WO2001098367A2

WO2001098367A2 - Neuroactive peptides for treatment of hypoxia and related conditions

Info

Publication number: WO2001098367A2
Application number: PCT/US2001/019839
Authority: WO
Inventors: Joseph R. Moskal; Hirotaka Yamamoto; Patrica A. Colley
Original assignee: Nyxis Neurotherapies, Inc.
Priority date: 2000-06-22
Filing date: 2001-06-22
Publication date: 2001-12-27
Also published as: CA2413974A1; EP1296999A2; AU2001271366A1; WO2001098367A3; JP2004500874A

Abstract

Compositions and methods are provided for the treatment of hypoxia. In a preferred embodiment, compositions and methods are provided for the treatment of the effects of hypoxia or ischemia on the central nervous system.

Description

NEUROACTIVE PEPTIDES FOR TREATMENT OF HYPOXIA AND RELATED CONDITIONS

(Case No. 95,1204-GG)

Reference to Related Applications

This application claims the benefit of U.S. Provisional application 60/213,614 filed June 22, 2000, which is incorporated by reference herein in its entirety.

Field of the Invention The instant invention is related to the field of neuroactive peptides, proteins, or amino acid compositions.

Description of the Related Art

It is now well known that the central nervous system (CNS) of mammals employs many neuroactive peptides to effect specialized signalling within the brain and spinal cord. Among the more well known neuroactive peptides are Somatostatin, Cholecystokinin, ND? , Substance P, Enkephalin, Νeuropeptide Y (ΝPY), Νeurotensin, TRH, CCK, and dynoφhin. (see generally The Biochemical Basis of Neurooharmacology, Cooper, Bloom and Roth, 5th ed., Oxford University Press, New York, 1986). The careful elucidation of the complex signalling pathways which operate in the CNS requires the identification and characterization of specific neuroactive peptides and their particular properties, as well as the characterization and localization of specific neurologically significant receptors. Identification of agonists and antagonists of CNS receptors, whether partial, complete, coordinately acting, or independently acting, is useful in that the more that is known of specific neuroactive peptides, the greater the range of manipulations that can be conducted on CNS receptor proteins, and the behavior of CNS receptor complexes. Significantly, the identification of unique agonists or antagonists allows for the fine characterization and localization of subsets of neuroactive receptors by their binding to these agonists or antagonists. By identifying neuroactive peptides, and using them to specifically perturb the behavior of known receptor complexes, more detailed understanding becomes available about the receptor complex, addition, new neuroactive peptides offer alternative means of altering the behavior of known CNS receptor complexes, or for the discovery of previously unknown receptor complexes or unknown behavior of known receptors.

The N-methyl-D-aspartate (NMDA) receptor, which has been implicated in neurodegenerative disorders, stroke-related brain cell death, convulsive disorders, and learning and memory, has been cloned from human tissue (see Hoffman, M., 1991,

Science, 254:801-2). In addition to being activated by the binding of NMDA, the NMDA receptors are activated by glutamate (Glu), and aspartate (Asp), as well as being competitively antagonized by D-2-amino-5-phosphonovalerate (D-AP5; D-APV), or non- competitively antagonized by phenylcyclidine (PCP), and MK-801. However, most interestingly, the NMDA receptor is coactivated by glycine (Gly). (Kozikowski et al., 1990, Journal of Medicinal Chemistry 33:1561-1571). The binding of glycine to an allosteric regulatory site on the NMDA receptor complex increases both the duration of channel open time, and most dramatically the frequency of the opening of the NMDA receptor channel. The NMDA receptor is considered central to long-term potentiation (LTP), which is the persistent strengthening of neuronal connections that is considered to underlie learning and memory (see Bliss and Collingridge, 1993, Nature 361:31-39, for review). Damage to the CNS, which may occur for example during a stroke, is thought to be caused by the overexcitement of cells which have the NMDA receptor, by flooding these cells with glutamate or aspartate, leading to the death of some 80% of such overexcited cells. The bulk of NMDA receptor-carrying cells are in the cortex and hippocampus regions of the brain, and after such overexcited killing of cells, patients are rendered incapable of learning new things, but can still recall items in long term memory. Human memory deficits associated with PCP abuse have been linked to the action of PCP, and is an expected consequence of the inhibition of calcium fluxes through the NMDA receptor. It is thought that drugs which can block, or otherwise alter the operation of the NMDA receptor may protect cells from overexcited killing, or NMDA receptor associated memory problems. Other drugs that interact with the NMDA receptor may enhance the ability of the cells to form LTP and thus enhance learning and memory. Because of the significance of the NMDA receptor, it would be most useful to have specific peptide agonists or antagonists which will allow for fine mapping of the tissue distribution, subtype characterization, and fine manipulation of NMDA receptors, and for characterization of the action of other agonists or antagonists on the NMDA receptor.

SUMMARY OF THE INVENTION The present invention relates to the treatment or prevention of a condition resulting in a lack of oxygen in the brain. The present invention provides peptides and amino acid compositions for treating hypoxia and ischemia and the effects of hypoxia or ischemia in the central nervous system.

In general, the present invention relates to DNA molecules and the polypeptides encoded thereby which are useful for modulation of certain receptors found within the brain, such as NMDA receptors. In a preferred embodiment, the DNA molecules encode members of the NT family of polypeptides, as shown in Table 1, below. In one embodiment, the polypeptides are useful for enhancing learning functions. In another embodiment, the polypeptides are useful for treatment of conditions resulting from hypoxia. In a preferred embodiment, the invention comprises a method for treating hypoxia comprising administering an effective amount of a peptide or amino acid composition, wherein the peptide composition comprises a peptide selected from the group consisting of Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ IDNO:3); Val-Tyr-Tyr-Ser-Gln-Gln-His- Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:4); Glu-Asp-Leu-Ala-Val-Tyr-Tyr-Ser-Gln-Gln- His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:5); Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr- Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu ( SEQ ID NO:9); yyss--GGllnn--GGllnn--HHiiss--TTyyrr--SSeerr--TThhrr--PPrroo--PPrroo--TThhrr--CQys (SEQ ED NO: 10);

Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Ser (SEQ ID NO: 11); Gln-Gln-His-Tyr-Ser (SEQ ID NO: 12); and Thr-Pro-Pro-Thr (SEQ ID NO: 13) and a pharmaceutically acceptable excipient. In a preferred embodiment, the invention comprises a method for treating the effects of hypoxia or ischemia on the central nervous system comprising administering an effective amount of such a peptide composition. hi a preferred embodiment, the peptide is Thr-Pro-Pro-Thr (SEQ ID NO: 13). In another embodiment, the peptide is cyclized. In another embodiment, the peptide comprises a substituted amino acid residue; preferably, the substituted amino acid residue is conservatively substituted. In another embodiment, the invention provides a DNA molecule encoding a peptide selected from the group consisting of: (SEQ ED NO.J); (SEQ ID NO:4); (SEQ ID NO:5); (SEQ ID NO:9); (SEQ ID NO:10) (SEQ ID NO:12); and (SEQ ID NO: 13) hi another embodiment, the present invention encompasses the use of a peptide selected from the group consisting of: (SEQ ID NO:3); (SEQ ID NO:4); (SEQ ID NO:5); (SEQ ED NO:9); (SEQ ID NO:10) (SEQ ID NO:12); and (SEQ ID NO: 13) in the manufacture of a medicament for the treatment of hypoxia.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows three graphs comparing results of testing for NMDA receptor binding glycine agonists using a pharmacological NMDA-specific function ³HMK-801 binding assay. FIG. 1 A shows activity of D-cycloserine, IB shows the activity of monoclonal antibody B6B21, and 1C shows the activity of peptide NT-3. Results are plotted as concentration of test component v. percentage of control binding of ³HMK-801 in the presence of 7-chlorokynurenic acid, a selective glycine site agonist.

