Quercetin-Containing Composition for Use in the Treatment of Amyotrophic Lateral Sclerosis - Patent application

Abstract

Effective amounts of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid for use in treating amyotrophic lateral sclerosis in a subject. Also disclosed is the combination for use in reducing, slowing or ameliorating the progression of amyotrophic lateral sclerosis or a symptom thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 USC §119(e) of U.S. Provisional Patent Application No. 63 / 190,697, filed May 19, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes. Summary of the Invention

[0002] The present disclosure relates to a method of treating amyotrophic lateral sclerosis (ALS), comprising administering to a subject in need thereof effective amounts of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid. [Brief description of the drawings]

[0003] The file of this patent contains at least one photograph with color drawing(s). Copies of this patent with color drawing(s) or photographs will be provided to the Office upon request and payment of the necessary fee.

[0004] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles, features and characteristics of the invention.

[0005] [Figure 1] Figure 1 shows an exemplary assessment of the improvement of astrocytosis by isoquercetin. Confocal images from spinal cord sections stained with GFAP and Nissl stain. Note that isoquercetin-treated rat spinal cords have significantly fewer astrocytes (white) surrounding motor neurons compared to vehicle-treated ones. Scale bars are 5 μm and 20 μm. [Diagram 2]1 shows an exemplary evaluation of the amelioration of microgliosis by isoquercetin. Confocal images of the microglial marker Iba1. Microglial cells are significantly reduced in isoquercetin-treated rat spinal cords compared to vehicle-treated. [Diagram 3] 1 shows an exemplary evaluation of the amelioration of motor neuron pathology by isoquercetin. Confocal images of spinal motor neurons stained with antibodies against misfolded SOD1 and ubiquitin. Note that both markers are significantly reduced in isoquercetin-treated rat spinal cords compared to vehicle-treated.

[0006] (definition) As used herein, "amyotrophic lateral sclerosis" or "ALS" refers to motor neuron disorders such as primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), pseudobulbar and progressive bulbar palsy (PBP), and frontotemporal dementia (FTD).

[0007] As used herein, "quercetin" refers to both quercetin aglycone and quercetin derivatives, such as quercetin-3-O-glucoside (also known as "isoquercetin" and referred to herein as "IsoQ", "IsoQ" or "Iso-Q"), quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9-O-glucoside, quercetin-3'-O-glucoside, quercetin-4'-O-glucoside, quercetin-3-O-glucoside, quercetin-4'-O-glucoside, quercetin-5 ... Rutinoside (also known as rutin), quercetin-3-O-[α-rhamnosyl-(1->2)-α-rhamnosyl-(1->6)]-I3-glucoside, quercetin-3-O-galactoside, quercetin-7-O-galactoside, quercetin-3-O-rhamnoside, quercetin-7-O-galactoside, quercetin-glycoside, 7-hydroxyflavone, and pharma- ceutin . Quercetin may also refer to isoquercetin or rutin, or components of rutin or isoquercetin, or metabolites of rutin or isoquercetin or quercetin, sulfated, glucuronidated, or methylated forms of rutin or quercetin, and pharma- ceutically acceptable salts thereof.

[0008] As used herein, "Vitamin B3" refers to various forms of Vitamin B3, including, but not limited to, niacinamide, nicotinic acid, nicotinamide, inositol hexaniacinate, or combinations thereof.

[0009] As used herein, "Vitamin C" refers to Vitamin C, including, but not limited to, L-ascorbic acid, D-ascorbic acid, or both, and salts thereof (e.g., sodium ascorbate), or combinations thereof.

[0010] As used herein, "Folic acid" refers to vitamin B, including, but not limited to, vitamin B9, folic acid, pteroylglutamic acid, and L-methylfolate, 5-MTHF (5-methyltetrahydrofolate), or combinations thereof.

[0011] As used herein, "thiol isomerases," including but not limited to "extracellular thiol isomerases" and "vascular thiol isomerases," are multifunctional enzymes that affect protein structure through oxidoreductase, isomerase, and chaperone activities. These enzymes are localized in high concentrations in the endoplasmic reticulum of all eukaryotic cells and perform essential functions in nascent protein folding by mediating disulfide bond formation. However, thiol isomerases can escape endoplasmic reticulum retention and be secreted and localized on the plasma membrane. Several thiol isomerases, including but not limited to protein disulfide isomerase (PDI), ERp57, and ERp5, are secreted from platelets and endothelial cells and localized on their membranes. These vascular thiol isomerases are released after vascular injury and are known to be involved in thrombus formation. Vascular thiol isomerases also act as redox sensors. Vascular thiol isomerases respond to the local redox environment and affect S-nitrosylation of surface proteins in platelets and endothelial cells. In addition to their role in protein folding, thiol isomerases can modify allosteric disulfide bonds in proteins inside and outside the cell, thereby controlling protein function. The process of disulfide bond formation and cleavage is tightly controlled and responds to redox conditions. PDI is the prototype of these thiol isomerases, has a molecular weight of 57,000 and contains 508 amino acids. It is encoded by the P4HB gene and consists of four thioredoxin-like domains ab-b'-a', where a and a' are catalytically active units with a CGHC motif in the active site and are preceded by a signal sequence."Thiol isomerases" include, but are not limited to, protein disulfide isomerase (PDI), PDIA2, PDIA3 (also known as glucose-regulated protein 58 kD (GRP58)), PDIA4, PDIA5, PDIA6, PDIALT, PPIA, thioredoxin (TRX), AGR2, AGR3, CASQ1, CASQ2, DNAJC10, P4HB, TMX1, TMX2, TMX3, TMX4, TXNDC5, and TXNDC12, as well as the following endoplasmic reticulum resident proteins: ERp5, ERp27, ERp57, ERp72, ERp44, ERp46, ERp29.

[0012] As used herein, a "thiol isomerase inhibitor compound" is an inhibitor of one or more thiol isomerases. Exemplary thiol isomerase inhibitor compounds include zafirlukast, montelukast, CGP-13501 (CAS Registry No. 56189-68-5), CGP-7930 (CAS Registry No. 57717-80-3), alosetron, balsalazide, benserazide, butaclamol, levadopa, mesalazine, oxcarbazepine, pharmaceutically acceptable salts, prodrugs, and / or solid forms thereof. As one or more inhibitors of thiol isomerases, one or more of these compounds, or a combination of two or more of these compounds, can be used as an antithrombotic agent, an anticoagulant, an antiinflammatory agent, an antiviral agent, a chemotherapeutic agent or an anticancer agent, or the like, or a combination thereof. Zafirlukast is a synthetic selective peptide leukotriene receptor antagonist (LTRA) with the chemical name 4-(5-cyclopentyloxycarbonylamino-1-methylindo-1-3-ylmethyl)-3-methoxy-No-tolylsulfonylbenzamide. We have found that zafirlukast is a broad-spectrum thiol isomerase inhibitor that inhibits platelet function, thrombus formation and cancer cell proliferation. The synthesis and pharmaceutical forms of zafirlukast are further described in U.S. Patent Nos. 4,859,692; 5,294,636; 5,319,097; 5,482,963; 5,583,152; 5,612,367; 6,143,775; 6,333,361; and 6,399,104, the contents of which are incorporated herein by reference in their entireties. Montelukast is a synthetic peptide leukotriene receptor antagonist (LTRA) with the chemical name [R-(E)]-1-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methylcyclopropaneacetic acid.The synthesis and pharmaceutical forms of montelukast and montelukast sodium are further described in US Pat. No. 5,565,473, which is incorporated herein by reference in its entirety.

