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  Pharmaceutical Patents  

 

Title:  Method and composition for protecting neuronal tissue from damage induced by elevated glutamate levels
United States Patent: 
7,404,951
Issued: 
July 29, 2008

Inventors:
 Teichberg; Vivian I. (Savyon, IL)
Assignee:
  Yeda Research And Development Co. Ltd. (Rechovot, IL)
Appl. No.:
 10/522,415
Filed:
 July 31, 2003
PCT Filed:
 July 31, 2003
PCT No.:
 PCT/IL03/00634
371(c)(1),(2),(4) Date:
 January 26, 2005
PCT Pub. No.:
 WO2004/012762
PCT Pub. Date:
 February 12, 2004


 

Outsourcing Guide


Abstract

A method of reducing extracellular brain glutamate levels. The method comprises administering to a subject in need thereof a therapeutically effective amount of an agent capable of reducing blood glutamate levels thereby reducing extracellular brain glutamate levels.

Description of the Invention

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided a method of reducing extracellular brain glutamate levels, the method comprising administering to a subject in need thereof a therapeutically effective amount of an agent capable of reducing blood glutamate levels thereby reducing extracellular brain glutamate levels.

According to another aspect of the present invention there is provided a pharmaceutical composition for reducing extracellular brain glutamate levels, the pharmaceutical composition comprising, as an active ingredient, an agent capable of reducing blood glutamate levels and a pharmaceutically acceptable carrier.

According to yet another aspect of the present invention there is provided a pharmaceutical composition for reducing extracellular brain glutamate levels, the pharmaceutical composition comprising, as an active ingredient, pyruvate and oxaloacetate in a concentration suitable for reducing blood glutamate levels and a pharmaceutically acceptable carrier.

According to still another aspect of the present invention there is provided an article-of-manufacture comprising packaging material and a pharmaceutical composition identified for reducing extracellular brain glutamate levels being contained within the packaging material, the pharmaceutical composition including, as an active ingredient, an agent capable of reducing blood glutamate levels and a pharmaceutically acceptable carrier.

According to an additional aspect of the present invention there is provided a method of reducing extracellular brain glutamate levels in a subject in need thereof, the method comprising: (a) obtaining a blood sample; (b) contacting the blood sample with an agent capable of reducing glutamate levels of cells present in the blood sample to thereby obtain glutamate depleted blood cells; and (c) introducing the glutamate depleted blood cells into the subject, thereby reducing extracellular brain glutamate levels thereof.

According to yet an additional aspect of the present invention there is provided a pharmaceutical composition for reducing extracellular brain glutamate levels, the pharmaceutical composition comprising, as an active ingredient, oxaloacetate diethylester capable of reducing blood glutamate levels and a pharmaceutically acceptable carrier Alternative active ingredients of the pharmaceutical composition include, but are not limited to, oxaloacetate, pyruvate, NAD.sup.+, NADP.sup.+, 2-oxohexanedioic acid, 2-oxo-3-sulfopropionate, 2-oxo-3-sulfinopropionic 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, .alpha.-ketoisocaproate, .alpha.-ketoisovalerate, .alpha.-keto-.beta.-methylvalerate, succinic semialdehyde-(-4-oxobutyrate), pyridoxal phosphate, pyridoxal phosphate precursors and 3-oxoisobutanoate.

According to further features in preferred embodiments of the invention described below, the agent is at least one glutamate modifying enzyme and/or a modification thereof (e.g. an ester thereof).

According to still further features in the described preferred embodiments 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.

According to still further features in the described preferred embodiments the transaminase is selected from the group consisting of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, acetylornithine transaminase, ornithine-oxo-acid transaminase, succinyldiaminopimelate transaminase, 4-aminobutyrate transaminase, (s)-3-amino-2-methylpropionate transaminase, 4-hydroxyglutamate transaminase, diiodotyrosine transaminase, thyroid-hormone transaminase, tryptophan transaminase, diamine transaminase, cysteine transaminase, L-Lysine 6-transaminase, histidine transaminase, 2-aminoadipate transaminase, glycine transaminase, branched-chain-amino-acid transaminase, 5-aminovalerate transaminase, dihydroxyphenylalanine transaminase, tyrosine transaminase, phosphoserine transaminase, taurine transaminase, aromatic-amino-acid transaminase, aromatic-amino-acid-glyoxylate transaminase, leucine transaminase, 2-aminohexanoate transaminase, ornithine(lysine) transaminase, kynurenine-oxoglutarate transaminase, D-4-hydroxyphenylglycine transaminase, cysteine-conjugate transaminase, 2,5-diaminovalerate transaminase, histidinol-phosphate transaminase, diaminobutyrate-2-oxoglutarate transaminase, and udp-2-acetamido-4-amino-2,4,6-trideoxyglucose transaminase.