FIG. 2 shows the results of testing peptide NT-13 as a partial agonist in a pharmacological NMDA-specific function assay as measured by ³HMK-801 binding.

FIG. 3 shows the results of testing peptide NT-13 as a partial agonist in voltage- clamp experiments in an oocyte expression system, an electrophysiological NMDA- specific function assay. FIG. 3A shows the dose-dependent effects of NT-13 on NMDA currents by voltage-clamp experiments. FIGS. 3B-D demonstrate that peptide NT-13 has characteristics of a partial glycine agonist. FIG. 3B shows that NT- 13 mimics glycine, but is not as effective. FIG. 3C shows that NT-13 inhibits the standard NMDA+glycine current. FIG. 3D shows that the NT- 13-induced NMDA current is blocked by 7- chlorokynurenic acid, which is a selective glycine site antagonist.

FIG. 4 shows the results of testing peptide NT-13 as a partial agonist in a behavioral NMDA-specific function assay compared with cerebral spinal fluid control (CSF) and mAb B6B21. FIG. 4 shows that peptide NT-13 produced some cognitive enhancement in the Morris water maze task as measured by short latencies to swim to the submerged platform (FIG. 4A), path lengths to the platform (FIG. 4B), and decreased average distance to the target during the probe trial on Day 8 of training (FIG. 4C). FIG. 5 shows the results of eyeblirik conditioning in aging rabbits after treatment with mAb B6B21. Inset shows data for acquisition as maximum CRs achieved.

FIG. 6 illustrates the effects of NT-13 on the hypothalmus in hypoxic gerbils.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "hypoxia" relates to a deficiency of oxygen reaching the tissues of the body. Similarly, "ischemia" refers to any condition associated with an inadequate flow of oxygenated blood to a part of the body. Hypoxia and ischemia can occur any time that blood flow to a tissue is reduced below a critical level. This reduction in blood flow can result from the following non-limiting conditions: (i) the blockage of a vessel by an embolus (blood clot); (ii) the blockage of a vessel due to atherosclerosis; (iii) the breakage of a blood vessel (a bleeding stroke); (iv) the blockage of a blood vessel due to vasoconstriction such as occurs during vasospasms and possibly, during transient ischemic attacks (TIA) and following subarachnoid hemorrhage. Further conditions in which hypoxia and ischemia may occur include (i) myocardial infarction (when the heart stops, the flow of blood to organs is reduced and ischemia results); (ii) trauma; and (iii) cardiac and neurosurgery (blood flow needs to be reduced or stopped to achieve the aims of surgery). The effects of hypoxia or ischemia in the central nervous system can include temporary or permanent loss of function as well as loss of neurons. The instant invention provides certain specific neuroactive peptides which are characterized by the ability to bind to the NMDA receptor. The instant invention provides for specific polypeptides or amino acid compositions which bind to the NMDA receptor at the glycine co-agonist site, and effect at least the same biological activity from the NNDA receptor as the binding of glycine. Polypeptides or amino acid compositions of the instant invention may be purified from natural tissues, fluids, or cells. A preferred embodiment of the instant invention provides for the chemical synthesis of the polypeptides or amino acid compositions of the instant invention using conventional biochemistry methods, or molecular biology techniques. As the polypeptides or amino acid compositions of the instant invention are useful for the isolation and characterization of NMDA receptor activity and tissue localization, the instant invention provides for stabilized polypeptides or amino acid compositions wherein the backbone has incorporated modified peptides such that stability is enhanced, or via addition of framework modifications such that the three-dimensional conformation of the peptide fragment is stabilized or enhanced. The instant invention also provides for cyclized polypeptides or amino acid compositions.

An additional benefit of the polypeptides or amino acid compositions of the instant invention is that the small size will enhance the ability to cross the blood-brain barrier, and are thus suitable for in vivo administration and detection. Thus the instant invention encompasses polypeptides or amino acid compositions of the instant invention coupled to radioactive markers, MRI markers, metal ion markers, enzymatic markers, chemiluminescent markers, or any such marker which will allow for the detection of the polypeptide or amino acid compositions. The instant invention also encompasses pharmaceutical formulations of the polypeptides or amino acid compositions of the instant invention, in suitable pharmaceutical carriers such that they can he administered to a living subject. Such administration can be i.p., i.v., i.m., or by any other appropriate means. In instances where the polypeptides or amino acid compositions of the instant invention are used for detection of NMDA receptors using in vitro screening such as tissue section and staining, the attached marker can also be, in addition to the suitable markers above, proteins, antibody, avidin, biotin, and any other such marker which allows for the detection of the, presence of polypeptide or amino acid compositions in screening assays or staining procedures.

The instant invention also provides for methods of detecting NMDA receptors using the polypeptides or amino acid compositions of the instant invention and an appropriate marker for detection of the bound receptor and polypeptide or amino acid composition. Such methods can be practiced in vitro and in vivo depending on the conditions for detection used. Certain procedures which can be employed using such methods include, and are not limited to, MRI, CAT scan, X-ray, Sonogram, and other such non-invasive detection methodologies. Where invasive procedures are contemplated, ie. biopsy or tissue section, the use of standard immunological screening methods can be used to detect the presence of bound receptor/polypeptide, or such binding can be specifically visualized via immunological staining or other such detection means utilizing the wide range of available marker/detection systems. The instant invention therefore also provides for a method for modifying the biological activity of a NMDA receptor comprising said NMDA receptor contacting with a polypeptide or amino acid composition of the instant invention. Specifically encompassed are peptides having the amino acid sequences as listed in Table 1 below, that are predicted to be effective by assay data in the following examples.