[0013] As used herein, the term "pharmaceutical agent" or "compound" refers to a chemical entity or biological product, or a combination of chemical entities or biological products, that is administered to a human to treat or prevent or control a disease or condition.

[0014] As used herein, the term "active agent" refers to a compound, element, or mixture that, when administered to a patient, alone or in combination with other compounds, elements, or mixtures, imparts a direct or indirect physiological effect to the patient. The indirect physiological effect may occur via a metabolic product or other indirect mechanism. When the active agent is a compound, salts of the free compound, solvates (including hydrates), crystalline forms, non-crystalline forms (i.e., amorphous), and any polymorphs of the compound are included. All forms are contemplated herein, regardless of the method used to obtain them.

[0015] As used herein, the term "pharmaceutically acceptable salts" includes derivatives of the disclosed compounds modified by making the parent compound into its inorganic and organic salts, acid addition salts, or base addition salts. The salts of the compounds of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of a suitable base (such as hydroxides, carbonates, bicarbonates of Na, Ca, Mg, or K) or by reacting the free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are usually carried out in water or in an organic solvent, or a mixture of both. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, acetonitrile are used where possible. The salts of the compounds of the present invention further include solvates of the compounds of the present invention and salts of the compounds of the present invention.

[0016] Examples of pharma- ceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic acid salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylate, mesylate, esylate, besylate, sulfanilate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulfonate, ethanedisulfonate, oxalate, isethionate, HOOC-(CH2)n-COOH (n=0-4), and the like. Lists of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0017] As used herein, the term "about" immediately preceding a numerical value means a range of plus or minus 10% of that numerical value, for example, "about 50" means 45 to 55, and "about 25,000" means 22,500 to 27,500, unless the context of this disclosure indicates otherwise or is contradictory to such an interpretation.

[0018] As used herein, the term "dosage form" refers to a unitary administration of an active agent. Examples of dosage forms include tablets, capsules, injections, suspensions, solutions, emulsions, creams, ointments, suppositories, inhalants, transdermals, etc. An exemplary dosage form is a solid oral dosage form.

[0019] As used herein, the term "pharmaceutical compositions" refers to compositions that contain at least one active agent or a pharma- ceutically acceptable salt thereof, and at least one other substance, such as a carrier. Pharmaceutical compositions meet the US FDA GMP (Good Manufacturing Practice) standards for human or non-human drugs. Pharmaceutical compositions can be formulated into dosage forms.

[0020] As used herein, the term "carrier" applied to a pharmaceutical composition refers to a diluent, excipient, or vehicle in which an active agent is provided. As used herein, the term "carrier" applied to a pharmaceutical composition refers to a diluent, excipient, or vehicle in which an active agent is provided. Classes of carriers include, for example, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers are listed in one or more classes, for example, vegetable oils are used as lubricants in some formulations and as diluents in other formulations. Exemplary pharma- ceutical acceptable carriers include sugar, starch, cellulose, powdered tragacanth, malt, gelatin, talc, and vegetable oils. The pharmaceutical composition may include any additional active agent that does not substantially inhibit the activity of the active agent. The amount of carrier used with the compound is sufficient to provide a practical amount of material for administration per unit dose of the active agent. Types of carriers include, for example, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, lubricants, preservatives, stabilizers, surfactants, tableting agents, wetting agents, etc. Some carriers are listed in one or more classes, for example, vegetable oils are used as lubricants in some formulations and as diluents in others. Exemplary pharma- ceutically acceptable carriers include sugar, starch, cellulose, powdered tragacanth, malt, gelatin, talc, and vegetable oils. The pharmaceutical composition may contain any additional active agent that does not substantially inhibit the activity of the active agent.

[0021] As used herein, the term "patient" or "subject" refers to a human or non-human animal in need of medical treatment. Medical treatment includes treating, preventing, slowing, ameliorating, or prophylactic treatment of an existing condition, such as a disease or disorder, or diagnostic treatment in patients with a family history of ALS (research suggests that approximately 10% of ALS patients have a significant family history of ALS, suggesting a strong genetic predisposition, and a causative mutation has now been found in more than half of these cases). Sporadic ALS, on the other hand, is considered a complex trait with an estimated heritability of 40-50%.) In some embodiments, the patient is a human patient.

[0022] As used herein, the terms "administer," "administering," or "administration" refer to the direct administration of a compound or composition to a subject.

[0023] As used herein, the term "providing" means to give, manage, sell, distribute, transfer (whether for profit or not), manufacture, compound, or dispense.

[0024] As used herein, the term "providing a thiol isomerase inhibitor compound or a pharma- ceutically acceptable salt thereof with at least one additional therapeutic agent" means that the active agent or a pharma- ceutically acceptable salt thereof and the additional active agent(s) are provided simultaneously in a single dosage form, simultaneously in separate dosage forms, or provided in separate dosage forms for administration separated by a time period within which both the active agent or a pharma- ceutically acceptable salt thereof and the at least one additional active agent are present in the patient's bloodstream. The active agent or a pharma- ceutically acceptable salt thereof and the additional active agent do not need to be prescribed to the patient by the same medical practitioner. The additional active agent or agent does not require a prescription. Administration of the active agent or a pharma- ceutically acceptable salt thereof, or the at least one additional active agent, can be by any suitable route, such as, for example, oral tablet, oral capsule, oral liquid, inhalation, injection, suppository, or topical contact.

[0025] The term "treatment" as used herein includes providing an active agent or a pharma- ceutically acceptable salt thereof, either as the sole active agent or together with at least one additional active agent, sufficient to (a) prevent the disease or condition or symptoms of the disease or condition from occurring in a patient who may be predisposed to the disease or condition but has not yet been diagnosed with the disease or condition (e.g., a patient with a family history of ALS), (b) inhibit the disease or condition, i.e., arrest or delay its onset (in some embodiments, after diagnosis of ALS), and (c) ameliorate the disease or condition, i.e., cause regression of the disease or condition. "Treating" and "treatment" also refer to providing a therapeutically effective amount of an active agent or a pharma- ceutically acceptable salt thereof, either as the sole active agent or together with at least one additional active agent, to a patient suffering from a disease or condition that is affected by the activity of one or more active agents. By "a disease or condition affected by the activity of one or more active agents" is meant that one or more active agents are involved in the disease or condition.

[0026] As used herein, the term "effective amount" refers to an amount that results in measurable inhibition of at least one symptom or parameter of a particular disorder or pathological process. As used herein, the term "therapeutically effective amount" of the present composition is an amount that confers a therapeutic effect on the treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject sees or feels an effect, or the physician observes a change).

