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  Pharmaceutical Patents  

 

Title:  Treatment of injury to the brain by inhibition of acid sensing ion channels
United States Patent: 
8,030,442
Issued: 
October 4, 2011

Inventors:
 Simon; Roger P. (Portland, OR), Xiong; Zhi-Gang (Beaverton, OR)
Assignee:
  Morehouse School of Medicine (Atlanta, GA)
Appl. No.:
 11/943,546
Filed:
 November 20, 2007


 

Training Courses -- Pharm/Biotech/etc.


Abstract

Methods and compositions that inhibit acid sensing ion channels are provided for the prevention and treatment of brain injury, including injury caused by stroke or seizure. The methods and compositions of the invention are additionally effective for the reduction of acidosis in the brain.

Description of the Invention

SUMMARY OF THE EXEMPLARY EMBODIMENTS OF THE INVENTION

The present teachings provide methods and compositions for the prevention and treatment of brain injury. Specifically, the present teachings provide methods and compositions for the prevention and treatment of neuronal injury caused by acidosis of the brain.

The invention achieves these objects and satisfies additional objects and advantages by providing novel and surprisingly effective methods and compositions for treating and preventing neuronal injury through the use of inhibitors of acid sensing ion channels (ASIC).

Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and are implicated in perception of pain, ischemic stroke, mechanosensation, learning and memory.

Mammalian subjects amenable for treatment with inhibitors of acid sensing ion channels according to the methods of the invention include, but are not limited to those suffering from or at risk for neuronal injury including those with a history of seizures, including epilepsy; with a history of or at risk for ischemia; stroke; traumatic brain injury; surgery; infection; acidosis; ischemia; activation of one or more acid-sensing ion channels (with or without acidosis/ischemia); at risk for an ischemic event; at risk for stroke including a hemorrhagic stroke, an ischemic stroke, or the result of global ischemia (e.g., cardiac arrest); those with high cholesterol; high blood pressure; heart disease; irregular heart rhythms, such as atrial fibrillation, phlebitis, congestive heart failure; or any other disease or symptom that increases the likelihood of a neuronal injury such as those diseases and conditions that put an individual at risk for a seizure or stroke.

These and other subjects are effectively treated, prophylactically and/or therapeutically, by administering to the subject a neuronal protective effective amount of an ASIC inhibitor. Inhibitors of ASIC family members, as used herein, are substances that reduce (partially, substantially, or completely block) the activity or one or more members of the ASIC family, that is, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4, among others. In some examples, the inhibitors may reduce the channel activity of one or more members, such as the ability of the members to flux ions (e.g., sodium, calcium, and/or potassium ions, among others) through cell membranes (into and/or out of cells). The substances may be compounds (small molecules of less than about 10 kDa, peptides, nucleic acids, lipids, etc.), complexes of two or more compounds, and/or mixtures, among others. Furthermore, the substances may inhibit ASIC family members by any suitable mechanism including competitive, noncompetitive, uncompetitive, mixed inhibition, and/or by changing a subject's pH, among others. In some embodiments, an ASIC inhibitor may be selective within the ASIC family of channels. In other embodiments, an ASIC inhibitor may be specific for a particular ASIC family member. An exemplary ASIC inhibitor is psalmotoxin 1 (PcTx1), a toxin from a Psalmopoeus cambridgei and variants of PcTx1. Such variants may possess at least 50% sequence identity counted over the full length alignment with the amino acid sequence of a native PcTx1 polypeptide EDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVPKTPKT (SEQ ID NO. 1) using the NCBI Blast 2.0, gapped blastp set to default parameters. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 74%, at least 75%, at least 77%, at least 80%, at least 90% or at least 95% amino acid sequence identity.

Within additional aspects of the invention, combinatorial formulations and methods are provided which employ an effective amount of an ASIC inhibitor compound such as PcTx1 and variants thereof in combination with one or more secondary or adjunctive active agent(s) that is/are combinatorially formulated or coordinately administered with an ASIC inhibitor to yield a neuronal protective response in the subject. Exemplary combinatorial formulations and coordinate treatment methods in this context employ the ASIC inhibitor in combination with one or more additional, neuronal protective or other indicated, secondary or adjunctive therapeutic agents. The secondary or adjunctive therapeutic agents used in combination with, e.g., an ASIC inhibitor in these embodiments may possess direct or indirect neuronal protective activity, alone or in combination with, e.g. PcTx1, or may exhibit other useful adjunctive therapeutic activity in combination with, e.g., PcTx1.

Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, an antagonist selective for a glutamate receptor, such as an NMDA-receptor inhibitor including, but not limited to, ketamine, dextromethorphan, memantine, amantadine, 2-amino-5-phosphonopentanoate (AP5), dizocilipine, phencyclidine, riluzole, and cis-4-[phosphonomethyl]-2-piperidine carboxylic acid; an alkalinizing agent, such as sodium bicarbonate; nitroglycerin; anticoagulant medications, such as warfarin, dicumarol, anisinidione, and heparin; tissue plasminogen activator; aspirin; and anti-platelet agents including, but not limited to, clopidogrel bisulfate.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The instant invention provides novel methods and compositions for preventing and/or treating neuronal injury in mammalian subjects, including individuals and in vitro, ex vivo, and in vivo mammalian cells, tissues, and organs.