TABLE 1

NT Peptide Family NT-1 (SEQ ID. NO:l):

Lys-Ala-Ser-Gln-Asp-Nal-Ser-Thr-Thr-Nal-Ala ΝT-2 (SEQ ID. NO:2):

Ser-Ala-Ser-Tyr-Arg-Tyr-Thr NT-3 (SEQ ID. NO:3): Gln-Gln-His-Tvr-Ser-Thr-Pro-Pro-Thr

NT-4 (SEQ ID. NO:4):

Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr ΝT-5 (SEQ ID. NO:5):

Glu-Asp-Leu-Ala-Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr NT-6 (SEQ ID. NO:6):

Ser-Val-Gln-Ala-Glu-Leu-Asp-Leu-Ala-Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser- Thr-Pro-Pro-Thr NT-7 (SEQ JX>. NOJ):

Phe-Thr-Ile-Ser-Ser-Val-Gln-Ala-Glu-Leu-Asp-Leu-Ala-Nal-Tyr-Tyr-Ser-Gln- Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr

ΝT-8 (SEQ ID. NO: 8):

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Phe-Gly-Gly-Gly NT-9 (SEQ ID. NO:9):

Gln-Gln-His-Tyr-Ser-Tr --Pro-Pro-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu NT-10 (SEQ ID. NO:10)

Cys-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Cys

NT-11 (SEQ ID. NO: 11): Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Ser

NT-12 (SEQ ID. NO:12):

Gln-Gln-His-Tyr-Ser NT-13 (SEQ ID. NO: 13): Thr-Pro-Pro-Thr NT-14 (SEQ ID. NO: 14): Thr-Pro-Pro NT-15 (SEQ ED. NO:15):

Pro-Pro-Thr NT-16 (SEQ ID. NO:16): Pro-Pro

NT-17 (SEQ ID. NO:17): Thr-Pro-Thr NT-18 (SEQ ID. NO:18): Thr

Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. Naturally occurring residues may be divided into classes based on common side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, He;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu; In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 26 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis."

4) basic: Asn, Gin, His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into one or more regions of an NT polypeptide. In making such changes, the hydropathic index of amino acids may be considered.

Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. The hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0J); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3J); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et ah, 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functionally equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions."

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the NT polypeptides, or to increase or decrease the affinity of the Secs-1 polypeptides described herein. Exemplary amino acid substitutions are set forth in Table I. Table I Amino Acid Substitutions

A skilled artisan will be able to determine suitable variants of the polypeptides as set forth in SEQ ID ΝOS.: 1-17 using well-known techniques. For identifying suitable areas of the molecule that may be changed without destroying biological activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a Secs- 1 polypeptide to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the Secs-1 molecule that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of a Secs-1 polypeptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. Also encompassed within the scope of the present invention are DNA molecules encoding the NT polypeptides. As shown below, peptides may be encoded by any of several DNA molecules using the codon table (Table 2) as is well known in the art:

Table 2 Codon Table

For instance, as shown in Table 3, the NT- 13 peptide may be encoded by a DNA molecule having the following sequence:

Table 3 DNA Sequences Encoding NT-13 (Thr-Pro-Pro-Thr)

Any combination of the codons encoding Thr-Pro-Pro-Thr may be employed to encode the NT-13 peptide. For example, NT-13 maybe encoded by ACT-CCT-CCT- ACT (SEQ ID NO:19); ACT-CCC-CCT-ACT (SEQ ID NO:20); ACT-CCA-CCT-ACT (SEQ ID NO:21); ACT-CCG-CCT-ACT (SEQ ID NO:22), and other combinations of the codons as provided by Table 3. Oligonucleotides encoding NT-13 or any of the other polypeptides provided by the instant invention may be designed and synthesized using standard techniques. In addition, DNA encoding the peptides may be incoφorated into an expression vector for expression of the polypeptides in vitro or in vivo, as is well known in the art. These techniques are applicable to any of the peptides described herein (i.e., SEQ ID NOS. 1-17) as well as variants therefrom.

The following Examples are for illustrative puφoses only and are not intended, nor should they be construed, as limiting the invention in any manner. Those skilled in the art will appreciate that variations and modifications can be made without violating the spirit or scope of the invention.

EXAMPLE 1 Monoclonal Antibody specific for NMDA receptors

Monoclonal antibodies were generated by immunizing mice with dentate gyrus tissue isolated from 5 day old neonatal rats following standard protocols (see Moskal and Schafmer, 1986, The Journal of Neuroscience, 6(7):2045-2053). After screening the hybridomas for binding to dentate gyrus tissue via histochemical methods, promising candidate clones were isolated. Among the isolated clones was monoclonal antibody B6E11 which was found to block the production of LTP in rat hippocampal slices, and to suppress established LTP in both area CAl of the hippocampus and the dentate gyrus (see Stanton, Sarvey and Moskal, 1987, Proceedings of the National Acadamy of Science, U.S.A., 84: 1684-1688). The monoclonal antibody B6E11 was found to be effective in blocking LTP when applied to the apical dendrites synapsing with the potentiating input, but not when applied to cell bodies or to the basal dendrites of CAl. In contrast, a second monoclonal antibody, G6E3, which was from the same panel, of the same immunoglobulin class, and bound to the target tissue in a similar fashion, did not have any effect on LTP. Another monoclonal antibody, B6B21, was found to enhance LTP by glycine-like modulation of the NMDA receptor (see Haring, Stanton, Scheideler and Moskal, 1991, Journal of Neurochemistry, 57(l):323-332). This unique monoclonal antibody was able to significantly elevate LTP when applied to CAl pyramidal cell apical dendrites in rat hippocampal slices. B6B21 also elevated binding of N-(-l-(2-thienyl)cyclohexyl)-3,4- ³Hpiperdine (³H-TCP) to the intra-channel PCP site. This effect was eliminated by the maximal saturation of the NMDA receptors with combined addition of maximal concentrations of glutamate, glycine, and magnesium. Most importantly, monoclonal antibody B6B21 reversed 7-chlorokynurenic acid inhibition of ³H-TCP binding, but had no effect on the inhibition of ³H-TCP binding by APN. The enhancement of the binding of ³H-TCP by monoclonal antibody B6B21 was increased by glutamate but not glycine. Hippocampus-dependent learning was found to be facilitated by the binding of B6B21, or the addition of D-cycloserine, both of which bind specifically to the ΝMDA receptor, in in vivo experiments utilizing a rabbit eyeblink conditioning test (see Thompson, Moskal and Disterhoft, 1992, Nature, 359:638-641). lhtraventricular (into the brain ventricle) infusions of B6B21 significantly enhanced acquisition rates in hippocampus-dependent trace eyeblink conditioning in rabbits, halving the number of trials required to reach a criterion of 80% conditioned responses. Peripherial injections of D-cycloserine, a partial agonist of the glycine site on the NMDA receptor which crosses the blood-brain barrier, also doubled the rabbits' learning rates.

Study of the monoclonal antibody B6B21 allowed us to generate a panel of polypeptides or amino acid compositions (Table 1) which allow for the mimicking of the activity of the mAb B6B21, and thus the glycine co-agonist effect.

EXAMPLE 2

Pharmacological NMDA Specific Activity: ³HMK-801 Assay

The peptides of the instant invention are capable of specific binding to the mammalian NMDA receptor at the glycine co-agonist site. Remarkably, the peptides of the instant invention do not require the presence of a glycine (Gly, G) amino acid. Because of the significant role the NMDA receptor plays in the mammalian brain, specific agonists are very useful for the fine mapping of NMDA receptor tissue distribution, and correlation with disease, injury, or other pharmacological effects. Specific small peptide agonists are particularly useful in that they can be further modified for enhanced bioavailabilty and for transport across the blood-brain barrier.