[0027] As used herein, a "therapeutically effective amount" of an active agent refers to an amount that, when administered to a patient, is effective to provide a therapeutic benefit, such as preventing, slowing, inhibiting, or ameliorating symptoms (in patients with a family history of ALS), for example, preventing mast cell activation and preventing the formation of mast cell activating cytokines in patients suffering from ALS. Therapeutically effective amounts vary depending on factors such as the patient's health, age, weight, and the ability of the compound to elicit a desired response in the patient. Dosages can also be adjusted to obtain optimal therapeutic effects. A therapeutically effective amount is also an amount in which the toxic or adverse effects (e.g., side effects) of the active agent are outweighed by the therapeutically beneficial effects.

[0028] As used herein, the term "synergistic effect" refers to an interaction or cooperation that produces a whole that is greater than the sum of the parts. As used herein, the effect of isoquercetin or quercetin with zafirlukast produces a greater effect than quercetin or zafirlukast alone.

[0029] The term "preventing" refers to preventing a particular disorder, disease or condition and / or preventing the recurrence of a particular disorder, disease or condition. In some embodiments, "preventing" refers to preventing a particular disorder, disease or condition and / or preventing the recurrence of a particular disorder, disease or condition in patients with a family history of ALS.

[0030] As used herein, the term "prognosis" means the expected course and outcome of a disease, particularly the chances of recovery.

[0031] As used herein, the terms "treat," "treatment," "treated," or "treating" refer to both therapeutic and (in the case of patients with a family history of ALS) prophylactic treatment, the purpose of which is to obtain a beneficial or desired clinical result, such as protecting against or delaying (e.g., attenuating or postponing onset) an undesirable physiological condition, disorder, or disease (partially or fully), or to obtain a beneficial or desired clinical result, such as partial or total restoration or inhibition of the decline of a parameter, value, function, or outcome that was or becomes abnormal. For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent or intensity or rate of onset of a condition, disorder or disease, stabilization (i.e., not worsening) of the condition, disorder or disease state, delay in onset or slowing of progression of the condition, disorder or disease, improvement of the condition, disorder or disease state, and remission or relapse (whether partial or total), whether or not it leads to immediate relief of actual clinical symptoms, or enhancement or amelioration of the condition, disorder or disease, or whether or not it prevents the spread of the condition, disorder or disease state. Treatment aims to elicit a clinically significant response without undue side effects. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other animals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0032] The terms "improving," "enhancing," "treating," and "lowering" refer to the administration of an effective amount of a composition of the invention to a subject in need of amelioration of one or more of the above conditions, or having one or more of the above disorders, or having one or more symptoms or predispositions to one or more of the above disorders or conditions, with the purpose of ameliorating one or more of these conditions, or for the purpose of preventing, curing, mitigating, relieving, ameliorating one or more of these disorders, or one or more of these symptoms or predispositions.

[0033] The term "administration" refers to oral or parenteral delivery of a composition of the invention to a subject in any appropriate form, such as food, beverage, tablet, capsule, suspension, and solution.

[0034] The term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrathoracic, intranasal, intrathecal, and intracranial injection, as well as a variety of infusion techniques.

[0035] "Effective amount" refers to a dose of a composition sufficient to provide a physical benefit (e.g., reducing fatigue and / or improving endurance caused by the disease) or a therapeutic benefit (e.g., slowing the progression of ALS disease). Both in vivo and in vitro tests can be performed to determine optimal routes of administration and dosages.

[0036] As used herein, the term "disease" is generally intended to be synonymous with, and used interchangeably with, the terms "disorder," "dysfunction," "syndrome," and "condition" (among medical conditions).

[0037] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a medical condition or disorder described in this disclosure. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule or dosage form having a fixed ratio of active ingredients, or in multiple separate capsules for each active ingredient. Furthermore, such administration also includes the sequential use of each type of therapeutic agent in the same patient, with delivery of the individual therapeutic agents separated by 1-24 hours, 1-7 days, or a week or more. In either case, the treatment regimen provides the beneficial effect of the combination of agents in treating the condition or disorder described herein.

[0038] The present disclosure is not limited to the specific embodiments described in this application, which are intended as examples of various aspects. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope thereof. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0039] As used herein, the singular forms "a," "an," and "the" include the plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by prior invention. As used herein, the term "comprising" means "including, but not limited to."

[0040] Although various compositions, methods, and devices are described in terms "comprising" (which may be interpreted as "including, but not limited to") various components or steps, the compositions, methods, and devices may also "consist essentially of" or "consist of" the various components or steps, and such terms should be interpreted as defining an essentially closed group of members.

[0041] With respect to the use of virtually any plural and / or singular term herein, those of skill in the art can interpret the plural to the singular and / or the singular to the plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly provided herein for clarity.

[0042] In general, those skilled in the art will understand that the terms used in this specification, and particularly in the appended claims (e.g., the appended claims body), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Those skilled in the art will further understand that if a particular number of claims introduced are intended, such intent will be explicitly set forth in the claim, and if not, no such intent exists. For example, as an aid to understanding, the appended claims below may use the introductory phrases "at least one" and "one or more" to introduce the claims. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim repeat with the indefinite article "a" or "an" limits a particular claim that includes such an introduced claim repeat to embodiments that include only one such repeat, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used at the beginning of a claim. Moreover, even if a specific number of an introduced claim repeat is explicitly recited, one of skill in the art will recognize that such repetition should be interpreted to mean at least the number recited (e.g., "two repetitions" without other modifiers means at least two repetitions, or two or more repetitions).Furthermore, when a phrase similar to "such as at least one of A, B, and C" is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a phrase similar to "such as at least one of A, B, or C" is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further understand that virtually any conjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, contemplates the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B," or "A and B."

[0043] In addition, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0044] As will be understood by those of skill in the art, all ranges disclosed herein encompass all possible subranges and combinations of subranges thereof for any purpose, including in terms of providing a written description. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will also be understood by those of skill in the art, all phrases such as "up to," "at least," etc. refer to ranges that are inclusive of the recited numbers and can then be broken down into subranges as described above. Finally, as will be understood by those of skill in the art, ranges include individual members. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, 5 cells.

[0045] The various features and functions disclosed above, or alternatives thereof, may be combined into many other different systems or applications. Various alternatives, modifications, variations, or improvements not presently foreseen or anticipated, may subsequently be made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

[0046] Various methods for the treatment of amyotrophic lateral sclerosis (ALS) or related disorders selected from primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), pseudobulbar and progressive bulbar palsy (PBP), multiple sclerosis (MS), frontotemporal dementia (FTD) and Huntington's disease are described herein. The methods include administering at least one pharmaceutical composition to a subject. The treatment can reduce or eliminate the symptoms of ALS. The treatment can prevent further progression of the symptoms of ALS (e.g., lung dysfunction and / or inability to fully expand the lungs). The treatment can slow further progression of the symptoms of ALS (e.g., lung dysfunction and / or inability to fully expand the lungs). Thiol isomerase inhibitors, particularly PDI inhibitors, can suppress or stop mast cell activation, reduce mast cell hyperactivity, maintain mitochondrial function, protect nerve cells from degeneration, and prevent or reduce the decline in lung function and / or inability to fully expand the lungs seen in ALS.

