Of note: The EDSS scale ranges from 0 to 10 in 0.5 unit increments that represent higher levels of disability. Scoring is based on an examination by a neurologist. EDSS steps 1.0 to 3.5 refer to measures of impairment in eight functional systems (FS): pyramidal - weakness or difficulty moving limbs, cerebellar - ataxia, loss of coordination or tremor, brainstem - problems with speech, swallowing and nystagmus, sensory - numbness or loss of sensations, bowel and bladder function, visual function, cerebral (or mental) functions and other. The term "Multiple Sclerosis Functional Composite" or "MSFC" refers to a performance measure that uses standardized procedures for testing human function, and consists of the Timed 25 Foot Walk (T25FW); the 9 Hole Peg Test (SHPT); and the Paced Auditory Serial Arithmetic Test (PASAT).

The term "Modified Fatigue Impact Scale" or "MFIS" refers to the validated specific subject- reported outcome measure developed to evaluate the impact of fatigue on the lives of people with MS. This instrument provides an assessment of the effects of fatigue in terms of physical, cognitive, and psychosocial functioning. The full-length MFIS, consists of 21 items while the abbreviated version has 5 items.

The term "SF-36" refers to the multi-purpose, short-form health survey with 36 questions which yields an 8-scale profile of functional health and well-being scores as well as psychometrically-based physical and mental health summary measures and a preference -based health utility index. It is a generic measure, as opposed to one that targets a specific age, disease, or treatment group. The survey is developed by and can be obtained from QualityMetric, Inc. of Providence, R.I.

An agent, which is capable of reducing blood glutamate levels and enhancing brain to blood glutamate efflux according to this aspect of the present invention includes any glutamate modifying enzyme.

As used herein "a glutamate modifying enzyme" is an enzyme, which utilizes glutamate as a substrate and produces a glutamate reaction product. A glutamate modifying enzyme can be a natural occurring enzyme or an enzyme which has been modified to obtain improved features, such as higher affinity to glutamate than to a modified glutamate, stability under physiological conditions, solubility, enhanced enantioselectivity, increased thermostability and the like as is further described hereinunder.

Numerous glutamate modifying enzymes are known in the art. For example, transaminases, which play a central role in amino acid metabolism and generally funnel a-amino groups from a variety of amino acids via the coupled conversion of glutamate into a-ketoglutarate or of a- ketoglutarate into glutamate. Exemplary transaminases include, but are not limited to, glutamate oxaloacetate transaminases (GOT) and glutamate pyruvate transaminases (GPT). Additional exemplary transaminases are described in PCT publication no. WO/2009/144699, incorporated herein by reference in its entirety.

Other examples of glutamate modifying enzymes include, but are not limited to, glutamate dehydrogenases, which generate ammonium ion from glutamate by oxidative deamination; decarboxylases such as glutamate decarboxylase; ligases such as glutamate-ethylamine ligase, glutamate-cysteine ligase; transferases such as glutamate N-acetyltransferase and N2-acetyl-L- ornithine, adenylyltransferase; aminomutases such as glutamate- 1-semialdehyde 2,1-aminomutase and glutamate racemase [Glavas and Tanner (2001) Biochemistry 40(21):6199-204)].

It will be appreciated that artificially modified enzymes can also be used according to this aspect of the present invention.

Modification of enzymes can be effected using numerous protein directed evolution technologies known in the art [for review see Kuchner and Arnold (1997) TIBTECH 15:523-530; Furukawa J Bacteriol. 2004 Aug;186(16):5189-96] and WO/2009/144699 all incorporated herein by reference.

Since modified glutamate (i.e., glutamate reaction product) can be reversibly modified (i.e., interconverted) to glutamate, the agent, according to this aspect of the present invention, preferably includes a modified glutamate converting enzyme which is incapable of converting the modified glutamate back into glutamate to thereby insuring continual metabolism of glutamate.

Examples of modified or modifiable glutamate converting enzymes include but are not limited to GPT, GOT, Glutamate decarboxylase and glutamate dehydrogenase. Modified glutamate converting enzymes can also include glutamate modifying enzymes artificially modified to possess lower affinity for glutamate reaction product than for glutamate. For example, the E. coli GOT (GenBank Accession No. D90731.1) is characterized by an affinity for glutamate of about 8 mM and an affinity for 2-ketoglutarate of about 0.2 mM. A human enzyme or a humanized enzyme characterized by such affinities is preferably used according to this aspect of the present invention such as described by Doyle et al. in Biochem J. 1990 270(3):651-7.