Polypeptides or amino acid compositions of the instant invention were tested for the ability to mimic the glycine co-agonist effect on the NMDA receptor using a previously validated [³H]MK-801 binding assay. This functional assay takes advantage of the fact that increased [³H]MK-801 binding by NMDA receptors can only occur upon receptor-binding channel opening. This is because the MK-801 binding site is located inside the ionophore of the NMDA receptor complex, and is thus only accessible upon the opening of the receptor-channel complex. Thus increased binding of [³H]MK-801 is directly correlated with increased channel opening, in this assay, triggered by the binding of the polypeptides or amino acid compositions of the instant invention to the glycine co- agonist binding site.

To further refine the assay, a selective antagonist of the glycine binding site of the NMDA receptor-channel complex is added to the assay. The normal action of 7- chlorokynurenic acid is to selectively bind to the glycine site, and inhibit NMDA receptor channel opening, and thus inhibit the binding of [³H]MK-801 tό the NMDA receptor complex. The addition of peptide NT-13 reversed the inhibition of binding of [³H]MK- 801, and thus correlates with the finding that the polypeptides or amino acid compositions of the instant invention bind to the glycine binding site.

Membrane Preparation

Crude synaptic membranes used in the assay were prepared using rat hippocampal tissue (male Sprague-Dawly rats) and extensively washed using the procedures described previously (Haring et al., 1991, J. Neurochem. 57:323-331). Briefly, tissue which has been stored at -80 degrees C is homogenized in ice cold 5 mMTris (pH 7.4) using a Brinkman Polytron RTM and then pelleted by centrifugation at 48,000 g for 20 minutes. The resulting supernatant is discarded, and the membranes washed three times in cold buffer. Pellets are then resuspended in 5 mM EDTA, 15 mM Tris (pH 7.4), and incubated for one hour at 37 degrees C. The membrane suspensions are then pelleted by centrifugation at 48,000 g for 20 min and stored at -80 degrees C until use in the assay. Receptor Binding Assay

Frozen pellets are thawed at room temperature and washed three times by resuspension in 5 mM Tris (pH 7.4) and centrifugation. Final pellets are suspended at concentrations of 2 to 3 mg/ml in 5 mM Tris buffer (pH 7.4). Binding reactions are initiated by the addition of 200 μg of freshly prepared membranes to reaction mixtures (about 1 ml final volume) containing 1 nM ³HMK-801 at 25 degrees C in the presence of a range of peptide concentrations and 60 μM 7-chlorokynurenic acid. Non-specific binding is determined using 10 μM unlabelled MK-801. Binding reactions are terminated by filtration through a Brandel 24-well cell harvester onto Whatman GF/B glass filters that have been presoaked in 0.25% polyethyleneimine for 30 minutes.

Data Analysis/Interpretation

Peptides which stimulate ³HMK-801 binding at concentrations equal to or less than the effective dose of D-cycloserine (at 10^"5) are deemed to be positive for ability to bind the glycine site of the NMDA receptor complex.

FIG. 1 shows data comparing the binding data using D-cycloserine (FIG. 1 A), mAb B6B21 (FIG. IB), and peptide NT-3 (FIG. 1C). FIG. 1C clearly shows that peptide NT-3 binds to the glycine site of the NMDA receptor in a fashion similar to mAb B6B21 and D-cycloserine. Data is reported as % control binding in the presence of 7- chlorokynurenic acid against concentration of tested material.

FIG. 2 shows the binding activity of peptide NT-13 as above. Data is reported as % control binding in the presence of 7-chlorokynurenic acid against concentration of tested material.

Table 4 below lists results from assay results using the peptides of the instant invention.

TABLE 4

10^"9 M 10^"8 M 10^"7 M 10^_oM 10^~5 M

NT-1* 97 +/- 2 100 +/- 2 73 +/- 6

NT-2* 80 +/- 9 109 +/- 15 106 +/- 4 77 +/- 12

NT-3 112 +/- 20 123 +/- 0 121 +/- 4 111 +/- 11 90 +/- 1

NT-4 106 +/- 2 106 +/- 6 116 +/- 2 110 +/- 1 109 +/- 10

NT-5 109 +/- 8 100 +/- 9 114 +/- 2 106 +/- 2 112 +/- 4

NT-6 95 +/- 6 101 +/- 9 101 +/- 15 96 +/- 1 91 +/- 7

NT-7 96 +/- 4 103 +/- 4 97 +/- 1 104 +/- 0 100 +/- 3

NT-8 105 +/- 7 110 +/- 5 108 +/- 4 107 +/- 6 115 +/- 6

NT-9 104 +/- 12 126 +/- 7 125 +/- 5 112 +/- 10 120 +/- 3

NT- 10 100 +/- 2 90 +/- 8 114 +/- 3 128 +/- 4 116 +/- 12

NT- 11 96 +/- 23 111 +/- 4 118 +/- 4 120 +/- 11 127 +/- 21

NT-12 95 +/- 21 104 +/- 10 95 +/- 7 106 +/- 9 106 +/- 2

NT-13 97 +/- 8 103 +/- 8 110 +/- 10 129 +/- 10 117 +/- 7

NT-14 89 +/- 6 101 +/- 1 96 +/- 5 98 +/- 8 95 +/- 4

NT-15 99 +/- 21 103 +/- 2 100 +/- 7 108 +/- 12 93 +/- 1

NT-16 83 +/- 9 89 +/- 8 97 +/- 6 103 +/- 3 117 +/- 12

NT-17 122 +/- 23 125 +/- 17 128 +/- 9 117 +/- 14 119 +/- 21

NT-18 88 +/- 21 104 +/- 33 101 +/- 4 103 +/- 6 123 +/- 19

*In the presence of 10 μM L-glutamate

Table 4 shows the effect of peptides NT-1 through NT- 18 on NMDA receptor activation as measured by [³H]MK-801 binding in rat hippocampus, reported as a percentage of control binding (+/-S.E.M.) at various concentrations of peptide. The data reported for peptides NT- 15 through NT- 18 and the experimental error for each point make it unlikely that any of these peptides have meaningful biological activity. It should be emphasized that peptide concentrations of 10^"5 M, approaching millimolar range (100 mM), for efficacious concentration are too high to be considered reliable indicators that the peptide is biologically active. At such a high concentration, it is likely that non- specific binding effects are magnified in relation to specific binding such that the binding equilibrium of the receptor/ligand is skewed. Thus, those peptides showing optimal binding activity candidates.

For example, NT-14 and NT- 15 are without effect at any concentration tested, NT- 17 is approximately equally active across a broad range of concentrations, while NT- 16 and NT-18 are most efficacious at 10^"5 M. By taking into account the concentration and the experimental error, it is reasonable to exclude these peptides as not being biologically active.

However, it is noted that D-cycloserine is a partial agonist at the glycine site of the NMDA receptor, and can act as a cognitive enhancer (Thompson, Moskal and Disterhoft, 1992, Nature, 359:638-641). Serine and threonine do share many structural features, such that it may be possible for threonine to have similar enhancing properties as serine. Thus it is possible to predict that replacing the threonine moieties of NT-3 and NT-13 with serine, will result in active peptides.

