DNP and DNP prodrug treatment of neuromuscular, neurodegenerative, autoimmune, developmental, concussion, dry eye disease, and / or metabolic diseases

Abstract

Compositions and methods for treating neuromuscular, neuromuscular degenerative, neurodegenerative, autoimmune, developmental, traumatic, hearing loss-related, and / or metabolic diseases including Spinal Muscular Atrophy (SMA) syndrome (SMA1, SMA2, SMA3, and SMA4, also known as Types I, II, III, and IV), Traumatic Brain Injury (TBI), concussion, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome. The compositions are selected from 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, bis 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs; bisprodrug, bioprecursor molecules, and combinations thereof.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 304,584, filed March 7, 2016, and U.S. Provisional Application No. 62 / 460,318, filed February 17, 2017, which are incorporated by reference in their entireties. TECHNICAL FIELD

[0003] The present invention relates to the development of disease mitigating treatments for reversing, slowing or preventing: neuromuscular, spinal muscular atrophy (SMA) syndrome (types I, II, III and IV), neuromuscular degeneration, neurodegeneration, autoimmune, developmental, traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, hearing loss related and / or metabolic diseases, including Wolfram syndrome, Wolcott-Rallison syndrome and conditions in pediatric, adult and geriatric populations. In one aspect, the treatment of the following diseases involves the use of one or more isomers of dinitrophenol (DNP): neuromuscular, neuromuscular degeneration, neurodegeneration, autoimmune, developmental, traumatic diseases of the CNS, hearing loss related, including spinal muscular atrophy (SMA) syndrome (SMA1, SMA2, SMA3 and SMA4, also known as types I, II, III and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use and / or metabolic diseases and conditions, including Wolfram syndrome and Wolcott-Rallison syndrome. The present invention also relates to the synthesis of a diastereomeric dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP) prodrug and the use of said prodrug and / or 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol or 3,5-dinitrophenol for the treatment of: neuromuscular, neuromuscular degeneration, neurodegeneration, autoimmune, developmental, traumatic diseases of the CNS, hearing loss related, including spinal muscular atrophy (SMA) syndrome (SMA1, SMA2, SMA3 and SMA4, also known as types I, II, III and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use and / or metabolic diseases and conditions, including Wolfram syndrome and Wolcott-Rallison syndrome.

[0004] BACKGROUND

[0005] There is currently no drug therapy that significantly alters the time course of insidious neuromuscular, neuromuscular degenerative, neurodegenerative, autoimmune, developmental, traumatic, hearing loss-related, and / or metabolic diseases to early death, and thus there is an unmet medical need. For example, due to the severity of the consequences of Batten disease, the current recommendation from the NCL- Foundation is palliative therapy as early as possible. Epilepsy is treated with valproate and lamotrigine, spasticity with baclofen and tetrahydrocannabinol, and myoclonic epilepsy with piracetam and zonisamide (leaflet issued by the NCL-Stiftung).

[0006] Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a neurodegenerative disease characterized by the loss of motor neurons in the brainstem, cerebral cortex, and spinal cord, which leads to progressive weakness and death within 3-5 years after onset. For ALS, various treatments have existed, and none has proven effective in inhibiting the progression to death, except riluzole. Riluzole prolongs life to a modest extent (a few months) and prolongs the time before patients need ventilatory support, but signs of hepatotoxicity (~10%) must be monitored.

[0007] Alzheimer's disease (AD) is a disease characterized by the loss of neurons in the brain, corresponding to a decline in cognition and premature death. Despite many drug therapies that have been tried, these drugs cannot significantly alter the life span of patients. Parkinson's disease, well known for its effects on Michael J. Fox, Mohamed Ali, and others, is a chronic killer of neurons that produce dopamine, called dopaminergic neurons. Current therapies focus on replacing dopamine, and there has been no new progress in quite some time.

[0008] Duchenne muscular dystrophy (DMD), a pediatric neuromuscular disease in young boys (X-chromosome-bearing), is a devastating disease that causes children to require leg braces by age 8 and wheelchairs by age 10. The loss of the myoglobin gene affects both the brain and muscles, the only two tissues that express the myoglobin gene. Myoglobin loss leads to osmotic swelling and rupture of mitochondria and muscle atrophy. Other forms of ataxia, and many other neurodegenerative / neuromuscular diseases, do not have treatments that significantly alter the progression of the disease to early death. Multiple sclerosis (MS) is an autoimmune disease of the CNS with an unknown cause. Once the disease develops, the myelin sheath (the insulator surrounding nerve fibers or axons) is attacked by immune cells, leading to neuronal dysfunction between the brain and body organs. The result is a wide range of neurological symptoms, such as involuntary movement of the limbs that impairs walking, visual / speech impairment, bladder and bowel dysfunction, etc. Although lifespan is only moderately shortened, MS typically begins in children aged 20-30 and progresses with worsening symptoms over time. Currently, there are many drugs for immunosuppression, but none of them "alleviate the disease" in the treatment of MS, nor do any drugs provide protection against disease progression.

[0009] In addition to neurodegenerative and autoimmune diseases, there is an unmet medical need for the treatment of developmental disorders such as Angelman syndrome (AS), a pediatric neurodevelopmental disorder associated with developmental delay, motor dysfunction, taciturnity, and epilepsy, caused by a condition called… Ubiquitin Ligase E3A (UBE3A) It is caused by mutations in the imprinting gene. Similarly, Rett syndrome is a pediatric degenerative disease that affects young girls (X-chromosome type, boys die in utero) and leads to death. Currently, there is no treatment to address the effects of the disease, including breathing problems, motor problems, and seizures.

[0010] In addition to available treatments for neurodegenerative, neuromuscular, autoimmune, and developmental diseases, there are very few pharmacotherapy options for epidemic metabolic diseases to eradicate the problem to address the effects of nutrient excess and subsequent obesity. Obesity is prevalent and difficult to treat. There are approximately 1 billion overweight adults in the world, of which over 300 million are clinically obese. In the US alone, 35% of Americans have a BMI over 30, and 1 in 400 have a BMI of 50 (~800,000 individuals). Obesity can lead to ectopic lipid accumulation (e.g., nonalcoholic fatty liver disease, NAFLD) and oxidative damage from reactive oxygen species. As a result, the risk of many other diseases is increased in obese individuals, including insulin resistance, sleep apnea, hypertension, kidney disease, inflammation, knee joint complications, depression, high blood pressure, cardiovascular disease, type 2 diabetes (T2D), and even some cancers. While even small amounts (~5%) of weight loss can yield metabolic benefits, it is difficult for most patients to achieve and maintain such weight loss. As a result, bariatric surgery is now considered the best method to reverse obesity. Despite increasing popularity, bariatric surgery often requires permanent anatomical changes to the gastrointestinal tract, which fundamentally forces reduced caloric intake. These procedures are associated with mortality (1 / 200) and short- and long-term morbidity, such as wound complications and venous thrombosis, as well as reactive hypoglycemia, micronutrient deficiencies, dumping syndrome, etc. These procedures pose additional challenges in extremely obese patients (BMI > 50 kg / m 2 ). Pharmacological approaches to treating obesity have focused on inducing malabsorption (e.g., orlistat) or satiety (e.g., rimonabant, sibutramine), but with very modest effects (~5% body weight) and with significant side effects (e.g., steatorrhea, depression, and MI, respectively). New pharmacological therapies are needed.

[0011] Thus, there is a significant need for improved treatments for neurodegenerative, neuromuscular, developmental, autoimmune, and / or metabolic diseases.

[0012] SUMMARY

[0013] A first aspect of the present invention provides a composition for treating a neurodegenerative disease, a neuromuscular disease, a neuromuscular degenerative disease, a developmental disease, an autoimmune disease, a traumatic disease of the CNS, a metabolic disease, and / or a disease associated with hearing loss due to aging, noise, concussion, traumatic brain injury (TBI), drug-induced and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as Types I, II, III, and IV), traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren’s syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising one or more isomers of dinitrophenol (DNP), i.e., 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP.

[0014] A second aspect of the present invention provides a composition for treating a neurodegenerative disease, a neuromuscular disease, a neuromuscular degenerative disease, a developmental disease, an autoimmune disease, a traumatic disease of the CNS, a metabolic disease, and / or a disease associated with hearing loss due to aging, noise, drug-induced and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as Types I, II, III, and IV), traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren’s syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising a prodrug selected from:

[0015] amino acid (AA) esters of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 to II-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 to IV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 to VI-10);

[0016] AA esters with methylene dioxides (formaldehyde equivalent) spacers (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 to II-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 to IV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 to VI-13);

[0017] Amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 to II-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 to IV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 to VI-17);

[0018] Amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 and II-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18 and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, Formulas VI-18 and VI-19);

[0019] Phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6);

[0020] 1,3 Diketone analogs I-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32 (Schemes 1-6);

[0021] Carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 to III-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6);

[0022] Benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes 1-6);

[0023] and combinations thereof.

[0024] A third aspect of the present application provides a composition for treating neurodegenerative diseases, neuromuscular diseases, neuromuscular degenerative diseases, developmental diseases, autoimmune diseases, traumatic diseases of the CNS, metabolic diseases, and / or diseases related to hearing loss due to aging, noise, drug-induced, and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising: a phosphate prodrug that alters the molecule by introducing a water-soluble prodrug moiety through conjugation with a free phenolic functional group.

[0025] Scheme 1

[0026]

[0027] Scheme 2

[0028]

[0029] Scheme 3

[0030]

[0031] Scheme 4

[0032]

[0033] Scheme 5

[0034]

[0035] Scheme 6

[0036]

[0037] A fourth aspect of the present application provides a composition for treating neurodegenerative diseases, neuromuscular diseases, neuro-muscular degenerative diseases, developmental diseases, autoimmune diseases, traumatic diseases of the CNS, metabolic diseases, and / or diseases associated with hearing loss due to aging, noise, drug-induced, and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising: a prodrug selected from a 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP "gemini" prodrug, wherein the prodrug is represented by Formula VII:

[0038] .

[0039] A fifth aspect of the present application provides a method of synthesizing a composition of a prodrug for treating neurodegenerative diseases, neuromuscular diseases, developmental diseases, autoimmune diseases, traumatic diseases of the CNS, metabolic diseases, and / or diseases associated with hearing loss due to aging, noise, drug-induced, and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome. In one embodiment, the method comprises: reacting 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and / or 3,5-dinitrophenol with 5-(tert-butyldimethylsilyloxy)isophthaloyl dichloride (2) in the presence of pyridine / dichloromethane to provide a precursor (3); and removing the TBDMS protecting group in acetone / HCl to provide the prodrug (Scheme 7).

[0040] Scheme 7

[0041]

[0042] A sixth aspect of the present application provides a composition for treating neurodegenerative diseases, neuromuscular diseases, neuromuscular degenerative diseases, developmental diseases, autoimmune diseases, traumatic diseases of the CNS, metabolic diseases, and / or diseases related to hearing loss due to aging, noise, drug-induced, and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising: a biological precursor of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP that releases 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol upon oxidative metabolism by cytochrome P-450, wherein the biological precursor is represented by Formulas VIII and IX (Scheme 8; including synthetic routes and oxidative metabolism):

[0043] Scheme 8

[0044]

[0045] A seventh aspect of the present application provides a composition for treating neurodegenerative diseases, neuromuscular diseases, neuromuscular degenerative diseases, developmental diseases, autoimmune diseases, traumatic diseases of the CNS, metabolic diseases, and / or diseases related to hearing loss due to aging, noise, drug-induced, and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising: DNP prodrugs and biological precursors with linkers comprising open functional groups delivered as a depot nanoparticle formulation that releases DNP at low doses in a slow, sustained manner compared to the dose and release of DNP alone.

[0046] The eighth aspect of the present invention provides a method for treating the following diseases: neurodegenerative diseases, neuromuscular diseases, neuromuscular degenerative diseases, developmental diseases, autoimmune diseases, traumatic diseases of the CNS, metabolic diseases, and / or diseases related to hearing loss due to aging, noise, drug-induced and / or hereditary hearing loss, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3 and SMA4, also known as types I, II, III and IV), traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjögren's syndrome, rheumatoid arthritis, post-LASIK surgery, antidepressant use, Wolfram syndrome, and W. The method for treating Olcott-Rallison syndrome includes: administering a dose of a composition to a patient requiring treatment, wherein the composition is selected from 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP; dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs; a gemini prodrug, a biological precursor molecule, or a combination thereof, wherein the dose of the active pharmaceutical ingredient is about 0.01 mg / L. The dose is approximately 50 mg / kg of the patient's body weight, or approximately 0.01 mg / kg of the patient's body weight, or approximately 25 mg / kg of the patient's body weight.

[0047] A ninth aspect of the application provides a method of treating traumatic brain injury (TBI), ischemic stroke, Huntington's disease (adult onset Huntington's disease, juvenile Huntington's disease), epilepsy (massive seizures, refractory seizures, atypical absence seizures, atonic seizures, clonic seizures, myoclonic seizures, tonic seizures, tonic-clonic seizures, simple partial seizures, complex partial seizures, secondary generalized seizures, febrile seizures, non-epileptic seizures, gelastic and dacrystic seizures, and absence seizures), multiple sclerosis (MS) (relapsing-remitting multiple sclerosis (RRMS), secondary-progressive MS (SPMS), primary-progressive MS (PPMS), and progressive-relapsing MS (PRMS)), lupus (systemic lupus erythematosus (SLE), discoid (cutaneous), drug-induced lupus (dil), and neonatal lupus), diabetes (type 1 diabetes, type 2 diabetes, young adult onset diabetes (MODY: MODY1, MODY2, MODY3, MODY4, MODY5, MODY6, MODY7, MODY8, MODY9, MODY10, MODY11)), non-alcoholic steatohepatitis (NASH), schizophrenia (paranoid schizophrenia, disorganized schizophrenia, catatonic schizophrenia, residual schizophrenia, schizoaffective disorder), myasthenia gravis (MG) (ocular myasthenia gravis, congenital MG, and generalized myasthenia gravis), rheumatoid arthritis (RA), Graves' disease, Guillain-Barre syndrome (GBS), muscular dystrophy (Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, and oculopharyngeal muscular dystrophy), severe burns, aging, amyotrophic lateral sclerosis (ALS), ataxia (Friedrich's ataxia, spinocerebellar ataxia 1 (SCA1), spinocerebellar ataxia 2 (SCA2), spinocerebellar ataxia 3 (SCA3), spinocerebellar ataxia 6 (SCA6), spinocerebellar ataxia 7 (SCA7), spinocerebellar ataxia 11 (SCA11), dentatorubral-pallidoluysian atrophy (DRPLA), and glutamine ataxia), Batten disease or neuronal ceroid lipofuscinosis (NCL) (infantile NCL (INCL), late-infantile NCL (LINCL), juvenile NCL (JNCL), or adult NCL by increasing energy expenditure and / or inducing BDNF mRNA expression and protein levels through treatment with DNP to reverse, slow, or prevent neuromuscular and / or neurodegeneration and / or muscle atrophy.(ANCL)), Alzheimer's disease (early onset Alzheimer's disease, late onset Alzheimer's disease, and familial Alzheimer's disease (FAD)), optic neuritis (ON), Leber's hereditary optic neuropathy (LHON), autism spectrum disorders (Asperger's syndrome, pervasive developmental disorder (PDD), childhood disintegrative disorder (CDD), and autism), Rett syndrome, Angelman syndrome, Leigh disease, Prader Willi syndrome, fragile X syndrome, depression (major depression, psychotic depression, postpartum depression, seasonal affective disorder, atypical depression, psychotic depression, bipolar disorder, premenstrual dysphoric disorder, situational depression), Parkinson's disease (idiopathic Parkinson's disease, vascular Parkinson's syndrome, Lewy body dementia, hereditary Parkinson's disease, drug-induced Parkinson's syndrome, juvenile Parkinson's disease, and atypical Parkinson's syndrome), Wolfram syndrome (and any associated conditions, such as diabetes issues, hearing, vision, ataxia, neurodegeneration, etc.), spinal muscular atrophy (SMA; types I, II, III, and IV), hearing loss due to noise (explosions and high noise), aging-related hearing loss, drug-induced hearing loss, and / or genetic hearing loss, concussions, dry eye keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolcott-Rallison syndrome, mitochondrial diseases, developmental disorders, metabolic syndrome (increased blood pressure, elevated blood glucose levels, excess waist circumference body fat, and abnormal cholesterol levels), and / or autoimmune diseases, the method comprising: administering to a patient in need of treatment a dose of 0.01 mg / kg to 50 mg / kg, wherein the composition is selected from 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP; a bisecting 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrug; a bisprodrug, a bioprecursor molecule for treating neurodegenerative, neuromuscular, developmental, autoimmune, and / or metabolic diseases and combinations thereof, wherein the dose of the composition is about 0.01 mg / kg to 50 mg / kg of body weight of the patient in need of treatment.

