WO2004089376A2

WO2004089376A2 - Identification and targeting of consensus sites by which dynorphin exerts its neurotoxic effects enables analgesia without neurotoxicity

Info

Publication number: WO2004089376A2
Application number: PCT/US2004/009990
Authority: WO
Inventors: Amina S. Woods; Toni S. Shippenberg; Lawrence Sharpe
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2003-04-01
Filing date: 2004-04-01
Publication date: 2004-10-21
Anticipated expiration: 2005-10-01
Also published as: WO2004089376A3

Abstract

Molecular decoyants are provided for the treatment of an organism which has been subjected to a dynorphin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, having a chemical structure which functionally resembles the ligand binding site of the endogenous NMDA receptor for the dynorphin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynorphin.

Description

IDENTIFICATION AND TARGETING OF CONSENSUS SITES BY WHICH DYNORPHIN EXERTS ITS NEUROTOXIC EFFECTS ENABLES ANALGESIA

WITHOUT NEUROTOXICITY

Field of the Invention

Molecular decoyants are provided for the treatment of an organism which has been subjected to a dynorphin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, having a chemical structure which functionally resembles the ligand binding site of the endogenous NMDA receptor for the dynorphin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin.

Background of the invention Dynorphin A and other opioid peptides derived from dynorphin are potent analgesics that are synthesized in the brain and periphery, and released in response to pain, inflammation and nerve injury. Unlike morphine, they do not produce respiratory depression or constipation, side effects that limit the therapeutic use of currently available opiate analgesics. A major limitation, however, in the use of dynoφhins for the treatment of pain has been the non-opioid actions of these peptides. hi addition to their opioid receptor-mediated actions that result in antinociception, the dynoφhins activate the NMDA receptor. As a consequence of this non-opioid action, the intrathecal administration of dynoφhin A and other dynoφhin-derived peptides produces ischemia, and motor paralysis. In addition to the foregoing, spinal cord injury and neuropathic pain stimulate the release of dynoφhins, which release results in antinociception. As a consequence of their interactions with the NMDA receptor, the dynoφhins produce neurodegeneration, hyperalgesia, and allodynia. Prevention of the non-opioid receptor-mediated actions of the dynoφhins is thus not only important for the development of effective analgesics that lack the side effects that limit the use of moφhine but also may be critical for the prevention and treatment of spinal cord injury, stroke and ischemia. Summary of the Invention A preferred embodiment provides a molecular decoyant for treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, having a chemical structure which functionally resembles the ligand binding site of the endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin, the decoyant being a substance selected from the group consisting of: (a) a substance which is a portion of the NR1 subunit of the endogenous NMDA receptor for the dynoφhin, the portion being (i) sufficiently small so as not to elicit an autoimmune response against the endogenous NMDA receptor when administered in vivo, and (ii) not substantially larger than the smallest size needed to retain the elements of the binding site of the endogenous NMDA receptor which are essential for retaining the ability of the endogenous NMDA receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity permitting the competition with binding of the dynoφhin to the endogenous NMDA receptor and not having the elements of the binding site of the endogenous opioid receptor which are essential for retaining the ability of the endogenous opioid receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity so as not to permit a competition with binding of the dynoφhin to the endogenous opioid receptor; (b) a substance having a chemical structure which substantially corresponds to that of the substance of (a), synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against the endogenous NMDA receptor when administered in vivo; and (c) a substance having a chemical structure consisting essentially of the chemical structure of the substance of (a) or (b).

Brief Description of the Drawings Figure A illustrates molecular decoyants for the treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, having a chemical structure which functionally resembles the ligand binding site of the endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin.

Figure 1 illustrates a mass spectrum of a solution containing an equimolar mixture of Dynoφhin (1-17) (MH⁺ = 2148.5), Dynoφhin (2-17) (MH⁺ - 1985.3), KVNSEEEEEDA peptide (MH⁺ = 1279.3) and noncovalent complexes of Dynoφhin (1- 17) and KVNSEEEEEDA peptide (MH⁺ = 3426.8) and Dynoφhin (2-17) and KVNSEEEEEDA peptide (MH⁺ = 3263.6). The insert shows a model of the interaction of Dynoφhin (1-17) and the KVNSEEEEEDA peptide. Figure 2 illustrates that addition of either 10 μM dynoφhin (1-17) (A) or dynoφhin

(2-17) (B) to the bathing solution increased the amplitude of glutamate-evoked currents to 165 ± 23% of control values. The co-application of increasing concentrations of either D- or L- KVNSEEEEEDA reduced the potentiation of glutamate currents evoked by the dynoφhin peptides (C and D). Figure 3 illustrates that D-KVNSEEEEEDA and its analog L-KVNSEEEEEDA attenuated Dynoφhin A(2-17)- and ischemia/reperfusion-induced toxicity in primary cortical cultures and in vivo. (A) Dynoφhin A(2-17) dose-dependently enhanced lactate dehydrogenase (LDH) levels. Addition of D-KVNSEEEEEDA by itself or a mixture of Dynoφhin A and D-KVNSEEEEEDA induced less increase in LDH activity (p<0.05). (B) Dynoφhin A(2-17) dose-dependently decreased 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) optical density (p<0.05). Such a response was not found in cells pretreated with a mixture of Dynoφhin A and D-KVNSEEEEEDA (p<0.05). (C) hitracerebroventricular administration of D-KVNSEEEEEDA or L-KVNSEEEEEDA markedly reduced cortical infarction induced by middle cerebral arterial ligation. The volume of infarction was significantly decreased in animals pretreated with D- KVNSEEEEEDA or L-KVNSEEEEEDA, as compared to those pretreated with vehicle. The volume of infarction = (sum of the infarction area in 7 consecutive brain slices (mm²)) x (thickness of the slice, 2 mm). (D) The area of the largest infarction in a slice from each rat was significantly diminished by KVNSEEEEEDA analogs. (E) The total number of infarcted slices was significantly dimimshed by KVNSEEEEEDA analogs. (F) Pretreatment with KVNSEEEEEDA increases locomotor activity in stroke rats. Animals were tested 48 hours after ischemia/reperfusion. The motor activity was examined 0-30 min after placing the animals in the chamber. Horizontal activities are measured by the sum of the total number of beam interruptions that occurred in the horizontal during the 30 min testing period. Total distance traveled and movement time were increased in the D- and L-KVNSEEEEEDA-pretreated rats, as compared to the vehicle-treated controls. Pretreatment with D- or L-KVNSEEEEEDA significantly reduced ischemia-induced body asymmetry as examined by an elevated body swing test (*p<0.05, One way ANOVA + Newman-Keuls' test).

Figure 4. A. Neuronal survival. B. Tactile allodynia. C. Paralysis. D. Rota-Rod. All graphs show a drastic improvement in animals given the KVNSEEEEEDA peptide in the presence of dynoφhin. Figure 5 A illustrates that KVNSEEEEEDA attenuates dynoφhin-induced hind limb paralysis as calculated by a 5-point paralysis rating scale.

Figure 5B illustrates the ability of KVNSEEEEEDA to reverse paralysis (closed diamonds) when given post paralysis. *, significant difference relative to Dyn 75 nmol (open square). ^A, significant difference relative to Dyn 100 nmol. Figure 6 illustrates that KVNSEEEEEDA prevents the expression of antinociceptive tolerance to moφhine. % MPE - 10 min after moφhine i.t.