A broad range of mammalian subjects, including human subjects, are amenable to treatment using the formulations and methods of the invention. These subjects include, but are not limited to, human and other mammalian subjects presenting with those suffering from or at risk for neuronal injury including those with a history of seizures including, but not limited to, epilepsy; stroke including, but not limited to a major ischemic attack, a transient ischemic attach, and a hemorrhagic event; traumatic brain injury; surgery; infection; acidosis; ischemia; activation of one or more acid-sensing ion channels (with or without acidosis/ischemia); at risk for an ischemic event; at risk for stroke including a hemorrhagic stroke, an ischemic stroke, or the result of global ischemia (e.g., cardiac arrest); those with high cholesterol; metabolic disorder; hypoxia; high blood pressure; heart disease; irregular heart rhythms, such as atrial fibrillation, phlebitis, congestive heart failure; or any other disease or symptom that increases the likelihood of a neuronal injury such as those diseases and symptoms that put an individual at risk for a seizure or stroke.

Acidosis, as used herein, is any regional or global acidification of cells and/or tissue(s) of the body. The acidification may involve any suitable drop from (normal) physiological pH, such as about 0.1, 0.2, or 0.5 pH units, among others. In addition, the acidification may have any suitable cause, such as reduced blood flow (ischemia), increased metabolic activity (e.g., seizures), infection, a genetic defect, and/or the like.

Ischemia, as used herein, is a reduced blood flow to an organ(s) and/or tissue(s). The reduced blood flow may be caused by any suitable mechanism including a partial or complete blockage (an obstruction), a narrowing (a constriction), and/or a leak/rupture, among others, of one or more blood vessels that supply blood to the organ(s) and/or tissue(s). Accordingly, ischemia may be created by thrombosis, an embolism, atherosclerosis, hypertension, hemorrhage, an aneurysm, surgery, trauma, medication, or any other condition known to reduce blood flow. The reduced blood flow thus may be chronic, transient, acute, sporadic or any other characterization of fixed and/or variable reduced blood flow conditions.

Any suitable organ or tissue may experience a reduced blood flow in the ischemia being treated. Exemplary organs and/or tissues may include the brain, arteries, the heart, intestines, the eye (e.g., the optic nerve and/or retina), etc. Ischemia-induced injury (i.e., disease and/or damage) produced by various ischemias may include ischemic myelopathy, ischemic optic neuropathy, ischemic colitis, coronary heart disease, and/or cardiac heart disease (e.g., angina, heart attack, etc.), among others. Ischemia-induced injury thus may damage and/or kill cells and/or tissue, for example, producing necrotic (infarcted) tissue, inflammation, and/or tissue remodeling, among others, at affected sites within the body. Treatment according to aspects of the present teachings may reduce the incidence, extent, and/or severity of this injury.

Within the methods and compositions of the invention, one or more acid sensing ion channel (ASIC) inhibiting compound(s) as disclosed herein is/are effectively formulated or administered as a neuroprotective agent effective for treating and preventing brain injury and/or related disorders. In exemplary embodiments, PcTx1 is demonstrated for illustrative purposes to be an ASIC inhibiting effective agent in pharmaceutical formulations and therapeutic methods, alone or in combination with one or more adjunctive therapeutic agent(s). The present disclosure further provides additional, pharmaceutically acceptable neuroprotective compounds in the form of a native or synthetic compound, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as neuroprotective therapeutic agents within the methods and compositions of the invention.

ASICs belong to the degenerin/epithelial Na+ channel family of amiloride sensitive cation channels. They are widely distributed throughout the mammalian peripheral and central nervous system and have been implicated in many physiological and pathophysiological processes. Various ASIC subunits form homomultimeric and heteromultimeric channel complexes that vary in their expression within organs and are activated at different pH values (Krishtal, O. Trends Neurosci 26 477-483 (2003)).

Four of these subunits may form functional homomultimeric channels that are activated by acidic pH to conduct a sodium-selective, amiloride-sensitive, cation current. The pH of half-maximal activation (pH.sub.0.5) of these channels differs: ASIC1a, pH.sub.0.5=6.2 (Waldmann et al., 1997a); ASIC1.beta. (also termed ASIC1b), a splice variant of ASIC1a with a unique N-terminal, pH.sub.0.5=5.9 (Chen et al., 1998); ASIC2a, pH.sub.0.5=4.4 (Waldmann et al., 1999); and ASIC3, pH.sub.0.5=6.5 (Waldmann et al., 1997b). Neither ASIC2b nor ASIC4 can form functional homomeric channel (Akopian et al. 2000; Grunder et al. 2000 and Lingueglia et al. 1997), but ASIC2b has been shown to associate with other subunits and modulate their activity (Lingueglia et al., 1997). In addition to Na.sup.+ permeability, homomeric ASIC1a may flux Ca.sup.2+ (Waldmann et al. 1997a; Chu et al. 2002 and Yennolaieva et al. 2004). Of the six ASIC subunits cloned, ASIC1a, ASIC2a and ASIC2b, are expressed in brain neurons (Duggan, A., et al., J. Biol Chem 277:5203-5206 (2002); Lingueglia, E., et al., J. Biol. Chem. 272:29778-29783 (1997); Waldmann, R., et al., Nature 386:173-177 (1997); Waldmann, R., et al J. Biol. Chem. 271:10433-10436 (1996)).

Although the exact subunit composition of ASICs in native neurons has not been determined, both ASIC1a and ASIC2a subunits have been shown to be abundant in the brain (Price et al. 1996; Bassilana et al. 1997; Wemmie et al. 2002 and Alvarez de la Rosa et al. 2003).

Detailed functions of ASICs in both peripheral and central nervous systems remain to be determined. In peripheral sensory neurons, ASICs have been implicated in mechanosensation (Price et al. 2000 and Price et al. 2001) and perception of pain during tissue acidosis (Bevan and Yeats 1991; Krishtal and Pidoplichko 1981; Ugawa et al. 2002; Sluka et al. 2003 and Chen et al. 2002), particularly in ischemic myocardium where ASICs likely transduce anginal pain (Benson et al., 1999). The presence of ASICs in the brain, which lacks nociceptors, suggests that these channels may have functions beyond nociception. Indeed, recent studies have indicated that ASIC1a may be involved in synaptic plasticity, learning/memory, and fear conditioning (Wemmie et al. 2002 and Wemmie et al. 2003).

