[0028] [Figure 11C] Figure 11C shows circulating cTnl in porcine subjects monitored at intervals after cardiac arrest. Results are shown for porcine subjects treated with T3-nanoparticles, T3-nanoparticles with phosphocreatine, and epinephrine compared to controls.

[0029] [Figure 12A] 12A shows coronary sinus pH in porcine subjects monitored at regular intervals after cardiac arrest. Results are shown for porcine subjects treated with T3-nanoparticles, T3-nanoparticles with phosphocreatine, and epinephrine compared to controls.

[0030] [Figure 12B] 12B shows coronary sinus pCO2 in porcine subjects monitored at intervals after cardiac arrest. Results are shown for porcine subjects treated with T3-nanoparticles, T3-nanoparticles with phosphocreatine, and epinephrine compared to controls.

[0031] [Figure 13A]FIG. 13A shows the bioavailability of Nano-T3 in the brain of BALB / C mice compared to free T3.

[0032] [Figure 13B] FIG. 13B shows the bioavailability of Nano-T3 in the heart and lungs of BALB / C mice compared to free T3.

[0033] [Figure 14] FIG. 14 shows the effects of T3, Nano-T3, and epinephrine on rat pheochromocytoma (PC12) cells and T3-induced cells (HS5) under hypoxia on ATP levels.

[0034] [Figure 15] FIG. 15 shows the effects of T3, Nano-T3, and epinephrine on rat pheochromocytoma (PC12) cells and T3-induced cells (HS5) under hypoxia on troponin T release levels.

[0035] [Figure 16] FIG. 16 shows the neuroprotective effects of Nano-T3 and Nano-T3 / PCR compared to epinephrine on porcine brain tissue following cardiac arrest and resuscitation.

[0036] [Figure 17] FIG. 17 shows the effects of Nano-T3, Nano-T3 / PCR, and epinephrine on the release of neuron-specific enolase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The examples and figures set forth in the detailed description are merely examples and should not be used to limit the scope of the claims in their construction or interpretation.

[0038] Compositions and Related Methods Thus, in one aspect, the disclosure provides a composition (Composition 1) comprising nanoparticles of T3 and phosphocreatine (PCR) encapsulated or immobilized by a bioabsorbable polymer.

[0039] For example, Composition 1 may further have any of the following characteristics: 1.1 Composition 1, in which the nanoparticles comprise T3 (e.g., L-T3) covalently conjugated to a biodegradable polymer, and phosphocreatine (PCR) encapsulated within the biodegradable polymer. 1.2 Any of the preceding compositions wherein the polymer comprises poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA), e.g., PLGA which is a 50 / 50 copolymer of D,L-lactic acid and glycolic acid. 1.3 Any of the preceding compositions wherein the polymer comprises polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLA), e.g., PLGA, a 50 / 50 copolymer of D,L-lactic acid and glycolic acid, optionally conjugated with short chain polyethylene glycol (PEG, molecular weight 100-2,000 Daltons). 1.4 Any of the preceding compositions, wherein the average diameter of the T3 nanoparticles is about 50-1000 nm, e.g., 50-500 nm. Any of the preceding compositions, wherein the average diameter of the 1.5 T3 nanoparticles is about 100-300 nm, e.g., 200 nm. Any of the preceding compositions, wherein the zeta potential of the 1.6 T3 nanoparticles is between 0 and -20 mV. 1.7 Any of the preceding compositions, wherein the zeta potential of the T3 nanoparticles is between 0 and +20 mV. 1.8 Any of the preceding compositions wherein T3 is covalently bonded to a bioabsorbable polymer. 1.9 Any of the preceding compositions wherein the nanoparticles comprise a second pharmacologically active ingredient. 1.10 Any of the preceding compositions, wherein the molecular weight range of the PLGA is about 5,000-50,000 Daltons, preferably 6,000-8,000 Daltons. 1.11 Any of the preceding compositions, wherein the PLGA is conjugated to PEG having a molecular weight range of 5,000-20,000 Daltons, preferably 6,000-8,000 Daltons. 1.12 Any of the preceding compositions, wherein the PLGA is conjugated to PEG having a molecular weight range of about 6,000-8,000 daltons. 1.13 Any of the preceding compositions, wherein T3 is chemically conjugated to PLGA or PLGA-PEG for assembly of nanoparticles. 1.14 Any of the preceding compositions, wherein the composition is dispersed in a physiological sterile medium. 1.15 Any of the preceding compositions wherein the composition is dispersed in saline or dextrose. 1.16 Any of the preceding compositions wherein the biodegradable polymer comprises chitosan.