[0047] In one embodiment, the present invention describes a method for treating amyotrophic lateral sclerosis (ALS), comprising administering an effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid to a subject in need thereof. The composition of the present invention may be in various forms. In some embodiments, quercetin, vitamin B3, vitamin C, and optionally folic acid are administered as a single agent, and zafirlukast and optionally folic acid are administered separately. In some embodiments, quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid are administered separately. In some embodiments, quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid are administered as a single agent. In some embodiments, quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid are administered in one or more formulations, wherein any one of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid is administered in combination or alone.

[0048] The present invention is based, at least in part, on the unexpected finding that a composition comprising quercetin, vitamin B3, vitamin C, zafirlukast and optionally folic acid as active ingredients exhibits synergistic effects in treating ALS patients compared to treatment with isoquercetin or quercetin or zafirlukast alone. Zafirlukast, a broad-spectrum thiol isomerase inhibitor, and isoquercetin or quercetin act primarily on different thiol isomerases. In general, thiol isomerases damage the spinal cord through mast cell activation, mast cell abnormal activation, and mitochondrial dysfunction, leading to neuroinflammation, microglial activation and damage, and cell death. Zafirlukast protects motor neurons from neuroinflammation, which is one of the causes of neurodegeneration in ALS. Zafirlukast may also inhibit abnormal mast cell activity and inhibit the decline in lung function and inability to fully inflate the lungs seen in ALS patients. Quercetin and isoquercetin are known to prevent mitochondrial dysfunction and maintain mitochondrial biogenesis, another pathogenesis of neurodegeneration in ALS. Quercetin and isoquercetin also suppress the decline in lung function and / or inability to fully inflate the lungs observed in ALS patients, albeit by inhibiting a different thiol isomerase than zafirlukast. Furthermore, the inventors have observed the surprising result that these compounds act synergistically at low doses to efficiently protect motor units. Furthermore, a composition comprising quercetin, vitamin B3, vitamin C, zafirlukast and optionally folic acid as active ingredients can delay the onset of paralysis and extend survival in vivo in animal models of ALS. A composition comprising quercetin, vitamin B3, vitamin C, zafirlukast and optionally folic acid as active ingredients shows surprising and substantial improvement in the treatment of ALS subjects.

[0049] Pharmaceutical Compositions In some embodiments, a method for treating amyotrophic lateral sclerosis (ALS) comprises administering to a subject in need thereof an effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid, and the thiol isomerase inhibitor is zafirlukast, montelukast, CGP-13501, CGP-7930, alosetron, balsalazide, benserazide, butaclamol, levadopa, mesalazine, oxcarbazepine, pharma- ceutically acceptable salts thereof, prodrugs thereof, and / or solid forms thereof. In some embodiments, a method for treating ALS comprises administering to a subject in need thereof an effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid.

[0050] In some embodiments, the method of treating ALS comprises administering to a subject in need thereof an effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid, wherein the composition comprises about 250 mg to about 1000 mg of quercetin. In some embodiments, the composition comprises about 20 μg to about 3 g of vitamin B3. In some embodiments, the composition comprises about 200 μg to about 3 g of vitamin C. In some embodiments, the composition comprises about 1000 μg to about 3000 μg of folic acid. In some embodiments, the composition comprises about 5 mg to about 160 mg of zafirlukast. In some embodiments, the composition comprises about 5 mg to about 320 mg, about 80 mg to about 160 mg, or about 5 mg to about 80 mg of zafirlukast.

[0051] The weight ratio of quercetin, vitamin B3, vitamin C, folic acid, and zafirlukast in the composition of the present invention can be 1:0.02-1:0.2-2.5, or any ratio therebetween. For example, the weight ratio may be 1:0.04-0.5:0.3-2.0, 1:0.05-0.3:0.4-1.5, 1:0.05-0.2:0.5-1, and 1:0.1-0.2:0.5-1. Preferred ratios include about 1:0.02:1, about 1:0.04:1, about 1:0.08:1, about 1:0.05:1.5, and about 1:0.16:1. Typically, a subject may be administered an amount of the composition providing 100 mg-2 g (preferably, 250 mg-1 g) of quercetin once a day or periodically.

[0052] After digestion, quercetin derivatives are converted to quercetin aglycone and other active derivatives and absorbed into the body. The amount of quercetin refers to the amount of quercetin aglycone or quercetin portion of quercetin derivatives. Quercetin can be added to the composition in pure form or as a component of a mixture (e.g., plant extract). Examples of commercially available quercetin include QU995 (containing 99.5% quercetin) and QU985 (containing 98.5% quercetin) available from Quercegen Pharmaceuticals LLC (Boston, Massachusetts). Examples of commercially available isoquercetin include ISQ950AN (containing 95% or more isoquercetin) and ISQ995AN (containing 99.5% isoquercetin) available from Quercis Pharma AG (Zug, Switzerland).

[0053] The compositions of the present invention can be in various forms, including, but not limited to, soft chews, capsules, tablets, and the like. For example, the composition is a soft chew composition that includes quercetin, niacinamide, ascorbic acid, sodium ascorbate, sugar, corn syrup, sucralose, soy lecithin, corn starch, glycerin, palm oil, xylitol, carrageenan, FD&C Yellow#6, FD&C Yellow#5, and natural and / or artificial flavors. An exemplary serving (5.15 g) of this soft chew composition includes 250 mg of quercetin, 12.9 mg of vitamin B3 (i.e., niacinamide), and 382.8 mg of vitamin C (i.e., L-ascorbic acid and sodium ascorbate). Folic acid can be included in the soft chew or provided separately in an amount of about 1000 μg to about 3000 μg. Zafirlukast can be included in the soft chew or provided separately in an amount of about 5 mg to about 320 mg, about 80 mg to about 160 mg, or about 5 mg to about 80 mg. The subject can ingest the soft chew composition 1 to 8 times (e.g., 4 times) per day. The amount consumed can vary depending, for example, on the disorder or condition to be treated and the physical condition of the subject. Another exemplary composition of the soft chew includes 5.25% by weight quercetin, 0.25% by weight vitamin B3, and 7.81% by weight vitamin C (i.e., L-ascorbic acid and sodium ascorbate).

[0054] The composition of the present invention may further comprise one or more active ingredients, such as isoflavones (e.g., genistein or genistin), curcumin, resveratrol, isoquercetin, luteolin, epigallocatechin gallate (EGCG), CoQ10, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).These active ingredients may be added to the composition in pure form or as a component of a mixture (e.g., an extract from a plant or animal).The appropriate daily dosage of each of these ingredients may vary, for example, depending on the disorder or condition to be treated and the physical condition of the subject. Some exemplary daily dosages of these ingredients are: 20-2,500 mg (preferably 250-1,000 mg) of curcumin, 10-1,000 mg (preferably 100-500 mg) of resveratrol, 10-1,000 mg (preferably 100-250 mg) of isoquercetin, 50-1,000 mg (preferably 100-700 mg) of EGCG, 25-300 mg (preferably 50-100 mg) of genistin / genistein, 10-1,000 mg (preferably 100-200 mg) of luteolin, 50-1,000 mg (preferably 70-500 mg) of EPA, and 50-1,000 mg (preferably 80-700 mg) of DHA. In addition, sweeteners such as sorbitol, maltitol, hydrogenated glucose syrup and hydrogenated starch hydrolysate, high fructose corn syrup, cane sugar, beet sugar, pectin, sucralose, etc., can be added to provide sweetness, if necessary. The composition can also include amino acids, fatty acids, proteins, fibers, minerals, flavor enhancers, or colorants. Exemplary amino acids include glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, arginine, and the L- and D-forms thereof. Amino acids may be added to aid digestion. Exemplary fatty acids include omega-3 fatty acids (eg, linoleic acid), omega-6 fatty acids (eg, linoleic acid), omega-9 fatty acids (eg, oleic acid), sunflower oil, sunflower lecithin, soybean oil, and soybean lecithin.Amino acids may be added to aid digestion. Exemplary proteins include plant proteins such as soy protein and chia seed protein. Exemplary fibers include plant fibers such as soy fiber and chia seed fiber. These ingredients can be added to the composition in pure form or as ingredients in a mixture (e.g., extracts from plants or animals).