[0048] A tenth aspect of the application relates to a method of treating a neurodegenerative disease, a neuromuscular disease, a neuromuscular degenerative disease, a developmental disease, an autoimmune disease, a traumatic disease of the CNS, and / or a metabolic disease and / or a disease associated with hearing loss due to aging, noise, drug-induced and / or genetic hearing loss, including spinal muscular atrophy (SMA) syndrome (SMA1, SMA2, SMA3 and SMA4, also known as types I, II, III and IV), traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, comprising: providing instructions to administer an effective dose of DNP or a pharmaceutically acceptable salt thereof or any prodrug described herein for a period of time sufficient to achieve a reduction in symptoms of the disease, wherein an effective dose of DNP and / or a prodrug thereof in the range of 0.001 mg / kg body weight to 50 mg / kg body weight is indicated. BRIEF DESCRIPTION OF DRAWINGS

[0050] The foregoing brief summary of an embodiment of the application and the following detailed description thereof are better understood when read in conjunction with the appended drawings and figures.

[0051] Figure 1 Figure is a comparative effect of two compounds of the application to reduce or block paralysis progression, as monitored as a clinical score (for efficacy, comparing parent (2,4-DNP) to prodrug (II-38) in the EAE MS model, at equivalent exposure of 5 mpk parent vs. 80 mpk prodrug). Legend: diamond - placebo; square - 2,4-DNP 5 mpk; triangle - II-38 8 mpk; x - II-38 16 mpk; star - II-38 80 mpk. P values by ordinal regression: 2,4-DNP 5 mpk = 0.0192; II-38 8 mpk = 0.000954; II-38 16 mpk = 0.000124; II-38 80 mpk = 0.00014 (using ordinal package from cran project www.cran.r-project.org, results from logistic regression model).

[0052] Figure 2is a graph illustrating comparative effects of two compounds of the invention to prevent weight loss seen in placebo mice (EAE MS model comparing parent (2,4-DNP) to prodrug (II-38) for weight change at equivalent exposure of 5 mpk parent with 80 mpk prodrug). Legend: Diamond - Placebo; Square - 2,4-DNP 5 mpk; Triangle - II-38 8 mpk; X - II-38 16 mpk; Star - II-38 80 mpk. All doses significantly maintained weight. P values by ordinal regression: 2,4-DNP 5 mpk = 0.0188; II-38 8 mpk < 0.0001; II-38 16 mpk = 0.044; II-38 80 mpk < 0.0001.

[0053] Figure 3 is a graph illustrating comparative results of in vivo noise exposure experiments (including DNP (2,4-DNP) and a prodrug of DNP (II-38)); II-38 + noise threshold is 5 dB, which is about 20 dB lower (better) than the noise only group; DNP (2,4-DNP) + noise threshold is 15 dB, which is about 10 dB lower than noise only, indicating that DNP (2,4-DNP) can provide protection against noise-induced hearing loss. Legend: Circle - Control (0 dB); Triangle (pointing down) - II-38 (5 dB); Triangle (pointing up) - 2,4-DNP (15 dB); Circle (solid) - Noise (25 dB).

[0054] Figure 4 illustrates results of single crystal X-ray diffraction of 2,4-dinitrophenyl morpholine-4-carboxylate.

[0055] Figure 5 illustrates a chromatogram of II-38 eluting at 8.04 min.

[0056] Figure 6 illustrates the equilibrium (solubility) phase of II-38 in HC1 buffer pH 1.2.

[0057] Figure 7 illustrates chromatograms of 2,4-DNP and II-38 prodrug eluting at 6.854 and 8.023 min, respectively, in phosphate buffer pH 7.4.

[0058] Figure 8 illustrates stability of II-38 in PB buffer pH = 7.4.

[0059] Figure 9The chromatogram of II-38 eluted in SGF at 3.08 min is shown.

[0060] Figure 10A This demonstrates the stability of II-38 in SGF at pH=1.2.

[0061] Figure 10B This demonstrates the stability of DNP morpholine in SGF at pH=1.2.

[0062] Figure 11 The chromatogram of II-38 eluted in SIF at 2.187 min is shown.

[0063] Figure 12A This demonstrates the stability of II-38 at SIF pH=6.8.

[0064] Figure 12B This demonstrates the stability of DNP morpholine at SIF pH=6.8.

[0065] Figure 13 Illustration of II-38 in rat plasma and 2,4-DNP released from II-38. Legend: Rhombus - 2,4-DNP; Circle - II-38.

[0066] Figure 14 This diagram illustrates II-38 in human plasma and the 2,4-DNP released from II-38. Legend: Rhombus - 2,4-DNP; Circle - II-38.

[0067] Figure 15 This section describes the calibration standard for 2,4-DNP in acetonitrile.

[0068] Figure 16 Indicate the calibration criteria for 2,4-DNP in rat plasma, n = 5.

[0069] Figure 17 This describes the standard curve for II-38 in rat plasma, n=3.

[0070] Figure 18 This diagram illustrates the plasma concentrations and time of 2,4-DNP in rats following a single intravenous injection of 1 mg / kg (n = 3) or an oral dose of 5 mg / kg (n = 4). Legend: Rhomboid-shaped 2,4-DNP, intravenous 1 mg / kg; Round-shaped 2,4-DNP, oral 5 mg / kg.

[0071] Figure 19Mean plasma concentration versus time for II-38 and 2,4-DNP released from II-38 prodrug following oral administration of a dose of 8 mg / kg of II-38 (equivalent to 5 mg / kg of II-38). Legend: Diamond - II-38, oral: equivalent 5 mg / Kg; Square - 2,4-DNP released from II-38, oral.

[0072] Figure 20 Mean plasma concentration versus time for II-38 and 2,4-DNP released from II-38 prodrug following oral administration of a dose of 40 mg / kg of II-38 (equivalent to 25 mg / kg of II-38 formulated in methylcellulose). Legend: Diamond - 2,4-DNP released from II-38, oral; Square - II-38, oral: equivalent 25 mg / Kg (methylcellulose).

[0073] Figure 21 Mean plasma concentration versus time for II-38 and 2,4-DNP released from II-38 prodrug following oral administration of a dose of 40 mg / kg of II-38 (equivalent to 25 mg / kg of II-38 formulated in PEG-400). Legend: Diamond - 2,4-DNP released from II-38, oral; Square - II-38, oral: equivalent 25 mg / Kg (PEG-400).

[0074] Figure 22 Mean plasma concentration versus time for II-38 and 2,4-DNP released from II-38 prodrug following oral administration of a dose of 80 mg / kg of II-38 (equivalent to 50 mg / kg of II-38). Legend: Diamond - 2,4-DNP released from II-38, oral; Square - II-38, oral: equivalent 50 mg / Kg.

[0075] Figure 23Mean plasma concentration of 2,4-DNP and 2,4-DNP released from II-38 after administration of 2,4-DNP at a dose of 5 mg / kg and II-38 at a dose of 8, 40 (methylcellulose), 40 (PEG-400) and 80 mg / kg (equivalent to 5, 25 (methylcellulose), 25 (PEG-400) and 50 mg / kg of DNP), respectively. n = 4. Legend: Diamond - 2,4-DNP, oral 5 mg / Kg; Square (red) - 2,4-DNP released from II-38, equivalent to 5 mg; Triangle - 2,4-DNP released from II-38, equivalent to 25 mg / Kg (methylcellulose); Square (purple) - 2,4-DNP released from II-38, equivalent to 50 mg / Kg; Square (blue) - 2,4-DNP released from II-38, equivalent to 25 mg / Kg (PEG-400).

[0076] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0077] Definitions:

[0078] Hereinafter, unless otherwise defined, the term “prodrug” refers to a drug that is inactive or partially active, which is metabolically changed to an active drug in vivo.

[0079] Hereinafter, unless otherwise defined, the term “depot nanoparticle formulation” refers to a biodeliverable nanoparticle comprising a wide range of 1) bipartite 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP) prodrugs, 2) geminal prodrugs and 3) bioprecursor molecules for the treatment of neurodegenerative or metabolic diseases.

[0080] Hereinafter, unless otherwise defined, the term “about” refers to plus or minus 10% of the stated value. For example, “about 1 mg / kg” refers to 0.9 mg / kg to 1.1 mg / kg.

[0081] Hereinafter, unless otherwise defined, bipartite 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol or 3,5-dinitrophenol are represented by 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP; the prodrugs of the individual isomers are represented by Formulas I-VI; which are selected from:

[0082] Amino acid (AA) esters of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 to II-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 to IV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 to VI-10);

[0083] AA esters incorporating methylene dioxides (formaldehyde equivalent) spacers (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 to II-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 to IV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 to VI-13);

[0084] Amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 to II-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 to IV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 to VI-17);

[0085] Amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 and II-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18 and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, Formulas VI-18 and VI-19);

[0086] Phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6);

[0087] 1,3 dione analogs I-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32 (Schemes 1-6);

[0088] Carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 to III-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6);

[0089] Benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes 1-6);

[0090] and combinations thereof.

[0091] In one embodiment, the present application provides compositions and methods for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The complete lack of historical success is attributed to the fact that most of the pharmaceutical industry has focused on downstream events, such as plaques / tangles, while the problem can be upstream at the mitochondria, which causes high cellular stress. In a study in which a mouse model of Alzheimer's disease was treated with DNP under chronic treatment, short-term memory was significantly improved in the Morris water maze, with a significant impact on disease progression. Data from a recent study in which 6-OHDA was used to destroy dopaminergic neurons in a model of Parkinson's disease showed that treatment with DNP, with chronic treatment, had a positive protective effect.

[0092] In one embodiment, the present application provides compositions and methods for treating Huntington's disease. In a model of Huntington's disease, treatment with DNP for more than 17 weeks showed a protective effect on spiny neurons that receive dopamine, and thus the compounds and medicaments of the present application should be ideal for treating this disease. DNP will prevent muscle loss because it reduces mitochondrial permeability swelling by reducing calcium concentration within the mitochondria and inducing BDNF, which is a myokine (muscle protective agent) outside the brain.

[0093] In one embodiment, the present application provides compositions and methods for treating cognitive impairment associated with DMD in the CNS. 2,4-DNP will, for example, help cognitive impairment associated with DMD in the CNS.

[0094] In one embodiment, the present application provides compositions and methods for treating Angelman and Rett syndromes. Oxidative stress is thought to play a role in impairing neurodevelopment, however the fact that DNP prevents significant ROS formation, induces BDNF, reduces duration of seizure activity can have a therapeutic effect in subjects, including children, suffering from these diseases.

[0095] In one embodiment, the present application provides compositions and methods for treating obesity. The key or mechanism for effective weight loss or reduction of ectopic fat to improve insulin resistance, fatty liver disease, type 2 diabetes, cardiovascular disease, etc. without necessarily overt weight change is to unbalance the body in terms of energy in versus energy out, and to keep the energy expended. Thus, new pharmacological therapies focus on increasing energy expenditure, which addresses the phenotype of metabolic disease and over-nutrition, which can potentially have a dramatic impact on the life of individuals suffering from these diseases.

[0096] Preliminary studies have demonstrated that (1) the ability to reduce ROS and increase lifespan in wild-type mice treated with mitochondrial chemical uncouplers, and (2) improved metabolic profiles, indicating that chronic mitochondrial chemical uncoupling therapy is safe, and improves health outcomes at low doses (~1-50 mg / day). These preliminary studies have demonstrated the rationale for evaluating mitochondrial chemical uncouplers like DNP and prodrug formulations for extended release in representative animal models of neurodegenerative, neuromuscular, developmental, autoimmune, and / or metabolic diseases and other conditions with overt ROS production (e.g., during stroke or ischemic events).

[0097] In one embodiment, the compositions and methods of the present application involve pharmacological intervention that abrogates overt ROS production and improves the quality of the mitochondrial population through mitophagy, induces brain-derived neurotrophic factor (BDNF), increases cAMP and / or reprograms cellular expression. In one embodiment, the drugs of the present application thus become "disease-modifying." In one embodiment, the present application induces MOG 35-55 Seven days after myelin peptide, a prodrug of DNP (II-38) was given to a mouse model of multiple sclerosis, called EAE. The prodrug of DNP II-38 was given in multiple exposures to 2,4-DNP or DNP. Both compounds provided a significant effect in reducing or blocking paralysis progression, as monitored as a clinical score ( Figure 1 ), as well as preventing the weight loss seen in placebo mice ( Figure 2 ). Even though DNP has historically been used at high doses for weight loss in the 1930s, it is shown here that it can be paradoxically repositioned to prevent wasting associated with neurodegenerative / autoimmune diseases at low doses.

[0098] In one embodiment, the compositions described herein can be used to treat hearing loss. In one such embodiment, 2,4-dinitrophenol (DNP) can be used in a dosage range independently selected from the group consisting of: 1 mg / day to 50 mg / day; 1 mg / day to 45 mg / day; 1 mg / day to 40 mg / day; 1 mg / day to 35 mg / day; 1 mg / day to 30 mg / day; 1 mg / day to 25 mg / day; 1 mg / day to 20 mg / day; 1 mg / day to 15 mg / day; 1 mg / day to 10 mg / day; 1 mg / day to 5 mg / day; 1 mg / day to 4 mg / day; 1 mg / day to 3 mg / day; 1 mg / day to 2 mg / day; 2 mg / day to 50 mg / day; 2 mg / day to 45 mg / day; 2 mg / day to 40 mg / day; 2 mg / day to 35 mg / day; 2 mg / day to 30 mg / day; 2 mg / day to 25 mg / day; 2 mg / day to 20 mg / day; 2 mg / day to 15 mg / day; 2 mg / day to 10 mg / day; 2 mg / day to 5 mg / day; 2 mg / day to 4 mg / day; 2 mg / day to 3 mg / day; 0.5 mg / day to 10 mg / day; 0.5 mg / day to 9 mg / day; 0.5 mg / day to 8 mg / day; 0.5 mg / day to 7 mg / day; 0.5 mg / day to 6 mg / day; 0.5 mg / day to 5 mg / day; 0.5 mg / day to 4 mg / day; 0.5 mg / day to 3 mg / day; 0.1 mg / day to 10 mg / day; 0.1 mg / day to 9 mg / day; 0.1 mg / day to 8 mg / day; 0.1 mg / day to 7 mg / day; 0.1 mg / day to 6 mg / day; 0.1 mg / day to 5 mg / day; 0.1 mg / day to 4 mg / day; 0.1 mg / day to 3 mg / day.In one such embodiment, 2,4-dinitrophenol (DNP) can be used in a dosage range independently selected from the group consisting of: about 1 mg / day to about 50 mg / day; about 1 mg / day to about 45 mg / day; about 1 mg / day to about 40 mg / day; about 1 mg / day to about 35 mg / day; about 1 mg / day to about 30 mg / day; about 1 mg / day to about 25 mg / day; about 1 mg / day to about 20 mg / day; about 1 mg / day to about 15 mg / day; about 1 mg / day to about 10 mg / day; about 1 mg / day to about 5 mg / day; about 1 mg / day to about 4 mg / day; about 1 mg / day to about 3 mg / day; about 1 mg / day to about 2 mg / day; about 2 mg / day to about 50 mg / day; about 2 mg / day to about 45 mg / day; about 2 mg / day to about 40 mg / day; about 2 mg / day to about 35 mg / day; about 2 mg / day to about 30 mg / day; about 2 mg / day to about 25 mg / day; about 2 mg / day to about 20 mg / day; about 2 mg / day to about 15 mg / day; about 2 mg / day to about 10 mg / day; about 2 mg / day to about 5 mg / day; about 2 mg / day to about 4 mg / day; about 2 mg / day to about 3 mg / day; about 0.5 mg / day to about 10 mg / day; about 0.5 mg / day to about 9 mg / day; about 0.5 mg / day to about 8 mg / day; about 0.5 mg / day to about 7 mg / day; about 0.5 mg / day to about 6 mg / day; about 0.5 mg / day to about 5 mg / day; about 0.5 mg / day to about 4 mg / day; about 0.5 mg / day to about 3 mg / day; about 0.1 mg / day to about 10 mg / day; about 0.1 mg / day to about 9 mg / day; about 0.1 mg / day to about 8 mg / day; about 0.1 mg / day to about 7 mg / day; about 0.1 mg / day to about 6 mg / day; about 0.1 mg / day to about 5 mg / day; about 0.1 mg / day to about 4 mg / day; about 0.1 mg / day to about 3 mg / day.