[0040] In a further aspect, the present disclosure provides a method (Method 1) for preventing or treating a disease, disorder, or condition characterized by a deficiency of adenosine triphosphate (ATP), comprising administering therapeutically effective amounts of triiodothyronine (T3) and phosphocreatine (PCR) to a subject in need of prevention or treatment. 1.1 Method 1, wherein the disease, disorder or condition is selected from a cardiovascular disorder, a hypoxia-related disorder, or a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction or a disorder characterized by a dysfunction of ATP synthase), a neurodegenerative disorder, a respiratory disorder, obesity, a metabolic disorder, or diabetes. 1.2 Any of the preceding methods wherein the disease, disorder or condition is a cardiovascular disorder (e.g., atherosclerosis (e.g., coronary atherosclerosis), ischemia-reperfusion (I / R) injury, hypertension (e.g., essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension), diabetes, cardiac hypertrophy, myocardial ischemia, myocardial infarction, cardiac arrest, cardiomyopathy (e.g., pediatric cardiomyopathy), cardiac dysfunction, cardiogenic shock, left ventricular trabeculation syndrome, and heart failure (e.g., acute heart failure)). 1.3 Any of the preceding methods wherein the disease, disorder or condition is a disorder associated with hypoxia (e.g., hemorrhagic shock, organ failure (e.g., ARDS, sepsis, septic shock or hemorrhagic shock organ failure), hypoxia due to organ transplant, renal failure (e.g., chronic renal failure), cerebral edema, papilloma, spinal cord injury, stroke (e.g., ischemic stroke or hemorrhagic stroke), ischemia or ischemia reperfusion injury, traumatic brain injury (e.g., concussion), cerebral hypoxia, spinal cord injury, edema (e.g., cerebral edema), and anemia). 1.4 The disease, disorder or condition is a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction (e.g., a mitochondrial myopathy, e.g., Kearns-Sayre syndrome; Leigh syndrome; mitochondrial DNA depletion syndrome; mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); diabetes and deafness; maternally inherited deafness and diabetes; mitochondrial neurogastrointestinal encephalomyopathy; myoclonic epilepsy with ragged-red fibers; neuropathy, ataxia, and retinal pigment epilepsy). degeneration (NARP); Pearson syndrome), disorders characterized by dysfunction of ATP synthase (e.g., apical hypertrophic cardiomyopathy and neuropathy, ataxia, autism, Charcot-Marie-Tooth syndrome, encephalopathy, epilepsy with cerebral pseudoatrophy, epilepsy with episodic weakness, hereditary spastic paraplegia, familial bilateral striatal necrosis, Leber's hereditary optic neuropathy, mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS), motor neuron syndrome, periodic limb paralysis, schizophrenia, spinocerebellar ataxia, tetralogy of Fallot), any of the preceding ways. 1.5 Any of the preceding methods, wherein the disease, disorder or condition is a neurodegenerative disorder (e.g., Huntington's disease, Alzheimer's disease, Parkinson's disease) or a disorder characterized by cell membrane repolarization. 1.6 Any of the preceding methods wherein the disease, disorder or condition is a respiratory disorder (e.g., emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), apnea, and asthma). 1.7 Any of the preceding methods wherein the disease, disorder or condition is diabetes or a diabetic disorder (e.g., diabetic ulcers, gangrene and diabetic retinopathy). 1.8 Any of the preceding methods, wherein the disease, disorder or condition is a metabolic disorder (e.g., metabolic syndrome). 1.9 Any of the preceding methods wherein the disease, disorder or condition is obesity. 1.10 Any of the preceding methods, wherein the T3 and PCR are present in the form of nanoparticles, and the T3 and phosphocreatine (PCR) are encapsulated or immobilized by a bioabsorbable polymer. 1.11 Method 1.10, wherein the nanoparticles comprise T3 (e.g., L-T3) covalently conjugated to a biodegradable polymer, and phosphocreatine (PCR) encapsulated within the biodegradable polymer. 1.12 Any of methods 1.10-1.11, wherein the polymer comprises poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA), e.g., PLGA, which is a 50 / 50 copolymer of D,L-lactic acid and glycolic acid. 1.13 Any of methods 1.10-1.12, wherein the polymer comprises polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLA), e.g., PLGA, a 50 / 50 copolymer of D,L-lactic acid and glycolic acid, optionally conjugated with short chain polyethylene glycol (PEG, molecular weight 100-2,000 Daltons). 1.14 Any of methods 1.10-1.13, wherein the average diameter of the T3 nanoparticles is about 50-1000 nm, for example 50-500 nm. 1.15 Any of methods 1.10-1.14, wherein the average diameter of the T3 nanoparticles is about 100-300 nm, e.g., 200 nm. 1.16 Any of methods 1.10-1.15, wherein the zeta potential of the T3 nanoparticles is 0--20 mV. 1.17 Any of methods 1.10-1.16, wherein the zeta potential of the T3 nanoparticles is between 0 and +20 mV. 1.18 Any of methods 1.10-1.17, wherein T3 is covalently bonded to a bioabsorbable polymer. 1.19 Any of methods 1.10-1.18, wherein the nanoparticles comprise a second pharmacologically active ingredient. 1.20 Any of methods 1.10 to 1.19, wherein the molecular weight range of the PLGA is about 5,000 to 50,000 daltons, preferably 6,000 to 8,000 daltons. 1.21 Any of methods 1.10-1.20, wherein the PLGA is conjugated to PEG having a molecular weight range of 5,000-20,000 daltons, preferably 6,000-8,000 daltons. 1.22 Any of methods 1.10-1.21, wherein the PLGA is conjugated to PEG having a molecular weight range of about 6,000-8,000 daltons. 1.23 Any of methods 1.10-1.22, where T3 is chemically conjugated to PLGA or PLGA-PEG for construction of nanoparticles. 1.24 Any of methods 1.10-1.23, wherein the composition is dispersed in a physiological sterile medium. 1.25 Any of methods 1.10-1.24, wherein the composition is dispersed in saline or dextrose. 1.26 Any of methods 1.10-1.25, wherein the biodegradable polymer comprises chitosan. 1.27 Any of the preceding methods, wherein the T3 and PCR are administered by injection. 1.28 Any of the preceding methods, wherein T3 and PCR are administered by bolus injection. 1.29 Any of the preceding methods wherein the T3 and PCR are administered by infusion. 1.30 Any of the preceding methods in which T3 and PCR are administered by bolus injection followed by infusion. 1.31 Any of the preceding methods wherein T3 and PCR are administered orally. 1.32 Any of the preceding methods in which the subject is a human being. 1.33 Any of the preceding methods in which T3 and PCR are administered in conjunction with cardiopulmonary resuscitation (CPR) and / or defibrillation.

[0041] In a further aspect, the disclosure provides a method for preventing, reversing or interrupting apoptosis (Method 2), comprising administering therapeutically effective amounts of triiodothyronine (T3) and phosphocreatine (PCR) to a subject in need thereof. Further embodiments of Method 2 are provided in combination with any of Compositions 1 and beyond.

[0042] As used herein, T3 refers to the naturally occurring form of triiodothyronine, shown below: [ka] T3 is also used herein to refer to triiodothyronines modified at the hydroxyl group arising from the 4-position of the phenyl ring. For example, T3 as used herein refers to a thyronine having a modified linking group (e.g., a C 1-10 amine linking group).

[0043] For example, a method using a composition comprising an effective amount of T3 nano and particles of phosphocreatine (PCR), having the characteristics of any of compositions 1 or 1.1-1.16 above, can be used to treat acute cardiac dysfunction. Examples of cardiac conditions that can be treated include cardiac arrest, cardiogenic shock, and acute heart failure.