It is thought that ASIC1a, which has a pH for half maximal activation (pH.sub.0.5) of 6.2 (Waldmann, R., et al., Nature 386:173-177 (1997)) is the most likely ASIC activated in physiological and pathophysiological conditions. ASIC1a allows the passage of both Na.sup.+ and Ca.sup.2+ ions into the cells (Waldmann, R., et al., Nature 386:173-177 (1997); Wu, L. et al., J. Biol. Chem 279:43716-43724 (2004); (Yermolaieva, O., et al., PNAS USA 101: 6752-6757 (2004)) and is involved in both physiological (Wemmie, J. A., et al. J Neurosci 23:5496-5502 (2003); Wemmie, J. A., et al., Neuron 34:463-477 (2002); Wemmie, J. A., et al. Proc Natl Acad Sci USA 101:3621-3626 (2004)) and pathological conditions (Allen, N. J., and Attwell, D. J Physiol 543:521-529 (2002); Diarra, A., Sheldon, C., Brett, C. L., Baimbridge, K. G., and Church, J. Neuroscience 93:1003-1016 (1999); Obrenovitch, T. P. et al., J Neurophysiol 64:1125-1133 (1990); Deitmer, J. W., and Rose, C. R. 48:73-103 (1996); Li, P. A., and Siesjo, B. K. Acta Physiol Scand 161:567-580 (1997)). In particular, the studies described in the examples below demonstrate that activation of ASIC1a is largely responsible for acidosis-mediated, glutamate independent neuronal injury.

Recently it has been reported that a simple molecule, the proton, plays a pivotal role in the development of the ischemic damage through activation of Ca.sup.2+ permeable acid-sensing ion channel: ASIC1a (U.S. Provisional Patent Application No. 60/611,241 and PCT Patent Application Serial No. PCT/US2005/033171, Xiong, Z. G., et al. Cell 118:687-698 (2004); Yermolaieva, O., et al. Proc Natl Acad Sci USA 101:6752-6757 (2004) each of which is incorporated by reference in their entirety). It has been known for several decades that acidosis occurs after ischemia and it is associated with neuronal injury. The experiments herein show that acidosis may activate Ca.sup.2+-permeable acid-sensing ion channels (ASICs), which may induce glutamate receptor-independent, Ca.sup.2+-dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs may be resistant to acid injury, while transfection of Ca.sup.2+-permeable ASIC1a may establish sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene may protect the brain from ischemic injury and may do so more potently than glutamate antagonism. Thus, acidosis may injure the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca.sup.2+].sub.i (intracellular calcium), disclosing new potential therapeutic targets for stroke.

In particular, as previously reported (Back, T., Hoehn, M., Mies, G., Busch, E., Schmitz, B., Kohno, K., and Hossmann, Ann Neurol 47:485-492 (2000)), in the so-called penumbral region there is a initial pH alkalinization. Interestingly, during the development of the ischemic lesion, the core pH drops to values around 6.5. These levels are sufficient to activate ASIC1a channels, which have a pH.sub.0.5 at 6.2 (Waldmann, R. Adv Exp Med Biol 502:293-304 (2001); Yermolaieva, O., Leonard, A. S., Schnizler, M. K., Abboud, F. M., and Welsh, M. J. Proc Natl Acad Sci USA 101:6752-6757 (2004)).

The alkalosis phenomenon of the penumbra has been previously described as resulting from the reduction of lactate formation, the elevated phosphorylation of adenosine nucleotides consuming H.sup.+, and acceptance of protons by the Krebs cycle (Back, T., Hoehn, M., Mies, G., Busch, E., Schmitz, B., Kolmo, K., and Hossmann, Ann Neurol 47:485-492 (2000)). As shown in the experiments below, the alkalosis is transient and, after .about.2 h of reperfusion (3 h after middle cerebral artery occlusion), the infarct reaches the parietal cortex and pH in this region drops to .about.6.5, a level sufficient to activate ASIC1a.

Therefore the pH drop and activation of ASIC1a proceeds from the ischemic core to the peripheral ischemic region as infarction matures. This delayed acid expansion into cortical penumbral region may explain the long-term neuroprotective window affected by ASIC1a blockade, and thus the protection observed in the experiments herein with blockade 5 hours after stroke.

Any suitable ASIC inhibitor or combination of inhibitors may be used in the compositions and methods of the present invention. Inhibitors of ASIC family members, as used herein, are substances that reduce (partially, substantially, or completely block) the activity of one or more members of the ASIC family, that is, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4, among others. In some examples, the inhibitors may reduce the channel activity of one or more members, such as the ability of the members to flux ions (e.g., sodium, calcium, and/or potassium ions, among others) through cell membranes (into and/or out of cells). The substances may be compounds (small molecules of less than about 10 kDa, peptides, nucleic acids, lipids, etc.), complexes of two or more compounds, and/or mixtures, among others. Furthermore, the substances may inhibit ASIC family members by any suitable mechanism including competitive, noncompetitive, uncompetitive, mixed inhibition, and/or by changing a subject's pH, among others. The expression "ASIC inhibitor" may refer to a product which, within the scope of sound pharmacological judgment, is potentially or actually pharmaceutically useful as an inhibitor of ASIC, and includes reference to substances which comprise a pharmaceutically active species and are described, promoted, and/or authorized as an ASIC inhibitor.