[0099] In another such embodiment for treating hearing loss, a prodrug of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP described herein can be used at a dose range that achieves equivalent exposure (AUC) to DNP. In one such embodiment, a prodrug of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP described herein can be used at a dose range independently selected from: 20 mg / day to 800 mg / day; 20 mg / day to 750 mg / day; 20 mg / day to 700 mg / day; 20 mg / day to 600 mg / day; 20 mg / day to 700 mg / day; 20 mg / day to 600 mg / day; 20 mg / day to 500 mg / day; 30 mg / day to 800 mg / day; 30 mg / day to 700 mg / day; 30 mg / day to 600 mg / day; 30 mg / day to 500 mg / day; 30 mg / day to 400 mg / day; 30 mg / day to 360 mg / day; 30 mg / day to 300 mg / day; 30 mg / day to 250 mg / day; 30 mg / day to 200 mg / day; 30 mg / day to 150 mg / day; 30 mg / day to 100 mg / day; 35 mg / day to 360 mg / day; 40 mg / day to 300 mg / day; 50 mg / day to 250 mg / day; or 60 mg / day to 200 mg / day; 5 mg / day to 500 mg / day; 5 mg / day to 400 mg / day; 5 mg / day to 300 mg / day; 5 mg / day to 200 mg / day; 5 mg / day to 100 mg / day; 5 mg / day to 50 mg / day; 5 mg / day to 40 mg / day; or 5 mg / day to 30 mg / day.In another such embodiment, the prodrug of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP described herein can be used in a dosage range independently selected from about 20 mg / day to about 800 mg / day; about 20 mg / day to about 750 mg / day; about 20 mg / day to about 700 mg / day; about 20 mg / day to about 600 mg / day; about 20 mg / day to about 700 mg / day; about 20 mg / day to about 600 mg / day; about 20 mg / day to about 500 mg / day; about 30 mg / day to about 800 mg / day; about 30 mg / day to about 700 mg / day; about 30 mg / day to about 600 mg / day; about 30 mg / day to about 500 mg / day; about 30 mg / day to about 400 mg / day; about 30 mg / day to about 360 mg / day; about 30 mg / day to about 300 mg / day; about 30 mg / day to about 250 mg / day; about 30 mg / day to about 200 mg / day; about 30 mg / day to about 150 mg / day; about 30 mg / day to about 100 mg / day; about 35 mg / day to about 360 mg / day; about 40 mg / day to about 300 mg / day; about 50 mg / day to about 250 mg / day; or about 60 mg / day to about 200 mg / day; about 5 mg / day to about 500 mg / day; about 5 mg / day to about 400 mg / day; about 5 mg / day to about 300 mg / day; about 5 mg / day to about 200 mg / day; about 5 mg / day to about 100 mg / day; about 5 mg / day to about 50 mg / day; about 5 mg / day to about 40 mg / day; or about 5 mg / day to about 30 mg / day.

[0100] Apparent reactive oxygen species (ROS) can be generated by noise and certain ototoxic drugs that can destroy the hair cells of the ear, leading to temporary or permanent hearing loss. Exposure to blast and chronic noise not only destroys the inner ear, but also causes cell death in the hippocampus, inhibits neurogenesis and impairs memory function. Aging can also manifest as mitochondrial dysfunction, which leads to hearing loss. Emerging is that attempts to treat hearing loss by targeting downstream problems are not very effective. In one embodiment, the compositions and methods of the present invention involve targeting oxidative stress downstream of the mitochondria, which can be the cause of many diseases. Various attempts have been made to reduce cellular stress, such as administering antioxidants, however these drugs have limited tissue penetration to the brain and reduce ROS after they have been formed. We have observed that chronic treatment of wild type mice with a particularly low dose of 2,4-dinitrophenol results in the treated mice living longer than the untreated mice. While not wishing to be bound by theory, it is hypothesized that DNP modulates the mitochondrial membrane potential, having a significant impact on preventing ROS formation in isolated mitochondria and in treated wild type mice. DNP treatment 3 hours after ischemia reduces the volume of brain infarction by 40% by protecting the penumbra or "threatened tissue" and can provide similar benefits after blast to protect the inner hair cells from apparent cell death. DNP is accompanied by a known risk benefit because it was used at high doses (~300 mg) for weight loss in over 100,000 people 80 years ago, however more recently shown to improve cognition and learning in models of neurodegeneration at extremely low doses. Pharmacologically, which is pleiotropic, shown to provide broad neuroprotection by reducing ROS / mTOR and increasing protective factors such as cAMP, CREB and BDNF, can be used to treat hearing loss. While DNP has a toxic past and a pervasive bias of incorrect dogma, a 28 day toxicity study was performed and confirmed that low doses of DNP are non-toxic, with a safety factor of at least 10x, do not inhibit ion channels, CYP, Caco-2, etc. and provide significant therapeutic benefits in a number of CNS models representing various indications, indicating possible advantages for inner ear and central brain auditory mitochondrial dysfunction.

[0101] Chemical synthesis :

[0102] In one embodiment, prodrugs comprising a self-immolative spacer and a water solubilizing moiety are synthesized to maintain the prodrug in a soluble form in GI tract fluids and which will then gradually reconstitute to the parent drug without precipitation. In some embodiments, these compounds are 1,3 dione analogs I-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32 (Schemes 1-6). The increased solubility of the prodrug and the high membrane permeability of the well dispersed parent drug will provide it with a higher driving force for easy absorption through the intestinal lumen. The conversion of the prodrug to the parent drug involves chemical cleavage at the self-immolative spacer through an intramolecular cyclization-elimination reaction under physiological conditions. The conversion time can be tuned by modifying the structure of the solubilizing moiety, the bond length of the spacer, the pKa of the amine group, and the pH of the medium. Although the computer predicted bioavailability can be low, it can be much higher when the unique pH dependence and tunable hydrolysis mechanism are considered. In addition, the production of the parent drug is not dependent on enzyme action, which can be advantageous in dealing with genetic variability associated with enzymatic prodrug hydrolysis in plasma.

[0103] In other embodiments, one or more isomers of dinitrophenol, i.e., 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, and / or a broad range of 1) bis 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs, 2) geminal prodrugs, and 3) bio-precursor molecules are used to treat neurodegenerative or metabolic diseases.

[0104] Synthesis of bis 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs can be performed; the prodrugs are selected from:

[0105] Amino acid (AA) esters of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 to II-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 to IV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 to VI-10);

[0106] AA esters with methylene dioxides (formaldehyde equivalent) spacers (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 to II-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 to IV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 to VI-13);

[0107] Amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 to II-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 to IV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 to VI-17);

[0108] Amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 and II-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18 and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, Formulas VI-18 and VI-19);

[0109] Phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6);

[0110] 1,3 Diketone analogs I-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32 (Schemes 1-6);

[0111] Carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 to III-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6);

[0112] Benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes 1-6); and combinations thereof, wherein the prodrugs are represented by Formulas I-VI:

[0113] Scheme 1

[0114]

[0115] Scheme 2

[0116]

[0117] Scheme 3

[0118]

[0119] Scheme 4

[0120]

[0121] Scheme 5

[0122]

[0123] Scheme 6

[0124]

[0125] In some embodiments, a wide range of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs can be considered. In some embodiments, prodrugs represented by Formulas I-1 to I-40; II-1 to II-40; III-1 to III-40; IV-1 to IV-40; V-1 to V-40; and VI-1 to VI-40 are considered. Computational screening with ADME-Tox computers of Pharma Algorithms was performed to identify virtual "hits" (i.e., prodrugs from the 32 prodrugs listed that have acceptable "predicted" oral bioavailability and aqueous solubility). Virtual screening allowed for ranking of the 10 best prodrug molecules for synthesis and testing.

[0126] Amino acid (AA) esters of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 to II-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 to IV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 to VI-10); AA esters incorporating a methylene dioxides (formaldehyde equivalent) spacer (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 to II-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 to IV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 to VI-13); amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 to II-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 to IV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 to VI-17); amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 and II-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18 and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, Formulas VI-18 and VI-19); phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6); 1,3 dione analogs I-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32 (Schemes 1-6); carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 to III-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6); benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes 1-6); and combinations thereof.

[0127] Examples of ionizable amine-containing prodrugs have increased aqueous solubility compared to the parent compound. In addition, AA ester prodrugs have the potential to further increase oral bioavailability due to active absorption by carriers (e.g., the small peptide carrier PEPT1). For example, the valine-containing prodrugs valacyclovir and valganciclovir are substrates for the enzyme PEPT1. These AA-containing prodrugs are hydrolyzed to the parent drugs by aminopeptidases in the brush border membrane of the Gl tract. Prodrugs that permeate to the peripheral circulation by passive permeation and / or by active transport are hydrolyzed by various peptidases in the plasma.

[0128] Two types of phosphate ester prodrugs of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP of Formula I-20, I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6) are synthesized. Given the fact that predictions by ADME-Tox computers are based primarily on the physicochemical properties of the molecule, according to ADME-Tox computational data, the oral bioavailability of these prodrugs can be poor. Because phosphate is a highly polar and extensively ionized pre-moiety, phosphate ester prodrugs can have significantly reduced membrane permeability compared to the parent drug.

[0129] The rationale for the success of phosphate ester prodrugs (e.g., phosphate esters) of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP of Formula I-20, I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6) as oral prodrugs is: 1) oral absorption is not rate limited by dissolution because phosphate ester prodrugs are highly soluble in Gl tract fluids; 2) phosphate esters are sufficiently chemically stable to prevent precipitation of the parent drug in the Gl tract; 3) phosphate esters are rapidly hydrolyzed by the membrane-bound alkaline phosphatase that is abundant on the brush border surface of cells lining the small intestine (i.e., enterocytes). Thus, the more permeable parent drug will be released and readily cross the enterocyte membrane and enter systemic circulation.

[0130] Scheme 8

[0131]

[0132] Synthetic prodrugs are modified to ensure water solubility. In some embodiments, acceptable solubility is tested prior to administration in animals. If solubility is problematic, the structure of the prodrug is changed by conjugation with the free phenolic functionality, introducing a water-soluble prodrug moiety to the molecule. In other embodiments, a prodrug linker moiety is used that imparts water-soluble properties to the prodrug molecule. In some embodiments, the solution to any water solubility problems is to utilize soft alkyl ether prodrugs, which incorporate an ethyleneoxy group into the promoiety, such as alkyl oxycarbonylmethyl (AOCOM) and N -alkyl- N -alkyloxycarbonylaminomethyl (NANAOCAM) prodrugs. These prodrugs have been found to be useful for delivering phenolic drug molecules and have generally acceptable water solubility and membrane permeation properties because they are strongly associated with water molecules, having good lipid solubility.

[0133] Synthesis of "twin" prodrugs of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP

[0134] In one embodiment, a 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP “twin” prodrug can be prepared by reacting a 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP with triphosgene in the presence of K2CO3 in dichloromethane to give 2,4-dinitrophenyl chlorocarbonate, which is further reacted with a base such as morpholine, piperidine, piperazine, N -alkylpiperazine to give the DNP prodrug, as shown in Scheme 9.

[0135] Scheme 9

[0136]

[0137] In one embodiment, the 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP“gemini” prodrug is prepared by reacting 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP with 5-(tert-butyldimethylsilyloxy)isophthaloyl dichloride (2) in the presence of pyridine / dichloromethane to give the precursor (3); followed by removal of the TBDMS protecting group in acetone / HCl to give the prodrug (4), which upon hydrolysis in blood plasma will provide 2 equivalents of 2,4-DNP. Although the prodrug linker moiety in (4) is an ester group, other alternative linkers such as a hindered ester linker, a carbonate linker, a carbamate linker, a phosphate linker, and AOCOM and NANAOCAM based linkers can also be incorporated into the prodrug structure to achieve appropriate sustained release kinetics. The presence of the free phenolic group in (4) can also be conjugated with a suitable hydrophilic moiety for improved water solubility if this is deemed necessary (Scheme 10).

[0138] Scheme 10

[0139]

[0140] 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP bio-precursors

[0141] Bio-precursors of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP that can release 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP upon cytochrome P-450 oxidative metabolism can be used. Scheme 8 illustrates the design of two bio-precursors that can release 2 and 4 equivalents of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP upon Cyt P-450 oxidation at the benzylic carbons (oxidation sites are shown by arrows). This oxidation converts the benzylic CH2group to a CO group to give an ester moiety, which can then be cleaved by ester hydrolysis to give 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP. The use of bio-precursors is generally a good alternative to the prodrug approach, and can provide slow release of the metabolically activated 2,4-DNP.

[0142] Scheme 11

[0143]

[0144] Alternative conjugation strategies

[0145] In some embodiments, DNP prodrugs and bioprecursors with linkers containing open functional groups are used, which allow each of these entities to be conjugated to a nanoparticle, e.g., a dendrimer, to modulate the pharmacokinetics of the molecule, making "trickle" drug delivery possible. Such DNP prodrugs and bioprecursors are delivered as depot nanoparticle formulations, which release the DNP at low doses in a slow, sustained manner compared to dosing and release of DNP alone, to avoid potential toxicity issues.

[0146] Nanotechnology offers the opportunity to increase the bioavailability of a drug particle. Particle size reduction results in an increase in surface area, resulting in faster dissolution. In some embodiments, the reduction is by one order of magnitude. In other embodiments, this can be sufficient to result in an increase in bioavailability. However, faster dissolution can not be sufficient to overcome exposure to acid and enzymes in the intestine. In addition, such exposure can require higher doses of the drug, resulting in unnecessary and potentially undesirable subject exposure to breakdown products and creating a significant waste.

[0147] Depot nanoparticle formulations are specifically formulated to provide slow absorption of a drug from the site of administration, usually to maintain therapeutic levels of a drug in the patient's system for several days or weeks at a time. Alternatively, depot nanoparticle formulations can provide convenience to patients requiring long-term medication. By delivering the drug without exposure to the Gl tract, the potential problem of drug degradation is avoided. In addition, due to the infrequent dosing regimen and convenience, depot nanoparticle formulations can provide better compliance. Other features of a depot nanoparticle formulation that would improve patient compliance are good local tolerability at the injection site and ease of administration. Good local tolerability means minimal irritation and inflammation at the injection site; ease of administration refers to the size of the needle and length of time required to administer a certain dose of a particular drug formulation.

[0148] In vitro and in vivo evaluation of prodrugs / bio-precursors

[0149] The prodrugs and bioprecursors of the present invention are faced with a wide range of pH and enzymes when orally administered to a patient. In one embodiment, the prodrugs / bioprecursors are stable in the environment of the Gl tract, but release the parent drug in a sustained manner in the plasma after absorption from the Gl tract. Oral administration exposes the compound to a pH of 1-2 in the stomach, a pH of 4.5 at the beginning of the small intestine, a pH of 6.6 as an average for the small intestine, and a pH of 5-9 in the colon. Stability-indicating methods are performed in aqueous buffered solutions and simulated Gl fluids to determine the resilience of the 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs / bioprecursors in the Gl tract, as well as their susceptibility to enzymatic conversion to the parent drug in rat plasma. These are methods that can be used to evaluate the chemical stability of prodrug candidates in vitro.

[0150] Determined in aqueous buffer (37 o C, pH 1-9)

[0151] Determined in simulated gastric fluid (USP, 37 o C)

[0152] Determined in simulated intestinal fluid (USP, 37 o C)

[0153] Determined in rat plasma (37 o C)

[0154] Single compound dosing studies are performed on 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP prodrug / bio-precursor candidates to determine their clinical potential. Most promising preclinical prodrug / bio-precursor candidates are absorbed intact from the gastrointestinal tract and are enzymatically cleaved in plasma to provide the parent drug. The presence and identification of the prodrug and parent drug also provide important information about the mechanism of action of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, which is valuable in the consideration of structure-activity and structure optimization studies in the selection of new structural entities.