According to one embodiment, the glutamate modifying enzyme is a glutamate oxaloacetate transaminase (GOT) or a glutamate pyruvate transaminase (GPT).

According to one embodiment, the glutamate oxaloacetate transaminase (GOT) is a Glutamate Oxaloacetate Transaminase 1 (GOT1), also referred to as serum-GOTl.

The agent which reduces blood glutamate levels according to this aspect of the present invention can include one or more co-substrates of glutamate modifying enzymes, which can accelerate activity of the latter (V_max). These can be administered in order to enhance the rate of endogenous glutamate modifying enzymes or in conjunction with glutamate modifying enzymes (described hereinabove).

Co-substrates of glutamate-modifying enzymes include, but are not limited to, oxaloacetate, pyruvate, NAD⁺, NADP⁺, 2-oxohexanedioic acid, 2-oxo-3-sulfopropionate, 2-oxo-3-sulfmopropionic acid, 2-oxo-3-phenylpropionic acid, 3-indole-2-oxopropionic acid, 3-(4-hydroxyphenyl)-2- oxopropionic acid, 4-methylsulfonyl-2-oxobutyric acid, 3-hydroxy-2-oxopropionic acid, 5- oxopentanoate, 6-oxo-hexanoate, glyoxalate, 4-oxobutanoate, a-ketoisocaproate, a-ketoisovalerate, a-keto-P-methyl valerate, succinic semialdehyde-(-4-oxobutyrate), 3-oxoisobutanoate, 5- oxopentanoate, 6-oxohexanoate and their artificially modified derivatives (e.g., esters).

According to one embodiment, the glutamate modifying enzyme comprises glutamate oxaloacetate transaminase (GOT) and the co-substrate comprises oxaloacetate.

According to one embodiment, the glutamate modifying enzyme comprises glutamate pyruvate transaminase (GPT) and the co-substrate thereof comprises pyruvate.

Optionally, co-factors of modified glutamate converting enzymes can be used according to some embodiments of the present invention. Examples of co-factors of modified glutamate converting enzymes include, but are not limited, to lipoic acid and its precursors, thiamine pyrophosphate and its precursors, pyridoxal phosphate and its precursors and the like.

According to one embodiment, the glutamate modifying enzyme comprises glutamate oxaloacetate transaminase (GOT) and the co-factor thereof comprises pyridoxal phosphate.

According to one embodiment, the glutamate modifying enzyme comprises glutamate pyruvate transaminase (GPT) and the co-factor thereof comprises pyridoxal phosphate.

In some cases, the agent administered is modified in order to increase the therapeutic effect or reduce unwanted side effects.

Alternatively or additionally, to increase the life-time of the enzyme in the blood, the present invention also envisages repetitive administration of the enzyme (e.g., on a monthly basis), use of reagents which will increase the life-time in the circulation e.g. PEGylated enzyme, or the entrapment of the enzyme in suitable carriers that should cause a sustained increase of blood enzyme levels allowing an effective blood glutamate scavenging with lower concentrations of the co-factor.

Alternatively, recombinant or purified enzymes of the present invention may be attached to a heterologous polypeptide that increases the half-life of the enzyme in the serum. Examples of heterologous amino acid sequences that may be used in accordance with the teachings of the present invention include, but are not limited to, immunoglobulin, galactosidase, glucuronidase, glutathione - S-transferase (GST), carboxy terminal peptide (CTP) from chorionic gonadotrophin (CG) and chloramphenicol acetyltransferase (CAT) (see for example U.S. Publication No. 20030171551, incorporated herein by reference).

According to one embodiment, the glutamate oxaloacetate transaminase (GOT) is a recombinant GOT (rGOT).

According to one embodiment, the rGOT is rGOTl.

According to one embodiment, rGOTl chimeric protein is generated by fusing the human GOTl gene (without the first ATG codon) to the C-terminus of the gene encoding the Small Ubiquitin-like Modifier (SUMO) entity, as described in PCT publication no. WO/2016/157190, incorporated herein by reference in its entirety.

According to one embodiment, the SUMO-rGOT chimeric protein is expressed in a host cell (e.g. bacterial cell), after which the SUMO entity is cleaved from the fusion protein, by a SUMO protease and the rGOTl is isolated and purified, as described in PCT publication no. WO/2016/157190, incorporated herein by reference in its entirety.