Brief Description of the Sequences

Detailed Description of the Preferred Embodiment Dynoφhin A and its major biotransformation product, dynoφhin A(2-17), elicit a number of pathological effects including neurological dysfunction and neuronal cell death (Caudel, R.M. & Manes, A.J. 2000 Pain 87:235-239). These actions are not opioid receptor-mediated but are reduced by N-methyl-D-aspartate receptor (NMDA) antagonists suggesting that the neurotoxic effects of dynoφhin are NMDA receptor-mediated (Pohl, M. et al. 1997 Brain Research 749:18-28; Herman, B.H. & Goldstein, A. 1985 J Pharmacol Exp Ther 232:27-32; Caudle, R.M. & Isaac, L. 1987 Brain Research 435:1-6; Bakshi, R. & Faden, A.I. 1990 Brain Research 507:1-5; Caudle, R.M, & Isaac, L. 1988 Brain Research 443:329-332; Hauser, K.F. et al. 1999 Exp Neurol 160:361-375; Brauneis, U. et al. 1996 J Pharmacol Exp Ther 279:1063-1068; Caudle, R.M. & Dubner, R. 1998 Neuropeptides 32:87-95). Here we report the identification of a conserved sequence of the NR1 subunit of the NMDA receptor complex that binds dynoφhin peptides non-covalently. We show that synthetic peptides containing this sequence form stable complexes with dynoφhin, and prevent the potentiation of NMDA receptor-activated responses produced by dynoφhin A and dynoφhin (2-17). These peptides prevent dynoφhin-induced cell death and protect against ischemic brain injury. Furthermore, they reduce dynoφhin-evoked dysesthesias and motor neuron loss in the spinal cord. These data demonstrate that peptides which complex with dynoφhin and prevent its binding to the NR1 receptor subunit are neuroprotective, and, thus, they are envisioned as effective therapeutic agents for the treatment of nerve injury.

Header A. The opioid peptide, dynoφhin A, has been implicated in the pathogenesis of ischemic brain injury, and neuropathic pain. Dynoφhin A-derived peptides are elevated following brain or spinal cord trauma, and sustained exposure to these peptides is neurotoxic. Although opiates are potent analgesics, the intrathecal infusion of dynoφhin to experimental animals produces abnormal pain (e.g., allodynia), hindlimb paralysis and a loss of neurons in the spinal cord. These actions of dynoφhin are not opioid receptor- mediated since they are insensitive to opioid receptor antagonists. Moreover, truncated peptides that lack the N-terminus essential for opioid receptor binding retain full activity. In vitro studies have shown that dynoφhin peptides bind to the NMDA receptor and can potentiate NMDA-evoked currents in brain and heterologous expression systems. NMDA receptor antagonists reduce dynoφhin-induced tactile allodynia and paralysis and prevent dynoφhin-induced cell loss in cortical and spinal cord cultures. Although these findings suggest an involvement of the NMDA receptor complex in mediating both the pronociceptive and neurotoxic actions of dynoφhin, the mechanism by which dynoφhin and related peptides modulate NMDA receptor function is unknown. We report here that the presence of arginine 6, 7 and lysine 11, 13 in the dynoφhin molecule enables dynoφhin and related peptides to bind non-covalently to a conserved acidic sequence of the NR1 subunit (594-599, EEEEED) via salt bridging and cation-π interactions. We show that synthetic peptides containing this sequence form stable complexes with dynoφhin and prevent the potentiation of NMDA-mediated cation currents elicited by N-truncated dynoφhin in Xenopus oocytes expressing NR1A/2B subunits. The administration of these peptides to rats prevents ischemic brain injury and neuronal cell death in cortical cultures and attenuates the tactile allodynia, flaccid paralysis and loss of spinal cord motor neurons elicited by intrathecal infusion of dynoφhin or N-truncated dynoφhin.

Header B. Beginning with Figure A, the novel solution to the problem of treatment of an organism which has been subjected to dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, involves the identification of the molecular structure of the ligand binding site in the native receptor and the production of mimic ligand binding sites.

These mimic ligand binding sites functionally resemble the ligand binding site of the endogenous NMDA receptor for the dynoφhin, and do not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin.

The mimic ligand binding sites of the present invention retain the elements of the binding site of the endogenous NMDA receptor which are essential for retaining the ability of the endogenous NMDA receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity permitting the competition with binding of the dynoφhin to the endogenous NMDA receptor and not having the elements of the binding site of the endogenous opioid receptor which are essential for retaining the ability of the endogenous opioid R to selectively and specifically recognize and bind to the dynoφhin with an affinity so as not to permit a competition with binding of the dynoφhin to the endogenous opioid receptor. The mimic ligand binding sites thus act as decoys. Such substances have been denominated "molecular decoyants."

In one embodiment, the molecular decoyants are taken from a substance which is a portion of the NRl subunit of the endogenous NMDA receptor for the dynoφhin, the portion being sufficiently small so as not to elicit an autoimmune response against the endogenous NMDA receptor when administered in vivo., and not substantially larger than the smallest size needed to retain the elements of the binding site of the endogenous NMDA receptor which are essential for retaining the ability of the endogenous NMDA receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity permitting the competition with binding of the dynoφhin to the endogenous NMDA receptor and not having the elements of the binding site of the endogenous opioid receptor which are essential for retaining the ability of the endogenous opioid receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity so as not to permit a competition with binding of the dynoφhin to the endogenous opioid receptor.

In another embodiment, the molecular decoyants are taken from a substance having a chemical structure which substantially corresponds to that of the substance described above, synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against the endogenous NMDA receptor when administered in vivo. n another embodiment, the molecular decoyants are taken from a substance having a chemical structure consisting essentially of the chemical structure of the substance of either of the above alternatives.

It is known that natural receptors are rather large structures, comprising some hundreds of amino acids, and can be as large as a molecular weight of about 250,000. The specific binding site, however, is much smaller and this opens up the possibility of preparing artificial binding sites, which are effective, yet which comprise a much smaller number of amino acids, preferably less than 100, and which have therefore a considerably lower molecular weight and thus a correspondingly lower immunogenicity. It has been found possible to prepare such binding-site-mimicking molecular decoyants which are adapted to bind specific ligands with a size on the order of about 10 amino acids. Such rather small peptide structures can be prepared by physically dividing the endogenous receptor or they can be prepared synthetically by the preparative procedures of peptide chemistry, such as Merrifield synthesis, or of peptidomimetic chemistry, or by genetic engineering. This opens up the possibility of large scale production of such specific peptide structures and their use as active materials in the treatment of an organism which has been subjected to dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect.