ASIC inhibitors may be selective with the ASIC family. For example, an ASIC1a inhibitor may have inhibition that is substantially stronger on ASIC1a than on another ASIC family member(s) when compared (for example, in cultured cells) after exposure of each to the same (sub-maximal) concentration(s) of an inhibitor. The inhibitor may inhibit ASIC1a selectively relative to at least one other ASIC family member (ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC 4, etc.) and/or selectively relative to every other ASIC family member. The strength of inhibition for a selective inhibitor may be described by an inhibitor concentration at which inhibition occurs (e.g., an IC.sub.50 (inhibitor concentration that produces 50% of maximal inhibition) or a K.sub.i value (inhibition constant or dissociation constant)) relative to different ASIC family members. An ASIC1a-selective inhibitor may inhibit ASIC1a activity at a concentration that is at least about two-, four-, or ten-fold lower (one-half, one-fourth, or one-tenth the concentration or lower) than for inhibition of at least one other or of every other ASIC family member. Accordingly, an ASIC1a-selective inhibitor may have an IC.sub.50 and/or K.sub.i for ASIC1a inhibition that is at least about two-, four-, or ten-fold lower (one-half, one-fourth, or one-tenth or less) than for inhibition of at least one other ASIC family member and/or for inhibition of every other ASIC family member.

ASIC inhibitors in addition to being selective may also be specific for particular channels within the ASIC family. For example, an ASIC1a-selective inhibitor, in addition to being selective, also may be specific for ASIC1a. ASIC1a-specific inhibition, as used herein, is inhibition that is substantially exclusive to ASIC1a relative to every other ASIC family member. An ASIC1a-specific inhibitor may inhibit ASIC1a at an inhibitor concentration that is at least about twenty-fold lower (5% of the concentration or less) than for inhibition of every other ASIC family member. Accordingly, an ASIC1a-specific inhibitor may have an IC.sub.50 and/or K.sub.i for ASIC1a relative to every other member of the ASIC family that is at least about twenty-fold lower (five percent or less), such that, for example, inhibition of other ASIC family members is at least substantially (or completely) undetectable.

Any suitable ASIC inhibitor or combination of inhibitors may be used in the methods and compositions herein. For example, a subject may be treated with an ASIC1a-selective inhibitor and a nonselective ASIC inhibitor, or with an ASIC1a-selective inhibitor and an inhibitor to a non-ASIC channel protein, such as a non-ASIC calcium channel. In some examples, a subject may be treated with an ASIC1a-selective inhibitor and an inhibitor of a glutamate receptor. The glutamate inhibitor may selectively inhibit an ionotropic glutamate receptor (e.g., an NMDA receptor, an AMPA receptor, or a kainate receptor, among others) or a metabotropic glutamate receptor. Furthermore, the inhibitor may selectively inhibit an NMDA receptor that is, selectively relative to other receptors and/or relative to non-NMDA glutamate receptors.

In some embodiments, an ASIC inhibitor may be or may include a peptide. "Proteins", "peptides," "polypeptides" and "oligopeptides" as used herein are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term "amino terminus" (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal of the protein, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the protein. Similarly, the term "carboxy terminus" (abbreviated C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminus of a protein, or to the carboxyl group of an amino acid at any other location within the protein. In keeping with standard polypeptide nomenclature, the following abbreviations for amino acids may be used herein to describe various ASIC inhibitors in the compositions and the methods of the present invention.

An ASIC inhibitor may have any suitable number of amino acid subunits (also termed residues), generally at least about ten and less than about one-thousand subunits. In some examples, the peptide may have a cystine knot motif. A cystine knot, as used herein, generally comprises an arrangement of six or more cysteines. A peptide with these cysteines may create a "knot" including (1) a ring formed by two disulfide bonds and their connecting backbone segments, and (2) a third disulfide bond that threads through the ring. In some examples, the peptide may be a conotoxin from an arachnid and/or cone snail species. An exemplary peptide is PcTx1 (psalmotoxin 1), a toxin from a tarantula (Psalmopoeus cambridgei (Pc)) which has the amino acid sequence: EDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVGVPKTPKT (SEQ ID NO. 1, FIG. 10 (see Original Patent)). As shown in FIG. 10, PcTx1 may include six cystine residues that form cystine bonds 52, 54, and 56 to create a cystine knot motif 58. The peptide also may include one or more beta sheet regions 60 and a positively charged region 62. An N-terminal region 64 and a C-terminal region 66 may flank the cystine knot motif.

One of skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (less than about 30%, typically less than about 20%, typically less than about 10%, more typically less than about 5%, typically less than about 3%, typically less than about 1%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. "Conservative substitution" in the context of the subject invention is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure of the polypeptide to be substantially unchanged for these regions. For example, the amino acid residues arginine, histidine and lysine are hydrophilic, basic amino acid residues and may therefore be interchangeable. Similarly, the amino acid residue isoleucine, which is a hydrophobic amino acid residue, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the polypeptide.

The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Variants of PcTx1 will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Altschul et al. (1994) presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at the NCBI website. A description of how to determine sequence identity using this program is available at the NCBI website, as are the default parameters.

Variants of PcTx1 peptides are typically characterized by possession of at least 90%, 80%, 78%, 75%, 74%, 50% sequence identity counted over the full length alignment with the amino acid sequence of a native PcTx1 peptide using the NCBI Blast 2.0, gapped blastp set to default parameters. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 77%, at least 80%, at least 90% or at least 95% amino acid sequence identity. When less than the entire sequence is being compared for sequence identity, variants will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI website. Variants of PcTx1 peptides polypeptides also retain the biological activity of the native polypeptide.