According to still further features in the described preferred embodiments the dehydrogenase is a glutamate dehydrogenase.

According to still further features in the described preferred embodiments the decarboxylase is a glutamate decarboxylase.

According to still further features in the described preferred embodiments the ligase is a glutamate-ethylamine ligase.

According to still further features in the described preferred embodiments the transferase is selected from the group consisting of glutamate N-acetyltransferase and adenylyltransferase.

According to still further features in the described preferred embodiments the aminomutase is a glutamate-1-semialdehyde 2,1-aminomutase.

According to still further features in the described preferred embodiments the agent is at least one co-factor of a glutamate modifying enzyme.

According to still further features in the described preferred embodiments the co-factor is selected from the group consisting of oxaloacetate, pyruvate, NAD.sup.+, NADP.sup.+, 2-oxohexanedioic acid, 2-oxo-3-sulfopropionate, 2-oxo-3-sulfinopropionic 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, .alpha.-ketoisocaproate, .alpha.-ketoisovalerate, .alpha.-keto-.beta.-methylvalerate, succinic semialdehyde-(-4-oxobutyrate), pyridoxal phosphate, pyridoxal phosphate precursors and 3-oxoisobutanoate.

According to still further features in the described preferred embodiments the agent is a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate and/or a modification thereof.

According to still further features in the described preferred embodiments the modified glutamate converting enzyme is a modified glutarriate oxaloacetate transaminase (GOT).

Such a modified enzyme can be obtained via in vitro evolution of, for example, a human GOT sequence. For example, chemical or molecular mutagenesis can be used to generate mutated GOT sequences which exhibit enhanced activity in transforming glutamate into .alpha.-ketoglutarate and preferably little or no reverse activity [strategies for in vitro enzyme evolution is described by, for example, Moore et al., in J Mol. Biol. 1997 Sep. 26; 272(3):336-47, additional references are provided hereinbelow].

According to still further features in the described preferred embodiments the agent is a co-factor of a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate.

According to still further features in the described preferred embodiments the agent is selected from the group consisting of lipoic acid, lipoic acid precursor, pyridoxal phosphate, pyridoxal phosphate precursor, thiamine pyrophosphate and thiamine pyrophosphate precursor.

According to still further features in the described preferred embodiments the agent includes a glutamate modifying enzyme and a co-factor thereof.

According to still further features in the described preferred embodiments the agent includes a glutamate modifying enzyme and a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate.

According to still further features in the described preferred embodiments the agent includes a co-factor of a glutamate modifying enzyme and a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate.

According to still further features in the described preferred embodiments the agent includes a co-factor of a glutamate modifying enzyme, a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate and a co-factor thereof.

According to still further features in the described preferred embodiments the agent includes a glutamate modifying enzyme, a co-factor thereof, a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate and a co-factor thereof.

According to still further features in the described preferred embodiments the agent includes a glutamate modifying enzyme, a co-factor thereof, and a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate.

According to still further features in the described preferred embodiments the agent includes a glutamate modifying enzyme, a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate and a co-factor thereof.

According to still further features in the described preferred embodiments the agent includes a glutamate modifying enzyme and a co-factor of a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate.

According to still further features in the described preferred embodiments the agent includes a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate and a co-factor thereof.

According to still further features in the described preferred embodiments the agent includes a co-factor of a glutamate modifying enzyme and a co-factor of a modified glutamate converting enzyme being selected incapable of converting the modified glutamate into glutamate.

According to still further features in the described preferred embodiments the administering is effected at a concentration of the agent not exceeding 1 g/Kg body weight/hour.

According to still further features in the described preferred embodiments the agent is at least one inhibitor of a glutamate synthesizing enzyme.

According to still further features in the described preferred embodiments wherein the inhibitor is selected from the group consisting of gamma-Acetylenic GABA, GABAculine, L-canaline, 2-amino-4-(aminooxy)-n-butanoic acid, 3-Chloro-4-aminobutanoate, 3-Phenyl-4-aminobutanoate, Isonicotinic hydrazide;(S)-3-Amino-2-methylpropanoate, Phenylhydrazine; 4-Fluorophenyl)alanine, Adipate, Azaleic acid, Caproate, 3-Methylglutarate, Dimethylglutarate, Diethylglutarate, Pimelate, 2-Oxoglutamate, 3-Methyl-2-benzothiazolone hydrazone hydrochloride, Phenylpyruvate, 4-hydroxyphanylpyruvate, Prephenate and Indole pyruvate.