EXAMPLE 3

Electrophysiological NMDA Specific Activity Assay

Direct electrophysiological measurement of the effects of the peptides of the instant invention on NMDA receptor function is a powerful, unambiguous, and relatively cost and time efficient screening method. Peptides of the instant invention, first selected as favorable via the ³HMK-801 binding assay, are then subjected to electrophysiological screening.

Methods Electrophysiological recordings are made with two-electrode voltage- clamp techniques, and standard oocyte expression system preparations (see Leonard and Kelso, 1990, Neuron 4:53-60; Kelso et al., 1992, J. Physiology 449:705-718). Oocytes are isolated and injected with mRNA for mouse NMDA receptor subunits: 70 nl of zl (zeta 1) RNA is co-injected with an equal volume of el (epsilon 1) RNA. After incubation for two days, recordings are made while clamping the membrane potential at -80 mV. The standard recording solution contains 95 mM NaCl, 2 mM KC1, 3.8 mM BaCl₂, and 5 mM HEPES. Mg⁺² is omitted because it blocks NMDA currents at some potentials; Ca⁺² is omitted because it can trigger the oocyte's endogenous Ca⁺² -dependent chloride current. Responses are evoked by the continuous perfusion of 3-5 ml of NMDA containing solutions. The recording chamber has a volume of about 400 ml. In some experiments, 7- chlorokynurenic acid is added to the perfusion solution to demonstrate that the peptides or amino acid compositions tested are able to compete with, and act at the glycine binding site. Data Analysis/Interpretation

FIG. 3 A shows that peptide NT-13 has a dose-dependent effect on NMDA currents. Increasing concentrations cause increased currents.

FIG. 3B shows that peptide NT-13 enhances NMDA current, in the absence of glycine. Here, 100 μM NMDA with 10 μM glycine elicited a large current (about 284 nA, downward deflection of trace). In the next phase, 100 μM NMDA with 100 μM peptide NT-13 elicited a significant current of about 40% of the saturated glycine+NMDA response. In this same cell, NMDA alone showed only a negligible response (about 7 nA).

FIG. 3C shows that at high glycine concentrations, added peptide NT-13 reduces NMDA current, here a reduction to about 91% of the control response to NMDA and saturating glycine. This data further supports the conclusion that the peptide competes at the glycine binding site.

FIG. 3D shows that in the absence of glycine, the effect of peptide NT-13 was blocked by the selective addition of the antagonist 7-chlorokynurenic acid.

EXAMPLE 4

Behavioral NMDA Specific Activity Assay Morris water maze (MWM)

There are two types of hippocampus (HPC)-dependent associative learning; a temporal-dependent and a spatial-dependent. The Morris water maze (Morris, 1984, J. Neurosci. Meth. 11:47-60; Brandeis et al., 1989, Int. J. Neurosci.48:29-69) is a well- established paradigm for measuring spatial-dependent learning, and thus complements other behavioral assays such as eyeblink studies which measure temporal learning. Both types of learning are affected by aging and are dependent on NMDA receptor activation (Morris, 1989, J. Neurosci. 9:3040-3057).

Method of Approach

Adult male Sprague-Dawley rats are used in all experiments. A total of 8 to 10 animals per group are used. The number of animals per group was selected upon power function analysis of previous data obtained in similar work. Based upon the outcome of these prior studies, realistic estimates of error variance for biochemical and behavioral endpoints were obtained. Using this information and establishing the value as p<0.05, the sample size was selected which would provide acceptable levels of statistical power. Estimates were based upon suggestions presented in Keppel (Design and Analysis, Prentice Hall, NY, 1973). In all instances, studies were designed to maximize the amount of information derived from a minimum number of animals. Rats are implanted with stainless steel guide cannulae in both the left and right lateral ventricle of the brain. Behavioral testing begins after the rats have rested for two weeks. Fifteen minutes prior to each days training session in the water maze task, described below, rats are infused with peptide at appropriate concentrations, or with artificial cerebrospinal fluid control vehicle (aCSF). Solutions are infused at a rate of about 1.0 ml/min into the cerebral ventricles (i.c.v.) using a 30 gauge injection cannula connected by PE-10 tubing to a 10 ml Hamilton syringe mounted in a CMA Instruments precision infusion pump. A total of about 3.0 ml is infused into each ventricle. To promote diffusion, the injection cannula is left in place for a period of two minutes following infusion. Acute injection procedures are used so as to maintain a specific treatment-testing interval. A non-injected control group is included to assess the effects of the injection procedure itself on the performance.

Water Maze Task

Fifteen minutes following i.c.v. injection, rats are tested in a Morris water maze task. This task is widely used to assess the behavioral properties and neurobiological substrates of spatial memory in rats. It has become popular since it is a rapidly acquired behavior, and is not motivated by food deprivation. Numerous studies have shown that performance of this task is dependent upon intact hippocampal and septohippocampal cholinergic circuitry. Task performance is disrupted by (i) surgical, or (ii) pharmacological disruption of the HPC or its cholinergic innervation, and by (iii) aging. It is accepted that this task provides a sensitive and useful behavioral assay for the functional intergrity of the septohippocampal pathway and the HPC (Opello et al., 1993, Physiol. Behav. 54(6): 1227-1233).

The standard MWM task requires rats to learn to swim to a submerged platform in a circular pool containing opaque water. Four equally spaced points around the edge of the pool are designated as start positions, and divide the pool into four equal quadrants. The submerged escape platform is located in one of the four quadrants throughout training. Each training trial consists of placing the rat into the water at one of the four start positions. The rat is allowed to search for the submerged platform for up to 60 seconds, and is allowed to remain on the platform for 30 seconds. The rats are tested for two trials per day, for 15 consecutive days. Different start positions around the pool are presented in a random sequence. Latency to swim to the submerged platform serves as the measure of acquisition. These testing parameters provide a task that is sensitive to both treatment- induced improvements and impairments of spatial memory. A second component of testing involves the introduction of probe trials after trials 2, 14, 26 and 38. During the probe trials the escape platform is removed and the rat's behavior is videotaped for 30 seconds. The following measures are derived by an automated video tracking system: (i) percent of time spent swimming in the correct quadrant, (ii) average distance from the target during the probe trial, and (iii) swim speed. The use of this sequential probe trial procedure allows the assessment of different components of spatial learning; procedural memory, a form of memory not related to hippocampal functions, and declarative memory, a HPC-dependent process. It is important to evaluate these two forms of cognition since only declarative memory is compromised in aging, Alzheimer's disease, and in the AF64A model (Opello et al.,1993, Physiol. Behav. 54(6): 1227-1233).

Histological Analysis Following the completion of the behavioral studies, rats are sacrificed and prepared for histological examination. Analysis is done to determine if repeated injection of peptides produce any signs of excitotoxic damage in the HPC. Blocks of HPC will be drop fixed in a 0.1M phosphate buffer solution containing 10% formalin and 30% sucrose. Coronal sections are cut and stained with cresyl violet. Staining of the pyramidal cell layers in CA3 and CAl is measured with an image analysis system.

