HK1119391A - Therapeutic agents targeting the ncca-atp channel and methods of use thereof - Google Patents

Description

Targeted NCCa-ATPTherapeutic agents for channels and methods of use thereof

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No.60/610,758 filed on 18/2004 and U.S. provisional application No.60/698,272 filed on 11/7/2005, which are incorporated herein by reference in their entirety.

Statement regarding federally sponsored research or development

The invention is conducted, in part, using government support under grant No. ns048260, awarded by the National Institutes of health, and the Merit Review grant by the United States Department of refuelling military Affairs (United States Department of vehicles Affairs). The united states government has certain rights in this invention.

Statement regarding other sponsored research or development

The invention is carried out in part with the support of withdrawal from Christopher Reeves Party Foundation (CRPF). CRPF has certain rights in the invention.

Technical Field

The present invention relates to the fields of cell biology, physiology and medicine. More specifically, the present invention proposes a novel method of treating a patient comprising administering a drug targeting a unique non-selective cationic calcium-ATP channel (NC) found in astrocytes_Ca-ATPA channel). In particular embodiments, the therapeutic compound is an antagonist and would benefit from blocking and/or inhibiting NC_Ca-ATPUse in the treatment of a channel, such as the treatment of spinal cord injury. And also relates to a method of containing NC_Ca-ATPA composition of channels.

Background

NC_Ca-ATPChannel

A unique nonselective monovalent cation ATP-sensitive channel (NC) was identified initially in Naturally Reactive Astrocytes (NRA) and later in neurons and capillary vascular endothelial cells following stroke or traumatic brain injury as described herein_Ca-ATPChannels) (see international application WO03/079987 to Simard et al, and Chen and Simard, 2001, each of which is incorporated herein by reference in its entirety). Consider NC as_CaATPThe channel is a heteropolymeric structure composed of the regulatory subunit of the sulfonylurea receptor type 1(SUR1) and the pore-forming subunit, with K in pancreatic beta-cells_ATPThe channels are similar (Chen et al, 2003). NC (numerical control)_Ca-ATPThe pore-forming subunits of the channels remain uncharacterized.

The SUR confers sensitivity to antidiabetic sulfonylureas, such as glibenclamide (glibenclamide) and tolbutamide (tolbutamide), and is responsible for this by being called "K⁺Channel openers "activation of a group of chemically diverse agents such as diazoxide, pinadil (pinacidil) and cromaalin (Aguilar-Byran et al, 1995; Inagaki et al, 1996; Isomoto et al, 1996; Nichols et al, 1996; Shyng et al, 1997"). Molecularly different SURs bind to different pore-formations in various tissuesSubunits to form different Ks with distinguishable physiological and pharmacological characteristics_ATPA channel. K in pancreatic beta-cells_ATPThe channel is formed by SUR1 connected to Kir6.2, and the myocardium and smooth muscle K_ATPThe channels are formed by SUR2A and SUR2B, respectively, connecting Kir6.2 and Kir6.1 (Fujita et al, 2000). Although composed of distinct pore-forming subunits, NC_Ca-ATPChannels are also sensitive to sulfonylurea compounds.

Furthermore, and K_ATPDifferent channels, NC_Ca-ATPChannels conduct sodium, potassium, cesium and other monovalent cations with nearly equal capacity (Chen and Simard, 2001), further indicating NC_Ca-ATPThe characterization of channels and thus the affinity for certain compounds differs from K_ATPA channel.

Has been identified by intracellular Ca²⁺Other non-selective cation channels that are activated and inhibited by intracellular ATP, but not in astrocytes. In addition, NC expressed and found in astrocytes with respect to calcium sensitivity and adenosine sensitivity_Ca-ATPThe channels are physiologically different from the other channels (Chen et al, 2001).

Other intracellular Ca has been identified in endothelial cells²⁺Non-selective cation channels activated and inhibited by intracellular ATP (Cscan and Adam-Vizi, Biophysic journal, 85: 313-327, 2003), but these channels were not regulated by SUR1 and were not inhibited by glyburide.

Spinal cord injury

Contusion of the spinal cord is often exacerbated by secondary damage from tissue inflammation and swelling. Secondary injury that enlarges the area of irreversible damage should in principle be preventable, since it is produced under medical care in a delayed manner, but no effective treatment has yet been obtained. Secondary injury usually involves a region of potentially viable tissue called a penumbra surrounding the initial injury. The viability of the neural tissue in the penumbra is unstable and those tissues are prone to death.

Alterations in gene expression associated with inflammation are among the earliest and strongest responses after spinal cord injury (Bareyre and Schwab, 2003; Bartholdi and Schwab, 1997).

The inflammatory response is required for resolution of the pathogenic event, but toxicity of many of its by-products causes damage to nearby or collateral tissues. It is generally believed that inflammation can be detrimental because cytotoxic substances such as TNF α and NO may be released, and because inflammation promotes the formation of edema and swelling, which in turn contributes to tissue ischemia. Thus, a strong inflammatory response may cause an enlargement of the area of initial tissue death. Conversely, relieving the inflammatory response may reduce the overall extent of the damage.

One of the most potent inflammatory stimulators in spinal cord injury is blood extravasated from fractured capillaries following injury. It is widely accepted that blood is highly toxic to central nervous system tissues, including the spinal cord.

Cells die by apoptosis and necrosis. The distinction is important not for dead cells, but for cells in the surrounding tissue-penumbra-which may survive, albeit initially weakly. Necrotic death triggers an inflammatory response, whereas apoptotic death does not. The molecular mechanisms responsible for inflammation following necrotic cell death are not fully understood, but it is likely that necrotic death (unlike apoptotic death) is accompanied by release of intracellular molecules upon cell membrane lysis. These intracellular molecules, when released, activate other cells, particularly microglia, whose activation leads to the expression of chemokines that in turn attract inflammatory cells. Thus, a logical therapeutic goal is to reduce necrosis, even if it is simply converted to apoptosis, to reduce the release of intracellular molecules that initiate inflammation.

An important class of intracellular molecules that can initiate inflammation in necrotic death are Heat Shock Proteins (HSPs). Spinal cord injury causes activation of astrocytes and upregulation of developmentally regulated intracellular proteins including vimentin, nestin, and HSPs. HSP-32 and HSP-70 are of particular interest because of their upregulation in spinal cord injury (Song et al, 2001; Mautes et al, 2000; Mautes and Noble, 2000). In astrocytes, HSP-32 (heme oxygenase-1) is induced by blood and blood products and HSP-70 is induced by hypoxia or glucose deprivation (Regan et al, 2000; Matz et al, 1996; Lee et al, 2001; Currie et al, 2000; Xu and Giffard, 1997; Papadopoulos et al, 1996; Copin et al, 1995).

HSP-70 and HSP-32 activate microglia in vivo (Kakimura et al, 2002), which in turn release inflammatory chemokines that attract macrophages and polymorphonuclear leukocytes (PMNs). Thus, the adverse pathological events leading to inflammation-mediated secondary injury may result in part from necrotic death of astrocytes and release of HSP as well as from extravasated blood. Thus, the present invention relates to the reduction of necrotic death of reactive astrocytes and the reduction of extravasation of blood as an improved therapeutic strategy to treat spinal cord injury.

Brief description of the invention

The present invention relates to NC containing neuronal cells, glial cells, or endothelial cells_Ca-ATPTherapeutic compositions of antagonists of the channels.

The present invention relates to methods of reducing spinal cord injury in a patient in need thereof comprising administering NC of neuronal, glial, or endothelial cells_Ca-ATPAn antagonist of the channel. Antagonist inhibits (closes, blocks, inactivates, decreases biological activity) NC_Ca-ATPA channel. Spinal cord injury may include contusion of the spinal cord.

One embodiment of the invention includes a method of treating a patient having a spinal cord injury comprising effectively inhibiting NC in neuronal cells, glial cells, neuroendothelial cells, or a combination thereof_Ca-ATPThe compound of the channel is administered to the patient. The compound is used for closing, blocking, partially blocking and/or inactivating NC_Ca-ATPThe channel is effective to inhibit the channel, thereby reducing Na + influx and other monovalent ions into the cell, reducing water accumulation in the cell, and thus reducing cell swelling. Thus, the compounds of the invention are reducedReducing or inhibiting NC_Ca-ATPActivation of channels which reduces sodium ions (Na)⁺) Thereby reducing and/or preventing or reducing depolarization of the cell.

The patient may comprise a patient having or at risk of a spinal cord injury. Patients at risk may include those receiving surgical and/or radiation therapy. Other patients at risk may include patients with spinal cord disorders, e.g., segmental malformations, spinal cord compression resulting from any known type of disease or infection. For example, cushing's syndrome can result in the growth of epidural adipose tissue that compresses the spinal cord. Other diseases may include arthritic diseases of the spine.

The compositions of the present invention may be delivered parenterally or enterally. Examples of gastrointestinal administration include, but are not limited to, oral, buccal, rectal, or sublingual. Parenteral administration can include, but is not limited to, intramuscular, subcutaneous, intraperitoneal, intravenous, intratumoral, intraarterial, intraventricular, intracavity, intravesical, intrathecal, or intrapleural. Other modes of administration may also include topical, mucosal (i.e. intranasal) or transdermal.