According to one embodiment, the rGOT is comprised in a protein preparation comprising Glutamate Oxaloacetate Transaminase 1 (GOT1) polypeptide molecules, wherein 100 % of the GOT1 polypeptide molecules have an alanine at position 1 of the GOT1 polypeptide, and wherein the GOT1 polypeptide molecules constitute at least 95% of the proteins in the preparation.

According to one embodiment, the protein constitutes at least 98 % of the molecules in the preparation.

According to one embodiment, the rGOT is comprised in a pharmaceutical composition comprising the protein preparation as the active agent and a pharmaceutically acceptable carrier.

According to one embodiment, the rGOTl comprises an amino acid sequence at least 90 % homologous to SEQ ID NO: 3.

According to one embodiment, the rGOTl comprises an amino acid sequence at least 95 % homologous to SEQ ID NO: 3.

According to one embodiment, the rGOTl comprises an amino acid sequence at least 99 % homologous to SEQ ID NO: 3.

According to one embodiment, the rGOTl comprises the amino acid sequence as set forth in SEQ ID NO: 3.

According to one embodiment, the rGOTl comprises the nucleic acid sequence as set forth in SEQ ID NO: 1 or 2.

According to one embodiment, rGOTl comprises an enzymatic activity of at least 500 U/L per 1 mg of purified rGOT. Determination of enzymatic activity can be carried out using any method known in the art, such as by a Reflotron strip assay (Roche, Basel, Switzerland).

The agent which reduces blood glutamate levels and enhances brain to blood glutamate efflux of some embodiments of the invention can be administered to a subject using any one of several suitable administration modes which are further described hereinbelow with respect to the pharmaceutical compositions of the present invention.

The agent utilized by the method of the present invention can be administered to an individual subject per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described hereinabove along with other components such as physiologically suitable carriers and excipients, penetrants etc. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the preparation accountable for the biological effect (e.g., the glutamate modifying enzyme).

Hereinafter, the phrases "physiologically acceptable carrier" and pharmaceutically acceptable carrier" are interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration of the pharmaceutical composition of the present invention may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraosseus and intraocular injections.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

According to one embodiment, the route of administration is a peripheral route of administration.

According to one embodiment, the route of administration is a systemic mode of administration.

According to a specific embodiment, the route of administration is an intravenous (i.v.) route. Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (i.e. agent which reduces blood glutamate levels) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., advanced or progressive multiple sclerosis) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

According to one embodiment, an effective amount is an amount that reduces blood (e.g. plasma) glutamate levels and enhances brain-to-blood glutamate efflux.

According to one embodiment, an effective amount is an amount capable of reducing glutamate levels in the central nervous system (CNS) of the subject.

According to one embodiment, an effective amount is an amount capable of reducing glutamate levels in the cerebrospinal fluid (CSF) of the subject.

According to one embodiment, an effective amount reduces the glutamate levels in the CNS (e.g. CSF) by at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or by 100 %.

Obtaining a CSF sample may be effected using any method known in the art, e.g. by fine needle aspiration. Likewise, measuring the glutamate levels in the CSF may be effected using any method known in the art, e.g. using a UPLC/MS/MS system consisting of e.g. an Acquity UPLC chromatographic system and a Quattro Premier XE triple quad mass spectrometer; or using an ELISA kit e.g. Glutamate Assay Kit (such as the one commercially available from Abeam).

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).

Animal models for multiple sclerosis are readily available, such as the experimental autoimmune encephalomyelitis (EAE) mouse model.

Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

According to one embodiment, the glutamate modifying enzyme (e.g. rGOTl) is administered in a single daily administration.

According to one embodiment, the glutamate modifying enzyme (e.g. rGOTl) is administered in multiple daily administrations (e.g. two, three or more administrations per day).

According to one embodiment, the glutamate modifying enzyme (e.g. rGOTl) is administered in a single weekly or monthly administration.

According to one embodiment, the glutamate modifying enzyme (e.g. rGOTl) is administered in multiple weekly or monthly administrations (e.g. two, three or more administrations per week or month).

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

It will be appreciated that the therapeutic compositions of the invention may comprise, in addition to the agents described above, other known medications for the treatment of multiple sclerosis such as, but not limited to, immunosuppressants, immunomodulatory drugs, neuroprotective drugs and cognitive enhancing drugs. According to one embodiment, the agent which reduces blood glutamate levels is administered to the subject prior to, following to, or concomitantly with an agent for the treatment of multiple sclerosis.

