Compositions for improving cell viability and methods of use thereof

Abstract

To provide a pharmaceutical composition for use in improving cell viability by reducing mitochondrial dysfunction in a cell.SOLUTION: The present invention provides a pharmaceutical composition for use in a method of reducing mitochondrial dysfunction in a subject, the method comprising administering to the subject about 10 mg / kg body weight to about 30 mg / kg body weight of tauroursodeoxycholic acid (TUDCA) and about 30 mg / kg body weight to about 100 mg / kg body weight of sodium phenylbutyrate.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Patent Application No. 14 / 140,083, filed December 24, 2013, and Provisional Patent Application No. 61 / 804,690, filed March 24, 2013, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present invention relates to a method and composition for use in improving cell viability, particularly neuronal cell viability, more particularly to a method and composition for use in improving cell viability through reducing reactive oxygen metabolite-mediated oxidative damage in cells, regulating redox homeostasis in cells, or reducing mitochondrial dysfunction in cells.The present invention relates to the field of drug therapy, more particularly to the treatment of Alzheimer's disease and other amyloidosis-related pathologies. [Background technology]

[0003] Neurodegenerative diseases of the central nervous system (CNS) cause progressive loss of neuronal structure and function and are devastating for affected patients and their families. Among these neurodegenerative diseases are, for example, multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS) and stroke. Due to the complexity of the CNS, many of these diseases are unfortunately poorly understood to date.

[0004] Alzheimer's disease is the most common neurodegenerative disease and one of the largest medical problems in the United States. In 2012, an estimated 5.4 million Americans were affected by the disease, making it the sixth leading cause of death. Because aging is the greatest risk factor for Alzheimer's disease, the number of patients is expected to swell to 7.1 million by 2025 as the elderly American population grows. Other risk factors include certain genetic mutations, diabetes, and inflammation.

[0005] Alzheimer's disease is characterized by the aggregation of amyloid beta into plaques and the formation of neurofibrillary tangles mediated by various forms of phosphorylated tau protein. Some of the major symptoms of the disease include memory loss, difficulty completing and planning daily tasks, confusion about time or place, problems with language or speech, and personality changes.

[0006] Alzheimer's disease is the most common of a broad class of dementias, many of which are believed to be mediated by the formation of amyloid plaques, amyloid oligomers, and / or phosphorylated tau protein. These diseases include, but are not limited to, Pick's disease, multi-infarct dementia, Creutzfeldt-Jakob disease, dementia with Lewy bodies, mixed dementia, and frontotemporal dementia.

[0007] Currently approved drugs used to treat Alzheimer's disease either block NMDA-type glutamate receptors or are acetylcholinesterase inhibitors, the latter of which are only moderately effective in only 50% of patients and only under certain cognitive tests for about 6-12 months. Both classes of drugs are based on a model of increasing neural excitation generally to the depressed brain, are prone to many side effects, and do nothing to alter the pathology of the disease. Although several classes of drugs are known or proposed to treat neurodegenerative diseases, effective therapies are few or nonexistent. Therefore, improved therapies for treating neurodegenerative diseases are needed. Summary of the Invention

[0008] At least in part, the present invention is based on the discovery that a composition comprising a bile acid (e.g., tauroursodeoxycholic acid (TUDCA)) in combination with a phenylbutyric acid (PBA) (e.g., 4-phenylbutyric acid (4-PBA)) significantly reduces reactive oxygen metabolite-mediated oxidative damage in cells, resulting in improved cell viability (e.g., neuronal cell viability). As discussed in the following examples, the compounds TUDCA and 4-PBA were evaluated individually and in combination as protective agents against hydrogen peroxide-induced cell apoptosis. Hydrogen peroxide-induced cell apoptosis is believed to be caused through alterations in redox homeostasis, overproduction of reactive oxygen species, and mitochondrial dysfunction. The examples demonstrate that TUDCA and 4-PBA have a greater additive effect in protecting cells from hydrogen peroxide exposure, as measured by cell viability and / or cell death. This surprising discovery suggests that these drugs synergistically increase each other's effectiveness in alleviating the aforementioned pathologies.

[0009] The present invention provides a method and composition for use in improving cell viability, particularly the viability of neural cells, more particularly by reducing reactive oxygen metabolite-mediated oxidative damage in cells, regulating redox homeostasis in cells or reducing mitochondrial dysfunction in cells, and improving cell viability.The present invention further relates to the combined administration of bile acid (e.g., tauroursodeoxycholic acid (TUDCA)) and phenylbutyric acid (e.g., 4-PBA) for improving cell viability and treating at least one symptom associated with oxidative stress-related disease, preventing the onset of such disease or delaying the onset of such disease.

[0010] The present invention further provides a novel approach for reducing oxidative stress in neuronal cells and for treating at least one symptom associated with a disease associated with oxidative stress, including, but not limited to, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis, Pick's disease, multi-infarct dementia, Creutzfeldt-Jakob disease, dementia with Lewy bodies, frontotemporal dementia), for preventing the onset time of onset or delaying the onset of such diseases, comprising the administration of a composition comprising a combination of a bile acid (e.g., tauroursodeoxycholic acid (TUDCA)) and a phenylbutyric acid (e.g., 4-PBA), or an analog, derivative, pharmacological equivalent or salt thereof, in a pharmaceutical composition or formulation.