NC that can be administered to cells_Ca-ATPAn effective amount of a channel antagonist includes a dose of about 0.0001nM to about 2000 μ M. More specifically, the dose to be administered is from about 0.01nM to about 2000. mu.M; about 0.01 μ M to about 0.05 μ M; about 0.05 μ M to about 1.0 μ M; about 1.0 μ M to about 1.5 μ M; about 1.5 μ M to about 2.0 μ M; about 2.0 μ M to about 3.0 μ M; about 3.0 μ M to about 4.0 μ M; about 4.0 μ M to about 5.0 μ M; about 5.0 μ M to about 10 μ M; about 10 μ M to about 50 μ M; about 50 μ M to about 100 μ M; about 100 μ M to about 200 μ M; about 200 μ M to about 300 μ M; about 300 μ M to about 500 μ M; about 500 μ M to about 1000 μ M; about 1000 μ M to about 1500 μ M and about 1500 μ M to about 2000 μ M. Of course, all of these amounts are exemplary, and any amount in between these points is contemplated to be useful in the present invention.

NC as therapy_Ca-ATPThe effective amount of the channel antagonist or related compound will vary depending upon the host treated and the particular mode of administration. Hair brushIn one embodiment of the invention, NC_Ca-ATPThe dose range of the channel antagonist or related compound is from about 0.01 μ g/kg body weight to about 20,000 μ g/kg body weight. The term "body weight" is applicable when the animal is to be treated. When isolated cells are to be treated, "body weight" as used herein should be understood to mean "total cell weight". The term "total body weight" may apply to isolated cells and animal treatments. All concentrations and therapeutic levels expressed as "body weight" or simply "kg" in this application are considered to encompass similar concentrations of "total cell weight" and "total body weight". However, one skilled in the art will recognize the utility of various dosage ranges, such as 0.01 μ g/kg body weight to 20,000 μ g/kg body weight, 0.02 μ g/kg body weight to 15,000 μ g/kg body weight, 0.03 μ g/kg body weight to 10,000 μ g/kg body weight, 0.04 μ g/kg body weight to 5,000 μ g/kg body weight, 0.05 μ g/kg body weight to 2,500 μ g/kg body weight, 0.06 μ g/kg body weight to 1,000 μ g/kg body weight, 0.07 μ g/kg body weight to 500 μ g/kg body weight, 0.08 μ g/kg body weight to 400 μ g/kg body weight, 0.09 μ g/kg body weight to 200 μ g/kg body weight, or 0.1 μ g/kg body weight to 100 μ g/kg body weight. Furthermore, those skilled in the art will recognize that various dosage levels are useful, e.g., 0.0001. mu.g/kg, 0.0002. mu.g/kg, 0.0003. mu.g/kg, 0.0004. mu.g/kg, 0.005. mu.g/kg, 0.0007. mu.g/kg, 0.001. mu.g/kg, 0.1. mu.g/kg, 1.0. mu.g/kg, 1.5. mu.g/kg, 2.0. mu.g/kg, 5.0. mu.g/kg, 10.0. mu.g/kg, 15.0. mu.g/kg, 30.0. mu.g/kg, 50. mu.g/kg, 75. mu.g/kg, 80. mu.g/kg, 90. mu.g/kg, 100. mu.g/kg, 120. mu.g/kg, 140. mu.g/kg, 150. mu.g/kg, 160. mu.g/kg, 180. mu.g/kg, 200. mu.g/kg, 225. mu.g/kg, 250. mu.g, 300. mu.g/kg, 325. mu.g/kg, 350. mu.g/kg, 375. mu.g/kg, 400. mu.g/kg, 450. mu.g/kg, 500. mu.g/kg, 550. mu.g/kg, 600. mu.g/kg, 700. mu.g/kg, 750. mu.g/kg, 800. mu.g/kg, 900. mu.g/kg, 1mg/kg, 5mg/kg, 10mg/kg, 12mg/kg, 15mg/kg, 20mg/kg and/or 30 mg/kg. Of course, all of these dosages are exemplary, and it is contemplated that any dosage between these points may also be useful in the present invention. For NC_Ca-ATPAntagonists of the channel or compounds related thereto may be used at any of the dosage ranges or dosage levels described above.

Through type 1 sulfonylurea receptors (SUR1)Antagonist blocking or inactivating or inhibiting NC_Ca-ATPChannel and NC opening by SUR1 activator_Ca-ATPA channel. More specifically, antagonists of type 1 sulfonylurea receptors (SUR1) include K_ATPBlockers of channels, SUR1 activators including K_ATPAn activator of the channel. More specifically, NC of the invention_Ca-ATPChannel to potassium ion (K)⁺) With a single channel conductance of 20 to 50 pS. NC (numerical control)_Ca-ATPThe channels are also exposed to Ca in the physiological concentration range on the cytoplasmic side of the cell membrane²⁺Stimulation in a concentration range of 10^-8To 10^-5M。NC_Ca-ATPThe channel is also inhibited by cytoplasmic ATP in a physiological concentration range, wherein the concentration range is 10^-1To 10M. NC (numerical control)_Ca-ATPThe channels are also permeable to the following cations: k⁺、Cs⁺、Li⁺、Na⁺(ii) a To the extent that the permeability ratio between any two cations is above 0.5 and below 2.

By NC_Ca-ATPInhibitors of channels, NC_CaATPChannel blockers, sulfonylurea receptor type 1(SUR1) antagonists, SUR1 inhibitors, or compounds capable of reducing the magnitude of membrane current through a channel may inhibit the channel. More specifically, the SUR1 antagonist is selected from the group consisting of glibenclamide, tolbutamide, repaglinide (repaglinide), nateglinide (nateglinide), meglitinide (meglitinide), miglitzole (midglizole), LY 39364, LY389382, glyclazide, glimepiridide, estrogens, estrogen related compounds (estradiol, estrone, estriol, genistein, non-steroidal estrogens (e.g., diethylstilbestrol), phytoestrogens (e.g., coumestrol), zearalenol, and the like), and is known to inhibit or block K_ATPA compound of a channel. MgADP can also be used to inhibit channels. Can be used to block or inhibit K_ATPOther compounds of the channel include, but are not limited to tolbutamide, glyburide (1[ p-2[ 5-chloro-O-anisamide) ethyl]Phenyl radical]Sulfonyl radical]-3-cyclohexyl-3-urea); chlorpromazine (1- [ [ (p-chlorophenyl) sulfonyl)]-3-propylurea; glipizide (1-cyclohexyl-3- [ [ p- [2 (5-methylpyrazinamide) ethyl ] ethyl]Phenyl radical]Sulfonyl radical]Urea); or tolazamide (tolazamide) (benzenesulfonamide-N- [ [ hexa ] amidehydrogen-1H-aza-1 radical) amino]Carbonyl radical]-4-methyl).

In particular embodiments, the amount of SUR1 antagonist administered to the patient ranges from about 0.0001 μ g/kg/day to about 20 mg/kg/day, from about 0.01 μ g/kg/day to about 100 μ g/kg/day, or from about 100 μ g/kg/day to about 20 mg/kg/day. Still further, the SUR1 antagonist may be administered to the patient in the form of a treatment, where the treatment may include the amount of SUR1 antagonist or the dose of SUR1 antagonist administered daily (1, 2, 3, 4, etc.), weekly (1, 2, 3, 4, 5, etc.), monthly (1, 2, 3, 4, 5, etc.), and the like. Treatment may be administered such that the SUR1 antagonistic dose administered to the patient is in the range of about 0.0001 μ g/kg/treatment to about 20 mg/kg/treatment, about 0.01 μ g/kg/treatment to about 100 μ g/kg/treatment, or about 100 μ g/kg/treatment to about 20 mg/kg/treatment.

In certain embodiments, the antagonists treat adverse conditions associated with cytotoxicity and ionic edema of the central nervous system. Such conditions include trauma, spinal cord injury, i.e., secondary neuronal injury, such as, but not limited to, hemorrhagic transformation, immune system response, oxidative damage, calcium and excitotoxicity, necrosis and apoptosis, and/or axonal injury. By NC_Ca-ATPThe protective effect of inactivation and/or inhibition of the channel is associated with a reduction in edema, a reduction in cell death, a reduction in blood extravasation in the injury site, a reduction in reactive oxide production, a reduction in inflammation or inflammatory response, and/or a reduction in hemorrhagic conversion. Thus, the compounds of the present invention reduce these symptoms compared to the level of symptoms if no compound is administered.

In a particular embodiment, NC_Ca-ATPThe channels are blocked, inhibited or otherwise reduced in activity, such that the ion Na⁺And/or the influx of other monovalent ions through the channel is reduced, halted, reduced, and/or stopped. Antagonists may prevent or reduce depolarization of cells, thus mitigating Na⁺Osmotic changes due to influx and cell swelling due to depolarization of the cells. Due to the fact thatTo NC, this_Ca-ATPInhibition of the channel may reduce cytotoxic edema and cell death, e.g., necrotic death of cells, and thus, the antagonists of the invention may be used to reduce secondary injury associated with spinal cord injury.

Still further, the invention can include a method of reducing or reducing the incidence of a patient suffering from spinal cord injury comprising administering an effective amount of NC that inhibits and/or inactivates neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPA compound of a channel. The reduction in morbidity results in an improvement in the physical and/or motor outcome and/or sensation of the patient. Thus, an increase in the range of motion and/or an increase in sensation in the patient is an indicator of a decreased incidence. In further embodiments, an increase in the well-being of the patient is also an indicator that the incidence of the patient is reduced.

Still further, the invention can include a method of reducing blood and/or hemoglobin concentrations in or near or around a contusion site in a spinal cord injury patient comprising administering an effective amount of NC that inhibits neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPA compound of a channel.

Still further, another embodiment includes a method of reducing the size of a lesion in a spinal cord injury in a patient comprising administering an effective amount of NC that inhibits neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPA compound of a channel. The reduction in lesion size reduces the likelihood of contralateral involvement.