[0044] For example, without being bound by theory, it is believed that co-delivery of T3-nanoparticles and PCR may act rapidly to restore spontaneous circulation while maintaining normal levels of ATP in cardiomyocytes.

[0045] In one embodiment, the particles provide sustained release that allows T3 to affect gene expression. In another embodiment, T3 is covalently attached to a bioabsorbable polymer, which reduces genomic effects and enhances effects on integrin receptors.

[0046] The T3 nanoparticles of the invention, for example having the features of any of compositions 1 or 1.1-1.16 above, can be administered in combination with or adjunctive to conventional standard of care treatments for cardiac arrest, e.g., cardiopulmonary resuscitation defibrillation and epinephrine, or for diseases or disorders associated with hypoxia, e.g., myocardial hypoxia, stroke (e.g., ischemic stroke or hemorrhagic stroke), traumatic brain injury (e.g., concussion), ischemia (e.g., myocardial ischemia or retinal ischemia), hemorrhagic shock or edema (e.g., cerebral edema). They can be administered immediately after cardiac arrest, and optionally thereafter, e.g., 1-24 hours later, to maintain cardiac and neurological function.

[0047] Various methods are provided for synthesizing T3-nanoparticles. For example, a single emulsion process can produce chitosan-PLGA nanoparticles that encapsulate T3. In yet another example, a process that includes gelation / conjugation of preformed biodegradable polymers produces 1) chitosan nanoparticles that encapsulate T3, with and without glutaraldehyde as crosslinker; or 2) chitosan-PLGA nanoparticles that encapsulate T3. Other crosslinkers can be used.

[0048] In yet another example, a process involving chemically binding T3 to the surface of PLGA or PLGA-PEG nanoparticles produces 1) PLGA nanoparticles with immobilized T3, or 2) PLGA-PEG nanoparticles with immobilized T3, further comprising an active compound, such as phosphocreatine (PCr), within the nanoshell.

[0049] (Method of producing the composition of the present disclosure) In one embodiment, T3 is covalently linked to biodegradable polymer, for example, via hydroxy on phenyl moiety.Such composition can be formed by using the activated T3, in which phenolic hydroxy is activated and amino moiety is protected using a suitable linker.For example, in one embodiment, amino-protected T3 is formed by using the synthesis shown in scheme 1 below. [ka]

[0050] The amino-protected T3 is then attached to the nanoparticles, e.g., via the phenolic hydroxy, e.g., by using an activated linker group, e.g., a moiety that can be attached to an amine group on a bioabsorbable polymer, e.g., the amino moiety on chitosan.

[0051] Thus, in one embodiment, the present invention provides a compound having a phenolic hydroxy group of the formula [CH2-O-CH]-[CH2] n For example, the present invention provides an amino-protected, activated T3 substituted with an epoxide moiety represented by formula 1: [ka] formula 1 where n is an integer selected from 1 to 5 and R is an amino protecting group, such as butoxycarbonyl (BOC). The present invention provides a compound represented by the formula:

[0052] Thus, T3 may be, for example, a compound of the formula [CH2-O-CH]-[CH2] n The resulting compound can then be activated, for example, according to the synthesis shown in Figure 10, using an epoxyalkyl of -X (wherein n = 1-5, X is a halogen, e.g., bromine). The resulting compound can then be selectively deprotected, if necessary, to give, for example, [ka] As such, the carboxy moiety is released and the phenolic hydroxy (here with propylene oxide) is activated to give amino-protected (here with BOC) T3.

[0053] The activated T3 is then coupled to a bioabsorbable polymer, e.g., T3 with an epoxy linker moiety and an amino protecting group is reacted with a bioabsorbable polymer with an amino group, followed by deprotection to obtain nanoparticles covalently bound with T3, e.g., as shown in FIG. 11. The reaction may be carried out in the presence of a stabilizing agent, e.g., polyvinyl alcohol, e.g., PVA 1% w / v, in a suitable solvent, e.g., dimethylsulfoxide, e.g., DMSO (0.1% v / v) and acetic acid (0.1% v / v) (the solvent is subsequently removed by dialysis). The number of T3 moieties attached to the nanoparticles may vary based on the reaction conditions and the amount of reactants used, but the distribution of variation is low when these conditions are kept constant. Typically, the nanoparticles contain 20-200 T3 moieties, e.g., about 50 per nanoparticle. The amount of T3 in a batch may be assayed, e.g., by separating the nanoparticles by filtration or centrifugation, weighing, dissolving the T3 nanoparticles in a strong base, and measuring by HPLC, as described below.

[0054] In another embodiment, T3 is a C at the hydroxy of the phenyl moiety. 1-10 The polymer is covalently attached to a bioabsorbable polymer via an amine linking group. The process generally proceeds according to Scheme 2: [ka]

[0055] In the first step, T3 is dissolved in anhydrous methanol. Thionyl chloride is then added and the reaction is refluxed for 24 hours. The reaction is cooled to room temperature and the methyl-protected T3 is obtained in the form of a precipitated white powder, which is washed with methanol and ether.

[0056] In the second step, the methyl-protected T3 is dissolved in anhydrous methanol. An equal amount of triethylamine (TEA) is added to the solution and stirred for 30 minutes. Then, an equal amount of benzyl chloroformate (CBZ) is added and stirred at room temperature for 6 hours. Methanol is removed and the product is extracted with dichloromethane (DCM) and washed with acidic water, bicarbonate and brine.

[0057] In the third step, a mixture of CBZ-protected T3, tert-butyloxycarbonyl (BOC)-protected 3-bromopropylamine and potassium carbonate (5 equiv.) in acetone was heated to reflux for 24 h. The reaction was filtered and concentrated, and the crude was then purified by flash column chromatography on silica gel using n-hexane and ethyl acetate (9:1 to 7:3) to give the final product.

[0058] The BOC protecting group is removed, followed by the methyl protecting group, until T3 is protected only with the CBZ group. T3 is mixed with PLGA functionalized with N-hydroxysuccinimide (NHS) in TEA and dimethylsulfoxide (DMSO). The resulting product is T3 covalently bound to PLGA, and the CBZ protecting group is removed.