[0155] Pharmacokinetic studies are performed on the most promising prodrug / bio-precursor candidates (i.e., prodrugs / bio-precursors that show the greatest stability in the GI tract and provide sustained release of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP in vitro and in vivo in rat plasma) in Sprague-Dawley rats. Complete PK profiles are obtained for oral administration of the prodrug in rats cannulated in the jugular and femoral veins to determine half-life (t 1 / 2 ), maximum plasma concentration (t max ), time to maximum plasma concentration (t max ), volume of distribution (V ss ), area under the plasma concentration versus time curve from time 0 to infinity (AUC 0-∞) and bioavailability (F%), as well as other important PK parameters, such as protein binding. LC / MS / MS was used as an analytical method to determine the above pharmacokinetic parameters of the prodrugs, as well as the plasma concentration and release kinetics of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP produced in plasma from enzymatic conversion of the prodrugs to 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP.

[0156] Although DNP is orally bioavailable, with good distribution in rats and a half-life of about 6 hours, the prodrug approach allows for prolonged plasma residence time at lower concentrations of the parent drug (DNP) through appropriate design of the prodrug release profile.

[0157] In one embodiment, the composition for treating neuromuscular, neuromuscular degenerative, neurodegenerative, autoimmune, developmental, traumatic, hearing loss-related, and / or metabolic diseases is independently selected from 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP, a bisecting 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP or 3,5-DNP) prodrug; a geminal prodrug, a bioprecursor molecule, and combinations thereof.

[0158] In one embodiment, the dosage of any of the foregoing embodiments of the composition of DNPs, prodrugs of DNPs; gemini prodrugs, bioprecursor molecules, and combinations thereof for the treatment of neuromuscular, neuromuscular degenerative, neurodegenerative, autoimmune, developmental, traumatic, hearing loss-related, and / or metabolic diseases can be about 0.01 mg / kg to about 50 mg / kg of the body weight of the patient in need of treatment; about 25 mg / kg to about 100 mg / kg of the body weight of the patient in need of treatment; or about 25 mg / kg to about 100 mg / kg of the body weight of the patient in need of treatment. In one embodiment, the dosage of any of the foregoing embodiments of the composition for the treatment of neuromuscular, neuromuscular degenerative, neurodegenerative, autoimmune, developmental, traumatic, hearing loss-related, and / or metabolic diseases can be 0.01 mg / kg to 50 mg / kg of the body weight of the patient in need of treatment; 25 mg / kg to 100 mg / kg of the body weight of the patient in need of treatment; or 25 mg / kg to 100 mg / kg of the body weight of the patient in need of treatment. In one embodiment, the present application is directed to a pharmaceutical composition of a DNP or a pharmaceutically acceptable salt, solvate, hydrate, and / or prodrug thereof described herein comprising a unit dosage, wherein the unit dosage ranges from about 0.1 mg to about 3000 mg. In one embodiment, the present application is directed to a pharmaceutical composition of a DNP or a pharmaceutically acceptable salt, solvate, hydrate, and / or prodrug thereof described herein comprising a unit dosage, wherein the unit dosage ranges from 0.1 mg to 3000 mg.

[0159] In one embodiment, the dosage of any of the foregoing embodiments of the composition for the treatment of neuromuscular diseases, neuro-muscular degenerative diseases, neurodegenerative diseases, autoimmune diseases, developmental diseases, traumatic diseases of the CNS, hearing loss associated with aging, noise, drug-induced and / or genetic, and / or metabolic diseases, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, DNP, DNP prodrugs; twin prodrugs, biological precursor molecules, and combinations thereof, can be independently selected from the group consisting of: about 0.01 mg / kg to about 50 mg / kg body weight; about 0.01 mg / kg to about 40 mg / kg body weight; about 0.01 mg / kg to about 30 mg / kg body weight; about 0.01 mg / kg to about 20 mg / kg body weight; about 0.01 mg / kg to about 10 mg / kg body weight; about 0.01 mg / kg to about 5 mg / kg body weight; about 0.01 mg / kg to about 1 mg / kg body weight; about 0.05 mg / kg to about 50 mg / kg body weight; about 0.05 mg / kg to about 40 mg / kg body weight; about 0.05 mg / kg to about 30 mg / kg body weight; about 0.05 mg / kg to about 20 mg / kg body weight; about 0.05 mg / kg to about 10 mg / kg body weight; about 0.05 mg / kg to about 1.0 mg / kg body weight; about 0.05 mg / kg to about 0.1 mg / kg body weight; about 0.1 mg / kg to about 40 mg / kg body weight; about 0.1 mg / kg to about 50 mg / kg body weight; about 0.1 mg / kg to about 30 mg / kg body weight; about 0.1 mg / kg to about 20 mg / kg body weight; about 0.1 mg / kg to about 15 mg / kg body weight; about 0.1 mg / kg to about 12 mg / kg body weight; about 0.1 mg / kg to about 10 mg / kg body weight; about 0.1 mg / kg to about 9 mg / kg body weight; about 0.1 mg / kg to about 8 mg / kg body weight; about 0.1 mg / kg to about 7 mg / kg body weight; about 0.1 mg / kg to about 6 mg / kg body weight; about 0.1 mg / kg to about 5 mg / kg body weight; about 0.1 mg / kg to about 4 mg / kg body weight; about 0.1 mg / kg to about 3 mg / kg body weight; about 0.1 mg / kg to about 2 mg / kg body weight; 0.1 mg / kg to about 1.0 mg / kg body weight; about 0.3 mg / kg to about 20 mg / kg body weight; about 0.3 mg / kg to about 15 mg / kg body weight; about 0.3 mg / kg to about 12 mg / kg body weight; about 0.3 mg / kg to about 10 mg / kg body weight; about 0.3 mg / kg to about 9 mg / kg body weight; about 0.3 mg / kg to about 8 mg / kg body weight; about 0.3 mg / kg to about 7 mg / kg body weight; about 0.3 mg / kg to about 6 mg / kg body weight; about 0.3 mg / kg to about 5 mg / kg body weight; about 0.3 mg / kg to about 4 mg / kg body weight; about 0.3 mg / kg to about 3 mg / kg body weight; about 0.3 mg / kg to about 2 mg / kg body weight; about 0.3 mg / kg to about 1.0 mg / kg body weight; about 0.5 mg / kg to about 15 mg / kg body weight; about 0.5 mg / kg to about 12 mg / kg body weight; about 0.5 mg / kg to about 10 mg / kg body weight; about 0.5 mg / kg to about 9 mg / kg body weight; about 0.5 mg / kg to about 8 mg / kg body weight; about 0.5 mg / kg to about 7 mg / kg body weight; about 0.5 mg / kg to about 6 mg / kg body weight; about 0.5 mg / kg to about 5 mg / kg body weight; about 0.5 mg / kg to about 4 mg / kg body weight; about 0.5 mg / kg to about 3 mg / kg body weight; about 0.5 mg / kg to about 2 mg / kg body weight; about 0.5 mg / kg to about 1.0 mg / kg body weight; about 0.8 mg / kg to about 15 mg / kg body weight; about 0.8 mg / kg to about 12 mg / kg body weight; about 0.8 mg / kg to about 10 mg / kg body weight; about 0.8 mg / kg to about 9 mg / kg body weight; about 0.8 mg / kg to about 8 mg / kg body weight; about 0.8 mg / kg to about 7 mg / kg body weight; about 0.8 mg / kg to about 6 mg / kg body weight; about 0.8 mg / kg to about 5 mg / kg body weight; about 0.8 mg / kg to about 4 mg / kg body weight; about 0.8 mg / kg to about 3 mg / kg body weight; about 0.8 mg / kg to about 2 mg / kg body weight; about 0.8 mg / kg to about 1.0 mg / kg body weight; about 1 mg / kg to about 3.0 mg / kg body weight; about 1.5 mg / kg to about 3.0 mg / kg body weight; about 1.0 mg / kg to about 2.0 mg / kg body weight; about 2.0 mg / kg to about 3.0 mg / kg body weight; about 0.5 mg / kg to about 2.5 mg / kg body weight; about 0.5 mg / kg to about 2.0 mg / kg body weight.

[0160] In one embodiment, the dosage of any of the foregoing embodiments of the composition for the treatment of neuromuscular diseases, neuro-muscular degenerative diseases, neurodegenerative diseases, autoimmune diseases, developmental diseases, traumatic diseases of the CNS, hearing loss associated with aging, noise, drug-induced and / or genetic, and / or metabolic diseases, including Spinal Muscular Atrophy (SMA) syndrome (SMA1, SMA2, SMA3 and SMA4, also known as types I, II, III and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, can be independently selected from 0.01 mg / kg to 50 mg / kg body weight; 0.01 mg / kg to 40 mg / kg body weight; 0.01 mg / kg to 30 mg / kg body weight; 0.01 mg / kg to 20 mg / kg body weight; 0.01 mg / kg to 10 mg / kg body weight; 0.01 mg / kg to 5 mg / kg body weight; 0.01 mg / kg to 1 mg / kg body weight; 0.05 mg / kg to 50 mg / kg body weight; 0.05 mg / kg to 40 mg / kg body weight; 0.05 mg / kg to 30 mg / kg body weight; 0.05 mg / kg to 20 mg / kg body weight; 0.05 mg / kg to 10 mg / kg body weight; 0.05 mg / kg to 1.0 mg / kg body weight; 0.05 mg / kg to 0.1 mg / kg body weight; 0.1 mg / kg to 40 mg / kg body weight; 0.1 mg / kg to 50 mg / kg body weight; 0.1 mg / kg to 30 mg / kg body weight; 0.1 mg / kg to 20 mg / kg body weight; 0.1 mg / kg to 15 mg / kg body weight; 0.1 mg / kg to 12 mg / kg body weight; 0.1 mg / kg to 10 mg / kg body weight; 0.1 mg / kg to 9 mg / kg body weight; 0.1 mg / kg to 8 mg / kg body weight; 0.1 mg / kg to 7 mg / kg body weight; 0.1 mg / kg to 6 mg / kg body weight; 0.1 mg / kg to 5 mg / kg body weight; 0.1 mg / kg to 4 mg / kg body weight; 0.1 mg / kg to 3 mg / kg body weight; 0.1 mg / kg to 2 mg / kg body weight; 0.1 mg / kg to 1.0 mg / kg body weight; 0.3 mg / kg to 20 mg / kg body weight; 0.3 mg / kg to 15 mg / kg body weight; 0.3 mg / kg to 12 mg / kg body weight; 0.3 mg / kg to 10 mg / kg body weight; 0.3 mg / kg to 9 mg / kg body weight; 0.3 mg / kg to 8 mg / kg body weight; 0.3 mg / kg to 7 mg / kg body weight; 0.3 mg / kg to 6 mg / kg body weight; 0.3 mg / kg to 5 mg / kg body weight; 0.3 mg / kg to 4 mg / kg body weight; 0.3 mg / kg to 3 mg / kg body weight; 0.3 mg / kg to 2 mg / kg body weight; 0.3 mg / kg to 1.0 mg / kg body weight; 0.5 mg / kg to 10 mg / kg body weight; 0.5 mg / kg to 9 mg / kg body weight; 0.5 mg / kg to 8 mg / kg body weight; 0.5 mg / kg to 7 mg / kg body weight; 0.5 mg / kg to 6 mg / kg body weight; 0.5 mg / kg to 5 mg / kg body weight; 0.5 mg / kg to about 4 mg / kg body weight; 0.5 mg / kg to 3 mg / kg body weight; 0.5 mg / kg to 2 mg / kg body weight; 0.5 mg / kg to 1.0 mg / kg body weight; 0.8 mg / kg to 15 mg / kg body weight; 0.8 mg / kg to 12 mg / kg body weight; 0.8 mg / kg to 8 10 mg / kg body weight; 0.8 mg / kg to 9 mg / kg body weight; 0.8 mg / kg to 8 mg / kg body weight; 0.8 mg / kg to 7 mg / kg body weight; 0.8 mg / kg to 6 mg / kg body weight; 0.8 mg / kg to 5 mg / kg body weight; 0.8 mg / kg to 4 mg / kg body weight; 0.8 mg / kg to 3 mg / kg body weight; 0.8 mg / kg to 2 mg / kg body weight; 0.8 mg / kg to 1.0 mg / kg body weight; 1 mg / kg to 3.0 mg / kg body weight; 1.5 mg / kg to 3.0 mg / kg body weight; 1.0 mg / kg to 2.0 mg / kg body weight; 2.0 mg / kg to 3.0 mg / kg body weight; 0.5 mg / kg to 2.5 mg / kg body weight; 0.5 mg / kg to 2.0 mg / kg body weight.

[0161] In one embodiment, the dosage of any of the foregoing embodiments of the composition for the treatment of neuromuscular diseases, neuro-muscular degenerative diseases, neurodegenerative diseases, autoimmune diseases, developmental diseases, traumatic diseases of the CNS, hearing loss associated with aging, noise, drug-induced and / or genetic, and / or metabolic diseases, including spinal muscular atrophy (SMA) syndromes (SMA1, SMA2, SMA3, and SMA4, also known as types I, II, III, and IV), traumatic brain injury (TBI), concussions, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, can independently be from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 300 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 200 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 100 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 50 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 30 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 20 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 10 mg / day / 70 kg of body weight of the patient in need of treatment; from about 1 mg / day / 70 kg of body weight of the patient in need of treatment to about 5 mg / day / 70 kg of body weight of the patient in need of treatment.

[0162] In one embodiment, the dosage of any of the foregoing embodiments of the composition for the treatment of neuromuscular diseases, neuro-muscular degenerative diseases, neurodegenerative diseases, autoimmune diseases, developmental diseases, traumatic diseases of the CNS, hearing loss associated with aging, noise, drug-induced and / or genetic, and / or metabolic diseases, including Spinal Muscular Atrophy (SMA) syndrome (SMA1, SMA2, SMA3 and SMA4, also known as type I, II, III and IV), traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolfram syndrome, and Wolcott-Rallison syndrome, can independently be from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 300 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 200 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 100 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 50 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 30 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 20 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 10 mg / day / 70 kg of body weight of the patient in need of treatment; from 1 mg / day / 70 kg of body weight of the patient in need of treatment to 5 mg / day / 70 kg of body weight of the patient in need of treatment.

