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Link:  Pharm/Biotech Resources


Title:  Methods for modulating brain damage

United States Patent:  6,921,775

Issued:  July 26, 2005

Inventors:  Jensen; Frances E. (Chestnut Hill, MA); Volpe; Joseph (Brookline, MA); Rosenberg; Paul (Newton, MA); Follett; Pamela L. (Boxboro, MA)

Assignee:  Children's Medical Center Corporation (Boston, MA)

Appl. No.:  213393

Filed:  August 5, 2002

Abstract

Methods are disclosed for modulating brain damage mediated by non-NMDA ionotropic glutamate receptor antagonists, as topiramate, in conditions such as periventricular leukomalacia, cerebral palsy, mental retardation and neonatal stroke.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for modulating brain damage mediated by non-NMDA ionotropic glutamate receptors, particularly in fetal and neonatal brains. The invention is based, at least in part, on the discovery that hypoxia/ischemia-mediated brain injury is attenuated by the non-NMDA ionotropic glutamate receptor antagonists NBQX and topiramate. As a consequence of this inhibition, physical markers of brain damage such as white matter lesions, oligodendrocyte cell death, and myelin basic protein loss are decreased. These effects are attributable, at least in part, to excitotoxic oligodendrocyte injury mediated by nonNMDA ionotropic glutamate receptors. Inhibition of non-NMDA glutamate receptors is thought to prevent injury following hypoxic/ischemic insult by preventing the influx of glutamate, an excitatory amino acid which mediates neuronal cell death.

Accordingly, in one aspect, the present invention is directed to a method for modulating, e.g., inhibiting, glutamate-mediated neuronal cell death by modulating non-NMDA ionotropic glutamate receptors. The method includes treating a subject with a non-NMDA ionotropic glutamate receptor antagonist, such that disorders associated with glutamate-mediated neuronal cell death are treated.

In one aspect, the invention provides methods for treating periventricular leukomalacia (PVL), mental retardation, and/or neonatal stroke in a subject (e.g., a mammal, such as a human). The method further includes administering a non-NMDA ionotropic glutamate receptor antagonist and a pharmaceutically acceptable carrier to treat these conditions. The method includes antagonists such as 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX), topiramate, 1-(4aminophemyl)-4-methyl-7,8-methylene-dioxy-5H-2,3-benzodiazepine) (GYKI52466), kynurenic acid, 6-cyano-7nitroquinoxaline-2,3-dione (CNQX), LY377770, decahydroisoquinoline (LY293558), 6,7-Dinitroquinoxaline-2,3 -dione (DNQX), ASAP 187, 1-(4′-Aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one (CFM-2), and γ-Glutamylaminomethyl sulphonic acid (GAMS), or pharmaceutically acceptable salts thereof (e.g., NBQX disodium salt and CNQX disodium salt). In a preferred embodiment, the antagonist is NBQX. In another preferred embodiment, the antagonist is topiramate. The method further provides treating periventricular leukomalacia (PVL), mental retardation, and/or stroke in a neonate. The method still further provides treating periventricular leukomalacia (PVL), mental retardation, and/or stroke in a fetus by administering the antagonist to a pregnant mother.

In another aspect, the invention features a method for preventing one or more causes of cerebral palsy in a subject (e.g., a human). The method includes administering a non-NMDA ionotropic glutamate receptor antagonist and a pharmaceutically acceptable carrier to prevent cerebral palsy. The method further includes antagonists such as NBQX, topiramate, GYKI52466, kynurenic acid, CNQX, LY377770, LY293558, DNQX, ASAP 187, CFM-2, and GAMS, or pharmaceutically acceptable salts thereof (e.g., NBQX disodium salt and CNQX disodium salt). In another preferred embodiment, the antagonist is topiramate. The method further provides preventing one or more causes of cerebral palsy in a neonate. The method still further provides preventing one or more causes of cerebral palsy in a fetus by administering the antagonist to a pregnant mother.