According to one embodiment, the agent for the treatment of multiple sclerosis is administered to a subject at an early stage of the disease and the agent which reduces blood glutamate levels is administered to the subject at a later (e.g. at an advanced or progressive stage) of the disease.

According to one embodiment, the agent for the treatment of multiple sclerosis and the agent which reduces blood glutamate levels are co -administered to the subject at any stage of the disease. Co-administration may be carried out on the same day, on consequent days, or even a week or few weeks apart.

According to one embodiment, the agent which reduces blood glutamate levels and the agent for the treatment of multiple sclerosis are be provided in a kit where each component is packed separately or in a co -formulation.

According to one embodiment, the agent for the treatment of multiple sclerosis comprises an immunosuppressant, an immunomodulatory drug, a neuroprotective drug, or cognitive enhancing drug, or a combination thereof.

According to one embodiment, the therapeutic agent for the treatment of multiple sclerosis is selected from the group consisting of a beta interferon, glatiramer acetate (GA, Copaxone^®), fingolimod (Gilenya^®), natalizumab (Tysabri^®), mitoxantrone (Novantrone^®), teriflunimide (Aubagio^®), BG-12 (Tecfidera^®), alemtuzumab (Lemtrada^®), daclizumab (Zinbryta^®), ocrelizumab (Ocrevus^®), amantadine (Symmetrel^®), amitriptyline (Elavil^®), nortriptyline, modafinil (Provigil^®), and dalfampridine (Ampyra^®), or a combination thereof.

According to one embodiment, the therapeutic agent for the treatment of multiple sclerosis is an anti-inflammatory drug.

Exemplary anti-inflammatory agents include, but are not limited to, NSAIDs (Non- Steroidal

Anti-inflammatory Drugs), corticosteroids (such as prednisone) and anti-histamines. Antiinflammatory agents which may be used according to the present teachings include, but are not limited to, Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Dilluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone;

Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;

Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;

Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen;

Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen

Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;

Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride;

Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;

Momiflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine

Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan

Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam

Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium

Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;

Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;

Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin;

Zomepirac Sodium.

Exemplary immunosuppressants and immunomodulatory agents include, but are not limited to, interferons such as alpha or beta interferon (e.g., Avonex^® or Betaseron^®), glatiramer acetate, corticosteroid (e.g. systemic corticosteroid such as methylprednisolone (Solu-Medrol^®)), prednisone (Deltasone^®), azathioprine (AZA), cyclophosphamide, methotrexate and mitoxantrone, or a combination thereof.

According to one embodiment, the cognitive enhancing drug comprises an acetylcholine receptor agonist, an acetylcholinesterase inhibitor, a butyrylcholinesterase inhibitor, an N-methyl-D- aspartate (NMDA) receptor antagonist, an activity-dependent neuroprotective protein (ADNP) agonist, a serotonin 5-HT1A receptor agonist, a 5-HT4 receptor agonist, a 5-HT6 receptor antagonist, a serotonin 1A receptor antagonist, a histamine H3 receptor antagonist, a calpain inhibitor, a vascular endothelial growth factor (VEGF) protein or agonist, a trophic growth factor, an anti-apoptotic compound, an AMPA-type glutamate receptor activator, a L-type or N-type calcium channel blocker or modulator, a potassium channel blocker, a hypoxia inducible factor (HIF) activator, a HIF prolyl 4-hydroxylase inhibitor, an anti-inflammatory agent, an inhibitor of amyloid A.beta. peptide or amyloid plaque, an inhibitor of tau hyperphosphorylation, a phosphodiesterase 5 inhibitor, a phosphodiesterase 4 inhibitor, a monoamine oxidase inhibitor, pharmaceutically acceptable salts thereof, or a combination thereof.

Exemplary cognitive enhancing drug include, but are not limited to, donepezil (Aricept^®), rivastigmine (Exelon^®), galanthamine (Reminyl^®), memantine (Namenda^®), or a combination thereof.