An embodiment provides a method for the treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, comprising: administering to the organism, in a quantity sufficient to reduce the neurotoxic effect without preventing the analgesic effect, a molecular decoyant having a chemical structure which functionally resembles the binding site of the endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin, the decoyant being a substance selected from the group consisting of: (a) a substance which is a portion of the NRl subunit of the endogenous NMDA receptor for the dynoφhin, the portion being (i) sufficiently small so as not to elicit an autoimmune response against the endogenous NMDA receptor when administered in vivo, and (ii) not substantially larger than the smallest size needed to retain the elements of the binding site of the endogenous NMDA receptor which are essential for retaining the ability of the endogenous NMDA receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity permitting the competition with binding of the dynoφhin to the endogenous NMDA receptor and not having the elements of the binding site of the endogenous opioid receptor which are essential for retaining the ability of the endogenous opioid receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity so as not to permit a competition with binding of the dynoφhin to the endogenous opioid receptor; (b) a substance having a chemical structure which substantially corresponds to that of the substance of (a), synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against the endogenous NMDA receptor when administered in vivo; and (c) a substance having a chemical structure consisting essentially of the chemical structure of the substance of (a) or (b).

Another embodiment provides use of a molecular decoyant for the manufacture of a medicament for the treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, having a chemical structure which functionally resembles the ligand binding site of the endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin, the decoyant being a substance selected from the group consisting of: (a) a substance which is a portion of the NRl subunit of the endogenous NMDA receptor for the dynoφhin, the portion being (i) sufficiently small so as not to elicit an autoimmune response against the endogenous NMDA receptor when administered in vivo, and (ii) not substantially larger than the smallest size needed to retain the elements of the binding site of the endogenous NMDA receptor which are essential for retaining the ability of the endogenous NMDA receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity permitting the competition with binding of the dynoφhin to the endogenous NMDA receptor and not having the elements of the binding site of the endogenous opioid receptor which are essential for retaining the ability of the endogenous opioid receptor to selectively and specifically recognize and bind to the dynoφhin with an affinity so as not to permit a competition with binding of the dynoφhin to the endogenous opioid receptor; (b) a substance having a chemical structure which substantially corresponds to that of the substance of (a), synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against the endogenous NMDA receptor when administered in vivo; and (c) a substance having a chemical structure consisting essentially of the chemical structure of the substance of (a) or (b). In another embodiment, the method (or use) further comprises administering (or use of) the molecular decoyant together with dynoφhin to reduce the neurotoxic effect of the administered dynoφhin.

In another embodiment, the method (or use) further comprises administering (or use of) the molecular decoyant in the absence of dynoφhin to reduce the neurotoxic effect of non-administered dynoφhin. hi another embodiment, the method (or use) further comprises administering the molecular decoyant (or use of the molecular decoyant for administration) to a patient with stroke or spinal cord injury to reduce the neurotoxic effect of dynoφhin.

In another embodiment, the method (or use) further comprises administering the molecular decoyant (or use of the molecular decoyant for administration) to a patient in a neuropathic pain state, such as in AIDS, to reduce the neurotoxic effect of dynoφhin.

In another embodiment, the method (or use) further comprises administering the molecular decoyant (or use of the molecular decoyant for administration) to a patient at risk for antinociceptive tolerance to moφhine and other opiates to reduce the risk, hence allowing the relief of pain with minimal opiate dosage, thus making the patient comfortable while reducing the side effects of the opiate.

As described below, KVNSEEEEEDA has proven to be a molecular decoyant for treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, the treatment being for the reduction of the neurotoxic effect without preventing the analgesic effect, having a chemical structure which functionally resembles the binding site of the endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of the endogenous opioid receptor for the dynoφhin, the decoyant being a substance which is a portion of the NRl subunit of the endogenous NMDA receptor for the dynoφhin.

The fact that KVNSEEEEEDA has been proven to be a decoyant against dynoφhin is only a case in point for the general claim of molecular decoyants as therapeutic agents. hi the specific case of the NMDA receptor, KVNSEEEEEDA, or improved versions of this molecular decoyant, can serve as an antidote against neurotoxic effects of dynoφhin elicited through its binding to the NMDA receptor.

It should be understood that KVNSEEEEEDA is but an intermediate tool which, by its genetic manipulation or chemical processing, would allow those of ordinary skill in the art to design even more efficient dynoφhin decoyants. Thus, the present invention is intended to include not only the specific 11 amino acid sequence of KVNSEEEEEDA, but variations and derivatives thereof which maintain, and preferably improve, its functional or pharmacological characteristics. For example, modified peptide sequences can be readily prepared and tested by routine techniques for preferred dynoφhin-binding characteristics so as to more effectively compete against the NMDA receptor. Such modification may involve substitution, deletion or insertion of amino acids or their chemical modification. For example, longer lived decoyants may be obtained in this manner. As enzymatic degradation of the decoyants in vivo may cause some decoyants to be relatively short-lived, one method of preventing such degradation would be by making synthetic peptides containing D-amino acids. Alternatively, organic molecules, i.e., not proteinaceous, can be designed so as to satisfy the physico-chemical requirements of a decoyant which must form a functional interface with the dynoφhin.

One example of such a molecule is a peptidomimetic. A peptidomimetic is a compound containing non-peptidic structural elements that is capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic does not have peptide bonds, so it cannot be hydrolyzed by endopeptidases or exopeptidases that cleave peptides. Peptidomimetics, however, can be designed with side chains similar to those of peptides of interest. Hence they would be able to interact with the sites of interest on receptors, while at the same time act as inhibitors to cleaving enzymes.

As shown in this cartoon, the side chain is attached to a nitrogen atom, rather than a carbon atom, but the acetamide bond is preserved.

It should further be understood that the decoyants of the present invention can be modified by extending the polypeptide or by adding specific chemical moieties intended to aid in drug design or to permit the decoyants to be used for additional utilities. One such modification would be to extend the polypeptide by moieties intended to affect solubility, e.g., by the addition of a hydrophilic residue, such as serine. Furthermore, the decoyant could be extended for the puφose of stabilization and preservation of a desired conformation, such as by adding cysteine residues for the formation of disulfide bridges.

Another reason to modify the decoyants would be to make the decoyant detectable, even after administration. This might be done by radioiodination with a radioactive iodine isotope, directly, or by adding tyrosine for subsequent radioiodination. Such detectable decoyants could be used to detect the presence and/or location of dynoφhin.

A further reason for modifying decoyants would be for accelerated clearance of the conjugated dynoφhin from the body. For example, a decoyant linked to an asialoglyco- moiety would be expected to be cleared by the liver. Thus, for example, a decoyant mimicking the NMDA receptor site of dynoφhin and containing such an asialoglyco- moiety, or any other moiety which would aid in its clearance, could be used to inactivate and quickly remove excess dynoφhin after the therapy is completed in order to reduce neurotoxic effects.

Another reason for modifying decoyants would be to increase their efficiency that allows for their use in much lower concentrations. According to the Net Phos and other phosphorylation programs the likelihood of the serine residue in KVNSEEEEEDA being phosphorylated is 99.9 %. We have shown that the presence of a phosphate group doubles the potency of an acidic peptide. Therefore, adding a phosphate group to a molecular decoyant of the present invention is also within the scope of the invention. Proof of the effectiveness of KVNSEEEEEDA as a decoyant against dynoφhin in vivo establishes the operability of the general concept of the present invention. The first requirement of a decoyant in accordance with the present invention is that it be a mimic of the NMDA receptor, i.e., it must functionally resemble the binding site, although it may differ physically. The term "functionally resemble" means that the decoyant binds to the NMDA receptor-binding site on dynoφhin in a selective and specific manner and with reasonable affinity. Replacing the five adjacent glutamate residues with five adjacent aspartate residues would give a peptide with identical properties to the example and thus would functionally resemble the example, so long as the residues were acidic residues.