[0055] In some cases, pharmaceutical compositions can further comprise one or more exemplary fillers.Exemplary fillers include cellulose and cellulose derivatives such as microcrystalline cellulose, starch such as dry starch, hydrolyzed starch, and starch derivatives such as cornstarch, cyclodextrin, sugars such as powdered sugar and sugar alcohols such as lactose, mannitol, sucrose, and sorbitol, inorganic fillers such as aluminum hydroxide gel, precipitated calcium carbonate, carbonates, magnesium aluminosilicate, dibasic calcium phosphate, and sodium chloride, silicon dioxide, titanium dioxide, titanium oxide, dicalcium phosphate dihydrate, calcium sulfate, alumina, kaolin, talc, or combinations thereof. The filler is present in the composition in an amount of from about 20% to about 65% by weight, from about 20% to about 50% by weight, from about 20% to about 40% by weight, from about 45% to about 65% by weight, from about 50% to about 65% by weight, or from about 55% to about 65% by weight, or any value between these ranges, based on the total weight of the composition.

[0056] In some examples, the pharmaceutical composition further comprises one or more disintegrants. Examples of disintegrants include starch, alginic acid, cross-linked polymers such as cross-linked polyvinylpyrrolidone, croscarmellose sodium, potassium starch glycolate, sodium starch glycolate, clay, cellulose, starch, gum, or combinations thereof. The disintegrant is present in the composition in an amount of about 1% to about 10% by weight, about 1% to about 9% by weight, about 1% to about 8% by weight, about 1% to about 7% by weight, about 1% to about 6% by weight, or about 1% to about 5% by weight, or any value between these ranges, based on the total weight of the composition.

[0057] In some examples, the pharmaceutical composition further comprises one or more binders. Binders include, but are not limited to, celluloses such as hydroxypropyl cellulose, methyl cellulose, and hydroxypropyl methyl cellulose, starches such as corn starch, pregelatinized starch, and hydroxypropyl starch, waxes such as acacia, tragacanth, sodium alginate, natural and synthetic gums, synthetic polymers such as polymethacrylates, polyvinylpyrrolidone, and povidone, dextrin, pullulan, agar, gelatin, tragacanth, macrogol, or combinations thereof. The binder is present in the composition at about 0.5% to about 5% by weight, about 0.5% to about 4% by weight, about 0.5% to about 3% by weight, about 0.5% to about 2% by weight, or about 0.5% to about 1% by weight, or any value between these ranges, based on the total weight of the composition.

[0058] In some examples, the pharmaceutical composition further comprises one or more wetting agents. Wetting agents include, but are not limited to, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, polyoxamers, poloxamer 188, polyoxyethylene ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, polysorbates, cetyl alcohol, glycerin fatty acid esters (e.g., triacetin, glycerin monostearate, etc.), polyoxymethylene stearate, sodium lauryl sulfate, sorbitan fatty acid esters, sucrose fatty acid esters, benzalkonium chloride, polyethoxylated castor oil, and combinations thereof. The humectant is present in the composition in an amount of from about 0.1% to about 1% by weight, from about 0.1% to about 2% by weight, from about 0.1% to about 3% by weight, from about 0.1% to about 4% by weight, or from about 0.1% to about 5% by weight, or any value between these ranges, based on the total weight of the composition.

[0059] In some examples, the pharmaceutical composition further comprises one or more lubricants, including, but not limited to, stearic acid, magnesium stearate, calcium hydroxide, talc, corn starch, sodium stearyl fumarate, alkali metal salts and alkaline earth metal salts, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (PEG), methoxypolyethylene glycol, propylene glycol, sodium oleate, glyceryl, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (PEG), methoxypolyethylene glycol, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof. The lubricant is present in the composition in an amount of from about 0.1 wt. % to about 5 wt. %, from about 0.1 wt. % to about 4 wt. %, from about 0.1 wt. % to about 3 wt. %, from about 0.1 wt. % to about 2 wt. %, or from about 0.1 wt. % to about 1 wt. %, based on the total weight of the composition, or any value between these ranges.

[0060] In some examples, the pharmaceutical composition further comprises one or more glidants. Glidants include, but are not limited to, colloidal silicon dioxide, talc, sodium lauryl sulfate, native starch, and combinations thereof. The glidant is present in the composition at about 0.05% to about 1% by weight, about 0.05% to about 0.9% by weight, about 0.05% to about 0.8% by weight, about 0.05% to about 0.5% by weight, or about 0.05% to about 0.1% by weight, or any value between these ranges, based on the total weight of the composition.

[0061] In some examples, the pharmaceutical composition can be a tablet and further comprises a topcoat, such as, but not limited to, a hydroxypropyl methylcellulose coating or a polyvinyl alcohol coating, available under the trade name Opadry, such as Opadry White, Opadry II (Opadry is a registered trademark of BPSI Holdings LLC, Wilmington, Del., USA). The topcoat is present in the composition in an amount of about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, or about 1% to about 5% by weight, or any value between these ranges, based on the total weight of the composition.

[0062] In some examples, the pharmaceutical composition may further include one or more preservatives. Examples of preservatives include, but are not limited to, sodium benzoate, paraoxybenzoic acid esters, methyl, ethyl, butyl and propyl paraben, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, sorbic acid, benzalkonium chloride (BKC), benzethonium chloride, phenol, phenyl mercaptan nitrate, thimerosal, or combinations thereof. A preservative may be included in the liquid dosage form. The preservative is in an amount sufficient to extend the shelf life or storage stability, or both, of the liquid dosage form. The preservative is present in the composition in an amount of about 0.05% to about 1% by weight, about 0.05% to about 0.9% by weight, about 0.05% to about 0.8% by weight, about 0.05% to about 0.5% by weight, or about 0.05% to about 0.1% by weight, or any value between these ranges, based on the total weight of the composition.