Another embodiment of the invention includes increasing or improving preservation of myelinated long tracts (tracts) comprising administering an effective amount of NC in inhibitory neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPA compound of a channel.

Still further, another embodiment of the invention encompasses a method of reducing GFAP upregulation in a spinal cord injury patient comprising administering an effective amount of NC to inhibit neuronal cells, glial cells, endothelial cells or a combination thereof_Ca-ATPOf passagesA compound is provided.

Still further, another embodiment includes a method of reducing extravasation of blood from a spinal cord injury comprising administering an effective amount of NC to inhibit neuronal cells, glial cells, endothelial cells or combinations thereof_Ca-ATPA compound of a channel. The patient may be a patient having or at risk of a spinal cord injury, e.g., a patient undergoing surgery or radiation therapy. Thus, the compounds may be administered before, during or after surgery and/or radiation therapy.

Another embodiment of the invention encompasses a method of reducing edema in a spinal cord injury penumbra in a patient comprising administering an effective amount of NC that inhibits neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPA compound of a channel.

Still further, another embodiment of the invention encompasses a method of treating a patient at risk of spinal cord injury comprising administering an effective amount of NC in inhibitory neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPA compound of a channel. Patients at risk may include those receiving surgical and/or radiation therapy. Other patients at risk may include patients with spinal disorders, e.g., segmental malformations, spinal cord compression resulting from any known type of disease or infection, e.g., cushing's syndrome or arthritic disease of the spine.

Still further, another embodiment of the invention includes a method of diagnosing neuronal cell edema and/or cytotoxic injury in the spinal cord of a patient, comprising: an antagonist labeled SUR 1; administering a labeled SUR1 antagonist to the patient; measuring the level of the labeled SUR1 antagonist in the spinal cord of the patient, wherein the presence of the labeled SUR1 antagonist in the spinal cord of the patient indicates neuronal cell edema and/or cytotoxic injury in the spinal cord. The labeled antagonist may comprise a compound labeled with a fluorescent label and/or a radioactive label. The compound may include an inhibitor of SUR1, an antibody and/or nucleic acid molecule of SUR1, and the like.

Another embodiment includes a method of determining a posterior penumbra of a spinal cord injury in a patient, comprising: an antagonist labeled SUR 1; administering a labeled SUR1 antagonist to the patient; visualizing the labeled SUR1 antagonist in the spinal cord of the patient, wherein the presence of the labeled SUR1 antagonist indicates a penumbra after the spinal cord injury in the patient.

In particular embodiments, determining the penumbra indicates the location of neuronal damage and/or monitoring disease progression.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. Those skilled in the art will also realize that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

Brief Description of Drawings

For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:

FIGS. 1A-1C show scanning electron micrographs showing the appearance of freshly isolated reactive astrocytes (FIG. 1A) and bubbling 5 min (FIG. 1B) and 25 min (FIG. 1C) after exposure to 1mM sodium azide. Separate markers indicate that the cells are GFAP positive.

FIG. 2 shows phase contrast micrographs showing freshly isolated reactive astrocytes under control conditions and exposure to 1mM azideAppearance of foam after sodium. Bubbling is reproduced by diazoxide alone, which opens NC_Ca-ATPThe channel, and sodium azide-induced blistering is blocked by the channel-blocking glibenclamide. Separate markers indicate that the cells are GFAP positive.

FIG. 3 shows exogenous phosphatidylinositol-4, 5-bisphosphate (PIP)₂) Is added to cause NC_Ca-ATPActivation of the channel despite the presence of ATP in the bath solution (bath solution). Initially, channel activity was recorded in inside-out patches from R1 astrocytes using a membrane containing 1. mu.M Ca²⁺And 10 μ M ATP in bath sufficient to block channel activity. 50 μ M PIP₂The addition of (2) results in channel activation, reflecting a significant decrease in the affinity of the channel for ATP.

FIG. 4 shows NC in R1 astrocytes_Ca-ATPThe channel is inhibited by estrogen. The initial portion of the recordings showed active activity of many overlapping channels, recorded in cell-attached patches of R1 astrocytes obtained from females. Addition of 10nM of estrogen to the bath rapidly caused a strong inhibition of channel activity. The mechanism involved is thought to be associated with estrogen receptor mediated phospholipase C (PLC) activation, leading to membrane PIP₂Exhausted and reflect a significant increase in affinity for ATP.

FIGS. 5A-5B show Western blots demonstrating that R1 astrocytes from both males and females express estrogen receptor and SUR1, SUR1 being NC_Ca-ATPThe identity of the channel. Cell lysates were obtained from male (M) and female (F) gelatin sponge implants and studied at two dilutions (4x and 1x), using lysates from uterus as control. FIG. 5A demonstrates expression of ER α and ER β in astrocytes from both sexes, visualized using antibodies against the Estrogen Receptor (ER). Western blot showed that cells from both sexes also expressed SUR1, pancreatic tissue was used as a control (fig. 5B).

FIG. 6 shows NC in male R1 astrocytes_Ca-ATPThe channel is inhibited by estrogen. The first part of the recording shows active activity from many superimposed channels, the recordingIn cell-attached patches of R1 astrocytes obtained from males. Addition of 10nM of estrogen to the bath rapidly caused a strong inhibition of channel activity.

Figures 7A-7B show necrotic death of freshly isolated reactive astrocytes following sodium azide-induced blebbing. Cell death was assessed using Propidium Iodide (PI) to identify necrotic death (fig. 7A) and annexin V to identify apoptotic death (fig. 7B). 1 μ M glibenclamide strongly attenuated a significant increase in necrotic death induced by 1mM sodium azide (fig. 7A). Apoptotic death was minimal after exposure to sodium azide (fig. 7B).

FIGS. 8A-8B show immunofluorescence images of 1 cell in the penumbra (FIG. 8A) and 2 cells in the middle of cerebral stroke (FIGS. 8B, 8C), immunolabeled against SUR 1; the co-labeling of GFAP confirmed their identity as astrocytes; note the "bubble-like" pattern of the label.

Figures 9A-9B show images of the site of traumatic brain injury following induction of reactive astrocytic necrotic death by infusion of diazoxide. Sections were labeled with nuclear-labeled DAPI to reveal a small cell layer (fig. 9A), and immunolabeled with an anti-MMP-8 antibody to identify these cells as PMNs (fig. 9B).

Fig. 10A-10D show immunofluorescence (composite) images of spinal cord sections from control (fig. 10A) and 24 hours post severe bilateral thoracic spinal cord crush injury, labeling SUR1 (fig. 10A, 10B, 10D) or GFAP (fig. 10C). At high magnification, the single SUR 1-positive trace seen in (fig. 10B) corresponds to GFAP-positive astrocytes that correspond to reactive astrocytes (fig. 10D).

FIGS. 11A-11C show SUR1 upregulation in moderate-severity cervical hemispinal cord contusion (SCI; the same model used in all subsequent illustrations). Epifluorescence (epifluorescence) images of SUR 1-immune labeled spinal cord tissue from the control region (fig. 11A) and low (fig. 11B) and high (fig. 11C) magnifications from the contusion region are shown. The high magnification view demonstrates that SUR1 expression at 24 hours occurs primarily in the capillaries in this model.

FIGS. 12A-12H show SUR1 and vimentin upregulation in capillaries in SCI. Surface fluorescence images of spinal cord tissue of 2 rats 24 hours post-contusion labeled with SUR1 (fig. 12A, 12D) and co-labeled with vimentin (fig. 12B, 12E, 12F); the superimposed image is also displayed.

FIGS. 13A-B show the up-regulation of the transcription factor SP1, the major transcription factor known to regulate the expression of SUR 1. Epifluorescent immune images of spinal cord tissue from 2 rats immunolabeled against SP1, control intact spinal cord (fig. 13A), and spinal cord 24 hours post contusion (fig. 13B).

FIGS. 14A-14C show that glibenclamide treatment reduced bleeding conversion. Figures 14A and 14B show images of spinal cord cryosections 24 hours after contusion in rats treated with saline (figure 14A) and in rats treated with glibenclamide (figure 14B); note treatment with glibenclamide, minor bleeding and preservation of contralateral structures. FIG. 14C shows tubes containing spinal cord homogenate 24 hours post contusion, 2 rats treated with saline (left) and 2 rats treated with glibenclamide (right); differences in color were noted, reflecting a significant decrease in hemoglobin concentration with glibenclamide treatment.

Figure 15 shows the effect of glibenclamide treatment on the course of bleeding transformation time following Spinal Cord Injury (SCI). Extravasated blood in the lesion area was assessed at various times after SCI. At sacrifice, the intravascular blood was first removed by perfusion, and then a 5mm spinal segment including the bruised area was excised, weighed and homogenized in 9x tissue mass volume of distilled water. Blood content was quantified using Drabkin reagent (5 rats per group). Values are expressed as absorbance at 560nm or as microliters of blood, assuming a hematocrit of 40%. In saline treated animals, blood volume gradually increased with time after SCI, reaching a plateau 6 hours after injury (filled squares). In glibenclamide treated animals, the values at 45 minutes were similar to the control, but with time after SC I the increase in values was significantly lower than in the control (filled circles).

FIGS. 16A-16D show that glibenclamide treatment reduced lesion size, GFAP expression, and preserved contralateral long tracts. FIGS. 16A and 16B show epifluorescence images of spinal cord sections 24 hours post contusion in saline treated rats (FIG. 16A) and glibenclamide treated rats (FIG. 16B) with an immune labeling of Glial Fibrillary Acidic Protein (GFAP). Figures 16C and 16D show images of spinal cord sections 24 hours post contusion in saline treated rats (figure 16C) and glibenclamide treated rats (figure 16D) stained for myelin using eriochrome cyanine-R. Note the minor damage and protection of the contralateral structures (sparing) with glyburide.