A decoyant in accordance with the present invention should not be substantially immunogenic. Reduction of size is a means of diminishing the immunogenicity of a substance, but not all large molecules are as immunogenic as some small molecules. To be classified as a decoyant in accordance with the present invention, the substance must be substantially non-immunogenic in the system of the host, regardless of the size of the substance, although the smallest possible size is preferred. It is very important, however, that the decoyant not be sufficiently immunogenic to elicit an autoimmune response against the NMDA receptor when administered in vivo.

A decoyant in accordance with the present invention must comprise the essential elements of the binding site of NMDA receptor and not substantially more. For the puφose of this invention, the "essential elements" of a binding site are defined as those elements essential for the decoyant activity, i.e., ligand recognition and binding. A receptor consists of many residues, only a few of which are involved in ligand recognition and binding. However, as discussed above, the decoyants of the present invention may be further modified for puφoses of drug design. Thus, for example, the entire NRl subunit of the NMDA receptor would not qualify as a decoyant, being both immunogenic and also considerably longer than necessary. The NRl subunit does, however, contain the information needed for the design and construction of a decoyant, i.e., residues 590-600. The fact that some additional peptide units may also be present, for example to improve the solubility of the essential required sequence, would not remove the structure from the category of decoyant as long as it is still substantially non-immunogenic and it is still selective, specific and of reasonable affinity. Addition of sugar molecules could be a modification with the same effect. Thus, additions to the molecule for the piupose of drug design are not considered when determining whether the substance contains substantially more than the elements of the NMDA receptor which are required for binding to the dynoφhin. The second requirement of a decoyant in accordance with the present invention is that it not be a mimic of the opioid receptor, i.e., it must not functionally resemble the binding site, indeed, it may differ physically. The term "not functionally resemble" means that the decoyant does not bind to the opioid receptor-binding site on dynoφhin in a selective and specific manner and with reasonable affinity.

A decoyant is a drag designed to intercept an agent, e.g., dynoφhin, having an undesired effect. As long as dynoφhin exerts its neurotoxic effect only after binding to a non-opioid, e.g., NMDA receptor site, a decoyant can be designed in accordance with the present invention to prevent such binding and thereby eliminate such undesired effect. The decoyants of the present invention may be administered to an animal, including a human patient, in order to reduce the undesired effects of the dynoφhin for which it was designed. Such decoyants can be used not only for the treatment of humans, but also for the treatment of other animals, including mammals, poultry, fish, etc. The specific effective dosages for the treatment to reduce the neurotoxic effect of dynoφhin without preventing its analgesic effect can readily be empirically detennined by those of ordinary skill in the art without undue experimentation. However, those skilled in the art will understand that the dosage of decoyant will depend to some extent on the amount of dynoφhin in the system of the host. The ratio of decoyant to dynoφhin molecules is preferably in the range of 10:1 to 1:10. Preferably, the amount of dynoφhin in the bloodstream or cerebrospinal fluid of the host will be monitored and the decoyant dosage adjusted accordingly during the course of treatment.

Compositions within the scope of the present invention include compositions wherein the decoyant is present in an effective amount to achieve its intended puφose. Determination of the effective amounts is within the skill in the art. i addition to the decoyants of the present invention, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those which can be administered by injection, contain from about 0.1 to 99 percent, and preferably from about 25 to 85 percent by weight, of the active ingredient, together with the excipient.

Any conventional route of administration may be used for the decoyants of the present invention. Although the preferred mode of administration is by injection, e.g., intravenously, intradermally, intraperitoneally, intramuscularly, intrathecally, etc., they may also be administered orally, by suppository or by any other route.

Other non-conventional means of administration can be envisioned which are also intended to be comprehended within the scope of the present invention. For example, bacterial expression systems are known in the art which would actually secrete a soluble form of the expressed protein into the medium, and other viral expression systems are available. It could be contemplated that such a secreting expression system could be used to generate the decoyant from within the host rather than producing it ex vivo and administering it to the host. Obviously, the secreting system must be compatible with the host. The term "administration" as used in the present specification and claims is intended to include such in vivo secretion systems.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, dissolving, or lyophilizing processes. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. The decoyants of the present invention may also be administered in the form of liposomes, pharmaceutical compositions in which the active ingredient is contained either dispersed or variously present in coφuscles consisting of aqueous concentric layers adherent to lipidic layers. The active ingredient may be present both in the aqueous layer and in the lipidic layer, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.

Header C. Peptides or proteins containing two or more adjacent Arginines (Arg, R) form non- covalent complexes with those containing two or more adjacent glutamic (Glu, E) or aspartic acids (Asp, D) (Woods, A.S. & Huestis, M.A. 2001 JASMS 12:88-96; Woods, A.S. et al. 2002 JASMS 13:166-169). The side chain of Arg's functional group is a guanidinium (basic and positively charged), and that of Asp and Glu is a carboxyl group (acidic and negatively charged), thus resulting in Coulombic interaction (salt bridge formation). Dynoφhin (YGGFLRRIRPKLKWDNQ) (SEQ ID NO: 1) and N-truncated dynoφhin fragments are extremely basic peptides due to the presence of five basic residues, three Arg in positions 6, 7, 9 and two lysines (Lys, K) in positions 11, 13. Examination of the sequences of cloned NMDA receptor subunits revealed that the NRl subunit (GenBank 5 accession No. L05666, SEQ ID NO: 2) contains five adjacent Glu followed by an Asp residue (594-599 of SEQ ID NO: 2, EEEEED):

MSTMRLLTLALLFSCSVARAACDPKTVNIGAVLSTRKHEQMFREAVNQANKRHGS WKIQLNATSVTHKPNAIQMALSVCEDLISSQVYAJLVSHPPTPNDHFTPTPVSYTAG FYRIPVLGLTTRMSIYSDKSIHLSFLRTVPPYSHQSSVWFEMMRVYSWNHIILLVSD l o DHEGRAAQKRLETLLEERESKAEKVLQFDPGTKNVTALLMEAKELEARVΠLS ASE

DDAATVYRAAAMLNMTGSGYVWLVGEREISGNALRYAPDGLLGLQLINGKNESA HISDAVGVVAQAVHELLEKENITDPPRGCVGNTNIWKTGPLFKRVLMSSKYADGV TGRVEFNEDGDRKFANYSLMNLQNRKLVQVGra^GTHVjTNDRKTJWPGGE GYQMSTRLK TfflQEPFVYVKPTLSDGTCKEEFTVNGDPVKKVICTGPNDTSPGSP

15 RHTVPQCCYGFCroLLIJKLARTMNFTYEVHLVADGKFGTQERVNNSNKKEWNGM MGELLSGQADMIVAPLTINNERAQYIEFSKPFKYQGLT1LVKKEIPRSTLDSFMQPFQ STLWLLVGLSVHVVAVMLYLLDRFSPFGRFKVNSEEEEEDALTLSSAMWFSWGV LLNSGIGEGAPRSFSARILGMVWAGFAMITVASYTANLAAFLVLDRPEERITGINDPR LRNPSDKFIYATVKQSSVDIYFi QVELSTMYRHMEKHNYESAAEAIQAVRDNKL 0 HAFiWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMRKDSPWKQNVSLSILKSHEN GFMEDLDKTWVRYQECDSRSNAPATLTFENMAGVFMLVAGGIVAGIFLIFIEIAYK røKDARRKQMQLAFAAVNVWRKNLQQYHPTDITGPLNLSDPSVSTVV.