[0063] In some examples, the pharmaceutical composition may further comprise one or more flavoring agents. Examples of flavoring agents include, but are not limited to, synthetic flavor oils and flavoring aromatics and / or natural oils, extracts from plant leaves, flowers, fruits, etc., or any combination thereof. Other examples include cinnamon oil, wintergreen oil, peppermint oil, clove oil, bay oil, anise oil, eucalyptus, thyme oil, cedar leaf oil, nutmeg oil, sage oil, bitter almond oil, cassia oil, etc., or combinations thereof. Also useful as flavoring agents are citrus oils such as vanilla, lemon, orange, grape, lime, grapefruit, fruit essences such as apple, banana, pear, peach, strawberry, raspberry, cherry, plum, pineapple, apricot, strawberry flavor, tutti fruity flavor, mint flavor, or combinations thereof. The fragrance is present in the composition in an amount of from about 0.1% to about 5% by weight, from about 0.1% to about 4% by weight, from about 0.1% to about 3% by weight, from about 0.1% to about 2% by weight, or from about 0.1% to about 1% by weight, or any value between these ranges, based on the total weight of the composition.

[0064] Pharmaceutical compositions can generally be in any physical form suitable for use in treating a subject. These forms are referred to as unit dosage forms, such as individual pills or tablets. In some instances, pharmaceutical compositions are formulated as tablets, capsules, granules, powders, liquids, suspensions, gels, syrups, slurries, suppositories, patches, nasal drops, aerosols, injections, implantable sustained release formulations, or mucoadhesive films. In some instances, pharmaceutical compositions are formed as tablets, bilayer tablets, capsules, multiparticulates, drug-coated spheres, matrix tablets, or multi-core tablets. The physical form can be selected according to the desired method of treatment.

[0065] The pharmaceutical composition can be prepared by various conventional methods, such as conventional mixing, dissolving, granulating, dragee-making, suspending, emulsifying, encapsulating, encapsulating, or lyophilizing processes. The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate the processing of the active agent into a pharma-ceutically usable preparation. The appropriate formulation can be selected according to the selected route of administration.

[0066] When the above composition is in powder form, it can be conveniently used in the preparation of beverages, pastes, jellies, capsules, and tablets. Lactose and corn starch are commonly used as diluents for capsules and carriers for tablets. Lubricants such as magnesium stearate are commonly included in tablets.

[0067] The composition of the present invention can be a dietary supplement or a pharmaceutical preparation. Dietary supplements can include additional nutrients such as minerals or amino acids. The composition can also be a food. As used herein, the term "food" refers broadly to any type of liquid and solid / semi-solid material used to nourish humans and animals, to maintain normal or accelerated growth, or to maintain stamina or alertness. Examples of human foods include, but are not limited to, tea-based beverages, juices, coffee, milk, jellies, cookies, cereals, chocolates, snack bars, herbal extracts, dairy products (such as ice cream, yogurt, etc.), soy products (such as tofu), rice products, etc.

[0068] Treatment method for amyotrophic lateral sclerosis In some embodiments, a method for treating amyotrophic lateral sclerosis comprises administering to a patient in need thereof a therapeutically effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid. In some embodiments, a method for treating ALS comprises administering to a patient in need thereof a therapeutically effective amount of quercetin, vitamin B3, vitamin C, a thiol isomerase inhibitor, and optionally folic acid, wherein the extracellular thiol isomerase inhibitor is zafirlukast, montelukast, CGP-13501, CGP-7930, alosetron, balsalazide, benserazide, butaclamol, levadopa, mesalazine, oxcarbazepine, pharmaceutically acceptable salts, prodrugs, and / or solid forms thereof.

[0069] The diagnosis of ALS is made on the basis of clinical signs established by a neurologist based on the history of the disease, the topographical distribution of neuronal loss, and the findings of some characteristic cytological changes. The use of the described methods and pharmaceutical compositions can result in the slowing, alleviation, or elimination of the disease, symptoms, or other undesirable characteristics in a subject compared to a control population (e.g., without treatment with the described methods and materials). The use of the described methods and pharmaceutical compositions can result in the halting or slowing of the progression of the disease, symptoms, or other undesirable characteristics in a subject compared to a control population. Dysregulation of mast cells is associated with the pathophysiology of ALS. The use of the described methods and pharmaceutical compositions can result in the downregulation of mast cell-induced IL-17, tryptase, substance P, and histamine. This can result in improved lung function and prevent the loss of the ability to fully inflate the lungs. The amount of reduction can generally be any amount. For example, the reduction can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and in ideal situations, about a 100% reduction (complete elimination of the disease, symptoms, or other undesirable characteristic).

[0070] In some embodiments, a method for treating ALS includes inhibiting or preventing cytokine production in a patient, the method comprising administering a therapeutically effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid. As sentinel cells that sense danger in the form of pathogens and other types of insults early in inflammation, mast cells play a pivotal role in initiating and propagating immune responses. Dysregulated mast cell function is critically involved in the pathology of ALS. In one embodiment, a method for inhibiting or preventing cytokine production in a patient includes administering a therapeutically effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid to a patient in need thereof. In some embodiments, a therapeutically effective amount of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid can reduce or delay the onset of muscle weakness, loss of lung function due to loss of chest expansion ability.

[0071] In some embodiments, a method of treating amyotrophic lateral sclerosis (ALS), comprising administering therapeutically effective amounts of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid to a patient or subject in need thereof, wherein the method of treating results in at least one of: reduced neuroinflammation in the degenerating spinal cord, reduced number of reactive astrocytes, reduced number of enlarged astrocytes in the lumbar spinal cord, reduced microgliosis, and reduced number of ventral horn motor neurons expressing misfolded SOD1 and ubiquitin, as well as reduced markers of mast cell activity, reduced or delayed onset of muscle weakness, and loss of lung function due to loss of chest expansion.

[0072] The method of the present invention significantly reduces cholesterol and levels, increases hemoglobin levels, reticulocyte counts, and red blood cell mass, and also reduces markers of mast cell activity. In addition, the composition results in increased mitochondrial biogenesis or retention (e.g., in muscle and brain cells), reduced mitochondrial DNA damage and mitochondrial loss, or increased cytochrome C levels and citrate synthase activity. Thus, it is useful for treating diseases associated with mitochondrial defects in both humans and animals. The composition can also induce gene expression and activity of T-helper lymphocyte (Th-1) cytokines (e.g., interferon gamma) and downregulate T-helper lymphocyte 2 (Th-2) cytokines (e.g., interleukin 13). In addition, it can inhibit the expression and / or activity of one or more of the following three enzymes: matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), and cyclooxygenase 2 (COX2). It can also inhibit epidermal growth factor receptor-mediated pathways, such as those mediated by epiregulin. The composition can be used to prevent neurodegeneration by chelating excess iron accumulation in neuronal cells. It is also an activator of sirtuins, which can increase the expression of SIRT1, 2, 3, 4, PGC1α, citrate synthase, and cytochrome C, thereby increasing mitochondrial size, efficiency, and production.

[0073] The methods may also be used to treat diseases and disorders, such as neurological and neurodegenerative diseases (e.g., dyslexia, dyspraxia, autism, Asperger's disease, Alzheimer's disease, mild cognitive impairment, and Huntington's disease).

