Title:  Using glutathiane to protect neurons from injury

United States Patent:  6,525,017

Issued:  February 25, 2003

Inventors:  Lipton; Stuart A. (Newton, MA)

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

Appl. No.:  245827

Filed:  May 19, 1994

A method for identifying agents useful for protection of a human neuron from injury. The method includes the steps of providing a cell which has NMDA receptor; selecting a 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.

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 .mu.M, or between 2.5 and 20 .mu.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 which do not allow too great a level of superoxide or peroxide to accumulate in the CNS, which may be toxic to the neurons. The usefulness of these agents is determined by performing the above method in the presence of compounds which naturally break down useful naturally-occurring oxidizing compounds. For example, an inhibitor of a peroxidase is tested in the presence of a naturally occurring peroxidase and a peroxide.

To determine whether the NMDA receptor is oxidized by the agent in the method, any number of methods may be used. For example, it is possible to measure the electrophysiological response of the cell to NMDA, and to determine the survival of the cell after exposure to the agent in the presence or absence of glutamate. Survival can be measured by any of a number of techniques, examples of which are presented below.

The method of this invention is suitable for identifying oxidizing agents which decrease, at least temporarily, NMDA receptor activity in vivo, and thus protect a neuron from neuronal injury. The identified agents are useful for treatment of neurological illness including strokes, anoxia, and the degenerative diseases discussed above. Modulation of the NMDA site by these agents allows central neurons to be protected from death or injury caused by activation of the NMDA subtype of the glutamate receptor. The agents are also useful for in vitro treatment of neurons to increase their longevity, and reduce chances of death.

In a second aspect, the invention features a method for protecting a neuron of an organism from neuronal injury. The method includes identifying an organism susceptible to neuronal injury, providing a pharmacologically acceptable composition including an agent which causes oxidation of an NMDA receptor, and administering the agent to the organism in an amount sufficient to oxidize an NMDA receptor.

Organisms, e.g., human patients, susceptible to neuronal injury are identified by any of a number standard techniques. Such patients will include those discussed above which are susceptible to, or suffer from, strokes, anoxia and certain degenerative diseases. They will also include those patients which have no symptoms but are found to have abnormally high levels of glutamate or related compounds in the CNS. The agents which are identified by the method described above, or by any other method, may be used for treatment of these patients. Those skilled in the art will recognize how to determine, by routine experimentation, the amount of agent necessary to provide sufficient oxidation of NMDA receptors without causing significant deleterious or side effects to the patient. Generally, this amount will be a balance between a level of agent where the potential of causing such deleterious effects is significant and a level where the agent provides complete protection against injury to the NMDA receptor.

In a third aspect the invention features a pharmacologically acceptable therapeutic composition, which includes any one of the above agents which cause oxidation of the NMDA receptor, admixed in a buffer to allow administration of the composition to an organism.

By "causes oxidation" of an NMDA receptor is meant to include agents which directly cause oxidation of the NMDA receptor, as well as agents which cause the in vivo level of an oxidizing agent of NMDA receptor to be increased.

Applicants have discovered that oxidation of the NMDA receptor by oxidizing agents provides protection of neurons from injury caused by the effect of glutamate on that receptor. Thus, these agents can be used to protect neurons which have NMDA receptors in vivo, and specifically those present in the CNS of a human. These agents are particularly useful for treatment of humans suffering from strokes, anoxia or certain degenerative diseases.

Applicants have also discovered that both reduced and oxidized glutathione (0.5-10 mM) can protect against toxicity mediated at NMDA receptors by a mechanism not related to the site of oxidation discussed above. Thus glutathione can be used in vivo or in vitro as discussed in this application for those agents which act to oxidize the NMDA receptor.

Claim 1 of 4 Claims

What is claimed is:

1. A method for treating a human patient to limit NMDA receptor-mediated injury to CNS neurons by providing a pharmacologically acceptable composition comprising glutathione and administering said composition to said patient in an amount sufficient to limit said neuronal injury.
 

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