[0011] In one aspect, the present disclosure provides a method for reducing reactive oxygen metabolite-mediated oxidative damage in cells, comprising contacting cells with a bile acid or a pharmaceutically acceptable salt, analog, derivative or prodrug thereof and phenylbutyric acid (PBA) or a pharmaceutically acceptable salt, analog, derivative or prodrug thereof. In one aspect, the reactive oxygen metabolite-mediated oxidative damage is hydrogen peroxide (H2O2)-mediated damage.

[0012] In another aspect, the disclosure provides a method of regulating redox homeostasis in a cell, the method comprising contacting the cell with a bile acid, or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof.

[0013] In yet another aspect, the disclosure provides a method of reducing mitochondrial dysfunction in a cell, the method comprising contacting the cell with a bile acid, or a pharmaceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharmaceutically acceptable salt, analog, derivative, or prodrug thereof.

[0014] In one or more embodiments, the bile acid is selected from the group consisting of tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, or an analog, derivative, or derivative thereof. In some embodiments, the bile acid is selected from the group consisting of tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA).

[0015] In some embodiments, the cells are contacted with bile acids at a concentration of about 80 μM to about 120 μM.

[0016] In one or more embodiments, the PBA is 4-phenylbutyric acid, glyceryl (tri-4-PBA), phenylacetic acid, 2-POAA-OMe, 2-POAA-NO2, 2-NOAA, or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof.

[0017] In some embodiments, the cell is contacted with phenylbutyric acid at a concentration of about 0.8 mM to about 1.2 mM. In some aspects, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In other embodiments, the cell is a neuronal cell.

[0018] In certain aspects, the disclosure provides a method of treating a neurodegenerative disease associated with reactive oxygen metabolite-mediated oxidative damage in a subject, comprising identifying a subject experiencing a neurodegenerative disease associated with reactive oxygen metabolite-mediated oxidative damage, administering to the subject a bile acid, or a pharma-ceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharma-ceutically acceptable salt, analog, derivative, or prodrug thereof, wherein the amount of PBA administered in combination with the bile acid is reduced by 10%-55% compared to administration of PBA alone.

[0019] In yet another aspect, the disclosure provides a method of treating a neurodegenerative disease in a subject in need of treatment, comprising identifying a subject having at least one copy of the APOE ε4 allele, administering to the subject a composition comprising a bile acid, or a pharma-ceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharma-ceutically acceptable salt, analog, derivative, or prodrug thereof, thereby treating the neurodegenerative disease.

[0020] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the invention will become apparent from the following detailed description and figures, and from the claims. [Brief description of the drawings]

[0022] [Figure 1]FIG. 1 is a graph demonstrating the ability of UDCA and PBA in combination to increase cell viability as assessed by PrestoBlue following hydrogen peroxide exposure in rat cortical neuronal cultures. **** denotes significance of p<.0001 vs. control. ## denotes significance of p<.01 vs. hydrogen peroxide exposure. # denotes significance of p<.05 vs. hydrogen peroxide exposure. The combination provides significant protection, but each drug individually does not. NMDA exposure at 300 μM is used as a negative control. DFX (not shown) was not included in the plot as it appeared to interfere with the PrestoBlue measurements and produced erratic results. [Diagram 2] FIG. 2 is a graph demonstrating the ability of combined TUDCA and PBA to ameliorate cell death as assessed by LDH following hydrogen peroxide (H2O2) exposure in rat cortical neuronal cell cultures. **** denotes significance of p<10-4 vs. control. ## denotes significance of p<.01 vs. hydrogen peroxide exposure. # denotes significance of p<.05 vs. hydrogen peroxide exposure. The combination provides significant protection, but each drug individually does not. NMDA exposure at 300 μM is used as a negative control, while DFX is the positive control in the LDH study. [Diagram 3] FIG. 3 demonstrates the chemical structure of TUDCA (Formula I) with labeled carbons to aid in understanding the positions at which substitutions can be made. [Figure 4] FIG. 4 demonstrates the chemical structure of UDCA (Formula II) with labeled carbons to aid in understanding the positions at which substitutions can be made. [Diagram 5] Figure 5 demonstrates the chemical structure of sodium ion stabilized PBA (Formula III). Derivatives available for use under the present invention are described in the Background. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Diabetes, inflammation, aging, certain genetic mutations and many other conditions are risk factors for Alzheimer's disease. The common thread between these conditions is that they are all related to increased production of free radicals, imbalance in redox homeostasis and / or increased free radical-mediated damage to cells and tissues. Superoxide and other free radicals have been implicated in increasing the amount of pathogenic amyloid proteins and in agglomeration to increase amyloid plaques, oligomers and other species. Without being limited by theory, some have suggested that this aggregation is due to a process involving oxidation of amino acids in the amyloid chains, particularly the methionine 35 residue.

[0024] The incorporation of amyloid proteins into mitochondria has been implicated in the release of cytochrome c, oxygen radicals, and other free radicals, which have been shown to increase the endoplasmic reticulum stress response and also increase the aggregation of amyloid proteins.

[0025] Recent reports suggest that reactive oxygen metabolites may be involved in the pathology of Alzheimer's disease. Oxygen radicals as well as lipid and nitrogen radicals are precursors in the apoptotic pathways mediated by BAX, cytochrome c and JNK. Evidence also suggests that changes in the redox state resulting from these species cause increased proteolytic activity, which is critical in the pathology of many diseases caused at least in part by abnormal protein processing. Changes in the cellular redox state also cause a decrease in key antioxidant species, such as glutathione (GSH) and vitamin E.