Exemplary neuroprotective therapies for multiple sclerosis are described in Villoslada, P. Multiple Sclerosis and Demyelinating Disorders (2016) 1: 1, incorporated herein by reference.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral

(fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format. For example, SEQ ID NO: 1 is expressed in a DNA sequence format (e.g. , reciting T for thymine), but it can refer to either a DNA sequence that corresponds to an GOT1 nucleic acid sequence, or the RNA sequence of an RNA molecule nucleic acid sequence. Similarly, though some sequences are expressed in a RNA sequence format (e.g. , reciting U for uracil), depending on the actual type of molecule being described, it can refer to either the sequence of a RNA molecule comprising a dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisioned. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531 ; 5, 192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839, 153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,01 1,771 and 5,281,521 ; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

GENERAL MATERIALS AND EXPERIMENTAL PROCEDURES

Animals

Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by subcutaneous injection of a peptide consisting of the 35-55 amino acids of myelin oligodendrocyte glycoprotein (MOG), synthesis by Genscript (Piscataway, NJ, USA), 200 \ag in incomplete Freund's adjuvant enriched with 3.3 mg/ml heat- inactivated Mycobacterium tuberculosis (Sigma, St. Louis, MO,USA). Pertussis toxin (Sigma), 100 ng/mouse, was injected intraperitoneally immediately after the encephalitogenic injection and 48 hours later. Mice were examined daily and scored as follows: 0 - no disease, 1 - loss of tail tonicity, 2 - hind leg weakness, 3 - hind leg complete paralysis, 3.5 hind leg complete paralysis with hind body paresis, 4 - hind and foreleg paralysis, 5 - moribund or dead animals. rGOT treatment

Single administration

rGOT treatment was applied to mice with apparent clinical manifestations, scores 1-3 (termed suppression therapeutic treatment) starting at days 11-14 from disease induction, by daily intraperitoneal injections, 40 μg per mouse per day (2 mg/kg), in 0.1 ml phosphate Buffered saline (PBS). Control mice with identical clinical scores were similarly injected with PBS (untreated controls).

Dual administration

rGOT treatment was applied to mice with apparent clinical manifestations, score 3.0 (termed suppression therapeutic treatment) starting at days 11-14 from disease induction, by twice daily intraperitoneal injections (morning and evening), 40 μg per mouse per injection (4 mg/kg/day), in 0.1 ml phosphate Buffered saline (PBS). Control mice with identical clinical scores were similarly injected with PBS (untreated controls).

Combined treatment with rGOT and Oxaloacetate

rGOT treatment is applied to mice with apparent clinical manifestations, scores 1-3

(suppression therapeutic treatment) starting at days 11-14 from disease induction, by daily intraperitoneal injections, 20 μg per mouse per day (1 mg/kg), in 0.1 ml phosphate Buffered saline (PBS). Mice are also treated with Oxaloacetate via the drinking water of the rGOT treated animals. Control mice with identical clinical scores are similarly injected with PBS (untreated controls) and are not treated with Oxaloacetate.

Measurement of glutamate and rGOT enzymatic levels

One month after EAE induction, CSF and blood were extracted. Glutamate concentrations and rGOT enzymatic levels were determined. Plasma and CSF samples were prepared with an additional filtration step using 3 kDa Amicon Ultra filters (Millipore, Billerica, MA, USA) by centrifugation at 13,500 x g for 60 min at 4 °C after a 5x dilution using an aqueous solution. Analysis of glutamate in mouse plasma and CSF was performed with an UPLC/MS/MS system consisting of an Acquity UPLC chromatographic system and a Quattro Premier XE triple quad mass spectrometer, both from Waters (Waters, Milford, MA, USA), with the latter operating in the MS/MS mode. EXAMPLE 1

The effect of rGOT on EAE

The effect of a single administration of rGOT was examined in the animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis (EAE). Both test groups were taken from the same experiment, but in each graph treatment was initiated when the mice were at different clinical stages. In Figures 1A-C the mice had more severe symptoms (grade 2-3, namely paralysis of hind body) compared to only minor disease in Figures 2A-C (grade 1-1.5, namely only loss of tail tonicity). As seen in the graphs, rGOT is effective on both groups of mice, however, better results were obtained from the group with a more advanced disease of mice. Importantly, both groups of mice were treated after the onset of disease symptoms (i.e. after clinical disease appearance) and not as prevention or blocking treatment for MS.