[0059] The production of nanoparticles is generally described in applicant's own publications, U.S. Patent Application Publication No. 20110142947, WO 2011 / 159899, ​​and U.S. Patent No. 13 / 704,526, the contents of each of which are incorporated herein by reference in their entirety. The nanoparticles described herein may be produced by similar means.

[0060] Without wishing to be bound by theory, it is believed that nanoparticles containing T3 and phosphocreatine take the form shown in Figure 10. Briefly, a suitable linking group (e.g., C 1-10 T3 molecules bearing an amine linking group are covalently attached to the outer surface of the PLGA nanoparticles, and phosphocreatine is encapsulated within the PLGA nanoparticles.

[0061] In one example, T3 nanoparticles are made from T3 and the following components: [ka] In one example, the nanoparticles have these components in approximately the following amounts: [Table 1]

[0062] Nanoparticle content is confirmed using, for example, DLS, TEM, NMR, HPLC and LC / MS. Nanoparticle formulations can be sterilized using conventional means, for example, filtration, gamma radiation.

[0063] The above measurement (i.e., viscosity) may be performed by any means known in the art. For example, it is contemplated that the viscosity of the chitosan solution may be measured at room temperature using a Brookfield digital viscometer, such as a DV-11+Pro. In another example, it is contemplated that the viscosity may be measured using an Ubbelohde viscometer. In such an example, it is contemplated that the viscometer may be connected to a visco-clock to record the time of the solution passing through. EXAMPLES

[0064] Example 1: Cardiovascular protective effects of nanoparticles of T3 and phosphocreatine against ischemic injury This study aimed to investigate the positive protective role of T3, T3 nanoparticles, and T3 plus phosphocreatine (PCR) nanoparticles in hypoxia-mediated cardiac cell injury, as well as their effects on angiogenesis and neuroprotection under hypoxic conditions. The effects of T3 and T3 nanoparticles + PCR on angiogenesis were tested in the CAM model. The cardioprotective effect of T3 under hypoxia was tested using isolated neonatal cardiomyocytes treated with PBS (control), free T3 (3 μM), free PCR (30 μM), and T3 nanoparticles + PCR. Mitochondrial function and sarcomere integrity were tested using an ATP-bioluminescence assay, and cardiac troponin T levels were tested using flow cytometry. The effect of T3 nanoparticles on induced neuronal cells under hypoxia was also tested. Finally, T3 and T3 nanoparticles tagged with Cy7 dye were injected into the tail vein of mice to monitor the biodistribution in real time.

[0065] The results of the study are shown in Figure 1. T3 and T3 nanoparticles increased angiogenesis in the CAM model compared to the control group (approximately 3-fold). Under hypoxia, improvement of cardiac ATP was achieved only with the combination of T3 nanoparticles and PCR, while maintaining normal troponin T levels (p<0.001). T3 nanoparticles caused a significant increase in the neuroprotective markers PAX6 and DLX2 by approximately 60% and 40%, respectively. In vivo, thiamin 7 signal intensity was detected mainly in the mouse brain and heart within minutes of administration, indicating that the composition containing T3 nanoparticles with PCR was also unexpectedly able to cross the blood-brain barrier.

[0066] T3 nanoparticles act by activating the cell surface receptor αvβ3 and distribute to the cytoplasm but not the nucleus. Thus, the composition of T3 nanoparticles + PCR offers a potentially new treatment for controlling tissue damage in cardiac ischemia and resuscitation.

[0067] Example 2: Comparison of synthesis and size of different nanoparticle bases For the nanoparticles, several different potential polymer bases were prepared. PLGA-based nanoparticles containing T3 were prepared by dissolving 200 mg PLGA and 20 mg T3 in 1 mL DMSO. The solution was added dropwise to 40 mL 1% PVA under sonication. The emulsion was lyophilized to remove the DMSO. The product was then reconstituted in 20 mL PBS.

[0068] A similar method was carried out to prepare PLGA-based nanoparticles containing T3 and PCR. PLGA-based nanoparticles containing T3 were prepared by dissolving 250 mg PLGA and 25 mg T3 in 1 mL DMSO. The solution was added dropwise to a solution of 20 mL 1% PVA containing 45 mg PCR under sonication. The resulting emulsion was then added dropwise to 30 mL 1% PVA. The emulsion was lyophilized and reconstituted in 25 mL PBS.

[0069] PLGA-PEG-based nanoparticles were also prepared. A mixture of PLGA-PEG and T3 was dropped into a 1% solution of PVA. The contents were sonicated and freeze-dried to obtain PLGA-PEG-T3 nanoparticles. Optionally, PCR was added to the resulting emulsion before freeze-drying in a 1% solution of PVA to produce PLGA-PEG-T3 nanoparticles encapsulating PCR.

[0070] The particle size of each base was measured by dynamic light scattering and the results are summarized in Table 1 below. [Table 2] As shown in the table above, all forms of nanoparticles show similar average diameters. PLGA nanoparticles without T3 or PCR showed a diameter of 179 nm, which was similar to L-T3-phosphocreatine-PLGA (181 nm) and L-T3-PLGA nanoparticles (185 nm). PLGA-PEG base showed a slightly smaller but comparable diameter at 157 nm. Figures 2-5 show the particle size distribution of each base produced.

[0071] The release kinetics of PLGA and PLGA-PEG based nanoparticles were also examined. It was observed that PLGA nanoparticles had superior release rates for the active agent compared to PLGA-PEG based nanoparticles.

[0072] Example 3: Cardiovascular protective effect of the nanoparticles of the present disclosure against ischemic injury Studies were conducted to investigate the positive protective role of nanoparticles containing both T3 and PCR in hypoxia-mediated cardiac cell injury, as well as their effects on angiogenesis and neuroprotection under hypoxic conditions.