The present invention successfully addresses the shortcomings of the presently known configurations by providing methods and compositions for protecting neuronal tissue from damage induced by elevated glutamate levels

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compositions and methods using same for reducing the levels of extracellular glutamate in the brain of a subject. Specifically, the present invention can be used to treat acute and chronic brain diseases in which elevated levels of glutamate are detrimental to the subject, such as in ischemic conditions.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Abnormally high glutamate levels in brain interstitial and cerebrospinal fluids are the hallmark of several neurodegenerative conditions. Numerous approaches to reduce glutamate excitotoxicity include development of glutamate receptor antagonists, and up-regulation of glutamate transporters. While the first are limited by adverse and even lethal effects probably due to poor selectivity, none of the latter have been successful in providing a viable therapeutic approach for lowering glutamate levels.

While reducing the present invention to practice and while searching for a novel therapeutic modality to clinical conditions associated with elevated extracellular brain glutamate levels, the present inventor has uncovered that excess extracellular brain glutamate can be eliminated by increasing the brain to blood glutamate efflux.

These findings enable to generate highly efficient therapeutic compositions which can be utilized to treat clinical conditions characterized by elevated extracellular brain glutamate levels.

Thus, according to one aspect of the present invention, there is provided a method of reducing extracellular brain glutamate levels.

The method is effected by administering to a subject in need thereof, a therapeutically effective amount of an agent capable of reducing blood glutamate levels, thereby increasing brain to blood glutamate efflux and consequently reducing extracellular brain glutamate levels.

Preferred individual subjects according to the present invention are mammals such as canines, felines, ovines, porcines, equines, bovines, humans and the like.

An agent, which is capable of reducing blood glutamate according to this aspect of the present invention includes any glutamate modifying enzyme and/or a co-factor thereof or any artificially modified derivatives (e.g. esters).

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 .alpha.-amino groups from a variety of amino acids via the coupled conversion of glutamate into .alpha.-ketoglutarate or of .alpha.-ketoglutarate into glutamate.

Examples of transaminases include but are not limited to glutamate oxaloacetate transaminases, glutamate pyruvate transaminases, acetylomithine transaminases, ornithine-oxo-acid transaminases, succinyldiaminopimelate transaminases, 4-aminobutyrate transaminases, alanine transaminases (note: same as glutamate pyruvate transaminases, (s)-3-amino-2-methylpropionate transaminases, 4-hydroxyglutamate transaminases, diiodotyrosine transaminases, thyroid-hormone transaminases, tryptophan transaminases, diamine transaminases, cysteine transaminases, L-Lysine 6-transaminases, histidine transaminases, 2-aminoadipate transaminases, glycine transaminases, branched-chain-amino-acid transaminases, 5-aminovalerate transaminases, dihydroxyphenylalanine transaminases, tyrosine transaminases, phosphoserine transaminases, taurine transaminases, aromatic-amino-acid transaminases, aromatic-amino-acid-glyoxylate transaminases, leucine transaminases, 2-aminohexanoate transaminases, ornithine (lysine) transaminases, kynurenine-oxoglutarate transaminases, D-4-hydroxyphenylglycine transaminases, cysteine-conjugate transaminases, 2,5-diaminovalerate transaminases, histidinol-phosphate transaminases, diaminobutyrate-2-oxoglutarate transaminases, UDP-2-acetamido-4-amino-2,4,6-trideoxyglucose transaminases and aspartate transaminases (please note: same as glutamate oxaloacetate transaminases).

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].

Typically, directed enzyme evolution begins with the creation of a library of mutated genes. Gene products that show improvement with respect to the desired property or set of properties are identified by selection or screening, and the gene(s) encoding those enzymes are subjected to further cycles of mutation and screening in-order to accumulate beneficial mutations. This evolution can involve few or many generations, depending on the progress observed in each generation.

Preferably, for successful directed evolution a number of requirements are met; the functional expression of the enzyme in a suitable microbial host; the availability of a screen (or selection) sensitive to the desired properties; and the identification of a workable evolution strategy.