Figure 17 shows that glibenclamide treatment improved vertical probing after SCI contusion. The bar graph shows the number of seconds it takes to explore vertically (upright) every three minute observation period 24 hours after contusion, 6 rats treated with saline, and 5 rats treated with glibenclamide.

Detailed Description

I. Definition of

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are defined below.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but is also consistent with the meaning of "one or more," at least one, "and" one or more than one. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer only to alternatives or mutually exclusive choices, although the disclosure supports definitions indicating only alternatives and "and/or".

As used herein, the term "antagonist" refers to a substance that acts in vivo to reduce the physiology of another chemical or biological substanceAn active biological or chemical agent. The term antagonist includes, but is not limited to, small molecules, chemicals, proteins, peptides, nucleic acid molecules, and the like. In the present invention, antagonists block, inhibit, reduce and/or decrease NC in neuronal, glial, or neuroendothelial cells (e.g., capillary endothelial cells)_Ca-ATPThe activity of the channel. In the present invention, antagonists associate, bind, connect NC's of neuronal, glial, or neuroendothelial cells (e.g., capillary endothelial cells)_Ca-ATPChannels, so that NC_Ca-ATPChannel closure (inactivation, partial blocking, blocking or inhibition) means a reduced biological activity in terms of biological activity in the diseased state. In particular embodiments, the antagonist is associated with, binds to and/or links to the NC_Ca-ATPThe regulatory subunit of the channel, particularly SUR 1. Alternatively, antagonists are combined, conjugated and/or linked to NC_Ca-ATPPore-forming subunits of channels such that NC_Ca-ATPThe channel is closed (inactivated and/or inhibited). The terms antagonist or inhibitor may be used interchangeably.

As used herein, the term "depolarization" refers to an increase in the permeability of a cell membrane to sodium ions, wherein the potential difference across the cell membrane is reduced or eliminated.

As used herein, the terms "effective amount" or "therapeutically effective amount" are interchangeable and refer to an amount that results in amelioration or alleviation of the symptoms of a disease or disorder. One skilled in the art understands that an effective amount may improve the condition of a patient or subject, but may not be a complete cure for a disease and/or disorder.

As used herein, the term "endothelium" refers to a layer of cells that line the internal surfaces of body cavities, blood vessels, and lymphatic vessels or form capillaries.

As used herein, the term "endothelial cells" refers to cells of the endothelium or cells lining the surface of a body cavity such as a blood or lymph vessel or capillary vessel. In particular embodiments, the term endothelial cell refers to a neuroendothelial cell or an endothelial cell that is part of the nervous system, e.g., the central nervous system or the brain.

As used herein, the term "hemorrhagic transformation" refers to a pathological result that occurs in the capillaries following ischemia. Those skilled in the art know that hemorrhagic transformation is caused by catastrophic damage to the capillaries, during which all blood components extravasate into the surrounding tissue. Understanding these phases requires the identification of 2 cases according to Starling's law: (i) the driving force to "push" the substance into the tissue; and (ii) permeability pores that allow these substances to pass into the tissue.

As used herein, the term "inhibit" refers to a compound that blocks, partially blocks, interferes with, reduces, decreases or inactivates NC_Ca-ATPThe capacity of the channel. Thus, those skilled in the art understand that the term suppression includes NC_Ca-ATPComplete and/or partial loss of channel activity as shown by a reduction in cell depolarization, a reduction in sodium ion influx or any other monovalent ion influx, a reduction in water influx, a reduction in blood extravasation, a reduction in cell death, and improvements.

As used herein, the term "lesion" refers to any pathological or traumatic discontinuity in tissue or loss of function of a portion thereof. For example, an injury includes any injury associated with the spinal cord, such as, but not limited to, contusions, compression injuries, and the like.

The term "morbidity" as used herein is a diseased state. Still further, morbidity can also refer to the prevalence, or the proportion of patients or cases with disease in a given population.

The term "mortality" as used herein is a state of death or causing death. Still further, mortality may also refer to the rate of death, or the proportion of the number of deaths to a given population.

As used herein, the term "neuronal cell" refers to a cell that is the morphological and functional unit of the nervous system. The cells include nerve cell bodies, dendrites, and axons. The terms neuron (neuron), neural cell (neural cell), neuronal (neural), neuron (neuron) and neural cell (neural) may be used interchangeably. Neuronal cell types may include, but are not limited to, the typical neuronal cell body showing internal structure, the (Cajal) level cells from the cerebral cortex; martinotic cells, bipolar cells, unipolar cells, Pukinje cells and pyramidal cells of the cerebral cortex motor.

As used herein, the term "neurological" refers to everything related to the nervous system.

As used herein, the term "glial" or "glial cell" refers to a cell that is a non-neuronal cellular element of the nervous system. The terms glial, glial cell (neurogliac), and glial cell (neuroglial cell) may be used interchangeably. Glial cells may include, but are not limited to, ependymal cells, astrocytes, oligodendrocytes, or microglia.

As used herein, the term "reduce/abate" refers to a reduction in cell death, inflammatory response, hemorrhagic transformation, blood extravasation, and the like, as compared to treatment without the compounds of the present invention. Thus, one skilled in the art can determine the extent of reduction of any symptoms and/or conditions associated with spinal cord injury in which a patient has received the treatment of the present invention as compared to a situation that would occur without treatment and/or without intervention.

The term "preventing" as used herein refers to minimizing, reducing or inhibiting the risk of developing a disease state or a parameter associated with a disease state or progression or other abnormal or adverse condition.

As used herein, "spinal cord," "spinal nervous tissue associated with a spinal level," "nervous tissue associated with a spinal level," or "spinal cord associated with a spinal level or levels" includes any spinal nervous tissue associated with a spinal level or level, all of which are interchangeable. Thus, one skilled in the art will recognize that spinal cord tissue includes all neuronal cells, as well as any glial cells associated therewith. Those skilled in the art know that the spinal cord and its associated tissues are associated with the cervical, thoracic and lumbar spine. As used herein, C1 refers to cervical spine level 1, C2 refers to cervical spine level 2, and so on. T1 refers to thoracic vertebral level 1, T2 refers to thoracic vertebral level 2, and so on. L1 refers to lumbar vertebra segment 1, L2 refers to lumbar vertebra segment 2, and so on, unless specifically noted otherwise.

The term "patient" as used herein is understood to mean any mammalian subject to which a composition is administered according to the methods described herein. One of skill in the art recognizes mammalian patients, including but not limited to humans, monkeys, horses, pigs, cows, dogs, cats, rats, and mice. In particular embodiments, the methods of the invention are used to treat a human patient. In further embodiments, the patient is at risk of developing a spinal cord injury. Thus, patients may or may not be aware of their disease state or underlying disease state, and may or may not be aware that they require treatment (therapeutic or prophylactic treatment).

The term "treating" as used herein refers to administering a therapeutically effective amount of the composition to a patient such that the patient has an improvement in the disease or condition. The improvement is any observable or measurable improvement. Thus, those skilled in the art recognize that treatment may improve the condition of a patient, but may not be a complete cure for the disease. Treatment may also include treating a patient at risk of developing a disease and/or condition.

II. the invention

The present invention relates to therapeutic compositions and methods of using the same. In one embodiment, the therapeutic composition is a neuronal, glial, or neuroendothelial cell NC_Ca-ATPAn antagonist of the channel.

In particular embodiments, the therapeutic compounds of the present invention include neuronal, glial, or endothelial cell NC_Ca-ATPAn antagonist of the channel. Antagonists are contemplated for use in the treatment of adverse conditions associated with central nervous system cytotoxicity and ionic edema. Such disorders include trauma, spinal cord injury, i.e. secondary toNeuronal damage such as, but not limited to, hemorrhagic transformation, immune system response, oxidative damage, calcium and excitotoxicity, necrosis and apoptosis, and/or axonal damage. By suppressing NC_Ca-ATPThe protective effect of the channel is associated with a reduction in edema, a reduction in reactive oxide production, a reduction in inciting inflammation, and/or a reduction in hemorrhagic conversion.

In one aspect, NC_Ca-ATPThe channel is blocked, inhibited, or otherwise reduced in activity. In such instances, NC is administered and/or administered_Ca-ATPAn antagonist of the channel. Antagonist modulation of NC_Ca-ATPA channel such that flow through the channel is reduced, interrupted, reduced, and/or stopped. NC for neuronal cells, glial cells, endothelial cells, or combinations thereof_Ca-ATPChannel activity, antagonists may have reversible or irreversible activity. Antagonists may prevent or reduce depolarization of cells, thus reducing cell swelling resulting from changes in permeability caused by depolarization of cells. Thus, for NC_Ca-ATPInhibition of the channel may reduce cytotoxic edema and endothelial cell death, such as necrotic death of the cell.

In a preferred embodiment, the present invention provides a method of reducing spinal cord injury in a patient comprising administering neuronal, glial, or endothelial cells NC_Ca-ATPAn antagonist of a channel, wherein the antagonist binds to the channel. To NC_Ca-ATPBinding of the channel blocks Na⁺And water flows into the astrocytes, neuronal cells and endothelial cells, thereby reducing swelling at or around the lesion. More particularly, antagonists mitigate secondary damage from primary spinal cord injury, e.g., reduce the progression of pathologic involvement of capillaries, i.e., hemorrhagic transformation, reduce immune system response, reduce oxidative damage, reduce calcium and excitotoxicity, reduce necrosis and cell death, and/or mitigate axonal injury.