[0073] The effect of T3 and Nano-T3+Pcr on angiogenesis was tested in chick chorioallantoic membrane (CAM) model. It was observed that T3 and Nano-T3 enhanced angiogenesis in the CAM model compared to the control group (about 3-fold). The results are summarized in Table 2 below. [Table 3]

[0074] Both T3 and T3 nanoparticles showed significantly enhanced angiogenesis compared to samples treated with basic fibroblast growth factor: T3 alone showed a 37% improvement in observed branch points compared to bFGF, and T3 bound to PLGA nanoparticles showed a 61% improvement compared to samples treated with bFGF.

[0075] The cardioprotective effect of T3 under hypoxia was then tested using isolated neonatal cardiomyocytes treated with PBS (control), T3 (1–3 μM), T3 and PCR (5 μM), T3 nanoparticles (1–3 μM), T3 and PCR nanoparticles (5 μM), and epinephrine (0.5 μM). The cells were cultured for 24 h at 37 °C in a hypoxic incubator containing 4% oxygen, 5% CO2. After incubation, the cells were harvested and compared with those in normal conditions. Mitochondrial function and sarcomere integrity were tested using an ATP-bioluminescence assay, and cardiac troponin T levels were tested using flow cytometry after the addition of troponin T antibody conjugated with FITC.

[0076] Under hypoxia, improvement in cardiac ATP was achieved with NT3+Pcr while maintaining normal Troponin T levels (p<0.001). As shown in Table 3 below, epinephrine demonstrated cardiomyocyte contraction levels similar to those of hypoxic cells, while samples treated with T3 and Nano-T3 demonstrated contraction levels closer to those of cells under normal conditions. [Table 4]

[0077] These results are generally comprised of the levels of ATP and troponin observed after treatment with nanoparticle forms of T3 and PCR. Figure 6 shows the effect that T3 and PCR treatment have on myocyte ATP levels, and Figure 7 shows the effect that T3 and PCR have on troponin levels. In both cases, treatment with epinephrine showed ATP and troponin release levels close to untreated hypoxic cardiomyocytes, while treatment with T3 nanoparticles and both T3 and PCR nanoparticles showed results close to control cardiomyocytes under normoxic conditions. T3 and Nano-T3 have similar results, but Nano-T3 has better delivery.

[0078] The effect of T3 nanoparticles on hypoxia-induced neuronal cells was also examined. The results are summarized in Figures 8 and 9. The results show that ATP and lactate dehydrogenase release levels in PC12 neuronal cells treated with Nano-T3 and Nano-T3 plus PCR are similar to cells under normoxic conditions. Furthermore, it was observed that Nano-T3 caused a significant increase in the neuroprotective markers PAX6 and DLX2 by approximately 60% and 40%, respectively.

[0079] Example 4: Blinded, Randomized, Vehicle-Controlled Preclinical Study of a Novel Nanoparticulate Formulation of Triiodothyronine in Cardiac Arrest In another study, we evaluated the efficacy of two nanoparticle formulations of T3 designed to prolong signaling through cell membranes in a porcine cardiac arrest model. In this study, pigs were subjected to cardiac arrest for 7 minutes, followed by manual CPR for up to 20 minutes with defibrillation every 2 minutes as needed. Two minutes after the start of CPR, animals were blindly randomized (n=10 / group) to intravenous vehicle (blank PLGA nanoparticles), Nano-T3 (nanoparticles covalently linked to T3; 0.125 mg / kg), Nano-T3 and PCR (nanoparticles covalently linked to T3 with encapsulated PCR; 0.125 mg / kg), or epinephrine (0.015 mg / kg). In animals that achieved ROSC (unassisted systolic blood pressure >80 mmHg for 1 minute), further pharmacological support was limited to one additional dose of the drug of choice for hypotension. Hemodynamics, left ventricular (LV) function, plasma cardiac troponin I (cTnI) and other parameters were assessed at baseline and for up to 4 hours after ROSC.

[0080] Compared to vehicle, ROSC rates were higher in animals receiving Nano-T3, Nano-T3 and PCR, or epinephrine. Immediately after ROSC, animals treated with Nano-T3 had lower heart rate and LV dP / dt compared to animals treated with epinephrine. max 11A and 11B), but the differences were no longer seen 30-60 min after ROSC. Analysis of coronary sinus blood samples taken immediately after drug administration showed that Pro-Al 616 and Pro-Al 617, but not epinephrine, produced significant improvements in coronary sinus pH and PCO2 compared to vehicle within the first 5 min after ROSC (see Figures 12 and 13).

[0081] Although survival times were comparable between groups (Nano-T3: 122 ± 30 min, Nano-T3 and PCR: 119 ± 22 min, epinephrine: 116 ± 26 min), epinephrine resulted in a 2-fold higher concentration of cTnI, indicating more severe myocyte injury (C). Results showed that the tested nanoparticles achieved ROSC rates and post-ROSC survival superior to vehicle and comparable to epinephrine. However, the significant reduction in cTnI levels after ROSC compared to epinephrine suggests that resuscitation with nanoparticles containing T3 and PCR may result in more favorable clinical outcomes after cardiac arrest. Further electron microscopy analysis showed improvements after treatment with both Nano-T3 or Nano-T3 / PCR compared to free T3 alone.

[0082] Example 5: Neuroprotective Effects of Nano-T3 and Phosphocreatine in the Brain Quantification of brain injury biomarkers and histopathological evaluation of brain tissue will be performed using commercially available assays designed to quantitate circulating concentrations of human brain injury biomarkers including S100 calcium binding protein B (S100B), phosphorylated neurofilament-H (pNF-H), neuron-specific enolase (NSE) and creatine kinase brain band (CK-BB). The assays will be selected for use with samples collected from animals tested in the cardiac arrest model of Example 4.

[0083] Brain tissue samples are analyzed using light and electron microscopy. Light microscopy of formalin-fixed tissue samples and electron microscopy of glutaraldehyde-preserved tissue samples from the frontal cortex, motor cortex, parietal cortex, and hippocampus of all animal subjects. Successful neuroprotective effects are demonstrated by subjects showing improved mitochondrial structure and / or reduced damage.