[0074] Additionally, the method can be used to treat certain symptoms of the above diseases or disorders. For example, it can be used to reduce certain symptoms of ALS, including muscle weakness, especially in the legs, feet, and ankles, difficulty walking and performing daily activities, muscle wasting, pain (e.g., facial pain or pain of unknown origin), muscle spasms, arm, shoulder, and tongue spasms, electric shock sensations, loss of awareness of the location of body parts, loss of coordination (e.g., speech), slurred speech, difficulty swallowing, increased frequency of falls, loss of posture, tremors when performing fine movements, loss of ability to perform rapid alternating movements (e.g., rhythmic movements), and short- or long-term memory loss. The method can be used to improve lung function and chest expansion in ALS patients. Additionally, the method can be used as a dietary supplement to improve the quality of life of patients.

[0075] The above-mentioned methods can be preliminarily screened for efficacy in treating the above-mentioned conditions by in vitro assays, and then confirmed by animal experiments and clinical trials.Other suitable analytical and biological assays will be apparent to those skilled in the art.For example, the bioavailability of quercetin can be measured by carrying out pharmacokinetic studies, and can be evaluated by the area under the curve in the plasma-drug concentration-time curve.

[0076] The compounds and pharmaceutical compositions described herein can be administered in therapeutically effective dosages to treat the aforementioned conditions, disorders, and diseases.

[0077] The compounds and pharmaceutical compositions described herein can be administered in prophylactically effective doses to alleviate or prevent the mentioned conditions, disorders, and diseases.

[0078] Administration can be performed daily for several days (e.g., about 2 to about 7 days), several weeks (e.g., about 1 to about 4 weeks, specifically about 2 or about 3 weeks), or several months (e.g., about 1 to about 36 months, specifically about 2 to about 24 months, more specifically about 6 to about 12 months). In some embodiments, administration can be performed daily for the remainder of the patient's life.

[0079] The therapeutically effective amount ranges from about 0.001 pg / kg / day to about 500 mg / kg / day, preferably 0.01 pg / kg / day and 100 mg / kg / day. Those skilled in the art will appreciate that certain factors may affect the dosage required to effectively treat a patient, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the patient, and other diseases present. Furthermore, treatment of a patient with a therapeutically effective amount of a compound may include a single treatment or may include a series of treatments. It will also be appreciated that the effective amount of a compound used for treatment may increase or decrease over the course of a particular treatment.

[0080] The method of administering effective amounts of quercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid to a patient in need thereof may be performed once, twice, or three or more times daily. Within this embodiment, administration may be orally.

[0081] Administration can generally be performed by any method. Administration methods include infusion or injection of the composition. Exemplary administration methods include topical delivery, subcutaneous delivery, intravenous injection (IV) delivery, intramuscular injection (IM) delivery, intrathecal injection (IT) delivery, intraperitoneal injection (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, and combinations thereof. In some embodiments, administration includes oral delivery.

[0082] For injection, the pharmaceutical composition can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, physiological saline buffer, and / or in a specific emulsion. The solution can contain one or more formulating agents, such as suspending agents, stabilizing agents, and / or dispersing agents. In certain examples, the pharmaceutical composition is provided in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use. For transmucosal administration, one or more penetrants appropriate for the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art.

[0083] For oral administration, the composition is formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc., for oral ingestion by the patient to be treated. For example, for oral solid formulations, the composition can take the form of a powder, capsule, and tablet. For oral administration, the composition can take the form of a tablet, troche, etc., formulated in a conventional manner.

[0084] For administration by inhalation, the pharmaceutical compositions may be delivered in the form of an aerosol spray from pressurized packs or nebulizers using a suitable propellant, such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0085] The subject is generally a mammal. Examples of subjects include primates, humans, dogs, cats, mice, rats, cows, horses, pigs, rabbits, and ferrets. In some examples, the subject is a human. The terms "subject," "individual," or "patient" are used interchangeably and are intended to include humans and non-human animals as used herein. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, rats, cats, cows, horses, ferrets, chickens, amphibians, reptiles, and the like. Examples of mammals include non-human primates, sheep, dogs, cats, cows, ferrets, horses, and the like. Example 1

[0086] Example 1: Demonstration of neuroprotective effects following administration of isoquercetin in an ALS mouse model Amyotrophic lateral sclerosis (ALS) is a paralytic neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. Survival after diagnosis ranges from 1 to more than 5 years and is determined in large part by the rate of spread of motor neuron pathology. The progression of paralysis in rodent models of ALS appears to be regulated, at least in part, by glial cells that become reactive in the degenerating spinal cord, proliferate, and express inflammatory mediators (Barbeito, et al., 2004; Ilieva et al., 2009; Lobsiger, et al., 2007). The SOD1G93A mutant rat model of ALS is a well-characterized model of the disease (Howland, et al., 2002). This model is characterized by the rapid spread of paralysis after onset, which is associated with prominent activation of glial cells and the appearance of abnormal glial cells that actively proliferate around degenerated motor neurons (Howland, et al., 2002; Diaz, et al., 2011). Furthermore, abnormal glial cells show significant neurotoxicity to cultured motor neurons (Diaz, et al., 2011; Trias, et al., 2013), suggesting that they may directly contribute to the rapid spread of paralysis in ALS rats.

[0087] Pharmacological downregulation of abnormal glial cells and reactive microglia by the tyrosine kinase inhibitor masitinib slows the progression of paralysis in a rat model of ALS (Trias, et al., 2016). We reasoned that microglial activation in ALS spinal cord may be sensitive to a quercetin derivative (isoquercetin) orally administered together with vitamin C and niacin to SOD1G93A rats. Quercetin is a naturally occurring flavonol characterized by a phenylbenzo(y)pyrone-derived structure and has strong antioxidant, neuroprotective, and immunomodulatory properties. Although the bioavailability of quercetin is sometimes limited, its derivative, isoquercetin (quercetin-3-O-β-d-glucopyranoside), has an improved pharmacokinetic profile and is efficiently converted to quercetin in vivo (Stopa, et al., 2017). Several reports have provided evidence for the neuroprotective effects of quercetin in animal models of Alzheimer's disease and ALS (Jung, et al., 2010; Sabogal-Guaqueta, et al., 2015) (Lazo-Gomez and Tapia, 2017).

[0088] material and method: Animals: Male SOD1G93A offspring were used for breeding to maintain the line. Rats were housed in a central animal facility with a 12-h light / dark cycle and free access to food and water. Perfusion with fixatives was performed under 90% ketamine-10% xylazine anesthesia, and efforts were made to minimize animal pain, discomfort, and stress. All procedures with experimental animals were performed in accordance with national and international guidelines and approved by the Institutional Animal Committee for Animal Experimentation. Male hemizygous NTac:SD-TgN(SOD1-G93A) L26H rats (Taconic), developed by Howland et al. (2002), were mated with wild-type non-transgenic Sprague-Dawley female rats and bred locally.

[0089] Treatment with Isoquercetin:All rats were weighed and their locomotion was assessed daily. Onset of disease was defined as significant muscle atrophy accompanied by gait abnormalities, typically expressed as a subtle dragging of one hind limb. End stage was defined as loss of righting reflex or inability to reach for food or water.