Exemplary PcTx1 variants may include, for example, an N-terminal deletion 70 so that the resulting sequence is CIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVPKTPKT (SEQ ID NO:2); a partial C-terminal deletion 72 so that the resulting sequence is EDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVPKTPKT (SEQ ID NO:3); a full C-terminal deletion 74 so that the resulting sequence is EDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVP (SEQ ID NO:4); or an N/C terminal deletion 76 so that the resulting sequence is CIPKWKGCVNRHGDCCEGLECWKRRRSFEVC (SEQ ID NO:5). (FIG. 11 (see Original Patent)) Other derivatives of PcTx1 may include any deletion, insertion, or substitution of one or more amino acids in SEQ ID NO: 1, for example, while maintaining sequence similarity or identity of at least about 25% or about 50% with the original PcTx1 sequence.

When the peptides are relatively short in length (i.e., less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the peptides described herein. Techniques for solid phase synthesis are known to those skilled in the art.

Alternatively, the peptides described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide or protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide or protein in a host, isolating the expressed peptide or protein and, if required, renaturing the peptide or protein. Techniques sufficient to guide one of skill through such procedures are found in the literature.

When several desired protein fragments or peptides are encoded in the nucleotide sequence incorporated into a vector, one of skill in the art will appreciate that the protein fragments or peptides may be separated by a spacer molecule such as, for example, a peptide, consisting of one or more amino acids. Generally, the spacer will have no specific biological activity other than to join the desired protein fragments or peptides together, or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. Nucleotide sequences encoding for the production of residues which may be useful in purification of the expressed recombinant protein may be built into the vector. Such sequences are known in the art. For example, a nucleotide sequence encoding for a poly histidine sequence may be added to a vector to facilitate purification of the expressed recombinant protein on a nickel column.

Once expressed, recombinant peptides, polypeptides and proteins can be purified according to standard procedures known to one of ordinary skill in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of about 50 to 99% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.

One of skill in the art will recognize that after chemical synthesis, biological expression or purification, the desired proteins, fragments thereof and peptides may possess a conformation substantially different than the native conformations of the proteins, fragments thereof and peptides. In this case, it is often necessary to denature and reduce protein and then to cause the protein to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art.

Each PcTx1 variant or derivative may be tested for its ability to inhibit ASIC proteins selectively and/or for an effect, if any, on neuronal injury. Any suitable test system(s) may be used to perform this testing including any of the cell-based assay systems and/or animal model systems described elsewhere in the present teachings. The PcTx1 derivative also or alternatively may be tested in ischemic human subjects.

ASIC inhibiting compositions comprising PcTX1 and variants thereof, including pharmaceutical formulations of the invention, comprise a neuroprotective effective amount of an ASIC inhibiting compound, which is effective for prophylaxis and/or treatment of brain injury in a mammalian subject. Typically, a neuroprotective effective amount, of a compound will comprise an amount of the active compound which is therapeutically effective, in a single or multiple unit dosage form, over a specified period of therapeutic intervention, to measurably alleviate one or more symptoms of brain injury in the subject, and/or to alleviate one or more symptom(s) of stroke, seizure or related conditions in the subject. Within exemplary embodiments, these compositions are effective within in vivo treatment methods to alleviate brain injury.

ASIC inhibiting compositions of the invention typically comprise a neuroprotective effective amount or unit dosage of a PcTX1 peptide or variant thereof, which may be formulated with one or more pharmaceutically acceptable carriers, excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers, and/or other additives that may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantages for pharmaceutical use. Neuroprotective effective amounts of a PcTX1 peptide or variant thereof or other ASIC inhibiting compound (e.g., a unit dose comprising an effective concentration/amount of ASIC inhibiting compound) will be readily determined by those of ordinary skill in the art, depending on clinical and patient-specific factors. Suitable effective unit dosage amounts of the active compounds for administration to mammalian subjects, including humans, may range from 10 to 1500 mg, 20 to 1000 mg, 25 to 750 mg, 50 to 500 mg, or 150 to 500 mg. In certain embodiments, the neuroprotective effective dosage of an ASIC inhibiting compound may be selected within narrower ranges of, for example, 10 to 25 mg, 30 to 50 mg, 75 to 100 mg, 100 to 250 mg, or 250 to 500 mg. These and other effective unit dosage amounts may be administered in a single dose, or in the form of multiple daily, weekly or monthly doses, for example in a dosing regimen comprising from 1 to 5, or 2-3, doses administered per day, per week, or per month. In one exemplary embodiment, dosages of 10 to 25 mg, 30 to 50 mg, 75 to 100 mg, 100 to 250 mg, or 250 to 500 mg, are administered one, two, three, four, or five times per day. In more detailed embodiments, dosages of 50-75 mg, 100-200 mg, 250-400 mg, or 400-600 mg are administered once or twice daily. In alternate embodiments, dosages are calculated based on body weight, and may be administered, for example, in amounts from about 0.5 mg/kg to about 100 mg/kg per day, 1 mg/kg to about 75 mg/kg per day, 1 mg/kg to about 50 mg/kg per day, 2 mg/kg to about 50 mg/kg per day, 2 mg/kg to about 30 mg/kg per day or 3 mg/kg to about 30 mg/kg per day. In some embodiments, an ASIC inhibiting compound may be effective if given within a particular time period after the occurrence of a brain injuring event. For example, an ASIC inhibiting compound may be effective if given within 1, 2, 3, 4, 5, 6, 7 or more hours of the event. In other embodiments, an ASIC inhibiting compound may be administered prophalactically.

The amount, timing and mode of delivery of compositions of the invention comprising a neuroprotective effective amount of a ASIC inhibiting compound will be routinely adjusted on an individual basis, depending on such factors as weight, age, gender, and condition of the individual, the acuteness of the brain injury and/or related symptoms, whether the administration is prophylactic or therapeutic, and on the basis of other factors known to effect drug delivery, absorption, pharmacokinetics, including half-life, and efficacy.