[0026] Reactive oxygen metabolite-mediated disorders Recent studies have also begun to implicate reactive oxygen metabolite-mediated disorders as one of the major factors in neurodegenerative diseases. For example, hydrogen peroxide has been shown to affect mitochondria, cellular proteolytic status, amyloid-β production, redox homeostasis and many apoptotic pathways in cells. Hydrogen peroxide has also been shown to react with metal ions, e.g., iron and copper, via Fenton chemistry to form many reactive oxygen species. The reduction of antioxidants, e.g., vitamin E and glutathione, in multiple neurodegenerative pathologies suggests an increase in oxidative damage in these diseases.

[0027] PINK1 and Parkin (Parkinson protein 2, E3 ubiquitin protein ligase) are thought to regulate mitochondrial quality control, and mutations in these genes may be the cause of Parkinson's disease. Studies have shown that Parkin transcription increases after hydrogen peroxide is introduced into cells. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which induces Parkinson-like symptoms, also leads directly to the production of hydrogen peroxide. Some studies have demonstrated an increase of up to 90% in hydrogen peroxide levels in this disease.

[0028] In Alzheimer's disease, amyloid beta has been shown to directly produce hydrogen peroxide when in contact with metal ions, such as iron and copper, which are found in higher concentrations in the brains of patients with the disease. Hydrogen peroxide may also increase the processing of amyloid beta into its pathogenic form. Hydrogen peroxide and reactive oxygen-metabolite-mediated pathways are also believed to be the major pathways of cell death in this disease.

[0029] Huntington's disease also exhibits protein degradation products that may be caused by hydrogen peroxide, as well as widespread mitochondrial dysfunction. Increased 3-hydroxyquinoline has been implicated as a major pathological process in Huntington's disease and is known to directly induce hydrogen peroxide and hydroxyl radical production.

[0030] Hydrogen peroxide has also been shown to be a potential mediator in the pathology of ALS, and hydrogen peroxide scavengers are being considered as therapeutics in this field.

[0031] Regulation of redox homeostasis Redox homeostasis refers to the cell's attempt to manage reducing and oxidizing species in the cell to maintain a homeostatic redox state. Disruption of redox homeostasis can change the free energy requirements and allow processes to take place in the cell that do not occur under non-pathological conditions. Some processes that can be caused by a malfunction in regulating redox homeostasis include the aggregation of amyloid beta into plaques that characterize Alzheimer's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, senile plaques in Huntington's disease, plaques including amyloid plaques found in ALS, and tauopathy disorders. Under typical physiological conditions, these proteins do not aggregate, but it is believed that increased concentrations and atypical redox states alter the free energy of aggregation, ultimately leading to the formation of plaques observed in these pathologies. Altered redox states can also produce free radicals that interact with mitochondrial pathways to induce apoptosis. Thus, there is a need to develop drugs that can help regulate the redox state of cells. Because hydrogen peroxide induces a change in the redox state of cells, it can be used to cause redox-mediated injury to cells.

[0032] Mitochondrial dysfunction Mitochondrial dysfunction is widespread in neurodegenerative diseases. In Alzheimer's disease, cells have a severely reduced mitochondrial membrane potential, impaired mitochondrial glucose metabolism, and increased mitochondrial permeability. Mitochondria have been observed to mediate multiple apoptotic pathways, ultimately leading to neuronal cell death in Alzheimer's disease.

[0033] Both PINK1 and Parkin are mitochondrial quality control proteins. Mutations or absence of these proteins are strongly associated with Parkinson's disease. MPTP is a molecule used to induce the permanent symptoms of Parkinson's disease, acting through the disruption of mitochondrial complex I, leading to mitochondrial dysfunction, changes in the cellular redox state, and apoptosis.

[0034] Mutations in the Huntington gene and the resulting protein are believed to be the primary mediators of Huntington's disease and have been shown directly in cell culture to result in loss of mitochondrial membrane potential and reduced expression of critical oxidative phosphorylation genes. Huntington's disease pathology is also associated with a reduction in the number of mitochondria present in the central nervous system.

[0035] Mitochondrial mislocalization, impaired energy metabolism and apoptotic pathways are thought to mediate amyotrophic lateral sclerosis. Mitochondria from affected tissues have also been shown to overproduce reactive oxygen metabolites and leak them into the cytosol.

[0036] In many neurodegenerative diseases, mitochondria overproduce free radicals, cause a decrease in energy metabolism, increase permeability, decrease membrane potential, decrease antioxidants, leak metal ions into the cell, change the redox state of the cell, and drive the cell into a pro-apoptotic pathway. Thus, there is a need to develop drugs that can alter and attenuate the mechanisms of mitochondrial dysfunction.

[0037] APOE ε4 allele The genetics of Alzheimer's disease are complex. Mutations in at least four loci are associated with genetic susceptibility to AD (i.e., familial AD). Three genes are associated with early-onset AD: APP [β-amyloid precursor protein on chromosome 21], PS1 (presenilin 1) and PS2 (presenilin 2). The ε4 allele of the apolipoprotein E (APOE) gene on chromosome 19 is associated with late-onset AD. The association of APOE ε4 with AD appears to be strongest in individuals with onset before age 70 and weakens with age. Patients with at least one copy of the APOE ε4 allele have been shown to have particularly high levels of peroxidative metabolites and mitochondrial disorders. Treatment for this patient population may be particularly effective.