In yet another aspect, the invention features a method for treating grey and/or white matter injury in the brain of a perinatal subject. The method includes administering a non-NMDA ionotropic glutamate receptor antagonist and a pharmaceutically acceptable carrier to treat grey and/or white matter injury. The method further includes antagonists such as NBQX, topiramate, GYKI52466, kynurenic acid, CNQX, LY377770, LY293558, DNQX, ASAP 187, CFM-2, and GAMS, or pharmaceutically acceptable salts thereof (e.g., NBQX disodium salt and CNQX disodium salt). In a preferred embodiment, the antagonist is NBQX. In another preferred embodiment, the antagonist is topiramate. The method further provides preventing one or more causes of cerebral palsy in a neonate. The method still further provides preventing one or more causes of cerebral palsy in a fetus by administering the antagonist to a pregnant mother.

The invention further provides a method for identifying a compound capable of treating PVL, mental retardation, and/or stroke using a postnatal day seven (P7) rat pup. The method includes administering to a p7 rat, having been inflicted with hypoxic/ischemic injury, a test compound and assaying the ability of the test compound to modulate neonatal white matter injury.

In another aspect, the invention provides a method for identifying a compound capable of preventing one or more causes of cerebral palsy using a postnatal day seven (P7) rat pup. The method includes administering to a p7 rat, having been inflicted with hypoxic/ischemic injury, a test compound and assaying the ability of the test compound to modulate neonatal white matter injury.

Kits which include a pharmaceutical composition comprising a non-NMDA ionotropic glutamate receptor antagonist and a pharmaceutically acceptable carrier packed with instructions for use are also provided by the invention. In one embodiment, the kit is used to treat PVL, mental retardation, and/or neonatal stroke. In another embodiment, the kit is used to treat one or more causes of cerebral palsy. In still another embodiment, the kit is used to treat fetal grey and/or white matter injury in the brain. In yet another aspect, the pharmaceutical composition may be administered to a neonate or to a fetus via a pregnant mother. The kits include antagonists such as NBQX, topiramate, GYKI52466, kynurenic acid, CNQX, LY377770, LY293558, DNQX, ASAP 187, CFM-2, and GAMS, or pharmaceutically acceptable salts thereof (e.g., NBQX disodium salt and CNQX disodium salt).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for modulating brain damage mediated by non-NMDA ionotropic glutamate receptors, in particular in fetal and neonatal brains. The invention is based, at least in part, on the discovery that hypoxia/ischemia-mediated injury is attenuated by the non-NMDA ionotropic glutamate receptor antagonists NBQX and topiramate. As a consequence of this inhibition, white matter lesions, oligodendrocyte cell death, and myclin basic protein loss is decreased. These effects are attributable, at least in part, to excitotoxic oligodendrocyte injury mediated by non-NMDA ionotropic glutamate receptors.

Accordingly, in one aspect, the present invention is directed to a method for modulating, e.g., inhibiting, glutamate-mediated neuronal cell death by modulating non-NMDA ionotropic glutamate receptors. The method includes administering to a subject a non-NMDA ionotropic glutamate receptor antagonist in order to treat PVL, mental retardation, neonatal stroke, fetal grey matter injury, and/or fetal white matter injury, as well as to prevent one or more causes of cerebral palsy.

Pharmaceutically Acceptable Formulations

Pharmaceutical compositions, and packaged formulations, comprising a composition of the invention (e.g., a non-NMDA ionotropic glutamate receptor antagonist) and a pharmaceutically acceptable carrier are also provided by the invention. In the method of the invention, the non-NMDA ionotropic glutamate receptor antagonist can be administered in a pharmaceutically acceptable formulation. Such pharmaceutically acceptable formulation typically includes the non-NMDA ionotropic glutamate receptor antagonist as well as a pharmaceutically acceptable carrier(s) and/or excipient(s). As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and anti fungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Excipients include pharmaceutically acceptable stabilizers and disintegrants. The present invention pertains to any pharmaceutically acceptable formulations, including synthetic or natural polymers in the form of macromolecular complexes, nanocapsules, microspheres, or beads, and lipid-based formulations including oil-in-water emulsions, micelles, mixed micelles, synthetic membrane vesicles, and resealed erythrocytes. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Preferably, the route of administration is oral. Solutions or suspensions used for parenteral, intradetmal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a non-NMDA ionotropic glutamate receptor antagonist) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical formulation, used in the method of the invention, contains a therapeutically effective amount of the non-NMDA ionotropic glutamate receptor antagonist. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result. A therapeutically effective amount of the non-NMDA ionotropic glutamate receptor antagonist may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the non-NMDA ionotropic glutamate receptor antagonist (alone or in combination with one or more other agents) to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the non-NMDA ionotropic glutamate receptor antagonist are outweighed by the therapeutically beneficial effects. A non-limiting dosage range (i.e., an effective dosage) is from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the non-NMDA ionotropic glutamate receptor antagonist and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