An effective dose or multi-dose treatment regimen for the instant ASIC inhibiting formulations will ordinarily be selected to approximate a minimal dosing regimen that is necessary and sufficient to substantially prevent or alleviate neuronal damage or acidosis in the subject, and/or to substantially prevent or alleviate one or more symptoms associated with neuronal damage or acidosis in the subject. A dosage and administration protocol will often include repeated dosing therapy over a course of several days or even one or more weeks or years. An effective treatment regime may also involve prophylactic dosage administered on a day or multi-dose per day basis lasting over the course of days, weeks, months or even years.

Various assays and model systems can be readily employed to determine the therapeutic effectiveness of an ASIC inhibitor. Screening may involve any suitable assay system that measures interaction between ASIC proteins and the set of candidate inhibitors. Exemplary assay systems may include assays performed biochemically (e.g., binding assays), with cells grown in culture ("cultured cells"), and/or with organisms, among others.

A cell-based assay system may measure an effect, if any, of each candidate inhibitor on ion flux in the cells, generally acid-sensitive ion flux. In some examples, the ion flux may be a flux of calcium and/or sodium, among others. The assay system may use cells expressing an ASIC family member, such as ASIC1a, or two or more distinct sets of cells expressing two or more distinct ASIC family members, such as ASIC1a and another ASIC family member(s), to determine the selectivity of each inhibitor for these family members. The cells may express each ASIC family member endogenously or through introduction of foreign nucleic acid. In some examples, the assay system may measure ion flux electrophysiologically (such as by patch clamp), using an ion-sensitive or membrane potential-sensitive dye (e.g., a calcium sensitive dye such as Fura-2), or via a gene-based reporter system that is sensitive to changes in membrane potential and/or intracellular ion (e.g., calcium) concentrations, among others. The assay system may be used to test candidate inhibitors for selective and/or specific inhibition of ASIC family members, particularly ASIC1a.

Any suitable seizure/epilepsy model system(s) may be used to test candidate bioactive compositions and/or treatment regimens with the bioactive compositions. Accordingly, the candidate compositions may be tested in cell culture systems, tissue explant systems, and/or in whole animals. Furthermore, seizure-like activity or seizures may be induced by any suitable approach, including electrical stimulation, contact with a bioactive composition, a change in oxygen concentration (e.g., hypoxia or anoxia), and/or trauma, among others.

Candidate inhibitors also or alternatively may be tested in tissue-based assay systems. For example, candidate inhibitors may be tested on explants of brain tissue, such as hippocampal slices, among others.

One or more ASIC inhibitors may be administered to an ischemic subject(s) to test the efficacy of the inhibitors for treatment of ischemia. The ischemic subjects may be people or animals. In some examples, the ischemic subjects may provide an animal model system of stroke and/or epilepsy. Exemplary animal model systems include rodents (mice and/or rats, among others) with ischemia and/or seizure(s) induced experimentally. The ischemia and/or seizure(s) may be induced mechanically (e.g., surgically) and/or by administration of a drug, among others. In some examples, the ischemia may be induced by occlusion of a blood vessel, such as by constriction of a mid-cerebral artery.

Effectiveness of the compositions and methods of the invention may also be demonstrated by a decrease in the occurrence and symptoms of stroke or seizure including a decrease in abnormal synchronization of a group of brain cells, such that the brain cells exhibit normal electrical activity; there is a decrease in acidosis in the brain, a reduction or prevention of hemiparesis, hemiplegia, one-sided numbness, one-sided weakness, one-sided paralysis, temporary limb weakness, limb tingling, confusion, trouble speaking, trouble understanding speech, trouble seeing in one or both eyes, dim vision, loss of vision, trouble walking, dizziness, a tendency to fall, loss of coordination, sudden severe headache, noisy breathing, and/or loss of consciousness. In some embodiments, a reduction or elimination of symptoms may be determined by observation. In other embodiments, a reduction or elimination of symptoms may be determined by tests and/or instruments.

Effectiveness of the compositions and methods of the invention may also be demonstrated by an increase in the pH of the brain of a mammalian subject.

For each of the indicated conditions described herein, test subjects will exhibit a 10%, 20%, 30%, 50% or greater reduction, up to a 75-90%, or 95% or greater, reduction, in one or more symptom(s) caused by, or associated with, brain injury or condition in the subject, compared to placebo-treated or other suitable control subjects.

Within additional aspects of the invention, combinatorial ASIC inhibiting formulations and coordinate administration methods are provided which employ an effective amount of an ASIC inhibitor compound and one or more secondary or adjunctive agent(s) that is/are combinatorially formulated or coordinately administered with an ASIC inhibitor compound to yield a combined, multi-active agent neuroprotective (and/or acidosis reducing) composition or coordinate treatment method. Exemplary combinatorial formulations and coordinate treatment methods in this context employ the ASIC inhibitor in combination with the one or more secondary neuroprotective agent(s), or with one or more adjunctive therapeutic agent(s) that is/are useful for treatment or prophylaxis of the targeted (or associated) disease, condition and/or symptom(s) in the selected combinatorial formulation or coordinate treatment regimen. For most combinatorial formulations and coordinate treatment methods of the invention, an ASIC inhibiting compound is formulated, or coordinately administered, in combination with one or more secondary or adjunctive therapeutic agent(s), to yield a combined formulation or coordinate treatment method that is combinatorially effective or coordinately useful to treat or prevent brain injury and/or one or more symptom(s) of a brain injury or condition in the subject. Exemplary combinatorial formulations and coordinate treatment methods in this context employ an ASIC inhibiting compound in combination with one or more secondary or adjunctive therapeutic agents selected from, e.g., an antagonist selective for a glutamate receptor, such as an NMDA-receptor inhibitor including, but not limited to, ketamine, dextromethorphan, memantine, amantadine, 2-amino-5-phosphonopentanoate (AP5), dizocilipine, phencyclidine, riluzole, and cis-4-[phosphonomethyl]-2-piperidine carboxylic acid; an alkalinizing agent such as sodium bicarbonate; nitroglycerin; anticoagulant medications, such as warfarin, dicumarol, anisinidione, and heparin; tissue plasminogen activator; aspirin; and anti-platelet agents including, but not limited to, clopidogrel bisulfate.

