Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heavy metal containing doai
Reexamination Certificate
1998-08-27
2001-10-30
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heavy metal containing doai
C514S492000, C514S561000
Reexamination Certificate
active
06310093
ABSTRACT:
FIELD OF THE INVENTION
The invention is concerned with a treatment method for preventing neuronal death subsequent to injury, particularly in the central nervous system.
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BACKGROUND OF THE INVENTION
High extracellular levels of glutamate have been recognized as a significant biochemical consequence of various forms of neuronal injury, including ischemic, degenerative and traumatic brain damage. Further, it has been proposed that the elevated glutamate levels exacerbate the primary insult, possibly by acting at excitatory glutamate receptors. While it was previously believed that the source of the excess glutamate was neuronal stores, it is now recognized that at least some of the glutamate may be the result of enzymatic conversion of glutamine to glutamate by glutaminase.
U.S. Pat. No. 5,158,976 (Rosenberg) discloses the general idea of blocking glutaminase for neuroprotection, but does not appreciate the advantages of using membrane impermeant compounds, as described herein. The approach described by Rosenberg is directed to inhibiting intracellular glutamine metabolism and production of glutamate and does not appreciate that most of the extra glutamate in damaged neuronal tissue is produced by a glutaminase activity that is exposed to the extracellular milieu, presumably as a result of cell damage, as described herein. While Rosenberg describes the systemic toxicity that can ensue when glutaminase is inhibited systemically, it does not appreciate that such toxicity can be reduced considerably by selecting a glutaminase inhibitor that is impermeant to cell membranes and, preferably, specific for the mitochondrial form of the enzyme present in neuronal cells.
According to the discoveries underlying the present invention, membrane impermeant inhibitors having certain characteristics defined herein are both neuroprotective and selectively inhibit glutamate production by damaged neurons. Glutaminase in intact neurons, as well as glutamatergic transmission, are unaffected by such inhibitors, reducing systemic side effects and toxicity.
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 {fraction (1/10)} the concentration at which it inhibits or interferes with the function of other glutamine-utilizing enzymes, such as glutamine amidotransferas
Borin Michael
Elan Pharmaceuticals Inc.
Gorthey LeeAnn
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