[0038] In certain aspects, the present disclosure contemplates a method of treating a neurodegenerative disease in a subject in need of treatment, comprising identifying a subject having at least a copy of the APOE ε4 allele, administering to the subject a composition comprising a bile acid, or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharma-ceutically acceptable salt, analog, derivative, or prodrug thereof, thereby treating the neurodegenerative disease.

[0039] In certain aspects, the invention provides methods comprising contacting a cell with a bile acid. As used herein, a "bile acid" (e.g., a water-soluble bile acid derivative, a bile salt, or an amine-conjugated bile acid) is a bile acid that is substituted with a 3α-hydroxyl group and is typically located at C6, C7, or C8 of the sterol nucleus. 12It refers to a naturally occurring surfactant having a nucleus derived from cholanic acid, optionally substituted with other hydroxyl groups at the 1-position. Bile acid derivatives include, but are not limited to, derivatives formed with hydroxyl and carboxylic acid groups of bile acids with other functional groups, including, but not limited to, halogen and amino groups. Soluble bile acids may include aqueous preparations of the free acid form of bile acids combined with one of HCl, phosphoric acid, citric acid, acetic acid, ammonia or arginine.

[0040] In certain aspects, the methods of the invention include contacting a cell with a bile acid or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof. In one or more embodiments, the bile acid is tauroursodeoxycholic acid (TUDCA), as shown in formula I (Figure 3), with labeled carbons to aid in understanding positions that may be substituted.

[0041] In one or more embodiments, the bile acid is ursodeoxycholic acid (UDCA), as shown in Formula II (Figure 4), with labeled carbons to aid in understanding positions that may be substituted.

[0042] Physiologically relevant bile acid derivatives include any combination of substitution of hydrogen at the 3- or 7-position of TUDCA or UDCA, changes in the stereochemistry of the hydroxyl group at the 3- or 7-position, and pharma- ceutically acceptable salts, solvates, or amino acid conjugates thereof.

[0043] In some embodiments, the bile acid is a TUDCA compound of formula IV [ka]

[0044] [Wherein, R is —H or C1-C4 alkyl; R1 is -CH2-SO3R3 and R2 is -H; or R1 is -COOH and R2 is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3 or -CH2-S-CH2-COOH; R3 is -H or a residue of a basic amino acid] or a pharma- ceutically acceptable analog, derivative, prodrug or mixture thereof.

[0045] In addition to TUDCA and UDCA, the present invention also contemplates the use of bile acids, including, for example, chenodeoxycholic acid (also referred to as "chenodiol" or "enoic acid"), cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, cholic acid, or analogs, derivatives or derivatives thereof.

[0046] In certain aspects, the present invention provides a method comprising contacting a cell with phenylbutyric acid. Phenylbutyric acid (PBA) (FIG. 4, sodium salt) is an HDAC2 (histone deacetylase 2) inhibitor. Incorporation of PBA and derivatives results in differential gene expression that has been shown to exert a variety of effects. PBA has also been shown to act as a chemical chaperone with a variety of effects. One effect is a reduction in the production of pathogenic amyloid proteins. Another effect is an increase in neuroplasticity. PBA has also been shown to improve bile excretion. However, PBA is known to be cytotoxic to several different types of cells.

[0047] Physiologically relevant phenylbutyric acid (PBA) species include any hydrogen-deuterium replacement. Any salts, solvates, conjugates and pharmacologically relevant compounds are considered to be physiologically relevant. All phenylbutyric acid derivatives described in the prior art are also considered to be physiologically relevant. Other HDAC2 inhibitors are also considered as substitutes for phenylbutyrate. Phenylacetic acid, the active metabolite of PBA, may also be considered as a substitute.

[0048] In one or more embodiments, the PBA compound is 4-phenylbutyric acid (4-PBA). 4-PBA is a low molecular weight aromatic carboxylic acid. 4-PBA is defined herein to include not only 4-phenylbutyric acid as the free acid, but also its derivatives and physiologically acceptable salts thereof. In particular, "4-phenylbutyric acid" or "4-PBA" is defined as the free acid, but also as a pharma- ceutically acceptable salt, co-crystal, polymorph, hydrate, solvate, or prodrug form of 4-phenylbutyric acid. Most preferably, "4-phenylbutyric acid" or "4-PBA" is either the free acid or a pharma-ceutically acceptable salt of 4-PBA, such as its sodium salt. Analogs of 4-PBA include, for example, glyceryl (tri-4-PBA), phenylacetic acid, 2-POAA-OMe, 2-POAA-NO2, 2-NOAA. Physiologically acceptable salts of 4-phenylbutyrate include, for example, the sodium, potassium, magnesium or calcium salts.

[0049] Pharmaceutical Compositions and Methods of Administration In certain aspects, the methods described herein include the preparation and use of pharmaceutical compositions and medicaments that contain compounds identified as active ingredients by the methods described herein, including the pharmaceutical compositions themselves.

[0050] In some cases, the compositions disclosed herein can include other compounds, drugs and / or agents used to treat neurodegenerative diseases. For example, in some cases, the therapeutic compositions disclosed herein can be combined with one or more (e.g., 1, 2, 3, 4, 5, or less than 10) compounds.