[0163] In some embodiments, the pharmaceutical composition comprises a DNP selected from a DNP, 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, a bis-2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrug, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, including unit dosages independently selected from: about 0.1 mg to about 3000 mg; about 0.1 mg to about 1000 mg; about 0.1 mg to about 500 mg; about 0.1 mg to about 250 mg; about 0.1 mg to about 100 mg; about 0.1 mg to about 50 mg; about 0.1 mg to about 25 mg; about 0.1 mg to about 10 mg; about 0.1 mg to about 5 mg; about 0.1 mg to about 2.5 mg; about 0.1 mg to about 1 mg; about 0.1 mg to about 0.5 mg; about 0.1 mg to about 0.25 mg; about 0.1 mg to about 0.1 mg; about 0.1 mg to about 0.05 mg; about 0.1 mg to about 0.01 mg; about 0.1 mg to about 0.005 mg; about 0.1 mg to about 0.001 mg; about 0.1 mg to about 0.0005 mg; about 0.1 mg to about 0.0001 mg; about 0.1 mg to about 0.00005 mg; about 0.1 mg to about 0.00001 mg; about 0.1 mg to about 0.000005 mg; about 0.1 mg to about 0.000001 mg; about 0.1 mg to about 0.0000005 mg1 mg to about 100 mg; wherein the unit dose ranges from about 1 mg to about 50 mg; wherein the unit dose is about 1 mg; wherein the unit dose is about 2 mg; wherein the unit dose is about 3 mg; wherein the unit dose is about 4 mg; wherein the unit dose is about 5 mg; wherein the unit dose ranges from about 5 mg to about 10 mg; wherein the unit dose is about 6 mg; wherein the unit dose is about 7 mg; wherein the unit dose is about 8 mg; wherein the unit dose is about 9 mg; wherein the unit dose is about 10 mg; wherein the unit dose ranges from about 10 mg to about 15 mg; wherein the unit dose is about 11 mg; wherein the unit dose is about 12 mg; wherein the unit dose is about 13 mg; wherein the unit dose is about 14 mg; wherein the unit dose is about 15 mg; wherein the unit dose ranges from about 15 mg to about 20 mg; wherein the unit dose is about 16 mg; wherein the unit dose is about 17 mg; wherein the unit dose is about 18 mg; wherein the unit dose is about 19 mg; wherein the unit dose is about 20 mg; wherein the unit dose ranges from about 20 mg to about 30 mg; wherein the unit dose is about 25 mg; wherein the unit dose is about 30 mg; wherein the unit dose ranges from about 30 mg to about 40 mg; wherein the unit dose is about 35 mg; wherein the unit dose is about 40 mg; wherein the unit dose ranges from about 40 mg to about 50 mg; wherein the unit dose is about 45 mg; wherein the unit dose is about 50 mg; wherein the unit dose ranges from about 50 mg to about 100 mg; wherein the unit dose is about 75 mg; wherein the unit dose is about 100 mg; wherein the unit dose ranges from about 100 mg to about 200 mg; wherein the unit dose is about 150 mg; wherein the unit dose is about 200 mg; wherein the unit dose ranges from about 200 mg to about 300 mg; wherein the unit dose is about 200 mg; wherein the unit dose is about 250 mg; wherein the unit dose is about 300 mg; wherein the unit dose is about 350 mg; wherein the unit dose is about 400 mg; wherein the unit dose is about 450 mg; wherein the unit dose is about 500 mg; wherein the unit dose is about 750 mg; wherein the unit dose is about 1000 mg; wherein the unit dose is about 1500 mg; wherein the unit dose is about 2000 mg; wherein the unit dose is about 2500 mg; or wherein the unit dose is about 3000 mg.

[0164] In some embodiments, the pharmaceutical composition comprises a DNP selected from a DNP, 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, a bis 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrug, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, including unit dosages independently selected from: 0.1 mg to 3000 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to 1000 mg; wherein the unit dosage ranges from 0.1 mg to 500 mg; wherein the unit dosage ranges from 0.1 mg to1 mg to 100 mg; wherein the unit dose ranges from 1 mg to 50 mg; wherein the unit dose is 1 mg; wherein the unit dose is 2 mg; wherein the unit dose is 3 mg; wherein the unit dose is 4 mg; wherein the unit dose is 5 mg; wherein the unit dose ranges from 5 mg to 10 mg; wherein the unit dose is 6 mg; wherein the unit dose is 7 mg; wherein the unit dose is 8 mg; wherein the unit dose is 9 mg; wherein the unit dose is 10 mg; wherein the unit dose ranges from 10 mg to 15 mg; wherein the unit dose is 11 mg; wherein the unit dose is 12 mg; wherein the unit dose is 13 mg; wherein the unit dose is 14 mg; wherein the unit dose is 15 mg; wherein the unit dose ranges from 15 mg to 20 mg; wherein the unit dose is 16 mg; wherein the unit dose is 17 mg; wherein the unit dose is 18 mg; wherein the unit dose is 19 mg; wherein the unit dose is 20 mg; wherein the unit dose ranges from 20 mg to 30 mg; wherein the unit dose is 25 mg; wherein the unit dose is 30 mg; wherein the unit dose ranges from 30 mg to 40 mg; wherein the unit dose is 35 mg; wherein the unit dose is 40 mg; wherein the unit dose ranges from 40 mg to 50 mg; wherein the unit dose is 45 mg; wherein the unit dose is 50 mg; wherein the unit dose ranges from 50 mg to 100 mg; wherein the unit dose is 75 mg; wherein the unit dose is 100 mg; wherein the unit dose ranges from 100 mg to 200 mg; wherein the unit dose is 150 mg; wherein the unit dose is 200 mg; wherein the unit dose ranges from 200 mg to 300 mg; wherein the unit dose is 200 mg; wherein the unit dose is 250 mg; wherein the unit dose is 300 mg; wherein the unit dose is 350 mg; wherein the unit dose is 400 mg; wherein the unit dose is 450 mg; wherein the unit dose is 500 mg; wherein the unit dose is 750 mg; wherein the unit dose is 1000 mg; wherein the unit dose is 1500 mg; wherein the unit dose is 2000 mg; wherein the unit dose is 2500 mg; or wherein the unit dose is 3000 mg.

[0165] In some embodiments of the foregoing embodiments of the composition for treating a disease, the unit dose is an immediate release formulation. In some embodiments of the foregoing embodiments of the composition for treating a disease, the unit dose is an extended release formulation. In some embodiments of the foregoing embodiments of the composition for treating a disease, the unit dose is a sustained release formulation. In some embodiments of the foregoing embodiments of the composition for treating a disease, the unit dose is a controlled release formulation. In some embodiments of the foregoing embodiments of the composition for treating a disease, the unit dose is an oral dosage form. In some embodiments, the oral dosage form is a tablet. In some embodiments of the foregoing embodiments of the composition for treating a disease, the oral dosage form is a capsule. In some embodiments of the foregoing embodiments of the composition for treating a disease, the unit dose is a capsule without a filler. In some embodiments of the foregoing embodiments of the composition for treating a disease, the oral dosage form is a fast dissolving agent. In each of the foregoing embodiments, the disease can be independently selected from the group consisting of traumatic brain injury (TBI), concussion, ischemic stroke, Huntington’s disease (adult onset Huntington’s disease, juvenile Huntington’s disease), epilepsy (massive seizure, intractable seizure, atypical absence seizure, atonic seizure, clonic seizure, myoclonic seizure, tonic seizure, tonic-clonic seizure, simple partial seizure, complex partial seizure, secondary generalized seizure, febrile seizure, non-epileptic seizure, gelastic and dacrystic seizures, and absence seizure), multiple sclerosis (MS) (relapsing-remitting multiple sclerosis (RRMS), secondary-progressive MS (SPMS), primary-progressive MS (PPMS), and progressive-relapsing MS (PRMS)), lupus (systemic lupus erythematosus (SLE), discoid (cutaneous), drug-induced lupus (dil), and neonatal lupus), diabetes (type 1 diabetes, type 2 diabetes, young adult onset diabetes (MODY: MODY1, MODY2, MODY3, MODY4, MODY5, MODY6, MODY7, MODY8, MODY9, MODY10, MODY11)), nonalcoholic steatohepatitis (NASH), schizophrenia (paranoid schizophrenia, disorganized schizophrenia, catatonic schizophrenia, residual schizophrenia, schizoaffective disorder), myasthenia gravis (MG)(ocular myasthenia gravis, congenital MG and generalized myasthenia gravis), rheumatoid arthritis (RA), Graves' disease, Guillain-Barre syndrome (GBS), muscular dystrophy (Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy and oculopharyngeal muscular dystrophy), severe burns, aging, amyotrophic lateral sclerosis (ALS), ataxia (Friedreich's ataxia, spinocerebellar ataxia 1 (SCA1), spinocerebellar ataxia 2 (SCA2), spinocerebellar ataxia 3 (SCA3), spinocerebellar ataxia 6 (SCA6), spinocerebellar ataxia 7 (SCA7), spinocerebellar ataxia 11 (SCA11), dentatorubral-pallidoluysian atrophy (DRPLA) and glutamine ataxia), Batten disease or neuronal ceroid lipofuscinosis (NCL) (infantile NCL (INCL), late infantile NCL (LINCL), juvenile NCL (JNCL) or adult NCL (ANCL)), Alzheimer's disease (early onset Alzheimer's disease, late onset Alzheimer's disease and familial Alzheimer's disease (FAD)), optic neuritis (ON), Leber's hereditary optic neuropathy (LHON), autism spectrum disorders (ASD) (Asperger syndrome, pervasive developmental disorder (PDD), childhood disintegrative disorder (CDD) and autism), Rett syndrome, Angelman syndrome, Leigh disease, Prader Willi syndrome, fragile X syndrome, depression (major depression, psychotic depression, postpartum depression, seasonal affective disorder, atypical depression, psychotic depression, bipolar disorder, premenstrual dysphoric disorder, situational depression), Parkinson's disease (idiopathic Parkinson's disease, vascular Parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease and atypical parkinsonism), Wolfram syndrome (and / or any associated conditions, such as diabetes problems, hearing, vision, ataxia, neurodegeneration, etc.), spinal muscular atrophy (SMA; types I, II, III and IV), hearing loss due to noise (explosions and high noise), aging-related hearing loss, drug-induced hearing loss and / or genetic hearing loss, concussions, dry eye keratoconjunctivitis (dry eye disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK, antidepressant use, Wolcott-Rallison syndrome, mitochondrial diseases, developmental disorders, metabolic syndrome (increased blood pressure, elevated blood sugar levels, excess waist circumference body fat and abnormal cholesterol levels) and / or autoimmune diseases, by increasing energy expenditure and / or inducing BDNF with DNPmRNA expression and protein levels to reverse, slow, or prevent treatment of neurodegenerative, neuromuscular, developmental, autoimmune, and / or metabolic diseases and / or muscle atrophy.

[0166] In one embodiment, the present application provides methods of treating traumatic brain injury (TBI) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating concussion using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating ischemic stroke using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating severe burns using the embodiments of compositions and dosages described herein.

[0167] In one embodiment, the present application relates to methods of treating Huntington's Disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating adult-onset Huntington's Disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating juvenile Huntington's Disease using the embodiments of compositions and dosages described herein.

[0168] In one embodiment, the present application relates to methods of treating epilepsy using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating intensive seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating intractable seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating atypical absence seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating atonic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating clonic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating myoclonic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating tonic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating tonic-clonic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating simple partial seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating complex partial seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating secondary generalized seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating febrile seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating non-epileptic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating gelastic seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating crying seizures using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating absence seizures.

[0169] In one embodiment, the present application relates to methods of treating multiple sclerosis (MS) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating relapsing-remitting multiple sclerosis (RRMS) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating secondary-progressive MS (SPMS) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating primary-progressive MS (PPMS) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating progressive-relapsing MS (PRMS).

[0170] In one embodiment, the present application relates to methods of treating diabetes using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating type 1 diabetes using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating type 2 diabetes using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating maturity onset diabetes of the young (MODY) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY1 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY2 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY3 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY4 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY5 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY6 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY7 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY8 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY9 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY10 using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating MODY11 using the embodiments of compositions and dosages described herein.

[0171] In one embodiment, the present application relates to methods of treating nonalcoholic steatohepatitis (NASH) using the embodiments of compositions and dosages described herein.

[0172] In one embodiment, the present application relates to methods of treating muscular dystrophy using the embodiments of compositions and dosages described herein. In one such embodiment, for adults, such dosages range independently are: 10 mg / day to 150 mg / day; 20 mg / day to 150 mg / day; 30 mg / day to 150 mg / day; 40 mg / day to 150 mg / day; 50 mg / day to 150 mg / day; 60 mg / day to 150 mg / day; 70 mg / day to 150 mg / day; 80 mg / day to 150 mg / day; 90 mg / day to 150 mg / day; 80 mg / day to 100 mg / day; 90 mg / day to 100 mg / day; 91 mg / day to 100 mg / day; 92 mg / day to 100 mg / day; 93 mg / day to 100 mg / day; 94 mg / day to 100 mg / day; or independently are 90 mg / day; 91 mg / day; 92 mg / day; 93 mg / day; 94 mg / day; 95 mg / day; 96 mg / day; 97 mg / day; 98 mg / day or 99 mg / day. In one such embodiment, for adolescents, such dosages range independently are: 1 mg / day to 45 mg / day; 1 mg / day to 50 mg / day; 5 mg / day to 45 mg / day; 5 mg / day to 50 mg / day; 10 mg / day to 45 mg / day; 15 mg / day to 45 mg / day; 20 mg / day to 45 mg / day; 25 mg / day to 45 mg / day; 30 mg / day to 45 mg / day; 35 mg / day to 45 mg / day; 35 mg / day to 40 mg / day; or independently are in the range of 35 mg / day; 35 mg / day; 37 mg / day; 38 mg / day; 39 mg / day; 40 mg / day; 41 mg / day; 42 mg / day; 43 mg / day; 44 mg / day or 45 mg / day.In another such embodiment, for adults, such dosage ranges are independently: about 10 mg / day to about 150 mg / day; about 20 mg / day to about 150 mg / day; about 30 mg / day to about 150 mg / day; about 40 mg / day to about 150 mg / day; about 50 mg / day to about 150 mg / day; about 60 mg / day to about 150 mg / day; about 70 mg / day to about 150 mg / day; about 80 mg / day to about 150 mg / day; about 90 mg / day to about 150 mg / day; about 80 mg / day to about 100 mg / day; about 90 mg / day to about 100 mg / day; about 91 mg / day to about 100 mg / day; about 92 mg / day to about 100 mg / day; about 93 mg / day to about 100 mg / day; about 94 mg / day to about 100 mg / day; or independently about 90 mg / day; about 91 mg / day; about 92 mg / day; about 93 mg / day; about 94 mg / day; about 95 mg / day; about 96 mg / day; about 97 mg / day; about 98 mg / day; or about 99 mg / day. In another such embodiment, for adolescents, such dosage ranges are independently: about 1 mg / day to about 45 mg / day; about 1 mg / day to about 50 mg / day; about 5 mg / day to about 45 mg / day; about 5 mg / day to about 50 mg / day; about 10 mg / day to about 45 mg / day; about 15 mg / day to about 45 mg / day; about 20 mg / day to about 45 mg / day; about 25 mg / day to about 45 mg / day; about 30 mg / day to about 45 mg / day; about 35 mg / day to about 45 mg / day; about 35 mg / day to about 40 mg / day; or independently about 35 mg / day; about 35 mg / day; about 37 mg / day; about 38 mg / day; about 39 mg / day; about 40 mg / day; about 41 mg / day; about 42 mg / day; about 43 mg / day; about 44 mg / day; or about 45 mg / day. In one embodiment, the present application is directed to methods of treating Duchenne Muscular Dystrophy (DMD) using the embodiments of compositions and dosages described herein. In one embodiment, the present application is directed to methods of treating Becker Muscular Dystrophy using the embodiments of compositions and dosages described herein. In one embodiment, the present application is directed to methods of treating Myotonic Muscular Dystrophy using the embodiments of compositions and dosages described herein. In one embodiment, the present application is directed to methods of treating Congenital Muscular Dystrophy using the embodiments of compositions and dosages described herein. In one embodiment, the present application is directed to methods of treating Emery-Dreifuss Muscular Dystrophy using the embodiments of compositions and dosages described herein.In one embodiment, the present application relates to methods of treating facioscapulohumeral muscular dystrophy using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating limb-girdle muscular dystrophy using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating distal muscular dystrophy using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating oculopharyngeal muscular dystrophy using the embodiments of compositions and dosages described herein.

[0173] In one embodiment, the present application relates to methods of treating amyotrophic lateral sclerosis (ALS) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating ataxia using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating Friedreich's ataxia using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating Spinocerebellar ataxia 1 (SCA1) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating Batten disease. In one embodiment, the present application relates to methods of treating neuronal ceroid lipofuscinosis (NCL) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating infantile NCL (INCL) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating late infantile NCL (LINCL) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating juvenile NCL (JNCL) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating adult NCL (ANCL) using the embodiments of compositions and dosages described herein.

[0174] In one embodiment, the present application relates to methods of treating Alzheimer's disease using the embodiments of compositions and dosages described herein. In one such embodiment, such dosages range independently are: 1 mg / day to 45 mg / day; 5 mg / day to 45 mg / day; 10 mg / day to 45 mg / day; 15 mg / day to 45 mg / day; 20 mg / day to 45 mg / day; 25 mg / day to 45 mg / day; 30 mg / day to 45 mg / day; 35 mg / day to 45 mg / day; 35 mg / day to 40 mg / day or; independently are 31 mg / day; 32 mg / day; 33 mg / day; 34 mg / day; 35 mg / day; 36 mg / day; 37 mg / day; 38 mg / day; 39 mg / day or 40 mg / day. In another such embodiment, such dosages range independently are: about 1 mg / day to about 45 mg / day; about 5 mg / day to about 45 mg / day; about 10 mg / day to about 45 mg / day; about 15 mg / day to about 45 mg / day; about 20 mg / day to about 45 mg / day; about 25 mg / day to about 45 mg / day; about 30 mg / day to about 45 mg / day; about 35 mg / day to about 45 mg / day; about 35 mg / day to about 40 mg / day; or independently are about 31 mg / day; about 32 mg / day; about 33 mg / day; about 34 mg / day; about 35 mg / day; about 36 mg / day; about 37 mg / day; about 38 mg / day; about 39 mg / day or about 40 mg / day. In one embodiment, the present application relates to methods of treating early onset Alzheimer's disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating late onset Alzheimer's disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating familial Alzheimer's disease (FAD) using the embodiments of compositions and dosages described herein.