This sequence EEEEED is conserved in all species. We hypothesized that the adjacent guanidinium groups on Arg_{6; 7} of dynoφhin interact with adjacent carboxyl groups 5 of Glu on the NRl subunit resulting in the formation of a powerful salt bridge. To test this hypothesis we used mass-spectrometry to determine if, in solution, dynoφhin (1-17) or (2- 17) form a stable complex with an epitope of NRl containing the EEEEED sequence. Analysis of 0.3 μl of an equimolar solution of dynoφhins (1-17), (2-17) and the epitope of NRl (res. 590-600) containing the acidic residues KVNSEEEEEDA (final concentration of

30 222 fmol/μl of each), revealed molecular ions (MH⁺) at amu 1279.3 (NRl res. 590-600), 1985.3 (dyn 2-17), 2148.5 (dyn 1-17), 3263.6 corresponding to a complex of dynoφhin (2- 17) and KVNSEEEEEDA, and 3426.8 corresponding to a complex of dynoφhin (1-17) and KVNSEEEEEDA (Figure 1), confirming that peptides containing the epitope EEEEED form a non-covalent complex with both dynoφhin (1-17) or (2-17). Shorter fragments of dynoφhin that contain Arg 6 and 7 are also neurotoxic (Hauser, K.F. et al. 2001 Exp Neurol 168:78-87) and formed a salt bridge with the NRl epitope KVNSEEEEEDA. hi contrast, those fragments that lack Arg 6, 7, and are not neurotoxic, did not. To test if a larger epitope of NRl containing the KVNSEEEEEDA epitope would form a non-covalent complex with dynoφhin, a 51 amino acid epitope (res. 576-626) of the NRl subunit was synthesized. When equimolar solutions of dynoφhins (1-17) or (2-17) (666 fmol/μl) were added to this epitope, non-covalent complexes were observed at amu 7890 and 7726.8 amu, again confirming peptide-epitope complexing. A chymotryptic digest of dynoφhin (1-17), KVNSEEEEEDA (WS ept), and the 51 amino acid epitope of NRl (NMDA ept) revealed the presence of 20 complexed fragments (Table 1). Importantly, all complexes consisted of dynoφhin fragments containing the RR motif and KVNSEEEEEDA or the 51 residue epitope fragments containing two or more Glu residues indicating that the non-covalent complex formed is stable and resistant to disruption by enzymatic attack. Indeed, modeling of the secondary structure of dynoφhin and NRl epitope (Pole Bio-Lxformatique Lyonnais: http://pbil.ibcp. fr.cgi-bin/secpred_sopma.pl) revealed that both Arg _{; 7} and the EEEEED epitope are located on alpha helixes, an orientation that would further facilitate salt bridge formation. In addition, the presence of three aromatic residues in dynoφhin (Tyri, Phe₄, and Tφι₄) favor the formation of cation-π interactions (Woods, A. et al. 2003, ASAP Web Release Date 25-Jan-2003, J. Proteome Res., in press) with the Arg₅₈₈, Lys5₉₀ and Asn₅₉ residues of the NRl subunit, while the guanidinium in Arg₉ and the NH₂ groups in Lysn_; ι₃ of dynoφhin facilitate hydrogen bonding with the carboxyl groups of Glu and Asp that do not form a salt bridge with Arg_6, . Molecules involved in non-covalent interactions are in a constant flux of association and dissociation. However, in the case of dynoφhin and the NRl subunit two different types of interactions are involved: electrostatic and cation-π. The likelihood of simultaneous dissociation of two different types of interactions is remote. In addition, the Spartan program was used to calculate and model the surface potential of dynoφhin and KVNSEEEEEDA. The generated peptide models reflect the positive charges on dynoφhin and the negative charges on the KVNSEEEEEDA epitope thus supporting our finding, while emphasizing the dominance of the electrostatic interaction (Figure 1). Table 1. Non-covalent complexes detected after overnight digest with chymotrypsin

To determine whether KVNSEEEEEDA can prevent the interaction of dynoφhin peptides with the NRl subunit of the NMDA receptor in vitro, we used voltage clamp recording methods to measure glutamate-evoked currents in Xenopus oocytes expressing NR1A/NR2B receptors. In the presence of glycine, application of glutamate (50 μM) rapidly activated inward currents, an effect that was prevented by the NMDA receptor channel blocker MK-801. As previously reported for recombinant NMDA receptors, glutamate (50 μM) elicited little current in the absence of added glycine. However, the addition of either 10 μM dynoφhin A or dynoφhin (2-17) (Fig. 2 A and Fig. 2B) to the bathing solution increased the amplitude of glutamate-evoked currents to 165 + 23% of control values. The co-application of increasing concentrations of either D- or L- KVNSEEEEEDA reduced the potentiation of glutamate currents evoked by the dynoφhin peptides (Fig. 2C and 2D). KVNSEEEEEDA failed to modify glutamate-evoked currents in the absence of dynoφhin peptides. Similarly, it did not modify (³H)TCP binding to the NMDA receptor complex in cortical membranes indicating that the inhibitory effects of KVNSEEEEEDA did not result from a direct interaction with NMDA receptors. The neurotoxic actions of the dynoφhins have been attributed to the activation of NMDA receptors and resulting excitotoxicity. To determine whether KVNSEEEEEDA is neuroprotective in vitro, primary cortical cultures were exposed to dynoφhin (2-17) in the presence or absence of KVNSEEEEEDA. Exposure of cortical cultures to increasing concentrations of dynoφhin (2-17) caused significant cell death as indicated by increases in lactate dehydrogenase (LDH) activity and reduction in 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) concentrations. Neither the D- nor L-forms of KVNSEEEEEDA affected LDH or MTT levels. When, however, cultures were exposed to dynoφhin (2-17) in the presence of D- or L- KVNSEEEEEDA, the neurotoxic effects of dynoφhin A (2-17) were significantly reduced (Fig. 3 A and 3B).

Excessive NMDA receptor activation has been implicated in neuronal injury caused by stroke. Although direct-acting NMDA receptor antagonists reduce injury in experimental models of cerebral ischemia, neurotoxicity precludes their clinical use. Since dynoφhin A levels are increased during ischemic brain injury (R.T. Cheung & D.F. Cechetto 1995 J Comp Neurol 362:535-550) and dynoφhin A-related peptides enliance NMDA receptor-mediated responses, we next examined whether KVNSEEEEEDA can protect against nerve injury in rats using an established ischemia model. Rats received an intracerebro ventricular infusion of either L-KVNSEEEEEDA (10-150 nmol), D- KVNSEEEEEDA (150 nmol) or an equivalent volume of vehicle. Thirty min later, the carotid arteries and right middle cerebral artery were occluded for 60 min as previously described (Chen, S.T. et al. 1987 Neurology 37:1227-1229). The rats were killed 48 hr after reperfusion and brain injury volume was evaluated by 2,3,5-tripenyletrazolium chloride (TTC) staining. Infarction of the cortex was seen in all control animals. However, the size and infarct volume were significantly reduced in animals that had received KVNSEEEEEDA prior to occlusion (Fig. 3C-E). KVNSEEEEEDA pre-treatment also increased locomotor activity in stroke animals and reduced ischemia-induced body asymmetry as examined by the elevated body swing test (Fig. 3F).