III.NC_Ca-ATPChannel

The present invention is based in part on channel-specific NC_Ca-ATPDiscovery of channels, which is described in USPlease, publication No.20030215889, defined as a channel on astrocytes, is incorporated herein by reference in its entirety. More specifically, the invention further defines that the channel is expressed not only on astrocytes, but also on nerve cells, glial cells and/or neuroendothelial cells following central nervous system trauma such as spinal cord injury or other secondary neuronal injury associated with these events.

NC_Ca-ATPThe channel is exposed to calcium ions (Ca)²⁺) Activated and sensitive to ATP. Thus, the pathway is via intracellular Ca²⁺Non-selective cation channels that are activated and blocked by intracellular ATP. When opened by depletion of intracellular ATP, this channel is responsible for the abundance of Na⁺Complete depolarization by influx, which forms Cl^-Elevator gradient and H₂Osmotic gradient of O, resulting in cytotoxic edema and cell death. When the channel is blocked or inhibited, no large amount of Na is produced⁺Thus preventing cytotoxic edema.

Specific functional features will NC_Ca-ATPThe channels are distinguished from other known ion channels. These characteristics may include, but are not limited to 1) readily Na⁺、K⁺And non-selective cation channels through which other monovalent cations pass; 2) activation by an increase in intracellular calcium and/or by a decrease in intracellular ATP; 3) regulated by type 1 sulfonylurea receptor (SUR1), SUR1 was previously thought to be specific for K_ATPChannels are associated, such as those found in pancreatic beta cells.

More specifically, NC of the invention_Ca-ATPThe channels have potassium ions (K) of 20 to 50pS⁺) Single channel conductance. NC (numerical control)_Ca-ATPThe channels are also exposed to Ca on the cytoplasmic side of the cell membrane in physiological concentrations²⁺Wherein the concentration is in the range of 10^-8To 10^-5M。NC_Ca-ATPThe channel is also inhibited by cytoplasmic ATP in a physiological concentration range, wherein the concentration range is 10^-1To 10M. The following ions can also pass through NC_Ca-ATPA channel: k⁺、Cs⁺、Li⁺、Na⁺(ii) a Permeability ratio between any two cationsFor example, above 0.5 and below 2.

IV.NC_Ca-ATPInhibitors of channels

The invention includes inhibitors of the channel, e.g., antagonists of the channel. Examples of antagonists of the present invention may include the antagonists identified in US application publication No.20030215889, which is incorporated herein by reference in its entirety. Those skilled in the art recognize that NC_Ca-ATPThe channel is composed of two subunits, a regulatory subunit SUR1 and a pore-forming subunit. Those skilled in the art know that the nucleic acid sequence and amino acid sequence of SUR1 are readily available in GenBank, e.g., GenBank accession numbers L40624 (GI: 1311533) and AAA99237 (GI: 1311534), each of which is incorporated herein by reference in its entirety.

Inhibitors of SUR1

In particular embodiments, antagonists of sulfonylurea receptor-1 (SUR1) are useful for blocking channels. Examples of suitable SUR1 antagonists include, but are not limited to, glibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide, imiglizole, LY 39364, LY389382, glyclazide, glimepiride, estrogens, estrogen related compounds (estradiol, estrone, estriol, genistein, non-steroidal estrogens (e.g., diethylstilbestrol), phytoestrogens (e.g., coumestrol), zearalenol, and the like), and combinations thereof. In a preferred embodiment of the invention, the SUR1 antagonist is selected from the group consisting of glibenclamide and tolbutamide. Still further, another antagonist may be MgADP. Other antagonists include K_ATPAntagonists of the channel, such as, but not limited to, tolbutamide, glibenclamide (1[ p-2[ 5-chloro-O-anisamide) ethyl]Phenyl radical]Sulfonyl) -3-cyclohexyl-3-urea); chlorpromazine (1- [ [ (p-chlorophenyl) sulfonyl)]-3-propylurea; glipizide (1-cyclohexyl-3- [ [ p- [2 (5-methylpyrazinamide) ethyl)]Phenyl radical]Sulfonyl radical]Urea); or tolazamide (benzenesulfonamide-N- [ [ hexahydro-1H-aza)-1 radical) amino]Carbonyl radical]-4-methyl).

Inhibitors of SUR1 transcription and/or translation

In particular embodiments, the inhibitor may be a compound (protein, nucleic acid, siRNA, etc.) that modulates transcription and/or translation of SUR1 (regulatory subunit) and/or molecular entities including pore-forming subunits.

1. Transcription factor

Transcription factors are regulatory proteins that bind to specific DNA sequences (e.g., promoters and enhancers) and regulate the transcription of a region of coding DNA. Thus, transcription factors may be used to modulate the expression of SUR 1. Generally, transcription factors include a binding domain that binds to DNA (DNA binding domain) and a regulatory domain that controls transcription. In the case of regulatory domains that activate transcription, the regulatory domain is referred to as the activation domain. In the case of a regulatory domain that inhibits transcription, the regulatory domain is referred to as an inhibitory domain.

More specifically, transcription factors such as Sp1 and HIF1 α can be used to regulate expression of SUR 1. One skilled in the art recognizes that Sp1 and HIF1 α can modulate SUR1 expression. Thus, expression/activation of Sp1, which is normally induced by ischemia/hypoxia and/or hyperglycemia, can be prevented, which in turn would prevent expression of SUR1 (Chae YM et al, 2004, incorporated herein by reference). Accordingly, the present invention includes inhibitors or molecules that prevent Sp1 and/or HIF1 binding. Other such Sp1 inhibitors may include, but are not limited to, mithramycin.

Thus, it is contemplated that a candidate substance or SUR1 inhibitor may be a DNA-binding protein or transcription factor or molecule or compound that inhibits or interferes with the activity or binding of a transcription factor, such as Sp1 or HIF 1. It is suggested that SUR1 inhibitors may bind to regulatory elements located within a gene to alter transcription of the gene or may prevent binding of DNA-binding proteins or transcription factors such as Sp1 or HIF 1. The interaction of the putative SUR1 inhibitor with another compound, such as a protein, to form a complex, which interacts with DNA to alter transcription, such as to prevent or reduce transcription, is also contemplated in the present invention. It will be appreciated that the compound that interacts with the putative SUR1 inhibitor may be one or more than one compound.

2. Antisense and ribozyme

Antisense molecules that bind to translation or transcription initiation sites or splice junctions are ideal inhibitors. Antisense, ribozyme, and double-stranded RNA molecules target specific sequences to achieve reduction or elimination of specific polypeptides such as SUR 1. Thus, the construction of antisense, ribozyme, and double-stranded RNA and RNA interference molecules is contemplated and used to modulate SUR 1.

a. Antisense molecules

The antisense approach exploits the fact that nucleic acids are readily paired with complementary sequences. Complementary means that the polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. That is, larger purines will base pair with smaller pyrimidines to form a combination of guanine and cytosine pairings (G: C) and adenine and thymine pairings (A: T) in the case of DNA, or adenine and uracil pairings (A: U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methylcytosine, hypoxanthine and other bases in the hybridizing sequence does not interfere with pairing.

Targeting double-stranded (ds) DNA with a polynucleotide results in triple helix formation; targeting RNA will result in duplex formation. Antisense polynucleotides, when introduced into a target cell, will specifically bind their target polynucleotide and interfere with transcription, RNA processing, transport, translation, and/or stability. Antisense RNA constructs or DNA encoding such antisense RNA are used to inhibit gene transcription or translation or both in host cells, e.g., in host animals including human patients, in vitro or in vivo.

Antisense constructs are designed to bind to promoters and other control regions, exons, introns, and even exon-intron boundaries of genes. It is contemplated that the most effective antisense construct may include a region complementary to an intron/exon splice junction. Thus, antisense constructs having complementarity to a region within 50-200 bases of an intron-exon splice junction are used. It has been observed that some exon sequences can be included in a construct without seriously affecting its target selectivity. The amount of exonic material included varies depending on the particular exonic and intronic sequences used. One skilled in the art can readily test whether too much exon DNA is included simply by testing the construct in vitro to determine whether normal cell function is affected or whether expression of the relevant gene with complementary sequence is affected.

It is advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to produce a particular construct. For example, in cases where introns are required in the final construct, genomic cloning is required. The cDNA or synthetic polynucleotide may provide a more convenient restriction site for the remainder of the construct and, therefore, for the remainder of the sequence.

RNA interference

The use of double stranded RNA as an interfering molecule, e.g., RNA interference (RNAi), is also contemplated in the present invention. RNA interference is used to "knock down" or inhibit a particular target gene by simply injecting, bathing or feeding the double stranded RNA molecule to the target organism. This technique selectively "knockdown" gene function without the need for transfection or recombinant techniques (Giet, 2001; Hammond, 2001; Stein P et al, 2002; Svoboda P et al, 2001; Svoboda et al, 2000).

Another type of RNAi is commonly referred to as small interfering rna (sirna), which can also be used to inhibit SUR 1. The siRNA may comprise a double-stranded structure or a single-stranded structure whose sequence is "substantially identical" to at least a portion of the target gene (see WO04/046320, which is incorporated herein by reference in its entirety). "identity," as known in the art, is a relationship between two or more polynucleotide (or polypeptide) sequences, as determined by comparing the sequences. In the art, identity also refers to the degree of sequence relatedness between polynucleotide sequences, as determined by the match in nucleotide order between such sequences. Identity can be easily calculated. See, for example: computational Molecular Biology (in silico Molecular Biology), Lesk, a.m. editors, Oxford University Press, New York, 1988; biocomputing: the methods disclosed in information and Genome Projects (Biocomputerised: Informatics and Genome Projects), Smith, D.W.ea.academic Press, New York, 1993, and WO 99/32619, WO 01/68836, WO 00/44914 and WO 01/36646, which are hereby expressly incorporated by reference. There are a variety of methods for determining identity between two nucleotide sequences, a term well known in the art. Methods for determining identity are typically designed to produce the maximum degree of nucleotide sequence matching and are typically embodied as computer programs. Such procedures are available to those skilled in the relevant art. For example, the GCG program package (Devereux et al), BLASTP, BLASTN and FASTA (Atschul et al), and CLUSTAL (Higgins et al, 1992; Thompson et al, 1994).