[0084] Example 6: Neuroprotective effect of Nano-T3 against ischemic injury Tests were performed to evaluate the neuroprotective effect on hypoxic cells following administration of T3 nanoparticles (Nano-T3). HS5 cells were treated with T3 (0.5 μM) to induce neurogenesis, which was then monitored using flow cytometry. T3-induced cells and PC12 neuronal cells were treated with T3 (1–3 μM), Nano-T3 (1–3 μM) or epinephrine (0.5 μM). The cells were then cultured under hypoxia for 24 h. Results were obtained using ATP bioluminescence and LDH release assays.

[0085] Cy7 dye tagged T3 nanoparticles were injected into the tail vein of mice to monitor the biodistribution in real time. Cyamine 7 signal was detected using IVIS with excitation / emission maxima of 750 / 776 nm. IVIS images were acquired immediately before injection, immediately after injection, 1 hour after injection, 2 hours after injection, 3 hours after injection, 4 hours after injection, and 24 hours after injection to directly detect Cyamine 7 signal. As shown in Figures 13A and 13B, Nano-T3 was detected at significantly higher levels than T3 and control, which were barely detectable in either brain, heart or lung.

[0086] As shown in Figure 14, under hypoxia (4% oxygen, 5% CO2, 37°C for 24 hours), ATP levels increased approximately 1.5-fold in samples treated with Nano-T3 (P<0.001), whereas epinephrine-treated samples showed a slight increase and ATP levels decreased in controls. Correspondingly, LDH levels decreased approximately 6-fold in samples treated with Nano-T3 (P<0.001), whereas epinephrine-treated samples showed an increase (Figure 15). These results are consistent with those reported in Example 3 above.

[0087] Tests were also performed to detect neuroprotective markers (PAX6 and DLX2) using flow cytometry. The results showed that 48 hours after neuronal induction, PAX6 and DLX2 were increased compared to the control (P≦0.001). On the 7th day, PAX6 showed a significant decrease (45% less than the control) (**P≦0.001), while DLX2 showed a significant increase (90%) (+ P ≤ 0.001). However, cell samples treated with Nano-T3 showed an approximately 50% increase in PAX6 and DLX2 expression after 7 days of neuronal induction (PAX6 and DLX2 expression was 60% and 40%, respectively), indicating a neuroprotective effect.

[0088] Thus, Nano-T3 positively affected neurogenesis and cytoprotection through the induction of various neural transcription factors. These results suggest that Nano-T3, in addition to its cardioprotective and angiogenic effects mentioned above, is a potentially new therapeutic pathway for controlling tissue damage in cerebral ischemia.

[0089] Example 7: Histopathological analysis of hypoxic porcine brain tissue The effect of Nano-T3 and nanoparticles of T3 and phosphocreatine (Nano-T3 / PCR) on brain tissue damage after hypoxic insult was evaluated in pigs. Animals were subjected to cardiac arrest for 7 minutes. After this, animals were resuscitated with either Nano-T3, Nano-T3 / PCR or epinephrine. Brain tissue analysis was performed on resuscitated animals that survived for 4 hours. Normal pigs were used as controls. Brain tissue samples were excised from the frontal cortex, caudate nucleus, putamen, parietal cortex and hippocampus of each animal. Sections were then mounted on slides in formalin and paraffin and analyzed with an Aperio slide scanner. Neuronal damage was quantified as the number of cells damaged by ischemia. The results are summarized in Figure 16.

[0090] As shown, samples treated with Nano-T3 (i.e., labeled "616" in FIG. 16) and Nano-T3 / PCR (i.e., labeled "617" in FIG. 16) showed fewer cells damaged by ischemia than epinephrine-treated tissue samples from each brain region.

[0091] Example 8: Effect of Nano-T3 and Nano-T3 / PCR on the release of neuron-specific enolase Further studies were performed to analyze the effect of Nano-T3 treatment on the expression of neuron-specific enolase (NSE) in porcine plasma using the SimpleStep human neuron-specific enolase ELISA kit (Abcam ab217778), since the antibody used in the assay cross-reacts with porcine NSE.

[0092] Blood samples were collected from pigs subjected to cardiac arrest and resuscitation as described in Example 7. Seven minutes after cardiac arrest, animals were resuscitated with Nano-T3, Nano-T3 / PCR, or epinephrine. Two hours after ROSC, blood samples were collected from a peripheral vein into tubes containing EDTA and centrifuged for 15 minutes. Plasma samples were then used for NSE assay according to the manufacturer's instructions.

[0093] The results are summarized in Figure 17. As shown, samples treated with Nano-T3 (i.e., labeled "616" in Figure 17) and Nano-T3 / PCR (i.e., labeled "617" in Figure 17) showed a significant decrease in circulating NSE. Specifically, samples treated with epinephrine showed a 64% increase in NSE over baseline, while samples treated with Nano-T3 and Nano-T3 / PCR showed only a 15% and 5% increase, respectively.

[0094] The present invention includes the following aspects and embodiments. [1] A composition comprising nanoparticles of T3 and phosphocreatine (PCR) encapsulated or immobilized in a bioabsorbable polymer. [2] The composition of [1], wherein the nanoparticles comprise T3 (e.g., L-T3) covalently conjugated to a biodegradable polymer, and phosphocreatine (PCR) encapsulated within the biodegradable polymer. [3] The composition according to any one of [1] to [2], wherein the polymer comprises polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLA), for example, PLGA which is a 50 / 50 copolymer of D,L-lactic acid and glycolic acid. [4] The composition according to any one of [1] to [3], wherein the polymer comprises polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLA), e.g., PLGA which is a 50 / 50 copolymer of D,L-lactic acid and glycolic acid, optionally conjugated with short-chain polyethylene glycol (PEG, molecular weight 100 to 2,000 daltons). [5] The composition described in any one of [1] to [4], wherein the average diameter of the T3 nanoparticles is about 50 to 1000 nm, for example 50 to 500 nm. [6] The composition described in any one of [1] to [5], wherein the average diameter of the T3 nanoparticles is about 100 to 300 nm, for example 200 nm. [7] The composition described in any one of [1] to [6], wherein the zeta potential of the T3 nanoparticles is 0 to -20 mV. [8] The composition described in any one of [1] to [7], wherein the zeta potential of the T3 nanoparticles is 0 to +20 mV. [9] The composition described in any of [1] to [8], wherein T3 is covalently bonded to a bioabsorbable polymer.