Levels of dynoφhin A and related peptides increase significantly following spinal cord trauma and this increase is thought to contribute to neuronal death and neurodegeneration associated with secondary injury. To determine whether KVNSEEEEEDA also prevent cytotoxic actions of dynoφhin peptides in the spinal cord, we isolated neurons from the spinal cord of embryonic neurons and used time lapse photography to examine neuronal viability following exposure to dynoφhin (2-17) and KVNSEEEEEDA. Neuronal viability was assessed by repeatedly photographing the same neurons at 24 hrs using a Nikon Diaphot inverted microscope with phase contrast optics and neurons counted as previously described (Hauser, K.F. et al. 1999 Exp Neurol 160:361- 375). At 24-72 hrs in culture, large numbers of small multipolar cells that displayed a neuronal phenotype and NMDARl immunofluorescence were seen (Hauser, K.F. et al. 1999 Exp Neurol 160:361-375). Continuous exposure of cultures to 100 μM dynoφhin A(2-17) causes profound neuronal loss over time, an effect that is attenuated by NMDA receptor antagonists, but not opioid receptor antagonists (Hauser, K.F. et al. 1999 Exp Neurol 160:361-375). A significant increase in cell survival was observed in cultures exposed to equimolar concentrations of dynoφhin (2-17) and D-KVNSEEEEEDA (Fig. 4A).

The intrathecal injection of dynoφhin A or dynoφhin A(2-17) induces long-lasting allodynia, a condition in which normally non-noxious stimuli elicit pain. Higher doses or sustained exposure to these peptides induce hind-limb paralysis and a loss of neuronal cell bodies in the lumbosacral spinal cord. To determine whether KVNSEEEEEDA can protect spinal cord neurons in vivo, we examined whether the intrathecal infusion of KVNSEEEEEDA prevents the tactile allodynia elicited by dynoφhin (2-17). Rats were implanted with an intrathecal cannula as previously described (Yaksh, T.L. & Rudy, T.A.1976 Physiol Behav 17:1031-1036; Ren, K. 1999 Physiol Behav 67:711-716). Tactile allodynia was assessed 1, 3 and 7 days following the intrathecal infusion of dynoφhin A(2- 17) (5 nmol), KVNSEEEEEDA (30 - 250 nmol), or their combination using Von Frey filaments.

A significant increase in nociceptive threshold was apparent in all rats that received dynoφhin A(2-17). hi rats that received dynoφhin A(2-17) and KVNSEEEEEDA (250 nmol), nociceptive thresholds did not differ from that of vehicle-treated rats (Fig. 4B). To evaluate whether KVNSEEEEEDA can also prevent the motor dysfunction produced by a higher dose of dynoφhin (2-17), rats received an intrathecal infusion of dynoφhin (2-17) (75 nmol), KVNSEEEEEDA (30-250 mol) or their combination. The presence or absence of hind-limb paralysis was assessed over a 72 hr period and cumulative paralysis scores were calculated for each treatment group. All dynoφhin (2-17)-treated rats exhibited hind- limb paralysis within 5 min of infusion. When, however, dynoφhin A(2-17) was administered in combination with either D- or L- KVNSEEEEEDA, a dose-related reduction in the incidence and duration of flaccid paralysis was seen (Fig. 4C and Fig. 5A. The ability of KVNSEEEEEDA to protect against motor impairment was also assessed in studies examining the RotaRod performance of rats for three weeks after the intrathecal administration of dynoφhin A(2-17) and KVNSEEEEEDA (250 nmol). Although infusion of dynoφhin A(2-17) alone significantly impaired RotaRod performance, no such impairment occurred in animals that received dynoφhin A(2-17) with KVNSEEEEEDA (Fig. 4D).

A significant increase in nociceptive threshold was apparent in all rats that received dynoφhin A(2-17). In rats that received dynoφhin A(2-17) and KVNSEEEEEDA (250 nmol), nociceptive thresholds did not differ from that of vehicle-treated rats (Fig. 4B). To evaluate whether KVNSEEEEEDA can also prevent the motor dysfunction produced by a higher dose of dynoφhin (2-17), rats received an intrathecal infusion of dynoφhin (2-17) (75 nmol), KVNSEEEEEDA (30-250 mol) or their combination. The presence or absence of hind-limb paralysis was assessed over a 72 hr period and cumulative paralysis scores were calculated for each treatment group. All dynoφhin (2-17)-treated rats exhibited bind- limb paralysis within 5 min of infusion. When, however, dynoφhin A(2-17) was administered in combination with either D- or L- KVNSEEEEEDA, a dose-related reduction in the incidence and duration of flaccid paralysis was seen (Fig. 4C and Fig. 5 A). The ability of KVNSEEEEEDA to protect against motor impairment was also assessed in studies examining the RotaRod performance of rats for three weeks after the intrathecal administration of dynoφhin A(2-17) and KVNSEEEEEDA (250 nmol). Although infusion of dynoφhin A(2-17) alone significantly impaired RotaRod performance, no such impairment occurred in animals that received dynoφhin A(2-17) with KVNSEEEEEDA (Fig. 4D). When KVNSEEEEEDA was given 2 to 5 minutes after dynoφhin induced paralysis, it reversed dynoφhin-evoked tactile allodynia and dynoφhin-induced motor paralysis (Fig. 5B).

The motor dysfunction produced by high doses of dynoφhin A(2-17) is associated with a loss of neurons in the spinal cord. We performed immunocytochemical studies to identify specific neuronal subpopulations that are targeted by dynoφhin A(2-17) and determine whether neuronal death is prevented by intrathecal infusion of KVNSEEEEEDA. Intrathecal infusion of dynoφhin A(2-17) resulted in a selective loss of neurons expressing PKCγ in the dorsal horn as well as motor neuron loss. These effects were apparent for at least 3 days following infusion, hi rats that had received KVNSEEEEEDA in combination with dynoφhin A(2-17), sparing of PKCγ and motor neurons was seen. Chronic administration of opiates results in tolerance to their analgesic effects. Tolerance has been attributed to increased activity of spinal dynoφhin systems and the NMDA receptor dependent mechanisms. Consistent with this hypothesis, administration of dynoφhin antiserum or NMDA receptor antagonists attenuates antinociceptive tolerance to moφhine. Figure 6 shows that chronic exposure to moφhine (s.c. implantation of 75 mg moφhine pellet) results in tolerance to the analgesic effects of intrathecal moφhine. In animals, however, that received an intrathecal infusion of KVNSEEEEEDA 10 min prior to intrathecal moφhine, the expression of antinociceptive tolerance to moφhine was prevented. EXAMPLE 1

Peptides. The following epitopes of NRl were synthesized, KVNSEEEEEDA (res. 590-600 of SEQ ID NO: 2), and a larger (51-mer) epitope MLYLLDRFSP FGRFKVNSEE EEEDALTLSS AMWFSWGVLL NSGIGEGAPRS (res. 576-626 of SEQ ID NO: 2). The above peptides were made by the Sequencing and Synthesis Laboratory at the Johns Hopkins School of Medicine, Baltimore MD. Dynoφhins (1-17) and (2-17) were purchased from Sigma (Saint Louis, MO).