Examples of mutagenesis methods which can be used in enzyme directed evolution according to this aspect of the present invention include but are not limited to UV irradiation, chemical mutagenesis, poisoned nucleotides, mutator strains [Liao (1986) Proc. Natl. Acad. Sci. U.S.A 83:576-80], error prone PCR [Chen (1993) Proc. Natl. Acad. Sci. U.S.A 90:5618-5622], DNA shuffling [Stemmer (1994) Nature 370:389-91], cassette [Strausberg (1995) Biotechnology 13:669-73], and a combination thereof [Moore (1996) Nat. Biotechnol. 14:458-467; Moore (1997) J. Mol. Biol. 272:336-347].

Screening and selection methods are well known in the art [for review see Zhao and Arnold (1997) Curr. Opin. Struct. Biol. 7:480-485; Hilvert and Kast (1997) Curr. Opin. Struct. Biol. 7:470-479]. Typically, selections are attractive for searching larger libraries of variants, but are difficult to device for enzymes that are not critical to the survival of the host organism. Further more, organisms may evade imposed selective pressure by unexpected mechanisms. Less stringent functional complementation can be useful in identifying variants which retain biological activity in libraries generated using relatively high mutagenic rates [Suzuki (1996) Mol. Diversity 2:111-118; Shafikhani (1997) Biol. Techniques 23:304-310; Zhao and Arnold (1997) Curr. Opin. Struct. Biol. 7:480-485].

As described hereinabove, the agent according to this aspect of the present invention, can include one or more co-factors of glutamate modifying enzymes, which can accelerate activity of the latter (V.sub.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-factors of glutamate-modifying enzymes include but are not limited to oxaloacetate, pyruvate, NAD+, NADP+, 2-oxohexanedioic acid, 2-oxo-3-sulfopropionate, 2-oxo-3-sulfinopropionic 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, .alpha.-ketoisocaproate, .alpha.-ketoisovalerate, .alpha.-keto-.beta.-methylvalerate, succinic semialdehyde-(-4-oxobutyrate), 3-oxoisobutanoate, pyridoxal phosphate, 5-oxopentanoate, 6-oxohexanoate and their artificially modified derivatives (e.g., esters).

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 and GOT, and the like.

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.

Optionally, co-factors of modified glutamate converting enzymes can be included in the agent according to this aspect 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.

It will be appreciated that the agent according to this aspect of the present invention may also include inhibitors of glutamate synthesizing enzymes (e.g., phosphate activated glutaminase). Numerous inhibitors of glutamate producing enzymes are known in the art. Examples include but are not limited gabapentin which has been shown to modulate the activity of branched chain aminotransferases [Taylor (1997) Rev. Neurol. 153(1):S39-45] and aspirin at high doses (i.e., 4-6 g/day) a neuroprotective drug against glutamate excitotoxicity [Gomes (1998) Med. J. India 11:14-17]. Other inhibitors may be identified in the publicly available BRENDA, a comprehensive enzyme information system [www.brenda.uni-koeln.de/]. Examples include but are not limited to, gamma-Acetylenic GABA, GABAculine, L-canaline, 2-amino-4-(aminooxy)-n-butanoic acid; 3-Chloro-4-aminobutanoate; 3-Phenyl-4-aminobutanoate; Isonicotinic hydrazide;(S)-3-Amino-2-methylpropanoate; Phenylhydrazine; 4-Fluorophenyl)alanine; Adipate, Azelaic acid, Caproate, 3-Methylglutarate, Dimethylglutarate, Diethylglutarate, Pimelate, 2-Oxoglutamate; 3-Methyl-2-benzothiazolone hydrazone hydrochloride; Phenylpyruvate, 4-Hydroxyphenylpyruvate, Prephenate, Indole pyruvate and their artificially modified derivatives (e.g., esters). Although each of the components described hereinabove may comprise the agent of the present invention, it will be appreciated that for optimal blood-glutamate reducing activity, the agent may include a combination of the above described components (i.e., glutamate modified enzyme, co-factor thereof, modified glutamate converting enzyme and co-factor thereof).

As further illustrated in the Examples section which follows, for optimal brain-to-blood glutamate efflux the agent is preferably selected capable of reducing plasma glutamate levels rather than blood cell glutamate levels.

Thus, according to preferred embodiments of this aspect of the present invention the agent includes oxaloacetate and pyruvate. Preferably, the agent is administered at a dose not exceeding 1 g/kg.times.hour.