[0051] In some cases, the compositions disclosed herein may be used in combination with other agents, such as COX2 inhibitors, asthma medications, diabetes medications, other antioxidants, acetylcholinesterase inhibitors (e.g., donepezil, tacrine, rivastigmine, galantamine, physostigmine, neostigmine, huperzine A, icopezil (CP-118954, 5,7-dihydro-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo-[4,5-f]-1,2-benzisoxazol-6-one maleate), ER-127528 (4-[(5,6-dimethoxy- 2-Fluoro-1-indanone)-2-yl]methyl-1-(3-fluorobenzyl)piperidine hydrochloride), zanapezil (TAK-147; 3-[1-(phenylmethyl)piperidin-4-yl]-1-(2,3,4,5-tetrahydro-1H-1-benzazepin-8-yl)-1-propane fumarate), metrifonate (T-588; (-)-Ra-[[2-(dimethylamino)ethoxy]methyl]benzo[b]thiophene-5-methanol hydrochloride), FK-960 (N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide hydrate), TCH-346 (N-methyl-N-2-propynyldibenzo[b,f]oxepin-10-methanamine), SDZ-220-581 ((S)-a-amino-5-(phosphonomethyl)-[1,1'-biphenyl]-3-propionic acid) and combinations thereof), NMDA receptor antagonists (e.g., memantine, neramexane, rimantadine or amantadine, lipoxygenase inhibitors, leukotriene inhibitors, coconut oil, other HDAC inhibitors, statins, amantains, The therapeutic agents may include other compounds including amphetamines, other MAO inhibitors, metal chelators, BACE1-inhibitors, antibodies against amyloid beta, gamma secretase modulators, amyloid cleansers, phosphorylated tau antibodies, Aβ inhibitors, Aβ plaque removers, inhibitors of Aβ plaque formation, inhibitors of amyloid precursor protein processing enzymes, beta-amyloid converting enzyme inhibitors, beta-secretase inhibitors, gamma secretase modulators, nerve growth factor agonists and neurofibrillary tangle cleansers.

[0052] In some cases, the compositions disclosed herein can be formulated for use as or in pharmaceutical compositions. Such compositions can be formulated or adapted for administration to a subject by any route, for example, any route approved by the U.S. Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual, version number 004 (available at fda.give / cder / dsm / DRG / drg00301.htm). Pharmaceutical compositions may be formulated for oral, parenteral or transdermal delivery. The compounds of the present invention may be combined with other pharmaceutical agents. In some aspects, the present invention provides kits containing TUDCA and PBA compounds used in the present invention. The kits may include instructions for use for the physician and / or patient, syringes, needles, boxes, bottles, vials, etc. In certain aspects, the present invention provides methods and agents that are useful for preventing or treating neurodegenerative and related diseases, including multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), stroke, Pick's disease, multi-infarct dementia, Creutzfeldt-Jakob disease, Lewy body dementia, mixed dementia, frontotemporal dementia, etc. In particular, the present invention provides agents or pharmaceutical compositions that can be used to treat or prevent Alzheimer's disease and other amyloidosis-related pathologies, as well as to prevent complications of these conditions.

[0053] In some cases, the pharmaceutical composition can include an effective amount of bile acid and phenylbutyric acid, as described above. As used herein, the terms "effective amount" and "effective for treating" refer to an amount or concentration of one or more drugs that is effective within the context of its administration, and for a period of time (including acute or chronic administration and periodic or continuous administration), to obtain an intended effect or physiological outcome.

[0054] In some cases, the pharmaceutical composition can include a bile acid (e.g., TUDCA), a phenylbutyric acid (e.g., 4-PBA), and any pharma- ceutically acceptable carrier, adjuvant, and / or vehicle. In some cases, the medicament can further include one or more additional therapeutic agents in an amount effective to achieve modulation of the disease or symptoms of the disease.

[0055] Pharmaceutical compositions typically contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient together with the compound of the present invention, does not abolish its pharmacological activity, and is non-toxic when administered in a sufficient amount to deliver a therapeutic amount of the compound. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.

[0056] The pharmaceutical composition of the present invention may contain any conventional non-toxic pharma- ceutically acceptable carrier, adjuvant or vehicle.Optionally, the pH of the formulation may be adjusted with pharma- ceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0057] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0058] The pharmaceutical compositions may be in the form of liquid or powder for inhalation and / or nasal administration. Such compositions may be formulated using suitable dispersing or wetting agents (e.g., Tween® 80, for example) and suspending agents according to techniques known in the art. Sterile injectable preparations may be as sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution, and isotonic saline solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland, fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, and its glyceride derivatives, are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils, such as olive oil or castor oil, particularly in their polyoxyethylated form. These oil solutions or suspensions may contain a long chain alcohol diluent or dispersant or carboxymethylcellulose or similar dispersing agent commonly used in the formulation of pharma- ceutically acceptable dosage forms, such as emulsions and / or suspensions. Other commonly used surfactants, such as Tween® or Span®, and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0059] The pharmaceutical composition can be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions or solutions. For oral tablets, commonly used carriers include lactose and cornstarch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dry cornstarch. When aqueous suspensions and / or emulsions are orally administered, the active ingredient may be suspended or dissolved in an oil phase, combined with emulsifiers and / or suspending agents. If necessary, certain sweeteners and / or flavorings and / or colorings may be added.