Digest. A digest of the equimolar mixture of the KVNSEEEEEDA + the 51-mer + dynoφhin (1-17) was done as previously described (Woods, A.S. & Huestis, M.A. 2001 JASMS 12:88-96; Pole Bio-Informatique Lyonnais: http://pbil.ibcp.fr.cgi- bin/secpred_sopma.pl). Chymotrypsin (0.5 μg/μl) from Roche Diagnostics (Indianapolis, IN) was used, an overnight time point was added.

Mass Spectrometry. Spectra were acquired on a MALDI DE-Pro from Applied- Biosystem, equipped with a nitrogen laser (337 nm) as previously described (Woods, A.S. & Huestis, M.A. 2001 JASMS 12:88-96). Aliquots were also analyzed using a Q-TOF (Micromass, Manchester England) as previously described (Sobott, F. et al. 2002 J Biol Chem 277:38921-38929).

Animals and Drug Administration. Adult Sprague-Dawley rats (body weight = 336 + 7 g), purchased from the Charles River Laboratories, were anesthetized with chloral hydrate (0.4 g/kg, i.p.). Animals were placed in a stereotaxic frame. L-KVNSEEEEEDA (10 nmol in 10 μl, 30 nmol in 30 μl, 150 nmol in 30 μl), D-KVNSEEEEEDA (150 nmol in 30 μl) or vehicle (0.9% saline or water, 30 μl) were administered through a Hamilton syringe over 10 minutes intracerebrocortically. The coordinates were: 0.8 mm posterior to the bregma, 1.5 mm lateral to the midline, and -3.5 mm below dura surface. The speed of injection was controlled by a syringe pump at a speed of 2.5 μl/min. The needle was retained in place for 5 min after injection. A piece of bone wax (W810, Ethicon) was applied to the skull defect to prevent the leakage of the solution after injection.

Brain Ischemia/Keperfusion. Thirty minutes after intracerebral administration of KVNSEEEEEDA or vehicle, the anesthetized animals were subjected to cerebral ischemia. The ligation of the right middle cerebral artery (MCA) and bilateral common carotids (CCAs) was performed using methods previously described (Chen, S.T. et al. 1987 Neurology 37:1227-1229). The CCAs were ligated with non-traumatic arterial clips. The right MCA was ligated with 10-O suture. After 60 minutes of ischemia, the suture on the MCA and arterial clips on CCAs were removed to allow reperfusion. Core body temperature was monitored with a thermistor probe and maintained at 37°C with a heating pad during anesthesia. After recovery from the anesthesia, body temperature was further maintained at 37°C using a heat lamp.

Locomotor Behavioral Measurements. Animals were placed in an Accuscan activity monitor (Columbus, OH) 2 days after ischemia for behavioral recording. The monitor contained 16 horizontal and 8 vertical infrared sensors spaced 2.5 cm apart. The vertical sensors were situated 10.5 cm from the floor of the chamber. Each animal was placed in a 42 x 42 x 31 cm Plexiglas open box for one hour. Motor activities, such as distance traveled, vertical and horizontal movement were calculated by the number of beams broken by the animals from 0 to 30 min after placement in the chamber.

Triphenyltetrazolium Chloride (TTC) Staining. Two days after MCA ligation, some animals were sacrificed and perfused intracardially with saline. The brain tissue was then removed, immersed in cold saline for 5 minutes, and sliced into 2.0-mm thick sections. The brain slices were incubated in a 2% triphenyltetrazolium chloride (TTC, Sigma) dissolved in normal saline for 15 minutes at room temperature, and then transferred into a 5% formaldehyde solution for fixation. The area of infarction on each brain slice was measured double blind using a digital scanner and the Image Tools program (University of Texas Health Sciences Center, San Antonio). The total infarction volume in each animal was obtained from the product of average slice thickness (2 mm) and sum of the area of infarction in all brain slices.

Post-Stroke data. d-KVNSEEEEEDA was given within 30 min after middle cerebral artery ligation in 23 rats. Of these, 11 rats received 150 nmol (150 nmole/25 μl x 25 μl) and the other 12 rats received 416 nmol (500 nmol/30 μL x 25 μL) of d- KVNSEEEEEDA. We found that d-KVNSEEEEEDA, given after stroke, did not significantly reduce the volume of infarction (p=0.577, F(2,48)=0.556, One way ANOVA). However, d-KVNSEEEEEDA increased locomotor movement in stroke animals. There is a significant correlation between the log dose of d-KVNSEEEEEDA given and total distance traveled 2 days after stroke (P=0.039, R = 0.323, total distance traveled = 100.233 + (13.796 * log [KVNSEEEEEDA]). There is also a trend showing that movement time was enhanced in the stroke animals that received post-stroke d-KVNSEEEEEDA (P=0.062, R = 0.294, Movement time - 100.305 + (10.697 * log [d-KVNSEEEEEDA]). These data indicate that d-KVNSEEEEEDA, given early after ischemic insults, is envisioned as improving locomotor activity in stroke animals.

While the present invention has been described in some detail for piiφoses of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incoφorated by reference.

Claims

WHAT IS CLAIMED IS:

1. A molecular decoyant for treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, said treatment being for the reduction of said neurotoxic effect without preventing said analgesic effect, having a chemical structure which functionally resembles the ligand binding site of said endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of said endogenous opioid receptor for the dynoφhin, said decoyant being a substance selected from the group consisting of:

(a) a substance which is a portion of the NRl subunit of said endogenous NMDA receptor for said dynoφhin, said portion being (i) sufficiently small so as not to elicit an autoimmune response against said endogenous NMDA receptor when administered in vivo, and (ii) not substantially larger than the smallest size needed to retain the elements of the binding site of said endogenous NMDA receptor which are essential for retaining the ability of said endogenous NMDA receptor to selectively and specifically recognize and bind to said dynoφhin with an affinity permitting the competition with binding of said dynoφhin to said endogenous NMDA receptor and not having the elements of the binding site of said endogenous opioid receptor which are essential for retaining the ability of said endogenous opioid receptor to selectively and specifically recognize and bind to said dynoφhin with an affinity so as not to permit a competition with binding of said dynoφhin to said endogenous opioid receptor;

(b) a substance having a chemical structure which substantially corresponds to that of said substance of (a), synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against said endogenous NMDA receptor when administered in vivo; and

(c) a substance having a chemical structure consisting essentially of the chemical structure of the substance of (a) or (b).

2. The molecular decoyant of claim 1, whereby said portion of the NRl subunit of said endogenous NMDA receptor is residue 594-599.

3. The molecular decoyant of claim 1, wherein said molecular decoyant is a substance of (a).

4. The molecular decoyant of claim 3, wherein said molecular decoyant is a peptide chain expressed by cells genetically engineered by recombinant DNA techniques.

5. The molecular decoyant of claim 3, wherein said molecular decoyant is a synthetic peptide or peptidomimetic.

6. The molecular decoyant of claim 3, wherein said molecular decoyant includes the amino acid sequence EEEEED or KVNSEEEEEDA.