In certain embodiments the invention provides combinatorial neuroprotective (acidosis decreasing) formulations comprising an ASIC inhibiting compound and one or more adjunctive agent(s) having neuroprotective activity. Within such combinatorial formulations, the ASIC inhibiting compound and the adjunctive agent(s) having neuroprotective activity will be present in a combined formulation in neuroprotective effective amounts, alone or in combination. In exemplary embodiments, an ASIC inhibiting compound and a non-ASIC inhibiting agent(s) will each be present in neuroprotective amounts (i.e., in singular dosage which will alone elicit a detectable neuroprotective and/or acidosis decreasing response in the subject). Alternatively, the combinatorial formulation may comprise one or both of the ASIC inhibiting and non-ASIC inhibiting agents in sub-therapeutic singular dosage amount(s), wherein the combinatorial formulation comprising both agents features a combined dosage of both agents that is collectively effective in eliciting a neuroprotective (acidosis decreasing) response. Thus, one or both of the ASIC inhibiting and non-ASIC inhibiting agents may be present in the formulation, or administered in a coordinate administration protocol, at a sub-therapeutic dose, but collectively in the formulation or method they elicit a detectable neuroprotective response in the subject.

To practice coordinate administration methods of the invention, an ASIC inhibiting compound may be administered, simultaneously or sequentially, in a coordinate treatment protocol with one or more of the secondary or adjunctive therapeutic agents contemplated herein. Thus, in certain embodiments a compound is administered coordinately with a non-ASIC inhibiting agent, or any other secondary or adjunctive therapeutic agent contemplated herein, using separate formulations or a combinatorial formulation as described above (i.e., comprising both an ASIC inhibiting agent, and a non-ASIC inhibiting therapeutic agent). This coordinate administration may be done simultaneously or sequentially in either order, and there may be a time period while only one or both (or all) active therapeutic agents individually and/or collectively exert their biological activities. In some embodiments, the ASIC inhibitor and the non-ASIC inhibiting agent or other secondary or adjunctive therapeutic agent may be administered by the same or different routes of administration. A distinguishing aspect of all such coordinate treatment methods is that the ASIC inhibiting exerts at least some neuroprotective activity, which yields a favorable clinical response in conjunction with a complementary neuroprotective, or distinct, clinical response provided by the secondary or adjunctive therapeutic agent. Often, the coordinate administration of the ASIC inhibiting agent with the secondary or adjunctive therapeutic agent will yield improved therapeutic or prophylactic results in the subject beyond a therapeutic effect elicited by the ASIC inhibiting agent, or the secondary or adjunctive therapeutic agent administered alone. This qualification contemplates both direct effects, as well as indirect effects.

Within exemplary embodiments, an ASIC inhibiting agent will be coordinately administered (simultaneously or sequentially, in combined or separate formulation(s)), with one or more secondary neuroprotective agents, or other indicated therapeutic agents, e.g., selected from, for example, an antagonist selective for a glutamate receptor, such as an NMDA-receptor inhibitor including, but not limited to, ketamine, dextromethorphan, memantine, amantadine, 2-amino-5-phosphonopentanoate (AP5), dizocilipine, phencyclidine, riluzole, and cis-4-[phosphonomethyl]-2-piperidine carboxylic acid; an alkalinizing agent, such as sodium bicarbonate; nitroglycerin; anticoagulant medications, such as warfarin, dicumarol, anisinidione, and heparin; tissue plasminogen activator; aspirin; and anti-platelet agents including, but not limited to, clopidogrel bisulfate.

As noted above, in all of the various embodiments of the invention contemplated herein, the neuroprotective (acidosis decreasing) methods and formulations may employ an ASIC inhibiting agent or other therapeutic agent in any of a variety of forms, including any one or combination of the subject compound's pharmaceutically acceptable salts, isomers, enantiomers, polymorphs, solvates, hydrates, and/or prodrugs. In exemplary embodiments of the invention, PcTx1 is employed within the therapeutic formulations and methods for illustrative purposes.

The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended therapeutic or prophylactic purpose. Suitable routes of administration for the compositions of the invention include, but are not limited to, oral, buccal, nasal, aerosol, topical, transdermal, mucosal, injectable, slow release, controlled release, iontophoresis, sonophoresis, and including all other conventional delivery routes, devices and methods. Injectable methods include, but are not limited to, intravenous, intramuscular, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial, subcutaneous and intranasal routes.

The compositions of the present invention may further include a pharmaceutically acceptable carrier appropriate for the particular mode of administration being employed. Dosage forms of the compositions of the present invention include excipients recognized in the art of pharmaceutical compounding as being suitable for the preparation of dosage units as discussed above. Such excipients include, without intended limitation, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavorings, preservatives, buffers, wetting agents, disintegrants, effervescent agents and other conventional excipients and additives.

If desired, the compositions of the invention can be administered in a controlled release form by use of a slow release carrier, such as a hydrophilic, slow release polymer. Exemplary controlled release agents in this context include, but are not limited to, hydroxypropyl methyl cellulose, having a viscosity in the range of about 100 cps to about 100,000 cps or other biocompatible matrices such as cholesterol.