[0175] In one embodiment, the present application relates to methods of treating optic neuritis (ON) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating autism spectrum disorder (ASD) using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating Rett syndrome using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating Angelman syndrome using the embodiments of compositions and dosages described herein.

[0176] In one embodiment, the present application relates to methods of treating Parkinson's disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating idiopathic Parkinson's disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating vascular parkinsonism using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating Lewy body dementia using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating genetic Parkinson's disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating drug-induced parkinsonism using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating juvenile Parkinson's disease using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating atypical parkinsonism using the embodiments of compositions and dosages described herein.

[0177] In one embodiment, the present application relates to methods of treating Wolfram syndrome using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating diabetes problems associated with Wolfram syndrome using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating hearing problems associated with Wolfram syndrome using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating visual problems associated with Wolfram syndrome using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating ataxia associated with Wolfram syndrome using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating neurodegeneration associated with Wolfram syndrome using the embodiments of compositions and dosages described herein.

[0178] In one embodiment, the present application relates to methods of treating spinal muscular atrophy (SMA) type III using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating SMA type IV using the embodiments of compositions and dosages described herein.

[0179] In one embodiment, the present application relates to methods of treating hearing loss using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating hearing loss due to noise using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating hearing loss due to explosive noise using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating hearing loss due to high noise using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating age-related hearing loss using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating drug-induced hearing loss using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating genetic hearing loss using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating hearing loss due to concussions using the embodiments of compositions and dosages described herein. In one embodiment, the present application relates to methods of treating hearing loss due to traumatic brain injury (TBI) using the embodiments of compositions and dosages described herein.

[0180] In some embodiments, the unit dose is delivered intravenously. In some embodiments, the unit dose is delivered by intravenous drip with saline. In some embodiments, the unit dose is delivered by intravenous drip with saline, other drugs, vitamins, and / or nutraceuticals. In some embodiments, the unit dose is delivered subcutaneously. In some embodiments, the unit dose is delivered topically. In some embodiments, the unit dose is delivered transdermally. In some embodiments, the unit dose is in the form of a patch.

[0181] Doses can be given as a single daily dose, twice daily doses, three times daily doses, or more frequent. Doses can be given three times per week, twice per week, once per week, or less frequently. In one embodiment, the frequency of administration can be 1-5 times per day. In another embodiment, the frequency of administration can be 2-4 times per day. In another embodiment, the frequency of administration can be at least 3 times per day. In another embodiment, the frequency of administration can be twice per day. In another embodiment, the frequency of administration can be once per day. In another embodiment, the frequency of administration can be less frequent than once per day. In other embodiments, the frequency of administration can be once every 2 days or once every 3 days or once every 4 days or once every 5 days or once every 6 days. In another embodiment, the frequency of administration can be once per week. In another embodiment, the frequency of administration can vary over time, starting at a certain rate, such as once or twice per day, and then decreasing to a lower frequency, such as once every 2 days or once every 3 days or once per week, after the first day of treatment. In another embodiment, the frequency of administration can vary over time, starting at a certain rate, such as once or twice per day, and then decreasing to a lower frequency, such as once every 2 days or once every 3 days or once per week, after the first two or three days of treatment. In another embodiment, the frequency of administration can vary over time, starting at a certain rate, such as once or twice per day, and then decreasing to a lower frequency, such as once every 2 days or once every 3 days or once per week, after the first week of treatment. In another embodiment, the frequency of administration can be as needed when therapeutic treatment is required or desired.

[0182] From the disclosure included herein, it will be understood how to determine whether a subject needs additional and / or continued doses. It will also be understood that the selected frequency of administration can require adjustment of the dose of the active ingredient. Based on the disclosure included herein, it will also be understood that the selected dose of the active ingredient can require adjustment of the frequency of administration. The disclosure included herein, in conjunction with the skill in the art, will enable the skilled artisan to optimize the dose of the active ingredient and the frequency of administration of the active ingredient to treat a subject in need thereof.

[0183] Unit doses can also be adjusted for the size of the patient. In one embodiment, the amounts provided herein are based on a 60 kg patient. The same therapy can be provided to smaller or larger patients by reducing or increasing the dose size, respectively. To illustrate, a 20 kg pediatric patient will require a much smaller dose than a 60 kg adult patient.

[0184] Using formulation approaches, such as controlled release technology (polymer, liposome, etc.) to achieve the PK profile of DNP once a day. In some embodiments, DNP prodrugs and bioprecursors with linkers containing open functional groups are synthesized and these entities are each conjugated to nanoparticles, such as dendrimers, to modulate the pharmacokinetics of the molecule, making "trickle" drug delivery possible. Such DNP prodrugs and bioprecursors are delivered as depot nanoparticle formulations, which release DNP at low doses in a slow, sustained manner compared to the dosing and release of DNP alone, to avoid possible toxicity issues. In vitro stability, in vivo plasma release kinetics, and PK profiles are evaluated. In vivo studies are performed in Sprague-Dawley rats. LC / MS / MS is used to analyze plasma DNP released from various prodrug-nanoparticle formulations to determine the PK profile of DNP release in the rat model.

[0185] The following examples describe the application in more detail. These examples are provided for illustrative purposes only, and should in no way be taken as limiting the application. Example

[0186] Synthesis of 2,4-dinitrophenyl morpholine-4-carboxylate: 2,4-DNP (1 mmol) was reacted with triphosgene (0.5 mmol) in the presence of K2CO3 (1 mmol) in dichloromethane to get 2,4-dinitrophenyl chloro carbonate, which was further reacted with morpholine (1.1 mmol) at 0-5 °C to get DNP-morpholine prodrug.

[0187]

[0188] 2,4-dinitrophenyl morpholine-4-carboxylate analytical data: GC MS: M + , 297.1, HRMS: 298.0683 (M+H) + ; 1 H NMR (400 MHz, CDCl3): δ 3.57-3.59 (m, 2 H, CH2), 3.72-3.73 (m, 2H,CH2), 3.73-3.80 (m, 4H, 2 x OCH2), 7.53 (dd, 1H, Ar-H), 8.48-8.51 (dd, 1H, Ar-H), 8.94 (d, 1H, Ar-H) ppm . 13C NMR (75 MHz, CDCl3): δ 44.99, 45.69, 66.62,66.78, 121.94, 126.84, 129.18, 141.90, 144.85, 149.64, 151.10 ppm .

[0189] 2,4-Dinitrophenylpiperidine-1-carboxylate synthesis: 2,4-DNP (1 mmol) was reacted with triphosgene (0.5 mmol) in presence of K2CO3 (1 mmol) in dichloromethane to get 2,4-dinitrophenyl chlorocarbonate which was further reacted with piperidine (1.1 mmol) at 0-5 °C to get DNP-morpholine prodrug.

[0190]

[0191] 2,4-Dinitrophenylpiperidine-1-carboxylate analytical data: GC MS: M + , 295.1, 1 H NMR (400 MHz, CDCl3): δ 1.68 (m, 6 H, 3 xCH2), 3.49-3.51 (m, 2H, CH2), 3.64(m, 2H, CH2),7.52 (d, 1H, Ar-H), 8.45 (dd, 1H, Ar-H), 8.89 (d, 1H, Ar-H) ppm . 13 C NMR (75MHz, CDCl3): δ 24.05, 25.44, 25.67, 45.77, 46.67, 121.48, 126.52, 128.66,141.73, 144.22, 149.74, 150.66 ppm .

[0192] 2,4-Dinitrophenylpiperidine-1-carboxylate synthesis: 2,4-DNP (1 mmol) was reacted with triphosgene (0.5 mmol) in presence of K2CO3 (1 mmol) in dichloromethane to get 2,4-dinitrophenyl chlorocarbonate which was further reacted with piperidine (1.1 mmol) at 0-5 °C to get DNP-morpholine prodrug.

[0193]

[0194] Synthesis of 2,4-dinitrophenyl 4-(2-(piperidin-l-yl)ethoxy)benzoate: 4-(2-(piperidin-l-yl)ethoxy)benzoic acid (1 mmol) was refluxed with thionyl chloride (1.25 mmol) in dichloromethane to obtain its acyl chloride, 4-(2-(piperidin-l-yl)ethoxy)benzoyl chloride, which was further reacted with phenol (1 mmol) in the presence of triethylamine (2.0 mmol) to obtain the final product of 2,4-dinitrophenyl 4-(2-(piperidin-l-yl)ethoxy)benzoate.

[0195]

[0196] Analytical data of 2,4-dinitrophenyl 4-(2-(piperidin-l-yl)ethoxy)benzoate:

[0197] 1 H NMR (400 MHz, CDCl3); δ 1.60 (s, 2H), 1.96 (s, 4H), 3.08 (bs, 1H,4H), 3.32 (s, 2H, N-CH2), 4.56 (s, 2H, O-CH2), 7.04 (d, J = 8.8 Hz, 2H), 7.65(d, J = 8.8 Hz, 1H), 8.14 (d, J = 8.8 Hz, 2H), 8.56-8.58 (q, J= 2.4 Hz, 8.8Hz, 1 H), 8.99 (d, J = 2.8 Hz, 1H ) ppm . 1 H NMR (75 MHz, CDCl3); δ 22.42,23.59, 54.48, 56.51, 64.04, 114.95, 120.59, 121.78, 126.95, 129.11, 133.23,141.96, 145.01, 149.10, 162.91, 163.00 ppm .

[0198] Introduction: Pharmacokinetic analysis and bioavailability study of 2,4-DNP and its prodrug II-38 in male Sprague-Dawley male rats

[0199] 2,4-DNP is a biochemically active, uncoupling agent organic compound that inhibits energy (ATP) production in cells with mitochondria. It has been used to treat obesity, but its misuse as a diet pill has led to fatal serious side effects. The factor limiting the use of DNP in increasing doses is the excessive rise in body temperature due to heat generated during uncoupling. Thus, an overdose of DNP will lead to fatal overheating, with a rise in body temperature to a fatally high temperature that leads to death. Because of the subsequent serious side effects, the clinical dose is slowly escalated according to the patient's tolerance. Therefore, the design of a prodrug of 2,4-DNP with better PK will help to deliver lower doses of 2,4-DNP to the body and prevent overdosing. The II-38 prodrug was synthesized as a drug delivery system, with the ultimate goal of achieving the release of its parent drug (2,4-DNP) in live plasma. This study investigated the in vitro chemical and enzymatic stability of II-38, and the pharmacokinetic (PK) profile of II-38 and its parent compound (i.e., 2,4-DNP released from II-38).

[0200] Example 1: In vitro chemical and enzymatic stability study of II-38

[0201] To determine the stability of the II-38 prodrug in chemical buffers and simulated gastric and intestinal fluids adjusted to pH 1.2 and 7.4, and in rat and human plasma over a 24 h time course. When orally administered to a patient, the prodrug is exposed to a wide range of pHs. Oral dosing exposes the compound to pH 1-2 in the stomach, pH 4.5 at the beginning of the small intestine, pH 6.6 as the average pH of the small intestine, and pH 5-9 in the colon. These are the pHs that can be used to evaluate the chemical stability of a prodrug candidate such as II-38 in vitro. The following experiments were performed in aqueous solutions to determine the stability of the II-38 prodrug in the GI tract and susceptibility to enzymatic hydrolysis to the parent drug (2,4-DNP) in plasma:

[0202] pH: Stability in aqueous buffers (37 ᴏ C, pH 1.2 and 7.4)

[0203] GI: Stability in simulated gastric fluid (USP, 37 ᴏ C)

[0204] GI: Stability in simulated intestinal fluid (USP, 37 ᴏ C)

[0205] Plasma: Stability in rat and human plasma (37 ᴏ C)

[0206] HPLC chromatographic conditions

[0207] HPLC analysis was performed on an Agilent 1200 Infinity Series Quatpump equipped with a photodiode array detector and computer integration device. An Inertsil ODS-3 column, C18, 5 pm, 4.6 x 50 mm (GL Science Inc. Japan) protected by a guard column: Alltima C18, 5 pm, 4.6 x 7.5 mm (Grace Discovery Sciences, IL, USA) was used as stationary phase. An isocratic method with a mobile phase consisting of water / acetonitrile (70:30) each containing 0.05% formic acid was used. A flow rate of 0.9 mL / min was used, and detection was performed at a UV wavelength of 250 nm.

[0208] Stability of II-38 in hydrochloric acid (HCI) buffer pH 1.2

[0209] A hydrochloric acid pH 1.2 buffer was prepared. II-38 (2.5 mg) was dissolved in 0.125 mL of DMSO (5%). The reaction was initiated by slowly and gradually adding II-38 dissolved in DMSO to a total volume of 2.375 mL of HC1 buffer pH 1.2 (Table 1) in a Franz cell diffusion chamber pre-heated to 37°C. The solution was mixed every half hour for 30 seconds using a magnetic stirrer. Samples of 20 pL (20 pg / mL of II-38) were collected at 0, 1, 2, 4, 6, and 8 h. Each sample was mixed with acetonitrile, QS to 1 mL, vortexed for 30 seconds and centrifuged at 10,000 rpm at room temperature. A volume of 10 pL of supernatant was injected into the HPLC spectrometer to detect 2,4-DNP and II-38 in HC1 buffer pH 1.2.

[0210] Table 1: Formulation of II-38 in HC1 buffer pH 1.2

[0211]

[0212] As shown in Figure 8, no significant II-38 hydrolysis occurred within 8 h, as shown by the single chromatogram eluting at 8.04 min. The slope seen between 0 and 2 h represents the equilibrium (solubility) phase of II-38 in HC1 buffer. Figure 5 Figure 6

[0213] Stability of II-38 in phosphate buffer pH 7.4

[0214] ​​Formulation of II-38 in Phosphate Buffer (PB): To formulate 1 mg / mL of II-38 / PB, 2.6 mg of II-38 was formulated in 0.13 mL of DMSO (i.e. 5%) and added slowly and gradually to a pre-heated PB to a total volume of 2.47 mL in a Franz cell diffusion chamber pre-heated to 37°C (Table 2). The solution was mixed every half hour for 30 seconds using a magnetic stirrer. Samples of 20 μΐ^ (20 μg / mL of II-38) were collected at 0, 0.5, 1, 2, 4, 6 and 8 h. Each sample was mixed with acetonitrile, QS to 1 mL, vortexed for 30 seconds and centrifuged at 10,000 rpm at room temperature. A volume of 10 μΐ^ of supernatant was injected into the HPLC spectrometer.

[0215] Table 2: Formulation of II-38 in PB

[0216]

[0217] As shown in Figures Figure 7 , 8 there was no significant hydrolysis of II-38 in PB over 8 h, as shown by the single chromatogram eluting at 8.023 min.

[0218] Stability of II-38 in simulated gastric fluid (SGF) pH 1.2

[0219] To formulate 1 mg / mL of II-38 / SGF, 3.1 mg of II-38 was dissolved in 0.155 mL of DMSO (i.e. 5%) and added slowly and gradually to a pre-heated SGF to a total volume of 2.945 mL in a cell diffusion chamber pre-heated to 37°C (Table 3). The solution was mixed every half hour for 30 seconds using a magnetic stirrer. Samples of 20 μΐ^ (20 μg / mL of II-38) were collected at 0, 1, 2, 4, 6 and 8 h. Each sample was mixed with acetonitrile, QS to 1 mL, vortexed for 30 seconds and centrifuged at 10,000 rpm at room temperature. A volume of 10 μΐ^ of supernatant was injected into the HPLC spectrometer.