In some cases, the agent administered is modified in order to increase the therapeutic effect or reduce unwanted side effects. For example, administration of oxaloacetate diethylester is favorable over administration of oxaloacetate alone since oxaloacetate exerts its therapeutic potential at relatively high concentrations (see Example 21, FIGS. 27A-B and FIG. 32, see Original Patent) and requires full titration of its carboxyl moieties with sodium hydroxide at 3:1 stoichiometric ratio which presents unacceptable electrolyte load above safe levels.

The agent 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.

Although the administration route is selected so as to enable provision of the agent to the blood stream of the individual in some cases, such as brain surgery, administering can be effected by direct application of the agent to exposed brain tissue.

It is well known that glutamate levels increase significantly during brain surgery involving for instance brain retraction [Xu W, Mellergard P, Ungerstedt U, Nordstrom CH, Acta Neurochir (Wien) 2002 July; 144(7):679-83)], aneurysm surgery [Kett-White R, Hutchinson P J, Al-Rawi P G, Czosnyka M, Gupta A K, Pickard J D, Kirkpatrick P J, J. Neurosurg. 2002 June; 96(6):1013-9)] or surgery of benign lesions of the posterior fossa [Reinstrup P, Stahl N, Mellergard P, Uski T, Ungerstedt U, Nordstrom CH, Neurosurgery. 2000 September; 47(3):701-9; discussion 709-10]. In such cases, topical administration of the agent described above can be utilized to substantially reduce brain glutamate levels thereby substantially reducing the risk of its deleterious effects on brain tissue, including brain tissue edema and overall excitotoxicity.

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 (see Example 21 of the Examples section).

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, and/or cofactors thereof).

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 career 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 and intraocular injections.

Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body or by direct administration to brain tissues (e.g. topical) during, for example, open brain surgery.

Pharmaceutical compositions of the present 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 the present invention 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 invention 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 compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention 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 the present 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 preparations 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 the present 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 the present 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 effective to prevent, alleviate or ameliorate symptoms of disease 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.

For any pharmaceutical composition used by the treatment method of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

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. 1).

The Examples section, which follows provides further guidance as to suitable dosages.

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 or symptoms is achieved.

The amount of the pharmaceutical 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 including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Compositions of the present 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.

The agents of the present invention can be utilized in treating (i.e., reducing or preventing or substantially decreasing elevated concentrations of extracellular brain glutamate) of a variety of clinical conditions associated with elevated levels of extracellular brain glutamate such as brain anoxia, brain ischemia, stroke, perinatal brain damage, traumatic brain injury, bacterial meningitis, subarachnoid haemorhage, migraine, stress, hemorrhagic shock, epilepsy, acute liver failure, glaucoma, amyotrophic lateral sclerosis, HIV, dementia, amyotrophic lateral sclerosis (ALS), spastic conditions, open heart surgery, aneurism surgery, coronary artery bypass grafting and Alzheimer's disease.

It will be appreciated that fast acting pharmaceutical compositions and administration routes described hereinabove are preferably used in treating brain anoxia, brain ischemia, stroke, perinatal brain damage, traumatic head injury, bacterial meningitis, subarachoid haemorhage, migraine, stress, hemorrhagic shock, epilepsy, acute liver failure, open heart surgery, aneurysm surgery, coronary artery bypass grafting. When continuous administration is required a continuous drug release is preferred provided that endogenous production in the depleted organ does not occur.

While reducing the present invention to practice, the present inventor has also uncovered that blood cells which are isolated from the body, depleted of glutamate and returned to the body are capable of inducing a decrease in blood glutamate levels and thereby a decrease of extracellular brain glutamate levels.

Thus according to another aspect of the present invention there is provided an additional method of reducing extracellular bran glutamate levels.

The method according to this aspect of the present invention is based upon the rational that glutamate depleted blood cells, upon transfusion into a host subject, are capable of rapidly pumping plasma glutamate towards the original cell/plasma glutamate concentration ratio (i.e., substantially 4:1), thereby promoting brain-to-blood (i.e., brain-to-plasma) glutamate efflux and reducing extracellular brain glutamate concentration (see Example 15).

The method according to this aspect of the present invention is effected by treating blood samples derived from a subject with glutamate reducing agents such as those described hereinabove, isolating cells from the blood sample and returning the cells to the subject.

Preferably the blood sample according to this aspect of the present invention is obtained from the subject for further autologous transfusion. This reduces the risk of infectious diseases such as hepatitis, which can be transferred by blood transfusions.

It will be appreciated that matching blood type (i.e., matching blood group) samples from syngeneic donors can be used for homologous transfusion although non-matching blood type samples from allogeneic donors may also be used in conjunction with a deantigenation procedure. A number of methods of deantigenation of blood group epitopes on erythrocytes (i.e., seroconversion) are known in the art such as disclosed in U.S. Pat. Nos. 5,731,426 and 5,633,130.

