Using glutathiane to protect neurons from injury

Details Using glutathiane to protect neurons from injury Using glutathiane to protect neurons from injury

1994-05-19

2003-02-25

Kunz, Gary (Department: 1647)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S019300

Reexamination Certificate

active

06525017

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention concerns agents, and methods of their use, which protect a neuron of a human patient from injury, especially injury caused by the presence of excess glutamate or related compounds.
Glutamate is known to be a broad spectrum agonist of neuron activity, with efficacy at at least three subtypes of excitatory amino acid receptors, namely kainate, quisqualate, and N-methyl-D-aspartate (NMDA). It is present at high concentrations in mammalian central nervous systems (CNS) and is toxic to central neurons. It is thought to play a role in neuronal injury, and to mediate a variety of brain insults, including hypoxia, physical trauma, and Alzheimer's disease.
Certain glutamate antagonists can attenuate the acute neuronal injury produced by hypoxia, ischemia, and hypoglycemia. This protective effect on central neurons indicates that the antagonists may have clinical therapeutic utility in hypoxic brain injury. Choi, U.S. Pat. No. 4,806,543, describes a method for reducing adverse effects of neurotoxic injury by administering an enantiomer of an analgesic opioid agonist or antagonist. Such compounds are said to be useful for treatment of any animal species having NMDA receptors.
Hahn et al. (Proceedings National Academy of Science USA, 85:6556, 1988; not admitted to be prior art to the present invention) state that, in the mammalian central nervous system, glutamate is thought to be the major excitatory neurotransmitter that acts at the three receptor subtypes. Excessive stimulation of the NMDA subtype has been implicated in the pathophysiology of neuronal degeneration caused by a variety of conditions; these include anoxia, ischemia, hypoglycemia, seizures, and several neurodegenerative diseases, such as Huntington's disease, and the amyotrophic lateral sclerosis-Parkinsonism-dementia complex found on Guam.
SUMMARY OF THE INVENTION
In a first aspect, the invention features a method for identifying agents useful for protection of a neuron of an organism from injury. The method includes the steps of providing a cell which has an NMDA receptor; selecting an agent potentially useful for oxidation of the NMDA receptor; treating the cell with the agent; and determining whether the NMDA receptor is oxidized by the agent.
By protection of a neuron from neuronal injury is meant that the agent is able to either totally reverse, or at least partially reverse, the effect of excess glutamate on that neuron. Alternatively, the agent is able to reduce the effect of glutamate on the neuron, and thereby significantly increase the chances of that neuron surviving in the presence of glutamate or a related substance.
The term organism is intended to include any animal to which an agent of the invention can be administered for the indicated purpose, including both medicinal and veterinary purposes. Use in mammals and birds of all types is preferred, with use in humans being a primary utility.
The NMDA receptor is that molecule found in human neurons which interacts with NMDA to induce neuron excitation. Cells which include an NMDA receptor include any cell which has an NMDA receptor analogous to that present on human neurons in the CNS. Generally such cells are neurons and can be isolated from any organism, including humans. Examples of such cells include rat retinal ganglion cells, cultured mammalian central neurons, such as rat cortical neurons, cells of an intact chick retina, and glial cells.
The step of selecting an agent potentially useful for oxidation of the NMDA receptor is a step well understood by those skilled in the art. Generally, this step involves choosing any agent which is able to act as an oxidizing agent, and thus may act as such at the NMDA site. One example of an agent useful in the invention is DTNB (generally used in vitro at 0.5-10 mM). The agent is generally used in vivo, thus the agents screened generally will be those which have minimal adverse side effects on the organism to which they are to be administered. Those skilled in the art will readily recognize the meaning of this phrase, but generally it indicates that the effect of adding the agent to an organism to protect a neuron from injury causes minimal other effects to that organism, such as cell death, disruption of other physiological functions, and gross comfort of the organism. Preferably, agents useful in this invention will have little, if any, side effects on the organism to which they are administered.
Useful agents need not be oxidizing agents in their own right, and include those agents which will be acted upon in vivo to produce oxidizing agents at the in vivo site of the NMDA receptor which is to be protected from neuronal injury. Thus, a substrate for an enzyme naturally occuring within the organism, or separately provided, which is acted upon by that enzyme to produce a product which acts as an oxidizing agent for the NMDA receptor is useful in this invention. Indeed, if the product itself is not an oxidizing agent, but can be subsequently acted upon by another enzyme to produce an oxidizing agent, it is also useful in this invention. It is understood by those of ordinary skill in the art that such agents, which are not themselves oxidizing agents, are only useful if the enzymes or other compounds which must preferably act upon the administered agent naturally occur within the organism in close proximity to the NMDA receptor. For example, agents such as superoxides and peroxides are potentially useful as agents to oxidize an NMDA receptor in vivo. Thus, putrescene, the substrate for diamine oxidase, is a useful agent in this invention since it will cause production of peroxides which may oxidize an NMDA receptor. Other examples of agents which are potentially useful for oxidation of an NMDA receptor include substrates of xanthine oxidase (e.g., xanthine-ring containing substances), amino acid oxidases (e.g., D-serine, the substrate for a D-amino acid oxidase), lysine oxidase, monoamine oxidase (e.g., dopa may be used to generate peroxide in the CNS after it crosses the blood-brain barrier, is metabolized to dopamine, and is later acted upon by monoamine oxidase to generate peroxide), tyramine-containing foods or drugs, and other agents which may generate catecholamines in the CNS.
As discussed above, it is readily apparent to those skilled in the art how to select agents potentially useful for oxidation of the NMDA receptor. However, it must be noted that, in the method of this invention, the step of treating the cell with the agent means provision of the agent along with any of the enzymes or other products necessary to produce the oxidizing agent which will act in vivo on the NMDA receptor. For example, if putrescene is to be used in the method of the invention, both putrescene and diamine oxidase must be provided at the treating step. Alternatively, the product of such a combination, which is produced in vivo, for example, hydrogen peroxide, can be tested by the method of the invention and, if found to oxidize an NMDA receptor, that agent may be produced in any manner suitable in vivo. Thus, if hydrogen peroxide in low dose (e.g., maximally at 25 &mgr;M, or between 2.5 and 20 &mgr;M) is determined to be a suitable agent for protection of neurons, the hydrogen peroxide may be produced in vivo by any manner of known techniques, for example, by direct administration of hydrogen peroxide, or by use of the substrate-enzyme combinations discussed above.
Other agents which may be selected for testing in the method of this invention include those which prevent removal of oxidizing agents which occur naturally in vivo. For example, agents which prevent removal or destruction of superoxides and peroxides generated in vivo will result in a higher in vivo concentration of these superoxides or peroxides, thus causing greater oxidation of the NMDA receptor. Those skilled in the art will readily recognize that one example of such agents includes inhibitors of enzymes known to break down these oxidizing compounds. Such inhibitors must be chosen and used at concentrations wh

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