[0220] Table 3: Formulation of II-38 in SGF pH 1.2

[0221]

[0222] As shown in Figures Figure 9 , 10A and 10B, II-38 was stable in SGF, suggesting in vivo stability of II-38 under acidic conditions in gastric fluids for increased systemic bioavailability.

[0223] Stability of II-38 in simulated intestinal fluid (SIF) pH 6.8

[0224] Formulation of II-38 in SIF buffer pH 6.8: To prepare 1 mg / mL of II-38 / SIF, 2.6 mg of II-38 was dissolved in 0.13 mL of DMSO (i.e. 5%) and added slowly and gradually to a total volume of 2.47 mL of pre-heated SIF (Table 4) in a cell diffusion chamber pre-heated to 37 °C. The solution was mixed every half hour for 30 seconds using a magnetic stirrer. Samples of 20 μL (20 μg / mL of II-38) were collected at 0, 0.5, 1, 2, 4, 6 and 8 h Figure 12A and 12B ). Each sample was mixed with acetonitrile, QS to 1 mL, vortexed for 30 seconds and centrifuged at 10,000 rpm at room temperature. A volume of 10 μL of supernatant was injected into the HPLC spectrometer. Figure 11

[0225] Table 4: Formulation of II-38 in SIF

[0226]

[0227] Stability of II-38 in rat and human plasma

[0228] Formulation of II-38 in rat plasma: To prepare 1 mg / mL of II-38 / rat plasma, 2.4 mg of II-38 was prepared in DMSO (5%) and added slowly and gradually to a total volume of 2.28 mL of pre-mixed rat plasma (80% + PBS 15%) in a Franz cell diffusion chamber pre-heated to 37 °C. The solution was mixed every half hour for 30 seconds using a magnetic stirrer. Samples of 20 μL (20 μg / mL of II-38) were collected at 0, 1, 3, 4, 5, 20, 23 and 24 h Figure 13 and 14 ). Each sample was mixed with acetonitrile, QS to 1 mL, vortexed for 30 seconds and centrifuged at 10,000 rpm at room temperature. A volume of 10 μL of supernatant was injected into the HPLC spectrometer.

[0229] Table 5: Formulation of II-38 in rat plasma

[0230]

[0231] Detection of 2,4-DNP and II-38 in human plasma using HPLC

[0232] ​Formulation of II-38 in human plasma: To prepare 1 mg / mL of II-38, 2.3 mg of II-38 in DMSO (5%) was slowly and gradually added to a pre-mixed human plasma (80% + PBS 15%, see Table 6) in a Franz cell diffusion chamber pre-heated to 37°C to a total volume of 2.185 mL. The solution was mixed every half hour for 1 minute using a magnetic stirrer. Samples of 20 μL (20 μg / mL of II-38) were collected at 0, 0.5, 1, 2, 4, 6, and 8 h. Each sample was mixed with acetonitrile, QS to 1 mL, vortexed for 30 seconds and centrifuged at 10,000 rpm at room temperature. A volume of 10 μL of supernatant was injected into the HPLC spectrometer.

[0233] Table 6: Formulation of II-38 in human plasma

[0234]

[0235] SUMMARY

[0236] The data from the stability studies of II-38 in different enzyme-free aqueous buffers clearly indicate that II-38 does not show significant hydrolysis in acidic and basic buffer media. It can be concluded that II-38 is sufficiently stable in the Gl tract when administered orally to be absorbed as a single chemical entity. However, the hydrolysis of II-38 in 80% rat and human plasma predicts that II-38 will be hydrolyzed in systemic circulation to produce its parent drug, 2,4-DNP. The carbamate linkage in II-38 is cleaved in plasma to produce 2,4-DNP at a slow rate, which allows II-38 to be used as a chemical reservoir of 2,4-DNP for a long period of time.

[0237] Example 2: Pharmacokinetic analysis of 2,4-DNP and II-38

[0238] The oral bioavailability and plasma pharmacokinetic (PK) properties of II-38 prodrug and 2,4-DNP released from II-38 prodrug were investigated in Sprague-Dawley (S-D) male rats after a single i.v. injection of II-38 at 1.6 mg / kg (equivalent to 1 mg / kg of 2,4-DNP) and oral doses equivalent to 5, 25, and 50 mg / kg of 2,4-DNP. Blood samples were obtained up to 24 hours.

[0239] Animals

[0240] All procedures involving the use of rats were in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Guide for the Care and Use of Laboratory AnimalsThe guidelines described by the University of Arkansas for Medical Sciences (UAMS) Institutional Animal Care and Use Committee were followed. Male Sprague-Dawley rats weighing 250 - 300 grams were housed three per cage with ad libitum access to food and water in the Division of Laboratory Animal Resources, UAMS. Rats were anesthetized with isoflurane (2-5%) / oxygen (1.5-2.0 L / min) and surgically implanted with catheters in the jugular vein (i.v. dosing) and femoral vein (blood sample collection). Three - 4 days after surgery, rats were observed for signs of local infection at the surgical site, yellowing of the fur, presence of blood around the nose or eyes, signs of reduced appetite, and signs of reduced or absent excretory activity. After suitable evidence of recovery from surgery was obtained, IV or oral dosing was performed and blood samples (0.15 mL) were collected at 0, 5, 15, 30, 45, 60, 120, 240, and 480 min. Plasma samples were prepared and analyzed using LC / MS / MS spectroscopy.

[0241] 2,4-DNP dose preparation

[0242] The 2,4-DNP was dissolved in a phosphate buffered saline solution pH 7.4 containing 5% DMSO and 20% PEG-400 and filtered through a 0.2-μm filter. For the oral route, rats were gavaged with 5 mg / kg from a stock solution containing 0.625 / mL of 2,4-DNP in a total volume of 8 mL / kg. For i.v. injection, a dose of 1 mg / kg was used from a stock solution containing 1.25 mg / mL using a total volume of 0.8 mL / kg.

[0243] II-38 dose preparation

[0244] For i.v. injections, a total volume of 0.8 mL / kg was used at a dose of 1.6 mg / kg (equivalent to 1 mg / kg of 2,4-DNP). For the oral route, stock solutions of II-38 containing equivalent to 1, 5 and 25 mg / mL of 2,4-DNP were prepared and rats were administered with final doses equivalent to 1, 5 and 25 mg / kg of 2,4-DNP, respectively. Oral doses of 8 mg / kg of II-38 (equivalent to 5 mg / kg) were dissolved in a phosphate buffer solution pH 7.4 containing 5% DMSO and 20% PEG-400, while 40 mg / kg (equivalent to 25 mg / kg) doses were formulated with 0.5% methocel containing 1 % DMSO. High doses of II-38 (equivalent to 50 mg / kg) were formulated with water containing 1 % DMSO and 40% PEG-400. Because of the different vehicles used in the formulation of II-38, the dose equivalent to 25 mg / kg of 2,4-DNP was repeated using a vehicle composed of DMSO, PEG-400 and water (1 %: 40%: 59%). For formulation differences, the latter dose was used to compare the PK data of 2,4-DNP released from II-38.

[0245] Oral and i.v. animal experiments with 2,4-DNP

[0246] Sprague-Dawley male rats (weight 250-300 g) cannulated in the jugular and femoral vein were treated with 2,4-DNP at a single oral dose of 5 mg / kg (n = 4) or a single i.v. dose of 1 mg / kg (n = 3). For each i.v. and oral experiment, blood samples (0.15 mL) were collected at 0, 5, 15, 30, 45, 60, 120, 240 and 480 min. The withdrawn blood was replaced with heparinized saline (0.15 mL). Blood samples were centrifuged at 10,000 rpm for 10 min at room temperature and plasma samples were prepared and analyzed using a sensitive LC / MS / MS spectroscopy (see below).

[0247] Oral and i.v. animal experiments with II-38

[0248] Sprague-Dawley male rats (weight 250-300 g, n = 4) cannulated in the jugular vein and femoral vein were treated with II-38 at single oral doses of 8, 40 and 80 mg / kg (equivalent to 5, 25 and 50 mg / kg of 2,4-DNP, respectively) or injected with a single i.v. dose of 1.6 mg / kg of II-38 (equivalent to 1 mg / kg of 2,4-DNP, n = 2). Blood samples (0.15 mL) were collected at 0, 5, 15, 30, 45, 60, 120, 240 and 480 min. Plasma samples were prepared and concentrations of 2,4-DNP, II-38 and 2,4-DNP released from II-38 were quantified using the LC / MS / MS spectroscopy assay described below.

[0249] Preparation of plasma samples and extraction procedure

[0250] To isolate the analytes from rat plasma, 50 μΐ^of control or treated plasma were fortified with 10 μΐ^of 2,4-DNP-d3 (10 μg / mL) as an internal standard, to which 0.3 mL of methanol followed by 0.3 mL of acetonitrile were added. The mixture was vortex-mixed for 30 s and centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was transferred to a 5 mL glass tube and evaporated to dryness at 37 °C under nitrogen. The pellet was reconstituted with 50 μΐ^of acetonitrile, vortex-mixed for 30 s followed by sonication for 1 min. After centrifugation at 1000 rpm for 10 min at room temperature, 5 μΐ^of the supernatant was injected onto the column and the analytes were quantified by LC / MS / MS spectroscopy.

[0251] LC / MS / MS analysis

[0252] A sensitive liquid chromatography / tandem mass spectrometry (LC / MS / MS) assay was used to quantify 2,4-DNP and II-38 in rat plasma as follows:

[0253] 1- Plasma concentrations of 2,4-DNP following a single intravenous injection of 1 mg / kg or oral administration of 5 mg / kg; 2- Plasma concentrations of II-38 and 2,4-DNP released from II-38 following an intravenous injection of 1.6 mg / kg II-38 (equivalent to 1 mg / kg 2,4-DNP) or oral doses of 8, 40, and 80 mg / kg II-38 (equivalent to 5, 25, and 50 mg / kg 2,4-DNP, respectively). The mass spectrometer used was an Agilent quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. 2,4-DNP-d3 was used as an internal standard. 2,4-DNP, II-38, and 2,4-DNP-d3 were separated using an Alltima C18 column equipped with a guard column: Alltima C18, 5 µm, 4.6 x 7.5 mm (Grace Discovery Sciences, IL, USA), or an Alltima C18 column, 5 µm, 3.2 x 150 mm (Grace Discovery Sciences, IL, USA). The mobile phase consisted of water containing 0.005% formic acid as solvent A and acetonitrile containing 0.005% formic acid as solvent B. For the analysis of 2,4-DNP, II-38, and 2,4-DNP-d3 compounds, separation was performed using a gradient of 10–90% solvent B for 3.5 min, followed by 3.30 min at 90% B, and then equilibration back to the initial conditions for 3.20 min. The flow rate was 0.8 mL / min, and the column temperature was 30°C. The sample injection volume was 5 µL. The mass spectrometer was operated in negatively charged jet ionization mode with the optimal ion source settings determined using standards of 2,4-DNP, II-38, and 2,4-DNP-d3, at a collision energy of 15 V and a fragmentation energy of 75 V. MRM transition monitoring was performed as follows: 2,4-DNP- m / z 183.0 / 123.0, m / z 183.0 / 153.0, for 2,4-DNP-d3- m / z 186.0 / 126.0, m / z 186.0 / 156.0 and for II-38- m / z 297.2 / 183.2. Two separate standard curves were generated for 2,4-DNP and II-38 in rat plasma, and used to quantify 2,4-DNP and II-38, respectively.

[0254] Standard curve of 2,4-DNP in acetonitrile (ACN)

[0255] A stock solution of 2,4-DNP was prepared in acetonitrile. Eight standard curves were prepared at different concentrations. Calibration curves were obtained using quadratic least square regression of the area under the curve (AUC) against the 2,4-DNP concentration. The curves were linear in the range of 0.01–1 µg / mL, with a correlation coefficient R0. 2 = 0.9874 ( Figure 15 ).

[0256] Standard curve of 2,4-DNP and II-38 in plasma

[0257] Stock solutions of 2,4-DNP, II-38, and the internal standard (2,4-DNP-d3) were prepared in acetonitrile. Serially diluted solutions of 2,4-DNP and II-38 were prepared, and LC / MS / MS chromatograms of 2,4-DNP and II-38 calibration standards were generated for each analyte (n = 5) in the concentration range of 1–2000 ng / mL plasma. The calibration curves and chromatograms showed excellent linearity for 2,4-DNP and II-38 in the range of 20–2000 ng / mL and 200–1000 ng / mL, respectively, with correlation coefficients R0 for 2,4-DNP and II-38, respectively. 2 = 0.9988 and 0.9739 ( Figure 16 and 17 ).

[0258] Pharmacokinetics in plasma

[0259] Following intravenous bolus administration, plasma concentration-time curves for individual 2,4-DNP and II-38 prodrugs were calculated using a single-compartment open model and first-order elimination (Phoenix WinNonlin, Professional, version 6.2, Pharsight, Mountain View, CA).

[0260] Following oral administration, peak concentration (C0) was determined using a non-compartmental open model and first-order absorption analysis data. max ), T max and the area under the curve (AUC) from 0 to infinity 0-inf The actual apparent bioavailability of 2,4-DNP, II-38 prodrug, and 2,4-DNP released from II-38 was determined using Equation 1:

[0261]

[0262] AUC oral AUC i.v Dose oral and Dose i.v.The values ​​represent the AUC for oral and intravenous administration of 2,4-DNP or II-38 prodrugs, respectively. 0-inf And corresponding doses. The bioavailability of 2,4-DNP released from II-38 was obtained using Equation 1, from the AUC of 2,4-DNP released from II-38 by oral and intravenous administration.

[0263] The total apparent bioavailability of the II-38 prodrug was estimated in the same manner using AUC data for both the II-38 prodrug and 2,4-DNP. Equation 2 is a modified form of bioavailability expression that takes into account the time-dependent hydrolysis of the II-38 prodrug after administration to rats. The optimal estimate of total bioavailability also includes AUC data for 2,4-DNP released in plasma.

[0264] The release of 2,4-DNP from the II-38 prodrug occurs in an equimolar ratio. Since the prodrug is hydrolyzed to release 2,4-DNP at a 1:1 ratio, it is concluded that an equal amount of II-38 is hydrolyzed to form 2,4-DNP. The calculation of the oral bioavailability of the prodrug involves two different measures. The actual apparent bioavailability considers the time-dependent hydrolysis of the II-38 prodrug. To estimate the total apparent bioavailability of II-38, both systemic exposure to the II-38 prodrug and the 2,4-DNP it releases must be considered. This can be calculated using Equation 2:

[0265]

[0266] Plasma concentrations of 2,4-DNP following oral and intravenous administration versus time Figure 18 Provided in [the table]. Following oral administration, 2,4-DNP is rapidly absorbed, as indicated by plasma concentrations at 5 min. For the intravenous and oral routes, the half-lives of 2,4-DNP are 90 ± 9.9 min and 136.2 ± 10.9 min, respectively (Table 7).

[0267] In SD rats, oral administration of the II-38 prodrug at doses equivalent to 5, 25 (methylcellulose), 25 (PEG-400), and 50 mg / kg of 2,4-DNP resulted in the delivery of 2,4-DNP with peak plasma concentrations of 3.2 ± 0.2, 9.4 ± 0.6, 9.1 ± 0.84, and 15.2 ± 2.7 µg / mL, respectively, within 120 ± 16.9, 161.3 ± 39.4, 240, and 180 ± 34.6 min (Table 8). Notably, the PK profile of II-38 formulated in DMSO:PEG-400:water (1:40:59) solvent was completely comparable to that of the methylcellulose formulation.

[0268] Table 7: PK profile of 2,4-DNP after treatment with a single intravenous dose of 1 mg / kg (n = 3 rats) or a single oral dose of 5 mg / kg (n = 4 rats).