Thus, the siRNA comprises a nucleotide sequence substantially identical to at least a portion of a target gene, such as SUR1 or NC_Ca-ATPAny other molecular entity associated with the channel, such as a pore-forming subunit. Those skilled in the art will recognize that the nucleic acid sequence of SUR1 is readily available in GenBank, e.g., GenBank accession number L40624 (GI: 1311533), which is incorporated herein by reference in its entirety. Preferably, the siRNA comprises a nucleotide sequence identical to at least a portion of the target gene. Of course, when comparing an RNA sequence to a DNA sequence, an "identical" RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and in addition an RNA sequence will typically contain uracil where the DNA sequence contains thymine.

One skilled in the art will recognize that two polynucleotides of different lengths may be compared over the entire length of the longer fragment. Alternatively, the area may be smaller. To ultimately assess their usefulness in the practice of the present invention, sequences of the same length are typically compared. Preferably, the dsRNA used as siRNA has 100% sequence identity with at least 15 contiguous nucleotides of a target gene (e.g., SUR1), although dsRNA having 70%, 75%, 80%, 85%, 90% or 95% or more identity may also be used in the present invention. The siRNA that is substantially identical to at least a portion of the target gene may also be dsRNA in which one of the two complementary strands (or, in the case of self-complementary RNA, one of the two self-complementary portions) is identical to the sequence of the portion of the target gene or contains one or more insertions, deletions or single point mutations relative to the nucleotide sequence of the portion of the target gene. siRNA technology therefore has the property of being able to tolerate sequence variations that might be expected to result from genetic mutations, strain polymorphisms or evolutionary divergences.

There are several methods for preparing siRNA, such as chemical synthesis, in vitro transcription, siRNA expression vectors and PCR expression cassettes. Regardless of the method used, the first step in designing the siRNA molecule is to select an siRNA targeting site, which can be any site in the target gene. In a particular embodiment, the skilled person can manually select the target selection region of the gene, which can be the ORF (open reading frame) as the target selection region and can preferably be 50-100 nucleotides downstream of the "ATG" start codon. However, there are several readily available procedures for aiding in the design of siRNA molecules, such as siRNA Target Designer by Promega, siRNA Target Finder by genscript corp, siRNA retriever program by Imgenex corp, EMBOSS siRNA algorithm, siRNA program by Qiagen, Ambion siRNA predictor, Whitehead siRNA predictor, and Sfold. Thus, it is contemplated that any of the above procedures may be used to generate siRNA molecules that may be used in the present invention.

c. Ribozymes

Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains that have endonuclease activity (Kim and Cech, 1987; Forster and Symons, 1987). For example, many ribozymes accelerate phosphotransesterification reactions with high specificity, typically cleaving only one of several phosphoesters in oligonucleotide substrates (Cech et al, 1981; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind to the internal guide sequence ("IGS") of the ribozyme via specific base-pairing interactions prior to the chemical reaction.

Ribozyme catalysis is primarily observed as part of a sequence-specific cleavage/ligation reaction involving nucleic acids (Joyce, 1989; Cech et al, 1981). For example, U.S. patent 5,354,855 reports that certain ribozymes can act as endonucleases, with sequence specificity higher than that of known ribonucleases and similar DNA restriction enzymes. Sequence-specific ribozyme-mediated inhibition of gene expression is therefore particularly useful for therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990; Sioud et al, 1992). Most of this work involves modification of the target mRNA based on specific mutated codons cleaved by specific ribozymes. The preparation and use of other ribozymes specifically targeting a given gene will now be clear and easy based on the information contained herein and the knowledge of one of ordinary skill in the art.

Other suitable ribozymes include sequences from RNase P which have RNA cleavage activity (Yuan et al, 1992; Yuan and Altman, 1994), hairpin ribozyme structures (Berzal-Her ranz et al, 1992; Chrowrira et al, 1993), and delta hepatitis virus-based ribozymes (Perrotta and ben, 1992). The general design and optimization of ribozyme-directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988; Symons, 1992; Chowrira et al, 1994; and Thompson et al, 1995).

Other variables that are relevant to ribozyme design are the choice of cleavage sites on a given target RNA. Ribozymes are targeted to a given sequence by annealing to the site through complementary base pair interactions. Two sequences of homology are required for this targeting. These homologous sequence fragments flank the catalytic ribozyme structure defined above. Each homologous sequence can be 7 to 15 nucleotides in length. The only requirement for defining homologous sequences is that they are separated by specific sequences that act as cleavage sites on the target RNA. For hammerhead ribozymes, the cleavage site is a dinucleotide sequence on the target RNA, uracil (U) followed by adenine, cytosine, or uracil (A, C or U; Perriman et al, 1992; Thompson et al, 1995). The frequency with which such dinucleotides occur in any given RNA is statistically 3 out of 16.

The design and testing of ribozymes for efficient cleavage of target RNA is a well known process to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al (1994), and Lieber and Strauss (1995), each of which is incorporated by reference. The identification of effective and preferred sequences for use in the ribozyme targeting SUR1 is a matter of preparation and testing for a given sequence and is a common screening method known to those skilled in the art.

C. Method for screening inhibitors

Further embodiments of the invention may include identifying NCs_Ca-ATPMethods of channel inhibitors, such as antagonists. These assays may include random screening of large libraries of candidate substances; alternatively, assays may be used to focus on specific classes of compounds that are focused on to consider making them more likely to modulate NC_Ca-ATPThe function or activity of the channel or the structural properties of the expression.

Functional representation one skilled in the art can test mRNA expression, protein activity or channel activity, more specifically, modulators inhibit or block NC_Ca-ATPThe capacity of the channel. Thus, compounds screened according to the invention include, but are not limited to, binding to NC_Ca-ATPNatural or synthetic organic compounds, peptides, antibodies and fragments thereof, peptidomimetics (peptiomimetics) that block a channel (e.g., an antagonist).

About influencing NC_Ca-ATPScreening of compounds for channels, libraries of known compounds, including natural products or synthetic chemicals, and biologically active substances, including proteins, can be screened to select compounds that are inhibitors or activators. Preferably, such a compound is NC_Ca-ATPAntagonists, which comprise NC_Ca-ATPChannel inhibitors, NC_Ca-ATPChannel blockers, SUR1 antagonists, SUR1 inhibitors, and/or compounds capable of reducing the magnitude of membrane current through a channel.

Compounds can include, but are not limited to, small organic or inorganic molecules, compounds available in compound libraries, peptides, e.g., soluble peptides, including but not limited to members of a random peptide library (see, e.g., Lam, k.s. et al,1991, Nature 354: 82-84; houghten, R. et al, 1991, Nature 354: 84-86), and combinatorial chemically derived molecular libraries consisting of D-and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of a random or partially degenerate, directed phosphopeptide library; see, e.g., Songyang, Z. et al, 1993, Cell 72: 767 778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F (ab')₂And FAb expression library fragments and epitope-binding fragments thereof).

Other compounds that can be screened according to the invention include, but are not limited to, being able to cross the blood-brain barrier, enter suitable neural cells and affect NC_Ca-ATPChannel genes or some other involved NC_Ca-ATPSmall organic molecules that express genes for channel activity (e.g., by interacting with regulatory regions or transcription factors involved in gene expression); or influence NC_Ca-ATPActivity of channels or involving NC_Ca-ATPActive compounds of some other intracellular factors of channel activity.

To identify, prepare, produce, provide, make, or obtain an inhibitor, the NC is typically determined in the presence, absence, or both of a candidate substance_Ca-ATPChannel activity, wherein an inhibitor or antagonist is defined as down-regulating, decreasing, inhibiting, blocking or decreasing NC_Ca-ATPAny substance whose channel expresses or is active. For example, a method may generally comprise:

providing for suspected in vitro or in vivo inhibition of NC_Ca-ATPCandidate substances for channel expression or activity;

evaluation of candidate substances for inhibition of NC in vitro or in vivo_Ca-ATPThe ability of a channel to express or activate;

selecting an inhibitor; and

and (4) manufacturing the inhibitor.

In particular embodiments, an optional evaluation step can evaluate the specific binding of a candidate substance to NC in vitro or in vivo_Ca-ATPThe capacity of the channel;

in further embodiments, the NC may be provided in a cell or cell-free system_Ca-ATPChannel and NC_Ca-ATPThe channel contacts the candidate substance. Then, by evaluating the candidate substance pair NC_Ca-ATPThe effect of channel activity or expression is to select inhibitors. Once the inhibitor is identified, the method can further provide for the manufacture of the inhibitor.