Data Analysis/Interpretation

Overall treatment effects are assessed using either a one or two way analysis of variance (ANONA), depending on the occurrence of multiple factors or repeated measures, according to a mixed model AΝONA. Appropriate pair-wise comparisons are performed using Fisher's Least Significant Difference (LSD) test. Acceptable statistical significance is p<0.05, and all post-hoc tests are two-tailed. Results

FIG. 4 shows the results of testing peptide NT-13 as a partial agonist in a behavioral NMDA-specific function assay compared with artificial cerebral spinal fluid control (aCSF) and mAb B6B21. The results show that peptide NT-13 produced some cognitive enhancement in the Morris water maze task as measured by shorter latencies to swim to the submerged platform (FIG. 4A), decreased path lengths to the platform (FIG. 4B), and decreased average distance to the target during the probe trial on Day 8 of training (FIG. 4C). The data in FIG. 4A are presented as Escape Latency in seconds for each tested treatment. Here the rats treated with peptide NT-13 showed markedly improved escape latency time (about 10.75 seconds) as compared with CSF control (about 19 seconds), and mAb B6B21 treated animals (about 16.25 seconds). The data in FIG. 4B are presented as Path Length in centimeters for each tested treatment, where rats treated with peptide NT-13 showed decreased path length to target (about 300 cm), as compared to control CSF treated (about 420 cm) and mAb B6B21 treated animals (about 330 cm). The data in FIG. 4C are presented as Distance to Target in centimeters for each tested treatment, where the average distance from the target, once removed during the probe trial, is used as a measure for retention. Here rats treated with peptide NT-13 stayed very close to the location of the removed target, an average distance of about 31 cm, while control CSF and mAb B6B21 treated animals strayed about 36.25 cm and 35 cm, respectively.

These data demonstrate that treatment with the peptide of the instant invention can induce in vivo behavioral effects in mammals, specifically cognitive enhancement as demonstrated by improved performance in the Morris water maze task.

EXAMPLE 5

Behavioral NMDA Specific Activity Assay-Trace Eyeblink Trace Eyeblink Conditioning

Eyeblink or nictitating membrane conditioning has been adapted as a "model behavioral system" for use in the analysis of neural substrates of learning by several laboratories (Disterhoft et al., 1977, Brain Res. 137:127-143; Thompson, 1976, American Psychologist 31(3):209-227). Among the advantages of this system are the relative simplicity of the behavioral paradigm, the excellent control procedures available, the fact that associative learning is being analyzed, the ease of conditioned and unconditioned stimulus application and control, the ease of precise behavioral and neurophysiological measurement, and the extensive body of behavioral data which are available for this preparation (Goimezano, 1966, in Classical Conditioning, J. B. Sidowski ed., McGraw Hill, New York, pp.385-420; Gormezano et al., 1987, Classical Conditioning, Hillsdale, N.J.). The system has been correlated with several pathologies, including memory disorders related to aging (Solomon et al., 1988, Neurobiol. Aging 9:535-546), calcium deficiency and aging (Disterhoft et al., 1994, Annals NY Acad. Sci. 747:382-406), amnesia (Gabrieli et al., 1995, Behav. Neurosci. 109:819-827), and amnesic Korsakoff s patients and recovered alcoholics (McGlinchey-Berroth et al., 1995, Alcoholism: Clin. and Exp. Res. 19:1127-1132).

Method of Approach Female adult albino rabbits, Oryctolagus cuniculus, were surgically implanted with lateral ventricular guide cannulae bilaterally and fitted with restraining headbolts. Surgery was performed at least one week after arrival, and dosages for anesthesia were calculated according to weight (60 mg/kg ketamine-HCl, 10 mg/kg xylazine). Approximately 10 days after surgery, subjects were given a single, one hour session of habituation to the training environment.

Training began after two days of rest. Rabbits were restrained using snug bags with drawstrings at the front and rear and trained in separate sound attenuated chambers. The rabbits were placed in a padded plexiglass stock similar to that described by Gormezano et al. (1966, in Classical Conditioning, J. B. Sidowski ed., McGraw Hill, New York, pp. 385-420) with a bar attached for head restraint. The eyelids were held open with dress hooks and nictitating membrane extension was measured with an infared reflective sensor (Thompson et al., 1994, J. Neurosci. Meth., 54:109-117).

Prior to each training session, rabbit pairs received bilateral infusions of 5 μl of either B6B21 suspended in artificial cerebral spinal fluid (aCSF; 124 mM NaCl, 26 mM NaHC0₃, 3 mM KC1, 2.4 mM CaCl₂, 1.3 mM MgS0₄, 1.24 mM NaH₂ O₄, 10 mM D- glucose; pH 7.4), or aCSF alone at a rate of 1 μl/min/ventricle. Three concentrations of B6B21 were used; 0.3 μg/μl, 1.0 μg/μl or 3.0 μg/μl. The person conducting the experiment was blind as to the contents of the administered solution. Cannulated rabbits were trained in pairs counterbalanced among the four treatment groups with a maximum of six animals in each group.

Trace nictitating membrane conditioning began immediately after infusion. Training was given for 15 days with 80 trials/day (CS: 6 kHz, 90 dB, 100 msec, 5 msec rise/fall time; UCS: 3.5 psi tone, 150 msec). The trace interval was 500 msec to make the task dependent upon the hippocampus (Moyer et al., 1990, Behav. Neurosci. 104(2):243- 252). Trials were presented with a variable 30-60 sec intertrial interval and controlled by an IBM PC-compatible computer system (Akase et al., 1994, J. Neurosci. Method. 54:119-130; Thompson et al., 1994, J. Neurosci. Meth., 54:109-117).

Data Analysis/Interpretation

Overall treatment effects are assessed using either a one- or two-way analysis of variance (ANOVA), depending on the occurrence of multiple factors or repeated measures, according to a mixed model ANONA. Appropriate pair- wise comparisons are performed using Fisher's Least Significant Difference (LSD) test. Acceptable statistical significane is p<0.05, and all post-hoc tests are two-tailed.

Results In order to compare behavioral measures based upon the dosages of antibody

B6B21 received, rabbits were grouped according to total amount of B6B21 received each day; a CSF control, 1.5 μg, 3.0-5.0 μg, and 10.0-15.0 μg. The 1.5 μg B6B21 drug group was not included in the statistical analyses because it consisted of only one subject.