The invention, in another embodiment, provides a pharmaceutical composition consisting essentially of a non-NMDA ionotropic glutamate receptor antagonist and a pharmaceutically acceptable carrier, as well as methods of use thereof to modulate disorders associated with neuronal cell death e.g., PVL, mental retardation, neonatal stroke, cerebral palsy, fetal grey matter injury, and/or fetal white matter injury with the composition. By the term "consisting essentially of" it is meant that the pharmaceutical composition does not contain any other modulators of non-NMDA ionotropic glutamate receptors. In one embodiment, the pharmaceutical composition of the invention can be provided as a packaged formulation. The packaged formulation may include a pharmaceutical composition of the invention in a container and printed instructions for administration of the composition for treating a subject having a disorder associated with a non-NMDA ionotropic glutamate receptor, e.g., disorders associated with neuronal cell death. The instructions may include directions for the treatment of adults and/or children. Preferably, the instructions include directions for the treatment of neonates. In another preferred embodiment, the instructions include directions for the treatment of a fetus via the administration of the composition to the pregnant mother.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

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

Screening Assays

The ability of a non-NMDA ionotrophic glutamate receptor antagonist to produce a neurosalutary effect in a subject may be determined using any of a variety of art known assays. For example, the ability of a non-NMDA ionotrophic glutamate receptor antagonist to prevent cell damage, death, and/or function after an injury, e.g., an hypoxic/ischemic injury, may be determined histologically (e.g., by assaying tissue loss, immature OL loss, or MBP expression as set forth in the examples below).

Other tests that may be used to determine the ability of a non-NMDA ionotrophic glutamate receptor antagonist to produce a neurosalutary effect in a subject include standard tests of neurological function in human subjects or in animal models of brain injury such as memory tests (e.g., Morris water maze, T maze, spontaneous alternation test, and bar-pressing task); locomotor activity (e.g., vertical movements, sniffing, grooming, coordination, and spontaneous locomotor activity); exploratory activity (e.g., novel large cage test); anxiety (e.g., freezing test, hole-board test, elevated plus maze, forced swimming test); nociception (e.g., hot plate test); feeding motivation; and aggressive behavior. Examples of such tests can be found in Miyachi et al. (1994) Neurosci. Lett. 175:92-94; Vaillend et al. (1995) Behav. Genet. 25:569-579; Fiore et al. (1996) Exp. Parasitol. 83:46-54; Valentinuzzi et al. (1998) Learning & Memory 5:391-403; Andreatini et al. (1999) Braz. J. Med. Biol. Res. 32:1121-1126; Crabbe et al. (1999) Science 284:1670-1672; Rao et al. (1999) Psychopharm. 144:61-66; and U.S. Pat. No. 5,447,939 (1995).

Animal models suitable for use in the assays of the present invention include the immature rat model of hypoxia/ischemia (described in Follett et al. (2000) J. Neurosci. 20:9235-9241, set forth in Example 1). This animal model tests how well a compound can enhance the survival and sprouting of OL and MBP after hypoxic/ischemic injury. Accordingly, after administration of the non-NMDA ionotrophic glutamate receptor antagonist, the brains of these animals may be examined for cell damage and/or death. Alternatively, these animals may be evaluated for recovery of a certain function, e.g., the assays discussed supra.
 

Claim 1 of 5 Claims

1. A method for treating periventricular leukomalacia (PVL), mental retardation, and/or neonatal stroke in a human subject comprising administering to the human subject topiramate and a pharmaceutically acceptable carrier such that PVL, mental retardation, and/or neonatal stroke is treated.

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