[0060] Alternatively or additionally, pharmaceutical compositions can be administered by nasal aerosol or inhalation.Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and / or other solubilizing or dispersing agents known in the art.

[0061] In some aspects, the disclosure provides methods for using compositions, including pharmaceutical compositions, comprising a bile acid (e.g., TUDCA) and phenylbutyric acid (e.g., 4-PBA), as disclosed herein (shown below as "X"), in the following methods:

[0062] Disclosed herein is a substance X for use as a medicament in the treatment of one or more diseases or conditions (e.g., a neurodegenerative disease, referred to in the examples below as "Y"); the use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment of Y.

[0063] In some cases, the therapeutic compositions disclosed herein may be formulated for sale in the United States, import into the United States, and / or export from the United States.

[0064] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0065] Dosage In some aspects of the invention, TUDCA (tauroursodeoxycholic acid) and PBA (4-phenylbutyrate) are administered individually (i.e., in separate dosage forms). In some embodiments, TUDCA is administered in an amount of about 10 mg / kg body weight, about 15 mg / kg body weight, about 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, or about 40 mg / kg body weight. In some embodiments, PBA is administered in an amount of about 10 mg / kg body weight, about 30 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, or about 400 mg / kg body weight. The compounds can be administered individually or together and are included as part of a treatment regimen.

[0066] The present invention further provides dosing regimens such that the TUDCA and / or PBA dosage forms are administered individually or together as a single daily dose, on a daily basis, on a weekly basis, on a certain number of days basis, etc. Furthermore, patients may receive a particular dosage over a period of weeks, months, or years. For example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, etc.

[0067] The advantages of the compositions of the present invention include: (1) smaller solid dosage form size of PBA; and (2) lower dose of PBA required to obtain the same pharmacological effect. Thus, in certain aspects, the present invention provides an improved method for treating neurodegenerative diseases, comprising administering PBA in combination with bile acid at a reduced dosage compared to the treatment of neurodegenerative diseases with PBA (e.g., compared to the standard dosage of PBA). In some embodiments, the amount of PBA administered in combination with bile acid is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50% or about 55% compared to the dosage used when PBA is administered alone.

[0068] Treatment methods The methods described herein include methods for the treatment of disorders associated with cellular oxidative stress (e.g., reactive oxygen metabolite-mediated oxidative disorders in cells, redox imbalance in cells, or mitochondrial dysfunction in cells). In some embodiments, the disorder is a neurodegenerative disease (e.g., Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis). In general, the methods include administering a therapeutically effective amount of a bile acid (e.g., TUDCA) in combination with phenylbutyric acid (e.g., 4-PBA) as described herein to a subject (e.g., a mammalian subject, e.g., a human subject) in need of or determined to be in need of such treatment.

[0069] In some cases, the method can include the selection of a human subject who has or has had a condition or disease. In some cases, suitable subjects include, for example, subjects who have or have had a condition or disease, but for which the disease or aspects thereof have resolved and symptoms of the disease are currently alleviated (e.g., compared to other subjects (e.g., the majority of subjects) with the same condition or disease) and / or subjects who have survived with the condition or disease for an extended period of time (e.g., compared to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., compared to other subjects (e.g., the majority of subjects) with the same condition or disease).

[0070] The methods disclosed herein can be applied to a wide range of species, including humans, non-human primates (e.g., monkeys), horses, cows, pigs, sheep, deer, elk, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats and mice.

[0071] The terms "treat," "treating," "treatment," and the like, when applied to an isolated cell, include subjecting the cell to any type of process or condition or performing any type of operation or procedure on the cell. When applied to a subject, the term "treat" refers to providing medical or surgical attention, care, or management to an individual. An individual is usually ill or injured, or at increased risk of developing a disease compared to the average member of the population, and in need of such attention, care, or management.

[0072] In some embodiments, the term "treating" or "treatment" refers to administering an effective amount of a composition, such as a composition comprising bile acid and phenylbutyric acid, to a subject such that the subject has a beneficial or desired clinical outcome, such as a reduction in at least one symptom of the disease or an improvement in the disease. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of one or more symptoms, a reduction in the extent of the disease, a stabilized (i.e., not worsening) state of the disease, a delay or slowing of disease progression, an improvement or palliation of the disease state, remission (whether partial or complete), whether detectable or undetectable. Treating can refer to prolonging survival compared to expected survival in the absence of treatment. Thus, one of skill in the art will understand that a treatment improves the disease state, but may not be a complete cure for the disease. In some embodiments, a treatment can be a "prophylactic treatment" in which a composition disclosed herein (e.g., a composition comprising bile acid and phenylbutyric acid) is administered to a subject at risk of developing a neurodegenerative disease disclosed herein. In some aspects, treatment is "effective" if the progression of a disease is reduced or halted.

[0073] The term "subject" as used herein refers to any animal. In some cases, the subject is a mammal. In some cases, the term "subject" as used herein refers to a human (e.g., a male, female, or child).