EXAMPLE 2

The effect of rGOT treatment on glutamate levels and enzymatic levels in the CSF and blood of treated mice

To substantiate and expand the above findings, an additional experiment was performed using similar conditions (i.e. single administration of rGOT) as described in Example 1, above. Additionally, at the end of the experiment (one month after disease induction), the levels of glutamate and rGOT were measured in the cerebrospinal fluid (CSF) and in the blood serum.

rGot treatment was applied to mice with apparent clinical manifestations, scores 2-3 (for

Figures 3A-C) or score 1-1.5 (for Figures 4A-C), starting 11-14 days from disease induction, by daily intraperitoneal injections, 20 μg per mouse per day (1 mg/kg), in 0.1 ml phosphate Buffered saline (PBS). Control mice with identical clinical scores were similarly injected by PBS (untreated controls). As evident from the results, rGOT is effective in both groups of mice, however, better results were obtained from the group with a more advanced disease stage.

Next, the levels of glutamate and rGOT were analyzed in the CSF and blood of naive, non- treated EAE mice and rGOT treated EAE mice. As illustrated in Figures 5A-C, glutamate levels in the CSF of untreated EAE mice is significantly higher compared to that of naive control mice. In rGOT-treated EAE mice, CSF glutamate levels were significantly lower compared to that of untreated EAE mice. The levels of glutamate in the blood serum were not significantly affected by EAE induction or rGOT treatment.

Evaluation of rGOT levels one month after EAE induction revealed (Figures 6A-B) that rGOT levels in the CSF of rGOT-treated EAE mice is slightly higher compared to that of naive controls and compared to untreated EAE controls. However, rGOT concentration in the blood serum of rGOT-treated EAE mice is significantly higher compared to both naive mice and untreated EAE controls.

Taken together, these findings confirm the ability of rGOT-treatment to suppress clinical manifestations of severe EAE especially when treatment is initiated in the progressive disease stage. Furthermore, for the first time, the present inventors demonstrated that rGOT-treatment causes the efflux of the detrimental excess glutamate from the CNS into the blood circulation.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of treating advanced or progressive multiple sclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent which reduces blood glutamate levels and enhances brain to blood glutamate efflux, to thereby treat the advanced or progressive multiple sclerosis in the subject.

2. A therapeutically effective amount of an agent which reduces blood glutamate levels and enhances brain to blood glutamate efflux for use in treating advanced or progressive multiple sclerosis in a subject in need thereof.

3. A method of treating multiple sclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent which reduces blood glutamate levels and enhances brain to blood glutamate efflux and an agent for the treatment of multiple sclerosis, to thereby treat the multiple sclerosis in the subject.

4. A therapeutically effective amount of an agent which reduces blood glutamate levels and enhances brain to blood glutamate efflux and an agent for the treatment of multiple sclerosis for use in treating multiple sclerosis in a subject in need thereof.

5. The method of claim 3, or agent for use of claim 4, wherein the multiple sclerosis is an advanced or progressive multiple sclerosis.

6. The method of claim 1 or 5, or agent for use of claim 2 or 5, wherein the progressive multiple sclerosis comprises a primary-progressive MS (PPMS).

7. The method of claim 1 or 5, or agent for use of claim 2 or 5, wherein the progressive multiple sclerosis comprises a secondary -progressive MS (SPMS).

8. The method of claim 1 or 5, or agent for use of claim 2 or 5, wherein the progressive multiple sclerosis comprises a progressive-relapsing MS (RSPMS).

9. The method of claim 1 or 5, or agent for use of claim 2 or 5, wherein the advanced multiple sclerosis comprises a relapsing remitting MS involving neuronal damage.

10. The method of any one of claims 1, 3 or 5-9, wherein said administering is via a peripheral route.

11. The method of any one of claims 1, 3 or 5-10, wherein said administering is via an intravenous route.

12. The method of any one of claims 1, 3 or 5-11, wherein said administering is effected at least once a day.

13. The agent for use of any one of claims 2, 4-9, wherein said agent which reduces blood glutamate levels is for administration via a peripheral route.

14. The agent for use of any one of claims 2, 4-9 or 13, wherein said agent which reduces blood glutamate levels is for administration via an intravenous route.

15. The agent for use of any one of claims 2, 4-9 or 14, wherein said agent which reduces blood glutamate levels is for administration at least once a day.

16. The method of any one of claims 1, 3 or 5-12, or agent for use of any one of claims 2, 4-9 or 13-15, wherein the agent which reduces blood glutamate levels is at least one glutamate modifying enzyme.

17. The method or agent for use of claim 16, wherein the at least one glutamate modifying enzyme is selected from the group consisting of a transaminase, a dehydrogenase, a decarboxylase, a ligase, an aminomutase, a racemase and a transferase.