[0269] Route 2,4-DNP, i.v. 2,4-DNP, oral Parameters (units) Mean ± SEM Mean ± SEM AUC (min*pg / mL) 1345.9 ± 591.2 13288 ± 1634.1 t 1 / 2 (min)]]> 90 ± 9.9 136.2 ± 10.9 C max ( 0.1-10 µg / mL 11.5 ± 6.2 50.1 ± 7.4 t max (min)]]> 37.5 ± 3.8 Cl (mL / min / kg) 1.2 ± 0.6 Vss (mL / kg) 160.8 ± 135.1

[0270] Figure 18 Plasma concentrations of 2,4-DNP over time following a single IV injection or oral dose. Figure 19 , 20 21 and 22 show the mean plasma concentrations versus time profiles of II-38 and 2,4-DNP released from its II-38 prodrug after oral administration of equal doses of 5, 25 (methylcellulose), 25 (PEG-400), and 50 mg / kg of 2,4-DNP, respectively. Figure 23 The mean plasma concentrations and times of 2,4-DNP release from II-38 following oral administration of 5 mg / kg of 2,4-DNP were compared with those following oral administration of 8, 40 (methylcellulose), 40 (PEG-400), and 80 mg / kg of II-38 [equivalent to 5, 25 (methylcellulose), 25 (PEG-400), and 50 mg / kg of 2,4-DNP], n = 4. The carbamate bonds of the II-38 prodrug underwent significant in vivo cleavage to produce 2,4-DNP as a hydrolysis product of the II-38 prodrug. The bioavailability of 2,4-DNP released from II-38 and II-38 itself are provided in Tables 8, 9, and 10, respectively.

[0271] Table 8: PK profiles of 2,4-DNP released from II-38 after treatment with an oral dose of 5 mg / kg of 2,4-DNP and after treatment with a single oral dose of II-38 (equivalent to 5, 25 (methylcellulose), 25 (PEG=500), and 50 mg / kg of 2,4-DNP, respectively), n = 4 rats

[0272]

[0273] Table 8.1: PK profile of M101 released from M201 (M101-morpholine) following oral administration of M101 and treatment with a single oral dose of M201 (equivalent to 5, 25, and 50 mg / kg of M101, respectively).

[0274]

[0275] Table 9: PK profile of II-38 after treatment with a single oral dose of II-38 (equivalent to 5, 25 (methylcellulose), 25 (PEG=500), and 50 mg / kg of 2,4-DNP, respectively), n = 4 rats

[0276]

[0277] Table 10: PK profile of II-38 and 2,4-DNP released from II-38 after a single intravenous injection of 1.6 mg / kg II-38 (equivalent to 1 mg / kg 2,4-DNP), n = 2 rats

[0278]

[0279] Discussion

[0280] II-38 prodrug was synthesized as a drug delivery system with the ultimate goal of releasing the parent drug (2,4-DNP) into rat plasma. In vitro stability studies of II-38 (Chapter 1) provided no evidence of 2,4-DNP formation from II-38 within a 24-hour timeframe in chemical buffers adjusted to pH 1.2 and 7.4. Similarly, the II-38 prodrug was stable within a 24-hour timeframe in simulated gastric and intestinal fluids prepared using the USP method (neither II-38 nor 2,4-DNP are shown in the figure after 24 hours). Therefore, it is predictable that orally delivered II-38 will be absorbed into systemic circulation via the GI pathway as a stable entity and can be hydrolyzed under enzymatic conditions within systemic circulation to release 2,4-DNP into plasma.

[0281] II-38 was evaluated in vivo to investigate the PK profile and relative bioavailability of 2,4-DNP released from II-38, and to determine the time course of plasma levels of 2,4-DNP released from II-38 after a single oral dose of 8, 40, and 80 mg / kg of II-38 (equivalent to 5, 25, and 50 mg / kg of 2,4-DNP, respectively) or an intravenous bolus dose of 1.6 mg / kg (equivalent to 1 mg / kg of 2,4-DNP), and to compare these data with those of 2,4-DNP administered in rats as a single oral dose of 5 mg / kg or an intravenous injection of 1 mg / kg.

[0282] Mean concentrations and time profiles of 2,4-DNP released from II-38 prodrug and II-38 after oral administration. Figure 19 , 20(21 and 22) It can be noted that the carbamate linker in the prodrug undergoes hydrolysis to produce 2,4-DNP. The II-38 prodrug exhibits slow dissolution in plasma to its parent drug, 2,4-DNP, providing sustained release of 2,4-DNP. Notably, the hydrolysis of II-38 is incomplete, as the II-38 prodrug, along with 2,4-DNP, is observed at all time points following oral and intravenous administration. Figure 19 , 20 21 and 22).

[0283] Because II-38 contains an equimolar amount of 2,4-DNP, it is assumed that an equimolar amount of II-38 must have been hydrolyzed in plasma unless the carbamate bonds undergo cleavage in the intestine and / or liver to release 2,4-DNP. Alternatively, 2,4-DNP may undergo metabolism in plasma after formation from II-38. Nevertheless, predictions regarding the in vivo stability of the II-38 prodrug are correct, as significant levels of 2,4-DNP were observed in all rats after oral and intravenous administration, as shown by bioavailability of 58.5 ± 8.1, 62.8 ± 5.8, 41 ± 5.1, and 50.1 ± 11.5 for 2,4-DNP released from orally administered II-38 at doses equivalent to 5, 25 (methylcellulose), 25, (PEG-400), and 50 mg / kg, respectively (Table 8).

[0284] The single-compartment model describes the effect of intravenous administration of 2,4-DNP and II-38. Peak plasma levels of released 2,4-DNP (C2) were observed. max The detection of 2.2 ± 1.1 µg / mL within 5 min indicates rapid cleavage of II-38. A non-compartmental model is sufficient to describe the pharmacokinetic (PK) of orally administered II-38 prodrug and its released 2,4-DNP. This suggests that at any time point, the II-38 prodrug exists at concentrations lower than that of 2,4-DNP. Figure 19 , 20 Tables 21 and 22 and 8 and 9. Therefore, the absorption of the II-38 prodrug was shown to be very rapid, followed by rapid cleavage and sustained release of 2,4-DNP, as the latter was detected in plasma within 5 minutes after oral administration, and at t0.05 of 120–180 minutes. max The concentration of C reached 3.2 - 15.2 µg / mL. max For oral and intravenous administration of 2,4-DNP, the elimination half-life (t) is... 1 / 2The mean terminal half-lives were 136.3 ± 10.9 and 90.0 ± 9.9 min, respectively. These results are smaller than the extended mean terminal half-lives of 2,4-DNP released from II-38 at equivalent doses of 25 (methylcellulose), 25 (PEG-400), and 50 mg / kg (Table 2.2), which were 248.2 ± 45.3, 185.9 ± 18.1, and 334.1 ± 63.0 min, respectively, indicating that oral administration of II-38 resulted in a significant improvement in 2,4-DNP delivery compared to administration of 2,4-DNP alone.

[0285] Calculations showed that, compared with the 2,4-DNP released from its II-38 prodrug after oral administration of 2,4-DNP parent drugs equivalent to 5, 25 (methylcellulose), 25 (PEG-400), and 50 mg / kg, respectively, the levels of 2,4-DNP were increased by approximately 12.0-, 2.2-, 3.4-, and 1.4-fold, respectively, after oral administration of the 2,4-DNP parent drug. Figure 23 (See Table 8). Data from intravenous and oral administration of II-38 do indeed suggest that 2,4-DNP released from the II-38 prodrug may undergo other enzymatic activities occurring in different tissue compartments (including the liver) that are not plasma. In rat plasma, the II-38 prodrug and the 2,4-DNP it releases are freely partitioned to other metabolic sites, whereas in vitro plasma studies rely solely on plasma enzymes. However, due to its slow release, there is an extended half-life of 141.7–334.1 min and a delayed t-cycle of 120–240 min. max The release of 2,4-DNP from its prodrug implies prolonged exposure to the released 2,4-DNP and fewer unexpected peak values. In other words, the 2,4-DNP released after oral administration of II-38 is suitable for inhibiting 2,4-DNP due to rapid absorption and high plasma C60 levels. max Acute side effects caused by this.

[0286] Conclusions

[0287] In this study, oral administration of the II-38 prodrug resulted in significant release and gain of 2,4-DNP from II-38 over time, leading to prolonged exposure (AUC) and significant bioavailability of 2,4-DNP. Clearly, the II-38 prodrug was formulated to be stable and rapidly hydrolyzed in rat plasma.

[0288] Example 3: PK profile of 2,4-DNP released from DNP-piperidine

[0289] Table 11: PK profile of 2,4-DNP released from DNP-piperidine following treatment with a single oral dose of DNP-piperidine (equivalent to 5 mg / kg of 2,4-DNP) prepared in DMSO:PEG-400:PBS (5:20:75).

[0290]

[0291] Example 4: Treatment of Hearing Loss

[0292] In vivo noise exposure pilot-scale DNP (2,4-DNP) and DNP prodrug (II-38): Rats, N=6 / group: control, noise only, noise + 2,4-DNP (5 mpk), noise + II-38 (80 mpk). Noise exposure: 8 h, 105 dB, noise band 8–16 kHz. Treatment lasted 5 days, with noise starting qd via oral feeding. Endpoint: Composite action potential (CAP) recorded from the round window membrane 1 week after exposure. Threshold: A dB SPL intensity required to produce a 4 μV CAP amplitude.

[0293] Result: As Figure 3 As shown, the noise threshold for II-38+ was 5 dB, approximately 20 dB lower (better) than the noise-only group. These results indicate that the DNP prodrug (II-38) can provide protection against noise-induced hearing loss. Furthermore, as... Figure 3 As shown, the DNP (2,4-DNP)+noise threshold is 15 dB, which is about 10 dB lower than noise alone, indicating that DNP (2,4-DNP) can provide protection against noise-induced hearing loss.

[0294] To understand this invention, a description of embodiments thereof has been provided above. It will be understood that the invention is not limited to the specific embodiments described herein, but various modifications, rearrangements, and substitutions can be made without departing from the scope of the invention, as will now be apparent to those skilled in the art. Therefore, the following claims are intended to cover all such modifications and variations that fall within the true spirit and scope of the invention.

Claims

1. A composition for treating neurodegenerative diseases, autoimmune diseases, hearing loss related to aging, noise, drug-induced and / or hereditary hearing loss, metabolic diseases and / or Wolfram syndrome, said composition comprising a dinitrophenol (DNP) prodrug represented by the following formula: 。 2. The composition of claim 1, wherein the prodrug comprises a prodrug connector portion that imparts water-soluble properties to the prodrug molecule.

3. The composition of claim 2, wherein the water-soluble prodrug moiety is introduced into the molecule by conjugation with a free phenolic functional group.

4. The composition of any one of claims 1-3, wherein the prodrug is stable at pH 1-2, pH 4.5, or pH 5-9.

5. The composition of any one of claims 1-4, wherein the composition is administered orally.

6. The composition of any one of claims 1-5, wherein the neurodegenerative disease, autoimmune disease, hearing loss due to aging, noise, drug-induced and / or hereditary hearing loss, and / or metabolic disease is selected from multiple sclerosis, lupus, myasthenia gravis, Graves' disease, Graves-Bartholin's syndrome, muscular dystrophy, ataxia, Barthon's disease or neuronal ceroid lipofuscinosis, Alzheimer's disease, optic neuritis, Leber's hereditary optic neuropathy, autism spectrum disorder, Parkinson's disease, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss and / or hereditary hearing loss, mitochondrial disease, sleep apnea disease, hypertension, kidney disease, hypertension, obesity, inflammatory diseases, and diseases leading to knee complications.

7. The composition of claim 6, wherein Multiple sclerosis is selected from relapsing-remitting multiple sclerosis, secondary-progressive MS, primary-progressive MS, and progressive-relapsing MS. Lupus is selected from systemic lupus erythematosus, discoid lupus, drug-induced lupus, and neonatal lupus. Myasthenia gravis is selected from ocular myasthenia gravis, congenital MG, and generalized myasthenia gravis. Muscular dystrophy is selected from Duchenne muscular dystrophy, Beck muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Ehrlich-Deutsche muscular dystrophy, facial-shoulder-humeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, and oculopharyngeal muscular dystrophy. Ataxia is selected from Friedreich's ataxia, spinocerebellar ataxia 1, spinocerebellar ataxia 2, spinocerebellar ataxia 3, spinocerebellar ataxia 6, spinocerebellar ataxia 7, spinocerebellar ataxia 11, dentate nucleus, rubra, globus pallidus, hypothalamic nucleus atrophy and glutenin ataxia. Baten disease, or neuronal ceroid lipofuscinosis, is selected from infantile neuronal ceroid lipofuscinosis, late-infant neuronal ceroid lipofuscinosis, adolescent neuronal ceroid lipofuscinosis, and adult neuronal ceroid lipofuscinosis. Alzheimer's disease is selected from early-onset Alzheimer's disease, late-onset Alzheimer's disease, and familial Alzheimer's disease. Autism spectrum disorders are selected from Asperger's syndrome, generalized developmental delay, childhood disintegrative disorder, and autism. Parkinson's disease is selected from idiopathic Parkinson's disease, vascular Parkinson's syndrome, Lewy body dementia, hereditary Parkinson's disease, drug-induced Parkinson's syndrome, juvenile Parkinson's disease, and atypical Parkinson's syndrome. The noise that causes hearing loss is selected from explosions and high noise levels, and / or Metabolic syndrome is characterized by increased blood pressure, elevated blood sugar levels, excess waist circumference fat, and abnormal cholesterol levels.

8. Use of the composition in the preparation of medicaments for treating neurodegenerative diseases, autoimmune diseases, hearing loss due to aging, noise, drug-induced and / or hereditary hearing loss, metabolic diseases and / or Wolfram syndrome, The composition said composition comprises a dinitrophenol (DNP) prodrug represented by the following formula: ;and The dosage of the composition is: about 0.01 mg / kg of the patient's body weight to about 25 mg / kg of the patient's body weight; or about 0.01 mg / kg of the patient's body weight to about 50 mg / kg of the patient's body weight; or about 1 mg / 70 kg of the patient's body weight to about 200 mg / 70 kg of the patient's body weight.

9. The use of claim 8, wherein the neurodegenerative disease, autoimmune disease, hearing loss related to aging, noise, drug-induced and / or hereditary hearing loss, and / or metabolic disease is selected from: multiple sclerosis, lupus, myasthenia gravis, Graves' disease, Graves-Bartholin's syndrome, muscular dystrophy, ataxia, Barthon's disease or neuronal ceroid lipofuscinosis, Alzheimer's disease, optic neuritis, Leber's hereditary optic neuropathy, autism spectrum disorder, Parkinson's disease, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss and / or hereditary hearing loss, mitochondrial disease, sleep apnea disease, hypertension, kidney disease, hypertension, obesity, inflammatory diseases, and diseases leading to knee complications.

10. The use of claim 9, wherein Multiple sclerosis is selected from relapsing-remitting multiple sclerosis, secondary-progressive MS, primary-progressive MS, and progressive-relapsing MS. Lupus is selected from systemic lupus erythematosus, discoid lupus, drug-induced lupus, and neonatal lupus. Myasthenia gravis is selected from ocular myasthenia gravis, congenital MG, and generalized myasthenia gravis. Muscular dystrophy is selected from Duchenne muscular dystrophy, Beck muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Ehrlich-Deutsche muscular dystrophy, facial-shoulder-humeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, and oculopharyngeal muscular dystrophy. Ataxia is selected from Friedreich's ataxia, spinocerebellar ataxia 1, spinocerebellar ataxia 2, spinocerebellar ataxia 3, spinocerebellar ataxia 6, spinocerebellar ataxia 7, spinocerebellar ataxia 11, dentate nucleus, rubra, globus pallidus, hypothalamic nucleus atrophy and glutenin ataxia. Baten disease, or neuronal ceroid lipofuscinosis, is selected from infantile neuronal ceroid lipofuscinosis, late-infant neuronal ceroid lipofuscinosis, adolescent neuronal ceroid lipofuscinosis, and adult neuronal ceroid lipofuscinosis. Alzheimer's disease is selected from early-onset Alzheimer's disease, late-onset Alzheimer's disease, and familial Alzheimer's disease. Autism spectrum disorders are selected from Asperger's syndrome, generalized developmental delay, childhood disintegrative disorder, and autism. Parkinson's disease is selected from idiopathic Parkinson's disease, vascular Parkinson's syndrome, Lewy body dementia, hereditary Parkinson's disease, drug-induced Parkinson's syndrome, juvenile Parkinson's disease, and atypical Parkinson's syndrome. The noise that causes hearing loss is selected from explosions and high noise levels, and / or Metabolic syndrome is characterized by increased blood pressure, elevated blood sugar levels, excess waist circumference fat, and abnormal cholesterol levels.

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