[10] The composition described in any of [1] to [9], wherein the nanoparticles contain a second pharmacologically active ingredient.

[11] The composition according to any one of [1] to

[10] , wherein the molecular weight range of PLGA is approximately 5,000 to 50,000 daltons, preferably 6,000 to 8,000 daltons.

[12] The composition according to any one of [1] to

[11] , wherein the PLGA is conjugated to PEG having a molecular weight range of 5,000 to 20,000 daltons, preferably 6,000 to 8,000 daltons.

[13] The composition described in any of [1] to

[12] , wherein the PLGA is conjugated to PEG having a molecular weight range of approximately 6,000 to 8,000 daltons.

[14] The composition described in any of [1] to

[13] , wherein T3 is chemically conjugated to PLGA or PLGA-PEG for the construction of nanoparticles.

[15] The composition described in any of [1] to

[14] , wherein the composition is dispersed in a physiological sterile medium.

[16] The composition described in any of [1] to

[15] , wherein the composition is dispersed in saline or dextrose.

[17] The composition described in any one of [1] to

[16] , wherein the biodegradable polymer comprises chitosan.

[18] A method for preventing or treating a disease, disorder, or condition characterized by a deficiency of adenosine triphosphate (ATP), comprising administering to a subject in need of prevention or treatment therapeutically effective amounts of triiodothyronine (T3) and phosphocreatine (PCR).

[19] The method of

[18] , wherein the disease, disorder or condition is selected from a cardiovascular disorder, a hypoxia-related disorder, or a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction or a disorder characterized by a dysfunction of ATP synthase), a neurodegenerative disorder, a respiratory disorder, obesity, a metabolic disorder, or diabetes.

[20] The method of

[18] or

[19] , wherein the disease, disorder or condition is a cardiovascular disorder (e.g., atherosclerosis (e.g., coronary atherosclerosis), ischemia-reperfusion (I / R) injury, hypertension (e.g., essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension), diabetes, cardiac hypertrophy, myocardial ischemia, myocardial infarction, cardiac arrest, cardiomyopathy (e.g., pediatric cardiomyopathy), cardiac dysfunction, cardiogenic shock, left ventricular trabeculation syndrome, and heart failure (e.g., acute heart failure)).

[21] The method of

[22] or

[23] , wherein the disease, disorder or condition is a disorder associated with hypoxia (e.g., hemorrhagic shock, organ failure (e.g., ARDS sepsis, septic shock or organ failure due to hemorrhagic shock), hypoxia due to organ transplantation, renal failure (e.g., chronic renal failure), cerebral edema, papilloma, spinal cord injury, stroke (e.g., ischemic stroke or hemorrhagic stroke), ischemia or ischemia reperfusion injury, traumatic brain injury (e.g., concussion), cerebral hypoxia, spinal cord injury, edema (e.g., cerebral edema), and anemia).

[22] The disease, disorder, or condition is a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction (e.g., a mitochondrial myopathy, e.g., Kearns-Sayre syndrome; Leigh syndrome; mitochondrial DNA depletion syndrome; mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); diabetes and deafness; maternally inherited deafness and diabetes; mitochondrial neurogastrointestinal encephalomyopathy; myoclonic epilepsy with ragged-red fibers; neuropathy, ataxia, and retinitis pigmentosa). The method according to

[18] or

[19] , wherein the patient is a patient suffering from a disorder characterized by dysfunction of ATP synthase (e.g., apical hypertrophic cardiomyopathy and neuropathy, ataxia, autism, Charcot-Marie-Tooth syndrome, encephalopathy, epilepsy with cerebral pseudoatrophy, epilepsy with episodic weakness, hereditary spastic paraplegia, familial bilateral striatal necrosis, Leber's hereditary optic neuropathy, mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS), motor neuron syndrome, periodic paralysis, schizophrenia, spinocerebellar ataxia, tetralogy of Fallot).

[23] The method of

[18] or

[19] , wherein the disease, disorder or condition is a neurodegenerative disorder (e.g., Huntington's disease, Alzheimer's disease, Parkinson's disease) or a disorder characterized by cell membrane repolarization.

[24] The method of

[18] or

[19] , wherein the disease, disorder or condition is a respiratory disorder (e.g., emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), respiratory arrest and asthma).

[25] The method of

[18] or

[19] , wherein the disease, disorder or condition is diabetes or a disorder caused by diabetes (e.g., diabetic ulcer, gangrene and diabetic retinopathy).

[26] The method according to

[18] or

[19] , wherein the disease, disorder or condition is a metabolic disorder (e.g., metabolic syndrome).

[27] The method according to

[18] or

[19] , wherein the disease, disorder or condition is obesity.

[28] The method according to any one of

[18] to

[27] , wherein triiodothyronine (T3) and phosphocreatine (PCR) are in the form of a composition according to any one of [1] to

[17] .

[29] A method for preventing, redirecting or interrupting apoptosis, comprising administering therapeutically effective amounts of triiodothyronine (T3) and phosphocreatine (PCR) to a subject in need of such prevention, redirection or interruption.

[30] The method according to

[29] , wherein triiodothyronine (T3) and phosphocreatine (PCR) are in the form of a composition according to any one of [1] to

[17] . Other combinations and variations of the examples provided will be apparent based on this disclosure, and while it is not possible to provide specific examples of all of the many possible combinations and variations of the described embodiments, such combinations and variations may be included in the ultimately issued claims.

Claims

1. A composition comprising nanoparticles of T3 and phosphocreatine (PCR) encapsulated or immobilized in a biodegradable polymer.

2. The composition of claim 1, wherein the nanoparticles comprise T3 (e.g., L-T3) covalently conjugated to a biodegradable polymer and phosphocreatine (PCR) encapsulated within the biodegradable polymer.

3. The composition of claim 1 or 2, wherein the polymer comprises polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLA), such as a PLGA that is a 50 / 50 copolymer of D,L-lactic acid and glycolic acid.