7. The molecular decoyant of claim 3, wherein said organism is an animal.

8. The molecular decoyant of claim above, wherein said animal is a human.

9. The molecular decoyant of claim 1, wherein said molecular decoyant is a substance of (b).

10. The molecular decoyant of claim 9, wherein said molecular decoyant is a peptide chain expressed by cells genetically engineered by recombinant DNA techniques.

11. The molecular decoyant of claim 9, wherein said molecular decoyant is a synthetic peptide or peptidomimetic.

12. The molecular decoyant of claim 9, wherein said molecular decoyant includes an amino acid sequence substantially corresponding to EEEEED or

KVNSEEEEEDA.

13. The molecular decoyant of claim 9, wherein said organism is an animal.

14. The molecular decoyant of claim above, wherein said animal is a human.

15. A method for the treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, said treatment being for the reduction of said neurotoxic effect without preventing said analgesic effect, comprising:

(a) administering to said organism, in a quantity sufficient to reduce said neurotoxic effect without preventing said analgesic effect, a molecular decoyant having a chemical structure which functionally resembles the ligand binding site of said endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of said endogenous opioid receptor for the dynoφhin, said decoyant being a substance selected from the group consisting of: (b) a substance which is a portion of the NRl subunit of said endogenous NMDA receptor for said dynoφhin, said portion being (i) sufficiently small so as not to elicit an autoimmune response against said endogenous NMDA receptor when administered in vivo, and (ii) not substantially larger than the smallest size needed to retain the elements of the binding site of said endogenous NMDA receptor which are essential for retaining the ability of said endogenous NMDA receptor to selectively and specifically recognize and bind to said dynoφhin with an affinity permitting the competition with binding of said dynoφhin to said endogenous NMDA receptor and not having the elements of the binding site of said endogenous opioid receptor which are essential for retaining the ability of said endogenous opioid receptor to selectively and specifically recognize and bind to said dynoφhin with an affinity so as not to permit a competition with binding of said dynoφhin to said endogenous opioid receptor;

(c) a substance having a chemical structure which substantially corresponds to that of said substance of (a), synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against said endogenous NMDA receptor when administered in vivo; and

(d) a substance having a chemical structure consisting essentially of the chemical structure of the substance of (a) or (b).

16. Use of a molecular decoyant for the manufacture of a medicament for the treatment of an organism which has been subjected to a dynoφhin which exerts a neurotoxic effect only after first binding to an endogenous NMDA receptor and which exerts an analgesic effect only after first binding to an endogenous opioid receptor, said treatment being for the reduction of said neurotoxic effect without preventing said analgesic effect, having a chemical structure which functionally resembles the ligand binding site of said endogenous NMDA receptor for the dynoφhin, and which does not functionally resemble the ligand binding site of said endogenous opioid receptor for the dynoφhin, said decoyant being a substance selected from the group consisting of: (a) a substance which is a portion of the NRl subunit of said endogenous

NMDA receptor for said dynoφhin, said portion being (i) sufficiently small so as not to elicit an autoimmune response against said endogenous NMDA receptor when administered in vivo, and (ii) not substantially larger than the smallest size needed to retain the elements of the binding site of said endogenous NMDA receptor which are essential for retaining the ability of said endogenous NMDA receptor to selectively and specifically recognize and bind to said dynoφhin with an affinity permitting the competition with binding of said dynoφhin to said endogenous NMDA receptor and not having the elements of the binding site of said endogenous opioid receptor which are essential for retaining the ability of said endogenous opioid receptor to selectively and specifically recognize and bind to said dynoφhin with an affinity so as not to permit a competition with binding of said dynoφhin to said endogenous opioid receptor; (b) a substance having a chemical structure which substantially corresponds to that of said substance of (a), synthesized by chemical and/or recombinant DNA techniques and having the ability to selectively and specifically bind to the dynoφhin without eliciting an autoimmune response against said endogenous NMDA receptor when administered in vivo; and (c) a substance having a chemical structure consisting essentially of the chemical structure of the substance of (a) or (b).

17. The method of claim 15, wherein said method further comprises administering said molecular decoyant together with dynoφhin to reduce the neurotoxic effect of said administered dynoφhin.

18. The use of claim 16, wherein said use further comprises use of said molecular decoyant together with dynoφhin to reduce the neurotoxic effect of said administered dynoφhin.

19. The method of claim 15, wherein said method further comprises administering said molecular decoyant in the absence of dynoφhin to reduce the neurotoxic effect of non-administered dynoφhin.

20. The use of claim 16, wherein said use further comprises use of said molecular decoyant in the absence of dynoφhin to reduce the neurotoxic effect of non- administered dynoφhin.

21. The method of claim 15, wherein said method further comprises administering said molecular decoyant to a patient with stroke or spinal cord injury to reduce the neurotoxic effect of dynoφhin.

22. The use of claim 16, wherein said use further comprises use of said molecular decoyant for administration to a patient with stroke or spinal cord injury to reduce the neurotoxic effect of dynoφhin.

23. The method of claim 15, wherein said method further comprises administering said molecular decoyant to a patient in a neuropathic pain state to reduce the neurotoxic effect of dynoφhin.

24. The use of claim 16, wherein said use further comprises use of said molecular decoyant for administration to a patient in a neuropathic pain state to reduce the neurotoxic effect of dynoφhin.

25. The method of claim 15, wherein said method further comprises administering said molecular decoyant to a patient at risk for antinociceptive tolerance to moφhine to reduce said risk.

26. The use of claim 16, wherein said use further comprises use of said molecular decoyant for administration to a patient at risk for antinociceptive tolerance to moφhine to reduce said risk.

27. The method of claim 15 or use of claim 16, whereby said portion of the NRl subunit of said endogenous NMDA receptor is residue 594-599.

28. The method of claim 15 or use of claim 16, wherein said molecular decoyant is a substance of (a).

29. The method or use of claim 28, wherein said molecular decoyant is a peptide chain expressed by cells genetically engineered by recombinant DNA techniques.

30. The method of use of claim 28, wherein said molecular decoyant is a synthetic peptide or peptidomimetic.

31. The method or use of claim 28, wherein said molecular decoyant includes the amino acid sequence EEEEED or KVNSEEEEEDA.

32. The method of use of claim 28, wherein said organism is an animal.

33. The method or use of claim above, wherein said animal is a human.

34. The method of claim 15 or use of claim 16, wherein said molecular decoyant is a substance of (b).

35. The method of use of claim 34, wherein said molecular decoyant is a peptide chain expressed by cells genetically engineered by recombinant DNA techniques.

36. The method or use of claim 34, wherein said molecular decoyant is a synthetic peptide or peptidomimetic.

37. The method or use of claim 34, wherein said molecular decoyant includes an amino acid sequence substantially corresponding to EEEEED or KVNSEEEEEDA.

38. The method or use of claim 34, wherein said organism is an animal.

39. The method or use of claim above, wherein said animal is a human.

40. A pharmaceutical composition comprising a molecular decoyant in accordance with any of claims 1-14 in an amount effective to reduce the neurotoxic effect of dynoφhin without preventing its analgesic effect, and a pharmaceutically acceptable excipient.