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Title:  Method of preventing neuronal death

United States Patent:  6,310,093

Inventors:  Newcomb; Robert (Palo Alto, CA)

Assignee:  Elan Pharmaceuticals, Inc. (South San Francisco, CA)

Appl. No.:  141881

Filed:  August 27, 1998

Abstract

Disclosed is a method for preventing neuronal cell death subsequent to an injury to neuronal tissue, particularly central nervous tissue exposed to an ischemic or hypoxic event, to trauma or to a chronic neurodegenerative disorder. The method includes exposing the cells to a glutaminase inhibitor that is relatively impermeant to healthy, intact cell membranes, and which is preferably a specific inhibitor of the form of glutaminase that is widely present in brain tissue.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method of reducing damage to neuronal cells in a neuronal tissue of a subject who has experienced neuronal injury. The method includes administering a compound capable of selectively inhibiting extracellular glutaminase relative to glutaminase present in intact cells. Preferably, the selective inhibition of glutaminase is characterized by the compound's inability to permeate intact cell membranes, as evidenced by an inability of said compound to inhibit glutaminase when contacted with live primary cell cultures of neurons and glia and an ability to inhibit glutaminase when applied to crude membranes derived from said cell cultures.

In a preferred embodiment, the invention is directed to treatment of neuronal injury in the central nervous system. Such injury includes, but is not limited to, ischemic injury, such as focal ischemia due to stroke, traumatic injury that is the result of trauma, and chronic degenerative damage to the brain, such as from Alzheimer's disease.

Preferably, the compound comprises a reactive portion having a chemical affinity for the active site of glutaminase. The reactive portion is preferably a thiol-reactive moiety, as in the compound p-(chloromercuri)phenylsulfonic acid. Such compounds also include glutamate analogs; particularly preferred analogs are derived from 4fluoroglutamate. The compound may further include a bulky and/or polar portion effective to inhibit passage of the compound through cell membranes.

In another embodiment, a preferred compound has at least one anionic or highly polar group, selected from a nitro group, a carboxylate, and an oxide of sulfur or phosphorus. Of such compounds, those preferred are those which exhibit less inhibition of glutaminase in the presence of increased levels of phosphate, and which do not activate glutaminase in the presence of increased levels of phosphate. Particularly preferred compounds of this class have a significantly hydrophobic portion, such as an aromatic group or alkyl chain, preferably having at least six carbon atoms. Also preferred are those compounds which are gem-disubstituted with two such polar or anionic groups. In one embodiment, the compound has a gem-dinitro group, such as 1,1-dinitrooctane.

In another preferred embodiment, the method of the invention uses a compound which is further characterized by an inhibition potency that results in inhibition of at least 50% of extracellular glutaminase measured in the nerve tissue sample when the compound is present in the tissue at a concentration of less than 1 mM for a time period less than about 1 hour. In yet another embodiment, the compound is further characterized by having a selectivity for brain/kidney glutaminase. Selectivity, in this sense, indicates that the compound inhibits the brain/kidney glutaminase by 50% at a concentration that is no more than 1/10 the concentration at which it inhibits or interferes with the function of other glutamine-utilizing enzymes, such as glutamine amidotransferases and .gamma.-glutamyl transpeptidase.

With respect to administration, the compound can be administered either before, during or after the injurious event. In preferred embodiments of the invention, it is appreciated that the compound can be administered more than 2 hours following the event. The compound is preferably still efficacious when administered 6, 12, 18 or even 24 hours following the injury. According to an important feature of the invention, the compound can be administered by any of a number of parenteral routes, including intravenous, intraarterial, intrathecal, intracerebroventricular and the like. It is appreciated that the compound is able to cross the damaged blood brain barrier after neuronal injury.

In a related aspect, the invention includes a method for screening for neuroprotective compounds. The method is predicated on the observation that useful compounds cannot permeate or are highly resistant to permeation of intact neuronal membranes, but inhibit the glutaminase enzyme present in the membranes of damaged or injured cells. In view of these observations, compounds are tested in a glutaminase reaction mixture that consists essentially of membranes, particularly mitochondrial membranes derived from central nervous tissue. The reaction mixture also includes about a half-saturating amount of glutamine, usually greater than 0.1 mM, and a glutaminase-activating concentration of phosphate (generally greater than about 0.5 mM). Glutaminase activity in the system is compared in the presence and absence of the test compound, and the compound is selected for use in neuroprotection if the compound (i) inhibits glutamate production in the membrane preparation relative to control, and (ii) does not inhibit glutamate production in an intact cell preparation. The compound is tested in the intact cell membrane preparation by adding it to the cells at a concentration of between about 0.1 and 2 mM, washing the cells, and then damaging the cells and measuring glutaminase activity.

The method may comprise further steps of (i) exposing live neurons to the compound, (ii) measuring glutamate production in the culture medium, and (iii) selecting the compound if it does not inhibit glutamate production by the live neurons, relative to neurons which have not been exposed to the compound. Alternatively, further testing may comprise (i) measuring glutamatergic transmission in hippocampal brain slices exposed to the compound, and (ii) selecting the compound if it shows no effect on the field potential at a concentration equal to or greater than that required to inhibit glutaminase in neuronal cultures which have damaged, for example, by exposure to about 2 mM glutamine. These Further steps are especially useful if the compound is suspected to be a reversible in inhibitor of glutaminase.

Claim 1 of 16 Claims

It is claimed:

1. A method of reducing damage to neuronal cells in a neuronal tissue of a subject who has experienced neuronal injury, comprising

administering to the subject, in an amount effective to reduce such damage, a compound which selectively inhibits extracellular glutaminase relative to glutaminase present in intact cells,

wherein said compound is impermeable to intact cell membranes, such that said compound does not inhibit glutaminase when contacted with live intact cell cultures of neurons and glia, and inhibits glutaminase when applied to crude membranes derived from said cell cultures.



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