[0090] Immunohistochemical staining of rat spinal cord: After administration of 30 mg / kg / day isoquercetin for 15 days from the onset of paralysis, animals were deeply anesthetized and perfused transcardially with 0.9% saline and 4% paraformaldehyde in 0.1 M PBS (pH 7.2–7.4). Fixed spinal cords were removed, immersion-fixed for 24 h, and transversely sectioned (30 μm) on a Leica cryostat. For immunohistochemistry, serial sections were collected in 100 mM PBS. Free-floating sections were permeabilized with 0.3% Triton X-100 in PBS for 30 min at room temperature, passed through wash buffer, blocked with 5% BSA:PBS for 1 h at room temperature, and incubated overnight at 4 °C in a solution of 0.3% Triton X-100 and PBS containing primary antibodies. Primary antibodies used were rabbit anti-GFAP (1:500, Sigma), mouse anti-S100β (1:400, Sigma), rabbit anti-Iba1 (1:300, Abcam), mouse anti-CD68 (1:200, Abcam), and mouse anti-ChAT (choline acetyltransferase) (1:300, Millipore). After washing, sections were incubated with 1:1000 diluted secondary antibodies conjugated with Alexa Fluor 488 and / or Alexa Fluor 633 (1:1000, Invitrogen). Antibodies were detected by confocal microscopy using a ZEISS LSM 780 confocal microscope.

[0091] result:Isoquercetin administration after spinal cord paralysis reduces neuroinflammation in the degenerating spinal cord. We investigated whether post-paralysis treatment with isoquercetin would reduce the number of reactive astrocytes in the degenerating spinal cord. Astrocytes were identifiable as large GFAP-positive cells located around motor neurons as previously described. When rats were orally administered isoquercetin (30 mg / kg / day) for 15 days starting immediately after the onset of paralysis, isoquercetin (IsoQ) significantly reduced the number of enlarged astrocytes in the lumbar spinal cord compared to vehicle-treated rats (Figure 1; scale bars 5 μm and 20 μm).

[0092] Isoquercetin administration after paralysis significantly reduced microgliosis. Microgliosis was assessed by assessing the number of Iba1+ expressing cells in the ventral horn of the lumbar spinal cord compared to vehicle-treated animals. Notable was a reduction in the enlarged Iba1+ microglial cells surrounding motor neurons (Figure 2).

[0093] Isoquercetin ameliorates motor neuron pathology. Because motor neuron death is the main pathological feature of symptomatic rodent models and human ALS, we used the same experimental setup to investigate whether motor neuron pathology was affected by isoquercetin treatment. As shown in Figure 3, the number of ventral horn motor neurons expressing misfolded SOD1 and ubiquitin was measured 15 days after the onset of paralysis in vehicle-treated SOD1G93A rats and was clearly reduced in isoquercetin-treated rats compared to vehicle-treated rats. Misfolded SOD1 and ubiquitin are considered reliable markers of motor neuron pathology in ALS (Light, et al., 1991).

[0094] Isoquercetin is a flavonoid with pleiotropic antioxidant, anti-inflammatory, and neuroprotective properties (Lazo-Gomez and Tapia, 2017; Jung, et al., 2010; Sabogal-Guaqueta, et al., 2015). Here, we provide evidence that isoquercetin therapeutically modulates neuroinflammation associated with ALS progression. Notably, administration of isoquercetin to already paralyzed rats clearly reduced astrocytosis and microgliosis in the degenerating spinal cord. Such a therapeutic approach is attractive in the clinical setting of ALS, where drug treatment is initiated only after the onset of obvious motor symptoms. Example 2

[0095] Example 2: Demonstration of synergistic neuroprotective effects of post-paralysis treatment with isoquercetin and zafirlukast in an ALS mouse model material and method: Treatment with isoquercetin and zafirlukast: Disease onset is determined for each animal when significant muscle atrophy is accompanied by an abnormal gait, typically expressed as a subtle dragging of one hind limb. End stage is defined by loss of righting reflex or inability to reach for food or water.

[0096] Immunohistochemical staining of rat spinal cord: After 15 days of treatment with a composition containing isoquercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid, spinal cords are removed from animals and prepared for immunohistochemistry as described in Example 1. Primary antibodies include rabbit anti-GFAP (1:500, Sigma), mouse anti-S100β (1:400, Sigma), rabbit anti-Iba1 (1:300, abcam), mouse anti-CD68 (1:200, abcam), mouse anti-ChAT (choline acetyltransferase) (1:300, Millipore). After washing, sections are incubated with 1:1000 diluted secondary antibodies conjugated with Alexa Fluor 488 and / or Alexa Fluor 633 (1:1000, Invitrogen). Antibodies are detected by confocal microscopy using a ZEISS LSM 780 confocal.

[0097] result: Treatment with a composition comprising isoquercetin, vitamin B3, vitamin C, zafirlukast, and optionally folic acid has a synergistic effect in reducing mast cell activation and neuroinflammation more than isoquercetin or zafirlukast alone. Inhibiting mast cell activation results in the inhibition or downregulation of IL-17, tryptase, substance P, and histamine. Post-paralysis treatment with the composition reduces neuroinflammation in the degenerating spinal cord as measured by a reduction in the number of reactive astrocytes in the degenerating spinal cord, identifiable as large GFAP-positive cells located around the motor neurons. Treatment with the composition also reduced the number of hypertrophied astrocytes in the lumbar spinal cord. Isoquercetin ameliorates motor neuron pathology. Motor neuron pathology is affected by treatment with the composition as measured by a reduction in the number of ventral horn motor neurons expressing misfolded SOD1 and ubiquitin 15 days after onset of paralysis compared to vehicle-treated rats.

Claims

1. A composition for treating amyotrophic lateral sclerosis, comprising effective amounts of quercetin, vitamin B3, vitamin C, and zafirlukast as active ingredients, the composition being administered to a subject in need thereof.

2. 10. The composition of claim 1, wherein the composition comprises about 250 mg to about 2500 mg of quercetin.

3. 10. The composition of claim 1, wherein the composition comprises about 20 μg to about 3 g of vitamin B3.

4. 10. The composition of claim 1, wherein the composition comprises about 200 μg to about 3 g of vitamin C.

5. 10. The composition of claim 1, further comprising folic acid.

6. 6. The composition of claim 5, wherein the composition comprises about 1000 μg to about 3000 μg of folic acid.

7. The composition of claim 1, wherein the composition comprises from about 5 mg to about 320 mg of zafirlukast.

8. 10. The composition of claim 1, wherein the composition comprises about 80 mg to about 160 mg of zafirlukast.

9. 10. The composition of claim 1, wherein the effective amount is sufficient to halt the progression of one or more symptoms of ALS.

10. A composition according to claim 1, in which IL-17 is downregulated.

11. The composition of claim 9, wherein one or more of the ALS symptoms are selected from the group consisting of muscle weakness, difficulty walking, difficulty performing daily activities, muscle wasting, pain, muscle spasms, convulsions, loss of awareness of the location of body parts, loss of coordination, slurred speech, difficulty swallowing, thrombosis, increased frequency of falls, loss of posture, tremors when performing fine movements, loss of ability to perform rapid alternating movements, impaired pulmonary function, loss of ability to fully inflate the lungs, and loss of ability to expand the chest.

12. The composition of claim 1, wherein said administration results in a reduction in misfolded SOD1, a reduction in misfolded ubiquitin, a reduction in astrocytosis, a reduction in microgliosis, or a combination thereof.

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