V. treatment of spinal cord injury

In other embodiments, the therapeutic compounds of the present invention comprise NCs of neuronal cells, glial cells, neuroendothelial cells, or combinations thereof_Ca-ATPAn antagonist of the channel. Antagonists are contemplated for use in treating adverse conditions associated with spinal cord injury. Such disorders include secondary injuries associated with spinal cord injury, such as, but not limited to, cellular edema, cell death (e.g., necrotic cell death), inflammation, oxidative damage, axonal injury, hemorrhagic transformation, and the like. Antagonist protected expression NC_Ca-ATPCells of the channel, which is advantageous for clinical treatment where ionic or cytotoxic oedema is formed, where capillary integrity is lost. By suppressing NC_Ca-ATPThe protective effect of the channel is associated with a reduction in ionic and cytotoxic edema. Therefore, NC is suppressed_Ca-ATPThe compounds of the channel are neuroprotective.

In one aspect, NC_Ca-ATPThe channel blocks, inhibits or otherwise reduces activity. In such instances, NC is administered and/or administered_Ca-ATPAn antagonist of the channel. Antagonist modulation of NC_Ca-ATPA channel such that the flow (ions and/or water) through the channel is reduced, halted, reduced and/or stopped. NC for neuronal cells, glial cells, neuroendothelial cells, or combinations thereof_Ca-ATPChannel activity, antagonists may have reversible or irreversible activity. Thus, for NC_Ca-ATPInhibition of the channel may reduce cytotoxic edema and endothelial cell death, which is associated with the formation of ionic edema and hemorrhagic transformation.

Thus, the present invention is useful in the treatment or alleviation of inflammation associated with spinal cord injury. According to a particular embodiment of the invention, the administration of an effective amount of the active compound can block the channel, while if it remains open it leads to swelling of neuronal cells and cell death, which leads to the initiation of an inflammatory response. A variety of SUR1 antagonists are suitable for blocking the channel. Examples of suitable SUR1 antagonists include, but are not limited to, glibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide, miglitol, LY 39364, LY389382, glyclazide, glimepiride, estrogens, estrogen related compounds, and combinations thereof. In a preferred embodiment of the invention, the SUR1 antagonist is selected from the group consisting of glibenclamide and tolbutamide. Another antagonist that may be used is MgADP. Other therapeutic "strategies" to prevent nerve cell swelling and cell death may be employed, including but not limited to methods to maintain the polarization state of nerve cells and methods to prevent strong depolarization.

In a further embodiment, NC_Ca-ATPInhibitors or antagonists of the pathway may be used to reduce or alleviate or eliminate bleeding episodes and/or extravasation of blood near or around the site of injury. With NC_Ca-ATPAdministration of channel antagonists due to NC_Ca-ATPThe opening of the channel, endothelial cell depolarization is eliminated, slowed, reduced or inhibited. Thus, elimination of cell depolarization leads to Na⁺Elimination or inhibition of inflow, which prevents changes in osmotic gradient, thus preventing water from flowing into endothelial cells and stopping cell swelling, blebbing and cytotoxic edema. Thus, preventing or inhibiting or reducing endothelial cell depolarization may prevent or reduce bleeding, transformation, and/or extravasation of blood near or around the injury site.

Thus, the use of antagonists or related compounds thereof can reduce mortality in spinal cord injury patients and/or rescue or prevent injury in the penumbra region, including tissue regions at risk of becoming irreversibly injured.

In which NC can be administered_Ca-ATPThe neuronal cells for the channel antagonist may include any cell that expresses SUR1, e.g., any neuronal cell, glial cell, or neuroendothelial cell.

Patients that may be treated with antagonists or compounds related thereto include those suffering from spinal cord injury. Other patients that may be treated with the antagonists of the invention include those at risk of or prone to spinal cord injury, such as patients undergoing spinal surgery or spinal radiation therapy. In such cases, the patient is treated with the antagonist of the invention or a related compound thereof prior to actual treatment. Pretreatment may include administration of the antagonist and/or related compound for months (1, 2, 3, etc.), weeks (1, 2, 3, etc.), days (1, 2, 3, etc.), hours (1, 2, 3, 4, 5,6, 7,8, 9, 10, 11, 12), or minutes (15, 30, 60, 90, etc.) prior to actual treatment or surgery or radiation therapy. Treatment with the antagonist and/or related compound can be continued during and after the treatment and/or surgery until the patient's risk of developing spinal cord injury is reduced, reduced or alleviated. Still further, other patients at risk of spinal cord injury may include those with segmental malformations and/or other spinal cord disorders or compression diseases such as arthritis or Cushing's disease.

NC that can be administered to cells_Ca-ATPAn effective amount of a channel antagonist includes a dose of about 0.0001nM to about 2000 μ M. More specifically, the dose administered is from about 0.01nM to about 2000 μ M; about 0.01 μ M to about 0.05 μ M; about 0.05 μ M to about 1.0 μ M; about 1.0 μ M to about 1.5 μ M; about 1.5 μ M to about 2.0 μ M; about 2.0 μ M to about 3.0 μ M; about 3.0 μ M to about 4.0 μ M; about 4.0 μ M to about 5.0 μ M; about 5.0 μ M to about 10 μ M; about 10 μ M to about 50 μ M; about 50 μ M to about 100 μ M; about 100 μ M to about 200 μ M; about 200 μ M to about 300 μ M; about 300 μ M to about 500 μ M; about 500 μ M to about 1000 μ M; about 1000 μ M to about 1500 μ M and about 1500 μ M to about 2000 μ M. Of course, all of these amounts are exemplary, and any amount in between these points is contemplated to be useful in the present invention.

The antagonist or related compound may be administered parenterally or parenterally. Parenteral administration includes, but is not limited to, intravenous, intradermal, intramuscular, intraarterial, intrathecal, subcutaneous or intraperitoneal administration, U.S. patent nos.6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each expressly incorporated by reference herein in its entirety). Alimentary canal administration includes, but is not limited to, oral, buccal, rectal, or sublingual administration.

Administration of the therapeutic compounds and/or treatments of the present invention can include systemic, local, and/or regional administration, e.g., topical (cutaneous, transdermal), via a catheter, implantable pump, etc. Alternatively, other routes of administration are contemplated, such as arterial infusion, intracavitary, intraperitoneal, intrapleural, intraventricular and/or intrathecal. One skilled in the art will know to determine the appropriate route of administration using standard methods and procedures. Other routes of administration are discussed elsewhere in the specification and are incorporated herein by reference.

The treatment method comprises the step of using an effective amount of the composition containing NC_Ca-ATPA composition of a channel antagonist or a compound related thereto for treating an individual. An effective amount is generally defined as an amount sufficient to detectably and reproducibly ameliorate, reduce, minimize or limit the extent of a disease or symptom thereof. More specifically, NC for prediction_Ca-ATPTreatment with channel antagonists or compounds related thereto will inhibit cell depolarization, inhibit Na⁺Influx, inhibition of osmotic gradient changes, inhibition of water influx into cells, inhibition of cytotoxic cellular edema, reduction of stroke size, inhibition of hemorrhagic transformation, and reduction of patient mortality.

NC to be used_Ca-ATPEffective amounts of channel antagonists or related compounds are those amounts effective to produce a beneficial effect in a recipient animal or patient, particularly for treatment of stroke. Such levels can be determined initially by reading the open literature, by performing in vitro tests, or by conducting metabolic studies in healthy laboratory animals. Prior to use in a clinical setting, it is beneficial to conduct confirmatory studies in animal models, preferably widely recognized animal models of the particular disease to be treated. The preferred animal model for use in certain embodiments is a rodent model becauseThey are economical to use, especially since it is widely accepted that the results obtained can be used as a predictor of clinical value and are therefore preferred.

As is well known in the art, active compounds such as NC_Ca-ATPThe specific dosage level of a channel antagonist or its related compounds for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The person responsible for administration will determine the appropriate dosage for the individual patient. In addition, for human administration, the formulations should meet sterility, pyrogenicity, general safety and purity standards, as required by FDA office of biological standards.

One skilled in the art will recognize that an effective amount of an antagonist or related compound thereof can be that amount necessary to achieve the desired result: reduction in inflammation, reduction in cell death, reduction in hemorrhagic transformation, reduction in extravasated blood, reduction in lesion size, reduction in cGFAP upregulation, and the like. The amount may also be an amount that maintains a reasonable blood glucose level in the patient, e.g., the amount of antagonist maintains a blood glucose level of at least 60mmol/l, more preferably, the blood glucose level is maintained in the range of about 60mmol/l to about 150 mmol/1. Thus, the amount prevents the patient from becoming hypoglycemic. If the glucose level is not normal, one skilled in the art would administer insulin or glucose depending on whether the patient is hypoglycemic or hyperglycemic.

Thus, in certain embodiments, the invention encompasses co-administration of NC_Ca-ATPAntagonists of the channel and glucose or related carbohydrates to maintain appropriate serum glucose levels. Suitable blood glucose levels range from about 60mmol/l to about 150 mmol/l. Thus, glucose or related carbohydrates are co-administered to maintain serum glucose within this range.