18. The method or agent for use of claim 16 or 17, wherein the at least one glutamate modifying enzyme is a glutamate oxaloacetate transaminase (GOT) or a glutamate pyruvate transaminase (GPT).

19. The method or agent for use of claim 18, wherein said glutamate oxaloacetate transaminase (GOT) is a Glutamate Oxaloacetate Transaminase 1 (GOT1).

20. The method or agent for use of claim 18 or 19, wherein said glutamate oxaloacetate transaminase (GOT) is a recombinant GOTl (rGOTl).

21. The method or agent for use of claim 20, wherein said rGOTl is comprised in a protein preparation comprising Glutamate Oxaloacetate Transaminase 1 (GOTl) polypeptide molecules, wherein 100 % of the GOTl polypeptide molecules have an alanine at position 1 of the GOTl polypeptide, and wherein the GOTl polypeptide molecules constitute at least 95 % of the proteins in the preparation.

22. The method or agent for use of claim 21, wherein the protein constitutes at least 98 % of the molecules in the preparation.

23. The method or agent for use of claim 20, wherein said rGOTl is comprised in a pharmaceutical composition comprising the protein preparation of claims 21 or 22 as the active agent and a pharmaceutically acceptable carrier.

24. The method or agent for use of any one of claims 21-23, wherein said GOTl comprises an amino acid sequence at least 90 % homologous to SEQ ID NO: 3.

25. The method or agent for use of any one of claims 21-23, wherein said GOTl comprises the amino acid sequence as set forth in SEQ ID NO: 3.

26. The method of any one of claims 1, 3, 5-12 or 16-25, or agent for use of any one of claims 2, 4-9 or 13-25, wherein said agent which reduces blood glutamate levels comprises a glutamate modifying enzyme and a co-substrate thereof.

27. The method or agent for use of claim 26, wherein the glutamate modifying enzyme comprises glutamate oxaloacetate transaminase (GOT) and the co-substrate thereof comprises oxaloacetate.

28. The method or agent for use of claim 26, wherein the glutamate modifying enzyme comprises glutamate pyruvate transaminase (GPT) and the co-substrate thereof comprises pyruvate.

29. The method of any one of claims 1, 3, 5-12 or 16-28, or agent for use of any one of claims 2, 4-9 or 13-28, wherein said agent which reduces blood glutamate levels comprises a glutamate modifying enzyme and a co-factor thereof.

30. The method or agent for use of claim 29, wherein the glutamate modifying enzyme comprises glutamate oxaloacetate transaminase (GOT) or a glutamate pyruvate transaminase (GPT) and the co-factor thereof comprises pyridoxal phosphate.

31. The method of any one of claims 1, 3, 5-12 or 16-30, or agent for use of any one of claims 2, 4-9 or 13-30, wherein said therapeutically effective amount is an amount of said agent capable of reducing glutamate levels in a cerebrospinal fluid (CSF).

32. The method of any one of claims 1, 3, 5-12 or 16-31, wherein said agent which reduces blood glutamate levels is administered to said subject in combination with an agent for the treatment of multiple sclerosis.

33. The agent for use of any one of claims 2, 4-9 or 13-31, further comprising the use of an agent for the treatment of multiple sclerosis.

34. The method of any one of claims 3 or 32, or agent for use of claim 4 or 33, wherein said agent for the treatment of multiple sclerosis is selected from the group consisting of an antiinflammatory drug, an immunosuppressant drug, an immunomodulatory drug, a neuroprotective drug and a cognitive enhancing drug.

35. The method or agent for use of claim 34, wherein said agent for the treatment of multiple sclerosis is selected from the group consisting of a beta interferon, glatiramer (Copaxone^®), fingolimod (Gilenya^®), natalizumab (Tysabri^®), mitoxantrone (Novantrone^®), teriflunimide (Aubagio^®), BG-12 (Tecfidera^®), alemtuzumab (Lemtrada^®), daclizumab (Zinbryta^®), ocrelizumab (Ocrevus^®), amantadine (Symmetrel^®), amitriptyline (Elavil^®), nortriptyline, modafinil (Provigil^®), and dalfampridine (Ampyra^®).

36. The method of any one of claims 1, 3, 5-12, 16-32 or 34-35, or agent for use of any one of claims 2, 4-9, 13-31 or 33-35, wherein the subject is a human subject.