The final results showed that B6B21 administration enhanced acquisition of the trace conditioned eyeblink response in aging rabbits in a dose dependent manner (FIG. 5). Using an identical trace conditioning protocol, Thompson et al., (1995, Νeurobiol. Aging 747:382-406) reported that 40% of 36+ month-old rabbits (n=50) reached a criteria of 80%) CRs within 25 days of training. The remaining 60% of the rabbits failed to perform at a level>30%, and were referred to as "severely impaired." The aCSF controls (n=2) in the present study performed in a similar fashion to Thompson's "severely impaired" animals, as did the one animal receiving 1.5 μg B6B21 (Low Dose; n=l, data not shown). None of the rabbits in the present study, who received 10.0-15.0 μg B6B21 (High Dose; n=3) reached 80% CRs within 15 days. However, rabbits receiving 3.0-5.0 μg B6B21 (Intermediate Dose; n=4) began to show greater acquisition on Day 5 as compared to all other groups, and showed greater acquisition than controls as measured by the maximum CRs achieved (Student t-test: t(4)=3.34, p<0.05, see FIG. 5 inset). The average percentage of CRs during the final week of training was generated for each group to measure learning. A one-way ANONA between control and experimental groups indicated significant differences between group means (F(3,12)=3.9, p<0.05, p=0.037). A Fisher's PLST post-hoc T-test comparison between control and 3.0-5.0 μg groups showed enhanced learning (P(T≤t) one-tail=0.03, p<0.05), whereas the higher dosage group did not show a significant difference in acquisition in the same post-hoc t- test (P(T≤t) one-tail=0.09).

While the overall number of animals used for statistical analyses is small, these results clearly demonstrate that mAb B6B21 significantly enhances the acquisition of trace eyeblink conditioning in aging rabbits in a dose-dependent fashion. These results show that there is reasonably expected success in developing biologically active B6B21 peptide mimetics.

EXAMPLE 6 Hypothetical Example Behavioral ΝMDA Specific Activity Assay-Trace Eyeblink in Rats

Method of Approach

The FI hybrid of Fisher 344xBrown Norway rats are used because of minimal age-related pathology (Bronson, 1990, in Genetic Effects on Aging II, Harrison, D. E. ed.,

Telford Press, Caldwell, N.J.). and age-related impairments in eyeblink conditioning (Weiss and Thompson, 1992, Neurobiol. Aging 13:319-323). Nine month old, virgin male rats are used in the experiments.

Rats are anesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg/kg body weight). The top of the head is shaved and cleaned with alcohol and betadine.

A stereotaxic device with a gas anesthesia adapter and atraumatic ear bars (to protect the ear drums) are used. Once the animal is carefully secured, a midline incision is made on the scalp. The skin of the periosteum is retracted and the skull cleaned and dried. A hole is drilled through the skull approximately 0.8 mm behind the bregma, and 1.3 mm to the right and left of the midline (level head coordinates). The dura is then pierced, and a 25- gauge cannula is lowered into each hole to a depth of 4.0 mm below the cortical surface (Tonkiss and Rawlins, 1991, Exp. Brain Res. 85:349-358). These guide cannulae are then cemented to the skull with dental acrylic. A strip connector is cemented to the skull anterior to the cannula. The connector contains a ground wire, two wires (Teflon™ coated stainless steel) which are implanted subdermally within the upper eyelid to measure EMG activity, and two wires which are implanted to deliver a periorbital shock. Subjects are given one week of recovery before habituation.

Fifteen minutes prior to each days training session, described below, rats are infused with peptide at appropriate concentrations, or with artificial cerebrospinal fluid control vehicle (aCSF). Solutions are infused at a rate of about 1.0 ml/min into the cerebral ventricles (i.c.v.) using a 30 gauge injection cannula connected by PE-10 tubing to a 10 ml Hamilton syringe mounted in a CMA Instruments precision infusion pump. A total of about 3.0 ml is infused into each ventricle. To promote diffusion, the injection cannula is left in place for a period of two minutes following infusion. Acute injection procedures are used so as to maintain a specific treatment-testing interval. A non-injected control group is included to assess the effects of the injection procedure itself on the performance.

Rats are placed in a small cage in a sound attenuated chamber that has a speaker and ventilation fan. A cable is then connected between the experimental equipment and the strip connector implanted on the head. The conditioning stimuli is controlled by software running on a PC compatible computer and electronic modules from Coulbourn Instruments (Akase et al., 1994, J. Neurosci. Meth. 54: 119-130). The EMG activity is amplified, filtered, and full wave rectified with a time constant of 45 ms (Skelton, 1988, Behav. Neurosci. 102:586-590). The signal is sent to a computer for data collection and analysis (Thompson et al., 1994, J. Neurosci. Meth. 54:109-117).

Eyeblink conditioning is done using modified procedures as reported by Weiss and Thompson (1992, Neurobiol. Aging 13:319-323). The rats are habituated to the conditioning apparatus for one 45 minute session prior to training sessions. Animals are trained daily in pairs for 15 days with either trace 500 paradigm or the unpaired control paradigm for psuedoconditioning. Rats are trace eyeblink conditioned using a tone conditioning stimulus (CS, 100 ms, 1 KHz, 85 dB, 5 ms rise/fall time) and a periorbital shock unconditioned stimulus (US, 150 ms, 2 mA AC). The stimulus free trace period is 500 ms to make the task dependent upon the hippocampus (Moyer et al., 1990, Behav. Neurosci. 104(2):243-252). Conditioned rats receive 80 trials with paired tones and shocks at a random interval (ITI) of 30-60 seconds. Control rats receive 160 trials with either a tone alone, or a shock alone, at a random ITI of 15-30 seconds. After the conditioning sessions each rat undergoes five days of extinction training consisting of 80 sessions of tone alone trials with a 30-60 second ITI.

Histological Analysis Following the completion of the behavioral studies, rats are sacrificed and prepared for histological examination. Analysis is done to determine if repeated injections of peptide produce any signs of excitotoxic damage in the HPC. Blocks of HPC will be drop fixed in a 0.1M phosphate buffer solution containing 10% formalin and 30% sucrose. Coronal sections are cut and stained with cresyl violet. Staining of the pyramidal cell layers in CA3 and CAl is measured with an image analysis system.

Data Analysis/Interpretation

The data is analyzed with ANOVAs (one- or two-way analysis of variance) of group (conditioned vs. controls) x dose (ACSF and 3 doses). The repeated measures will also be analyzed with ANOVAs. The ANOVA for acquisition will include 15 levels in the ANOVA. The ANOVA for extinction will include 5 levels. This factorial design yields 8 groups of animals with about 10 animals per group used for reliable statistical analysis.

EXAMPLE 7

Autoradiography Studies

The peptides of the instant invention allow for the detailed study of distribution of of the biologically active peptide and/or receptors in brain tissue.

Method of Study

By adapting the autoradiographic methods of Bekenstein et al. (1990, Brain Res. 126:397-425) to the peptides of the instant invention, the regional binding specificity throughout the hippocampal region of the brain, as well as other tissues is examined. In the initial studies, optimized binding conditions are developed in terms of preincubations and washings, methods of drying, equilibrium of time course, saturation, and pharmocology of the binding site at 23 degrees C. Naive rats are sacrificed, the brains rapidly removed, embedded in OCT, and frozen in 2-methyl butane at -20 degrees C prior to sectioning. Ten μm coronal sections are cut on a cryostat and thaw-mounted on poly-D- lysine coated coverslips. Serial sectioning permits assessment of binding gradients along the entire septo-temporal and dorsal-ventral extent of the hippocampus. In the event that gradients in binding within (rather than across) specific hippocampal subfields or cell populations are found, particular care is taken to limit Scatchard analyses to areas free from such gradients. Adjacent sections are used for Nissel stained histological examination and for determination of nonspecific binding. Individual sections are preincubated in several repeated volumes of 20 mM HEPES buffer (pH. 7.4) to reduce concentrations of endogenous or exogenous ligands. Tissue are incubated with an optimized concentration of ³H-peptide of the instant invention, in 20 mM HEPES buffer for varying periods of time, to determine equilibrium binding for saturation studies. For saturation experiments, brain sections are incubated with varying concentrations of ³H- peptide of the instant invention, in 20 mM HEPES buffer. Nonspecific binding will be determined with the addition of excess cold peptide.