[0074] In some cases, selecting a subject includes obtaining a sample from a subject (e.g., a potential subject) and testing the sample for an indication that the subject is suitable for selection. In some cases, the subject may be confirmed or identified, for example, by a medical professional, as having or having a condition or disease. In some cases, indication of a positive immune response to a condition or disease may be made from patient records, family history, and / or detecting indicators of a positive immune response. In some cases, multiple parties may be involved in selecting a subject. For example, a first party may obtain a sample from a potential subject and a second party may test the sample. In some cases, the subject may be selected and / or referred by a medical professional (e.g., a general practitioner). In some cases, selecting a subject may include obtaining a sample from a selected subject and storing the sample and / or using it in the methods disclosed herein. The sample may include, for example, a cell or a cell population.

[0075] In some cases, the treatment method can include single administration, multiple administration and repeated administration as required for the prevention or treatment of the disease or condition that the subject suffers from.In some cases, the treatment method can include evaluating the level of disease in the subject before, during and / or after treatment.In some cases, treatment can be continued until a reduction in the level of disease in the subject is detected.

[0076] The terms "administer", "administering" or "administration" as used herein refer to implanting, absorbing, ingesting, injecting or inhaling the drug of the present invention, regardless of the form. In some cases, one or more compounds disclosed herein can be administered topically (e.g., intranasally) and / or orally to a subject. For example, the methods herein include administering an effective amount of a compound or compound composition to achieve a desired or stated effect. The specific dose and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular compound used, age, weight, general health, sex, diet, time of administration, excretion rate, drug combination, severity and course of the disease, condition or symptom, the patient's predisposition to the disease, condition or symptom, and the judgment of the treating physician.

[0077] After administration, the subject can be evaluated to detect, assess or determine the level of disease in the subject. In some cases, treatment can be continued until a change (e.g., a reduction) in the level of disease in the subject is detected.

[0078] Upon improvement of a patient's condition (e.g., a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of a compound, composition or combination of the invention may be administered, if necessary. Thereafter, either the dosage or the frequency of administration, or both, may be reduced, depending on the symptoms, to a level at which the improved condition is maintained. However, the patient may require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. EXAMPLES

[0079] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0080] material and method: Compounds were tested at Charles River Labs Discovery Research Services Finland using a neuronal cell model exposed to hydrogen peroxide as described herein.

[0081] Cortical mixed cultures were prepared from E18 Wistar rat fetuses (Laboratory Animal Center, Kuopio, Finland). Cortices were dissected and tissue was cut into small pieces. Cells were isolated by incubation with DNase and papain for 15 min. Cells were harvested by centrifugation (1500 rpm, 5 min). Tissue was triturated with a pipette and cells were plated (3 × 10 cells / well) on poly-L-lysine-coated 48-well plates in MEM supplemented with 2 g / l glucose, 2 mM glutamine, 0.1 μg / ml gentamicin and 10% heat-inactivated horse serum (HS-HI) and heat-activated bovine serum (FBS-HI). 5 cells / cm 2 After 3 days in vitro, the medium containing MEM with both supplements and 5% serum was replaced on the cells. On day 6 in vitro, unwanted cell division was prevented by adding cytosine arabinoside (10 μM final concentration) for 24 hours. Cultures were refed with MEM with supplements and 5% HS-HI before experiments were performed.

[0082] On day 10 in vitro, wells in good condition were selected for the experiment. Test compounds were diluted in MEM with supplements and 5% HS-HI. After 24 hours, 300 μM NMDA 24 hours was used as a control for total neuronal cell death, and H2O2 1 hour was used to induce approximately 30-60% cell death. DFX (100 μM) was used as a potential positive control to inhibit cell death induced by H2O2. Wells treated with medium only served as 0 controls. Test compounds were pipetted onto the cells 24 hours before adding H2O2. After 60 minutes, medium was removed and medium containing compound was pipetted into the wells for 24 hours.

[0083] After 24 hours, the medium of all wells was collected and possible cell debris was removed by centrifugation (13000 rpm, 3 min). 100 μl aliquots were pipetted into the microtiter plate in duplicate and an equal volume of LDH reagent was pipetted into the wells. Absorbance at 340 nm was immediately measured using a 3 min kinetic measurement protocol on a Multiskan Ascent ELISA reader (Thermo, Finland). The change in absorbance / min was determined and is directly proportional to the LDH released (=cell death).

[0084] After the cell medium was collected for LDH measurement, 100 μl PrestoBlue solution (PrestoBlue reagent (Invitrogen, #A13261, lot 915815C) diluted 1:10 in medium) was added to the wells and incubated for 1 hour in a +37° C. CO2 incubator. Fluorescence at 560 nm excitation / 615 nm emission was measured using a Victor fluororeader (PerkinElmer).

[0085] The number of wells per compound concentration used was six (n=6). One concentration of 100 μM TUDCA and 1 mM 4-PBA was tested individually and together. Statistical analysis was calculated on Microsoft excel. The values ​​were analyzed by a two-tailed, equal variance t-test, comparing every condition to every other condition.

[0086] Example 1 We tested the combination of TUDCA and 4-PBA in a neuronal model exposed to hydrogen peroxide to explore the synergistic effects they may have. The neuronal model exposed to hydrogen peroxide was chosen for its correlation with oxidative stress and pathology in various neurodegenerative diseases. In this study, we evaluated efficacy based on cell viability and cell death.