Compositions of the invention will often be formulated and administered in an oral dosage form, optionally in combination with a carrier or other additive(s). Suitable carriers common to pharmaceutical formulation technology include, but are not limited to, microcrystalline cellulose, lactose, sucrose, fructose, glucose, dextrose, or other sugars, di-basic calcium phosphate, calcium sulfate, cellulose, methylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, dextrin, maltodextrin or other polysaccharides, inositol, or mixtures thereof. Exemplary unit oral dosage forms for use in this invention include tablets, which may be prepared by any conventional method of preparing pharmaceutical oral unit dosage forms can be utilized in preparing oral unit dosage forms. Oral unit dosage forms, such as tablets, may contain one or more conventional additional formulation ingredients, including, but not limited to, release modifying agents, glidants, compression aides, disintegrants, lubricants, binders, flavors, flavor enhancers, sweeteners and/or preservatives. Suitable lubricants include stearic acid, magnesium stearate, talc, calcium stearate, hydrogenated vegetable oils, sodium benzoate, leucine carbowax, magnesium lauryl sulfate, colloidal silicon dioxide and glyceryl monostearate. Suitable glidants include colloidal silica, fumed silicon dioxide, silica, talc, fumed silica, gypsum and glyceryl monostearate. Substances which may be used for coating include hydroxypropyl cellulose, titanium oxide, talc, sweeteners and colorants.

Additional compositions of the invention can be prepared and administered in any of a variety of inhalation or nasal delivery forms known in the art. The intra nasal route is recognized as providing a method for bypassing the blood brain barrier and directly delivering therapeutic drugs to the central nervous system. This form of administration may be particularly useful in instances of brain injury. Devices capable of depositing aerosolized purified ASIC inhibiting formulations in the sinus cavity or pulmonary alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Methods and compositions suitable for pulmonary delivery of drugs for systemic effect are well known in the art. Additional possible methods of delivery include deep lung delivery by inhalation. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, may include aqueous or oily solutions of ASIC inhibiting compositions and any additional active or inactive ingredient(s).

Further compositions and methods of the invention are provided for topical administration of an ASIC inhibiting compound for the treatment of brain injury. Topical compositions may comprise an ASIC inhibiting compound along with one or more additional active or inactive component(s) incorporated in a dermatological or mucosal acceptable carrier, including in the form of aerosol sprays, powders, dermal patches, sticks, granules, creams, pastes, gels, lotions, syrups, ointments, impregnated sponges, cotton applicators, or as a solution or suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil liquid emulsion. These topical compositions may comprise a ASIC inhibiting compound dissolved or dispersed in a portion of a water or other solvent or liquid to be incorporated in the topical composition or delivery device. It can be readily appreciated that the transdermal route of administration may be enhanced by the use of a dermal penetration enhancer known to those skilled in the art. Formulations suitable for such dosage forms incorporate excipients commonly utilized therein, particularly means, e.g. structure or matrix, for sustaining the absorption of the drug over an extended period of time, for example, 24 hours. Transdermal delivery may also be enhanced through techniques such as sonophoresis.

Yet additional ASIC inhibiting compositions of the invention are designed for parenteral administration, e.g. to be administered intravenously, intramuscularly, subcutaneously or intraperitoneally, including aqueous and non-aqueous sterile injectable solutions which, like many other contemplated compositions of the invention, may optionally contain anti-oxidants, buffers, bacteriostats and/or solutes which render the formulation isotonic with the blood of the mammalian subject; and aqueous and non-aqueous sterile suspensions which may include suspending agents and/or thickening agents. The formulations may be presented in unit-dose or multi-dose containers. Additional compositions and formulations of the invention may include polymers for extended release following parenteral administration. The parenteral preparations may be solutions, dispersions or emulsions suitable for such administration. The subject agents may also be formulated into polymers for extended release following parenteral administration. Pharmaceutically acceptable formulations and ingredients will typically be sterile or readily sterilizable, biologically inert, and easily administered. Such polymeric materials are well known to those of ordinary skill in the pharmaceutical compounding arts. Parenteral preparations typically contain buffering agents and preservatives, and injectable fluids that are pharmaceutically and physiologically acceptable such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like. Extemporaneous injection solutions, emulsions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as described herein above, or an appropriate fraction thereof, of the active ingredient(s).

In more detailed embodiments, compositions of the invention may comprise an ASIC inhibiting compound encapsulated for delivery in microcapsules, microparticles, or microspheres, prepared, for example, by coaceivation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules); or within macroemulsions.

Other detailed embodiments, the methods and compositions of the invention for employ prodrugs of ASIC inhibiting agents. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug in vivo. Examples of prodrugs useful within the invention include esters or amides with hydroxyalkyl or aminoalkyl as a substituent, and these may be prepared by reacting such compounds as described above with anhydrides such as succinic anhydride.

The invention disclosed herein will also be understood to encompass methods and compositions comprising ASIC inhibiting agents using in vivo metabolic products of the said compounds (either generated in vivo after administration of the subject precursor compound, or directly administered in the form of the metabolic product itself). Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes methods and compositions of the invention employing compounds produced by a process comprising contacting an ASIC inhibiting compound with a mammalian subject for a period of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabelled compound of the invention, administering it parenterally in a detectable dose to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur and isolating its conversion products from the urine, blood or other biological samples.

The invention disclosed herein will also be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing a brain injury or condition in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) ASIC inhibiting compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of brain injury, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in ASIC receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods. In exemplary embodiments, a ASIC inhibiting compound is isotopically-labeled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as .sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O, .sup.31P, .sup.32P, .sup.35S, .sup.18F, and .sup.36Cl, respectively. The isotopically-labeled compound is then administered to an individual or other subject and subsequently detected as described above, yielding useful diagnostic and/or therapeutic management data, according to conventional techniques.
 

Claim 1 of 22 Claims

1. A method of treating ischemic brain injury or seizures in a mammalian subject comprising administering a neuroprotective effective amount of an acid sensing ion channel peptide inhibitor comprising a cystine knot motif selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, and SEQ ID NO: 5.

 

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