Analysis/Interpretation

Tissue sections are apposed to ³H-Ultrofilm (Amersham) and coexposed with methacrylate embedded tritium standards as needed for linear exposure of the film. A BioRad Phosphorimage Analyser can be used for these studies. Quantitative densitometric analysis is performed on a Macintosh Hfx workstation with an 8-bit grey scale scanner, and public domain image analysis software (Image™ v. 1.29) developed at NIMH.

EXAMPLE 8 Treatment of Hypoxia Using NT-13

The present invention relates to the treatment or prevention of a condition resulting in a lack of oxygen in the brain. As used herein, the term "hypoxia" relates to a deficiency of oxygen reaching the tissues of the body. Similarly, "ischemia" refers to any condition associated with an inadequate flow of oxygenated blood to a part of the body. Hypoxia and ischemia can occur any time that blood flow to a tissue is reduced below a critical level. This reduction in blood flow can result from the following non-limiting conditions: (i) the blockage of a vessel by an embolus (blood clot); (ii) the blockage of a vessel due to atherosclerosis; (iii) the breakage of a blood vessel (a bleeding stroke); (iv) the blockage of a blood vessel due to vasoconstriction such as occurs during vasospasms and possibly, during transient ischemic attacks (TIA) and following subarachnoid hemorrhage. Further conditions in which hypoxia and ischemia may occur include (i) myocardial infarction (when the heart stops, the flow of blood to organs is reduced and ischemia results); (ii) trauma; and (iii) cardiac and neurosurgery (blood flow needs to be reduced or stopped to achieve the aims of surgery). The effects of hypoxia or ischemia in the central nervous system can include temporary or permanent loss of function as well as loss of neurons. The present invention provides peptides and amino acid compositions for treating hypoxia and ischemia and the effects of hypoxia or ischemia in the central nervous system.

As shown in Figure 6, the peptide NT-13 is useful for treating hypoxia. A gerbil model was used to demonstrate the effectiveness of NT-13 in providing for cell survival in the hippocampus following exposure to hypoxic conditions. The subject animal was pre-treated (15 minutes prior to clamping the carotid artery see below) by ventricular administration of a pharmaceutical composition comprising a carrier alone (i.e., saline), MK-801 or the NT-13 peptide at the indicated concentrations. The carotid artery of the gerbil was then clamped off for 30 minutes to temporarily prevent blood flow to the brain of the animal, thereby inducing hypoxia. Twenty-four hours later, carrier alone (i.e., saline), MK-801 or the NT-13 peptide at the indicated concentrations were administered 30 minutes before sacrifice. The animal was then sacrificed and sections of the CAl region of the hippocampus was stained in order to determine cell viability using a vital stain. As demonstrated by Figure 6, the saline control had no effect on hypoxia of the cells. MK-801 provided for cell survival following hypoxia. Dose-dependent survival was observed after administration of NT-13 at 1 mg/kg and 2 mg/kg doses. Thus, the NT- 13 peptide is useful for treating conditions resulting from hypoxia. The methodologies provided above are also applicable to the other NT peptides and polypeptides provided herein (i.e., SEQ ID NOS.: 1-12 and 14-17).

It will be understood that the specification and examples above are illustrative, and not meant by way of limitation. One of ordinary skill in the art will be able to understand and determine from the teaching of the instant invention that other specific embodiments may be within the spirit and scope of the invention.

Claims

We claim:

A method for treating hypoxia comprising administering an effective amount of a peptide or amino acid composition, wherein said peptide or amino acid composition comprises a peptide selected from the group consisting of:

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:3);

Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:4);

Glu-Asp-Leu-Ala-Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ

ID NO:5);

Gln-Gln-His-Tyr-Ser-Tlιr-Pro-Pro-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu ( SEQ

ID NO:9);

Cys-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Cys (SEQ ID NO: 10)

Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Ser (SEQ ID NO: 11);

Gln-Gln-His-Tyr-Ser (SEQ ID NO: 12); and

Thr-Pro-Pro-Thr (SEQ ID NO: 13), and a pharmaceutically acceptable excipient.

2. The method of claim 1 wherein the peptide is Thr-Pro-Pro-Thr (SEQ ID NO: 13).

3. The method of claim 1 wherein said peptide is cyclized. 4. The method of claim 1 wherein said peptide comprises a substituted amino acid residue.

The method of claim 4 wherein said substituted amino acid residue is conservatively substituted.

A DNA molecule encoding a peptide selected from the group consisting of:

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:3);

Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:4);

Glu-Asp-Leu-Ala-Val-Tyr-Tvr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ

ID NO:5);

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu ( SEQ

ID NO:9);

Cys-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Cys (SEQ ID NO: 10) Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Ser (SEQ ID NO: 11);

Gln-Gln-His-Tyr-Ser (SEQ ID NO: 12); and,

Thr-Pro-Pro-Thr (SEQ ID NO: 13).

The use of a peptide selected from the group consisting of:

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:3);

Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:4);

Glu-Asp-Leu-Ala-Val-Tyr-Tyr-Ser-Gln-Gln-His-Tvr-Ser-Thr-Pro-Pro-Thr (SEQ

ID NO:5);

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu (SEQ

ID NO:9);

Cys-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Cys (SEQ ID NO: 10)

Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Ser (SEQ ID NO: 11);

Gln-Gln-His-Tyr-Ser (SEQ ID NO: 12); and,

Thr-Pro-Pro-Thr (SEQ ID NO: 13) in the manufacture of a medicament for the treatment of hypoxia. 8. A method for treating the effects of hypoxia on the central nervous system comprising administering to a mammal in need of treatment an effective amount of a composition comprising a peptide selected from the group consisting of:

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:3);

Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ ID NO:4);

Glu-Asp-Leu-Ala-Val-Tyr-Tyr-Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr (SEQ

ID NO:5);

Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu (SEQ

ID NO:9);

Cys-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Cys (SEQ ID NO: 10)

Ser-Gln-Gln-His-Tyr-Ser-Thr-Pro-Pro-Thr-Ser (SEQ ID NO: 11); Gln-Gln-His-Tyr-Ser (SEQ ID NO: 12); and, Thr-Pro-Pro-Thr (SEQ ID NO: 13) and a pharmaceutically acceptable excipient.

9. The method of claim 8 wherein the peptide is Thr-Pro-Pro-Thr (SEQ ID NO: 13).