[0087] The combined use of TUDCA and 4-PBA resulted in a cell viability of 90.4%, a statistically significant improvement over the control cell viability of 48.6% (p<.003). (Figure 1, Table 1) Individual treatment with TUDCA and 4-PBA resulted in cell viability of 59% and 72%, respectively, which was not significantly different from the control (p<.414 and p<.071, respectively). (Figure 1, Table 1) Thus, the combination results in a 24% increase in efficacy over the sum of the efficacy of the individual drug treatments. NMDA at 300 μM was used as a negative control, resulting in approximately 0% cell viability. DFX resulted in a bizarre result of approximately 11% less cell viability than the hydrogen peroxide exposure condition. This result was excluded as it was believed that DFX interfered with the PrestoBlue measurement mode. Efficacy is calculated as the absolute difference in mean viability from the mean viability of the control.

[0088] The near return to control provided by the combination of molecules, as seen in the PrestoBlue study, is truly remarkable and highlights the potential for this combination as a treatment for diseases associated with hydrogen peroxide toxicity, improving survival and reducing cell death more than the sum of the benefits provided by each compound alone. [Table 1]

[0089] The combined use of TUDCA and 4-PBA resulted in a cell death rate of 31.5%, a statistically significant improvement over the control cell death rate of 62.5% (p<.021). (Figure 2) Individual treatment with TUDCA and 4-PBA resulted in cell death rates of 60.4% and 46.5%, respectively, which were not significantly different from the control (p<.808 and p<.162, respectively). (Figure 2) Thus, the combination results in a 71% increase in efficacy over the sum of the efficacy of the individual drug treatments. DFX and NMDA were used as positive and negative controls, respectively, resulting in 38.5% and 100% cell death, respectively. Efficacy is calculated as the absolute difference in mean viability from the mean viability of the control.

[0090] These surprising and synergistic results suggest that the combination of TUDCA and 4-PBA may be a novel, effective and powerful therapy against reactive oxygen metabolite-mediated oxidative damage, thereby reducing oxidative stress in neuronal cells and treating at least one symptom associated with diseases related to oxidative stress, and preventing the onset time of or delaying the onset of such diseases. [Table 2]

[0091] Other Aspects While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for reducing reactive oxygen metabolite-mediated oxidative damage in a cell, the method comprising: treating the cell with Bile acids or pharma- ceutically acceptable salts, analogs, derivatives or prodrugs thereof; Phenylbutyric acid (PBA) or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof The method comprises contacting the

2. 1. A method of modulating redox homeostasis in a cell, the method comprising: Bile acids or pharma- ceutically acceptable salts, analogs, derivatives or prodrugs thereof; Phenylbutyric acid (PBA) or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof The method comprises contacting the

3. 1. A method for reducing mitochondrial dysfunction in a cell, the method comprising: Bile acids or pharma- ceutically acceptable salts, analogs, derivatives or prodrugs thereof; Phenylbutyric acid (PBA) or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof The method comprises contacting the

4. 1. A method of treating a neurodegenerative disease associated with reactive oxygen metabolite-mediated oxidative damage in a subject, the method comprising: Identifying a subject experiencing a neurodegenerative disease associated with reactive oxygen metabolite-mediated oxidative damage; administering to said subject a bile acid, or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof. Including, A method in which the amount of PBA administered in combination with a bile acid is reduced by 10% to 50% compared to administration of PBA alone.

5. 1. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: Identifying a subject having at least one copy of the APOE 4 allele; administering to said subject a composition comprising a bile acid, or a pharma- ceutically acceptable salt, analog, derivative, or prodrug thereof, and phenylbutyric acid (PBA), or a pharma-ceutically acceptable salt, analog, derivative, or prodrug thereof, thereby treating the neurodegenerative disease. The method includes:

6. 6. The method of any one of claims 1 to 5, wherein the bile acid is selected from the group consisting of tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid or an analogue, derivative or derivative thereof.

7. The method according to any one of claims 1 to 6, wherein the bile acid is selected from the group consisting of tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA).

8. The PBA may be 4-phenylbutyric acid, glyceryl (Tri-4-PBA), phenylacetic acid, 2-POAA-OMe, 2-POAA-NO ２ 8. The method of any one of claims 1 to 7, wherein the compound is 2-NOAA or a pharma- ceutically acceptable salt, analog, derivative or prodrug thereof.

9. 9. The method of any one of claims 1 to 3 or 6 to 8, wherein the cells are contacted with the bile acid at a concentration of about 80 μM to about 120 μM.

10. 10. The method of any one of claims 1 to 3 or 6 to 9, wherein the cells are contacted with phenylbutyric acid at a concentration of about 0.8 mM to about 1.2 mM.

11. The method of any one of claims 1 to 3 or 6 to 10, wherein the cell is a mammalian cell.

12. The method of claim 11 , wherein the mammalian cell is a human cell.

13. The method according to any one of claims 1 to 3 or 6 to 11, wherein the cell is a neuronal cell.

14. The reactive oxygen metabolite-mediated oxidative damage is caused by hydrogen peroxide (H ２ O ２ 2. The method of claim 1, wherein the disorder is a .DELTA.)-mediated disorder.

15. 5. The method of claim 4, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Pick's disease, multi-infarct dementia, Creutzfeldt-Jakob disease, dementia with Lewy bodies (DLB), mixed dementia, and frontotemporal dementia.

16. 16. The method of claim 15, wherein the neurodegenerative disease is Alzheimer's disease.

Images