Blood samples are contacted with the agent of the present invention under conditions suitable for reducing blood glutamate levels to thereby obtain glutamate depleted blood cells (as described herein above and further in Examples 14-15 of the Examples section which follows).

Glutamate depleted blood cells are then separated from plasma by well known separation methods known in the art, such as by centrifugation (see Example 14 of the Examples section).

Once glutamate depleted cells are obtained they are suspended to preferably reach the original blood volume (i.e., concentration).

Suspension of glutamate depleted cells is effected using a blood substitute. "A blood substitute" refers to a blood volume expander which includes an aqueous solution of electrolytes at physiological concentration, a macromolecular oncotic agent, a biological buffer having a buffering capacity in the range of physiological pH, simple nutritive sugar or sugars, magnesium ion in a concentration sufficient to substitute for the flux of calcium across cell membranes. A blood substitute also includes a cardioplegia agent such as potassium ion in a concentration sufficient to prevent or arrest cardiac fibrillation. Numerous blood substitutes are known in the art. Examples include but are not limited to Hespan.RTM. (6% hetastarch 0.9% Sodium chloride Injection [Dupont Pharmaceuticals, Wilmington Del.]), Pentaspan (10% pentastarch in 0.9% Sodium chloride Injection [Dupont Pharmaceuticals, Wilmington Del.]) and Macrodex (6% Dextran 70 in 5% Dextrose Injection or 6% Dextran 70 in 0.9% Sodium chloride Injection [Pharmacia, Inc. Piscataway, N.J.]) and Rheomacrodex (10% Dextran 40 in 5% Dextrose Injection or 10% Dextran 40 in 0.9% Sodium chloride Injection [Pharmacia, Inc. Piscataway, N.J.]). These products are known to the medical community for particular FDA approved indications and are extensively described in the volume entitled Physicians' Desk Reference, published annually by Medical Economics Company Inc.

It will be appreciated that treated blood samples may be stored for future use. In this case, however, a sterile preservative anticoagulant such as citrate-phosphate-dextrose-adenine (CPDA) anticoagulant is preferably added to the blood substitute solution. Also added are a gram-negative antibiotic and a gram-positive antibiotic. Blood is then stored in sterile containers such as pyrogen-free containers at 4.degree. C.

Finally, glutamate depleted blood cell solution is transfused intravenously or intravascularly as a sterile aqueous solution into the host subject, to thereby reduce extracellular brain glutamate levels.

It will be appreciated that previous studies have emphasized the relative impermeability of erythrocytes to extracellular glutamate [Young (1980) Proc. R. Soc. Lond. B. Biol. Sci. 209:355-75; Pico (1992) Int. J. Biochem. Cell Biol. 27(8):761-5 and Culliford (1995) J. Physiol. 489(Pt3):755-65]. However, these were done in the presence of an unfavorable glutamate concentration gradient.

Thus, not only does the present invention exhibits erythrocytes permeability to glutamate, thereby explaining the still unclear blood pool of intracellular glutamate, but provides with a blood exchange strategy which can be utilized in emergency conditions such as stroke and head trauma, in which a rapid reduction in CSF/ISF glutamate is desired.

The above described methodology can be effected using currently available devices such as incubators and centrifuges (see the Examples section for further detail) or a dedicated device which is designed and configured for obtaining a blood sample from the subject, processing it as described above and returning glutamate depleted blood cells to the subject or to a different individual which requires treatment.

Such a device preferably includes a blood inlet, a blood outlet and a chamber for processing blood and retrieving processed blood cells. At least one of the blood inlet and outlet is connected to blood flow tubing, which carries a connector spaced from the device for access to the vascular system of the subject.

Blood treatment devices providing an extracorporeal blood circuit to direct blood to a treatment device from the individual subject, and then to return the blood to the individual subject are well known in the art. Such treatment devices include, but are not limited to hemodialysis units, plasmapheresis units and hemofiltration units, which enable blood flow across a unit, which carries a fixed bed of enzyme or other bioactive agent.
 

Claim 1 of 17 Claims

1. A method of reducing extracellular brain glutamate levels in a subject in need thereof, the method comprising intravenously administering to the subject a therapeutically effective amount of a glutamate modifying enzyme, thereby enhancing brain to blood glutamate efflux, thereby reducing extracellular brain glutamate levels.

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