4. 4. The composition of any one of claims 1 to 3, wherein the polymer comprises polylactic-co-glycolic acid (PLGA) or polylactic acid (PLA), such as PLGA, a 50 / 50 copolymer of D,L-lactic acid and glycolic acid, optionally conjugated with short chain polyethylene glycol (PEG, molecular weight 100-2,000 Daltons).

5. The composition according to any one of claims 1 to 4, wherein the average diameter of the T3 nanoparticles is between about 50 and 1000 nm, such as between 50 and 500 nm.

6. The composition according to any one of claims 1 to 5, wherein the average diameter of the T3 nanoparticles is about 100 to 300 nm, such as 200 nm.

7. The composition according to any one of claims 1 to 6, wherein the zeta potential of the T3 nanoparticles is between 0 and -20 mV.

8. The composition according to any one of claims 1 to 7, wherein the zeta potential of the T3 nanoparticles is between 0 and +20 mV.

9. The composition of any one of claims 1 to 8, wherein T3 is covalently attached to a biodegradable polymer.

10. The composition of claim 3, wherein the molecular weight range of the PLGA is about 5,000 to 50,000 daltons, preferably 6,000 to 8,000 daltons.

11. The composition of claim 4, wherein the PLGA is conjugated to PEG having a molecular weight range of 5,000 to 20,000 daltons, preferably 6,000 to 8,000 daltons.

12. 5. The composition of claim 4, wherein the PLGA is conjugated to PEG having a molecular weight range of about 6,000 to 8,000 daltons.

13. The composition of claim 4, wherein T3 is chemically conjugated to PLGA or PLGA-PEG for assembly of nanoparticles.

14. The composition of any one of claims 1 to 13, wherein the composition is dispersed in a physiological sterile medium.

15. The composition of any one of claims 1 to 14, wherein the composition is dispersed in saline or dextrose.

16. The composition of any one of claims 1 to 15, wherein the biodegradable polymer comprises chitosan.

17. A pharmaceutical agent for the prevention or treatment of a disease, disorder or condition characterized by a deficiency of adenosine triphosphate (ATP), including triiodothyronine (T3) and phosphocreatine (PCR).

18. The pharmaceutical of claim 17, wherein the disease, disorder or condition is selected from a cardiovascular disorder, a disorder associated with hypoxia, or a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction or a disorder characterized by a dysfunction of ATP synthase), a neurodegenerative disorder, a respiratory disorder, obesity, a metabolic disorder, or diabetes.

19. The pharmaceutical composition of claim 17 or 18, wherein the disease, disorder or condition is a cardiovascular disorder (e.g., atherosclerosis (e.g., coronary atherosclerosis), ischemia-reperfusion (I / R) injury, hypertension (e.g., essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension), diabetes, cardiac hypertrophy, myocardial ischemia, myocardial infarction, cardiac arrest, cardiomyopathy (e.g., pediatric cardiomyopathy), cardiac dysfunction, cardiogenic shock, left ventricular trabeculation syndrome, and heart failure (e.g., acute heart failure)).

20. The pharmaceutical composition of claim 17 or 18, wherein the disease, disorder or condition is a disorder associated with hypoxia (e.g., hemorrhagic shock, organ failure (e.g., organ failure due to ARDS sepsis, septic shock or hemorrhagic shock), hypoxia due to organ transplantation, renal failure (e.g., chronic renal failure), cerebral edema, papilloma, spinal cord injury, stroke (e.g., ischemic stroke or hemorrhagic stroke), ischemia or ischemia reperfusion injury, traumatic brain injury (e.g., concussion), cerebral hypoxia, spinal cord injury, edema (e.g., cerebral edema), and anemia).

21. The disease, disorder or condition is a disorder characterized by low cellular energy (e.g., a disorder characterized by mitochondrial dysfunction (e.g., a mitochondrial myopathy, e.g., Kearns-Sayre syndrome; Leigh syndrome; mitochondrial DNA depletion syndrome; mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); diabetes and hearing loss; maternally inherited hearing loss and diabetes; mitochondrial neurogastrointestinal encephalomyopathy; myoclonic epilepsy with ragged-red fibers; neuropathy, ataxia, and retinitis pigmentosa).

19. The pharmaceutical composition according to claim 17 or 18, wherein the patient is a patient suffering from a disorder characterized by insufficient functioning of ATP synthase (e.g., apical hypertrophic cardiomyopathy and neuropathy, ataxia, autism, Charcot-Marie-Tooth syndrome, encephalopathy, epilepsy with cerebral pseudoatrophy, epilepsy with episodic weakness, hereditary spastic paraplegia, familial bilateral striatal necrosis, Leber's hereditary optic neuropathy, mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS), motor neuron syndrome, periodic paralysis, schizophrenia, spinocerebellar ataxia, tetralogy of Fallot).

22. The pharmaceutical of claim 17 or 18, wherein the disease, disorder or condition is a neurodegenerative disorder (e.g., Huntington's disease, Alzheimer's disease, Parkinson's disease) or a disorder characterized by cell membrane repolarization.

23. The pharmaceutical composition of claim 17 or 18, wherein the disease, disorder or condition is a respiratory disorder (e.g., emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), respiratory arrest and asthma).

24. 19. The method of claim 17 or 18, wherein the disease, disorder or condition is diabetes or a disorder caused by diabetes (e.g. diabetic ulcer, gangrene and diabetic retinopathy).

25. The pharmaceutical composition of claim 17 or 18, wherein the disease, disorder or condition is a metabolic disorder (e.g., metabolic syndrome).

26. The pharmaceutical composition of claim 17 or 18, wherein the disease, disorder or condition is obesity.

27. A medicament according to any one of claims 17 to 26, wherein the triiodothyronine (T3) and phosphocreatine (PCR) are in the form of a composition according to any one of claims 1 to 16.

28. Medications for preventing, redirecting or interrupting apoptosis, including triiodothyronine (T3) and phosphocreatine (PCR).

29. The method of claim 28, wherein the triiodothyronine (T3) and phosphocreatine (PCR) are in the form of a composition according to any one of claims 1 to 16.

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