NC as therapy_Ca-ATPThe therapeutically effective amount of the channel antagonist or related compound will vary depending upon the host treated and the particular mode of administration. Hair brushIn one embodiment of the invention, NC_Ca-ATPThe dose range of the channel antagonist or related compound is from about 0.01 μ g/kg body weight to about 20,000 μ g/kg body weight. The term "body weight" is applicable when the animal is to be treated. When isolated cells are to be treated, "body weight" as used herein should be understood to mean "total cell weight". The term "total body weight" may apply to isolated cells and animal treatments. All concentrations and therapeutic levels expressed herein as "body weight" or simply "kg" are also considered to encompass similar concentrations of "total cell weight" and "total body weight". However, one skilled in the art will recognize the availability of various dosage ranges, for example, 0.01 μ g/kg body weight to 20,000 μ g/kg body weight, 0.02 μ g/kg body weight to 15,000 μ g/kg body weight, 0.03 μ g/kg body weight to 10,000 μ g/kg body weight, 0.04 μ g/kg body weight to 5,000 μ g/kg body weight, 0.05 μ g/kg body weight to 2,500 μ g/kg body weight, 0.06 μ g/kg body weight to 1,000 μ g/kg body weight, 0.07 μ g/kg body weight to 500 μ g/kg body weight, 0.08 μ g/kg body weight to 400 μ g/kg body weight, 0.09 μ g/kg body weight to 200 μ g/kg body weight, or 0.1 μ g/kg body weight to 100 μ g/kg body weight. Furthermore, those skilled in the art will recognize that various dosage levels are useful, e.g., 0.0001. mu.g/kg, 0.0002. mu.g/kg, 0.0003. mu.g/kg, 0.0004. mu.g/kg, 0.005. mu.g/kg, 0.0007. mu.g/kg, 0.001. mu.g/kg, 0.1. mu.g/kg, 1.0. mu.g/kg, 1.5. mu.g/kg, 2.0. mu.g/kg, 5.0. mu.g/kg, 10.0. mu.g/kg, 15.0. mu.g/kg, 30.0. mu.g/kg, 50. mu.g/kg, 75. mu.g/kg, 80. mu.g/kg, 90. mu.g/kg, 100. mu.g/kg, 120. mu.g/kg, 140. mu.g/kg, 150. mu.g/kg, 160. mu.g/kg, 180. mu.g/kg, 200. mu.g/kg, 225. mu.g/kg, 250. mu.g, 300. mu.g/kg, 325. mu.g/kg, 350. mu.g/kg, 375. mu.g/kg, 400. mu.g/kg, 450. mu.g/kg, 500. mu.g/kg, 550. mu.g/kg, 600. mu.g/kg, 700. mu.g/kg, 750. mu.g/kg, 800. mu.g/kg, 900. mu.g/kg, 1mg/kg, 5mg/kg, 0mg/kg, 12mg/kg, 15mg/kg, 20mg/kg and/or 30 mg/kg. Of course, all of these doses are exemplary, and any dose in between these points is contemplated to be useful in the present invention. For NC_Ca-ATPChannel antagonists, or compounds related thereto, may be administered in dosage ranges or dosage levels of any of the foregoing.

Will follow the general protocol for administration of therapeutic agentsTherapeutic NC of the invention_Ca-ATPAdministration of channel antagonist compositions to a patient or subjects, if any, taking into account the NC_Ca-ATPToxicity of channel antagonists. It is contemplated that the treatment cycle may be repeated as desired. Various standard therapies are also contemplated, as well as surgical interventions, which may be used in conjunction with the above treatments.

Treatment may include various "unit doses". A unit dose is defined as containing a predetermined quantity of a therapeutic composition (NC) calculated to produce a desired response in relation to its administration (e.g., appropriate route and treatment regimen)_Ca-ATPAntagonists of the channel or related compounds). The amount to be administered, as well as the particular route and formulation, is within the skill of those in the clinical arts. The patient to be treated is also important, in particular the condition of the patient and the required protection. The unit dose need not be administered as a single injection, but may comprise a continuous infusion over a set period of time.

Combination therapy

Within the scope of the invention, it is contemplated that NC may be used_Ca-ATPAntagonists of the channels or their related compounds are used in combination with other therapeutic agents to more effectively treat spinal cord injury. In some embodiments, it is contemplated that conventional therapies or agents, including but not limited to pharmacological therapeutic agents, may be combined with the antagonists of the invention or related compounds thereof.

Pharmacological Therapeutics and methods of administration, dosages, and The like, are well known to those skilled in The art (see, e.g., "Physicians Desk Reference," The pharmacological Basis of Therapeutics, "Remington's pharmacological Sciences," and The Merck Index, eleven Edition, of Goodman & Gliman, incorporated herein by Reference in relevant part), and may be combined with The present invention in accordance with The disclosure herein. Some variation in the dose must occur depending on the condition of the patient to be treated. In any event, the person responsible for administration will determine the appropriate dosage for an individual patient, and such individual determination is within the skill of one of ordinary skill in the art.

Non-limiting examples of pharmacological therapeutic agents that may be used in the present invention include anti-inflammatory agents. Anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory agents (e.g., naproxen, ibuprofen, celecoxib) and steroidal anti-inflammatory agents (e.g., glucocorticoids, dexamethasone, methylprednisolone).

Other agents that may be used in combination with the antagonists of the invention may include, but are not limited to, antioxidants, calcium blockers, drugs that control excitotoxicity, and drugs that enhance axonal signaling, such as 4-aminopyridine.

Still other agents that may be used in combination with the antagonists of the present invention may also include agents designed to promote regeneration through the use of trophic factors, and growth inhibitory substances.

Furthermore, non-pharmacological interventions may also be used in combination with the antagonists of the invention, such as transplantation, peripheral nerve transplantation, hypothermia (cooling).

When other therapeutic agents are used, the effective amount of the other therapeutic agent can be defined simply as when incorporated into the NC as long as the dosage of the other therapeutic agent does not exceed the previously recited toxicity level_Ca-ATPA channel antagonist or a related compound thereof is administered to an animal in an amount effective to reduce edema or to reduce secondary injury. This can be readily determined by monitoring the animal or patient and determining the physical and biochemical parameters of those health and disease states that indicate the success of a given treatment. Such methods are routine in animal testing and clinical practice.

To inhibit bleeding turnover, reduce oxidative stress, reduce cell death, reduce cell swelling, and the like using the methods and compositions of the invention, cells are typically contacted with NC_Ca-ATPAntagonists of the channels, or compounds related thereto, in combination with other therapeutic agents, such as anti-inflammatory agents and the like. These compositions are provided in a combined amount effective to inhibit hemorrhagic transformation, cell swelling, cell death, edema, and the like. The process involves contacting the cells with NC_Ca-ATPChannel antagonists or related compounds, while binding other therapeutic agents or factors. This can be achieved by: contacting the cell with a single composition or pharmaceutical preparation comprising two agents, or contacting the cell with two different compositions or preparations simultaneously, wherein one composition comprises NC_Ca-ATPAn antagonist of the channel or a derivative thereof, and the other comprising an additional agent.

Alternatively, the NC may be used at intervals ranging from minutes to hours to weeks to months before or after treatment with other agents_Ca-ATPChannel antagonists or compounds related thereto. In the case of separate application of the other agents to the cells, it is generally ensured that the effective time period between each delivery time does not expire, so that the agents are still able to exert a favorable combined effect on the cells. In such cases, it is contemplated that the cells will be contacted with the two treatment modalities within about 1-24 hours of each other, more preferably, within about 6-12 hours of each other.

Route of administration of formulations and compounds

The pharmaceutical compositions of the present invention comprise an effective amount of one or more NCs dissolved or dispersed in a pharmaceutically acceptable carrier_Ca-ATPInhibitors (antagonists) or related compounds or other agents of the channel. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, for example, if appropriate to a human. Those skilled in the art will appreciate from this disclosure that there is at least one NC_Ca-ATPThe preparation of Pharmaceutical compositions of modulators (antagonists and/or agonists) of channels or related compounds or other active ingredients, as exemplified in Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company, 1990, incorporated herein by reference. Further, for animal (e.g., human) administration, it will be understood that the formulation should meet sterility, pyrogenicity, general safety and purity standards, as required by FDA office of biological standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar materials, and combinations thereof, as known to those skilled in the art (see, for example, Remington's pharmaceutical Sciences, 18 th edition, Mack printing company, 1990, pp.1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated that it may be used in pharmaceutical compositions.

NC depending on whether administration is in solid, liquid or aerosol form, and whether sterility is required for such routes of administration as injection_Ca-ATPInhibitors (antagonists) or related compounds of the channel may include different types of carriers. The invention can be administered intravenously, intradermally, transdermally, intrathecally, intraventricularly, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, regionally, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, local perfusion bathing target cells directly, by catheter, by lavage, cream, liquid compositions (e.g., liposomes), or by other methods or any combination of the foregoing, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, Mack PrintingCompany, 1990, incorporated herein by reference).

Can be combined with NC_Ca-ATPInhibitors (antagonists) of the channel or related compounds are formulated into the composition as a free base, neutral or salt form. Pharmaceutically acceptable salts, including acid addition salts, such as those formed with the free amino groups of the protein composition, or with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric, or mandelic acid. The salts with free carboxyl groups may also be derived from inorganic bases, e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or hydrogen hydroxideIron melting; or an organic base such as isopropylamine, trimethylamine, histamine or procaine. When formulated, the solution is administered in a manner compatible with the agent and in a therapeutically effective amount. The formulations can be readily administered in a variety of dosage forms, such as those formulated for parenteral administration, such as injection solutions, or aerosols for delivery to the lungs, or for administration to the digestive tract, such as drug delivery capsules and the like.

Further in accordance with the present invention, compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier should be assimilable and include a liquid, a semi-solid, i.e., a paste, or a solid carrier. Administrable compositions useful in the practice of the methods of the present invention are suitable unless any conventional vehicle, agent, diluent or carrier is deleterious to the recipient or the therapeutic effectiveness of the composition contained therein. Examples of carriers or diluents include fats, oils, water, salt solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The composition may also include various antioxidants to prevent oxidation of one or more of the ingredients. In addition, the action of microorganisms can be prevented by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methyl paraben, propyl paraben), chlorobutanol, phenol, sorbic acid, mercury ethyl salicylate, or combinations thereof.

In accordance with the present invention, the composition is admixed with the carrier in any conventional and practical manner, i.e., by dissolution, suspension, emulsification, mixing, encapsulation, absorption, and the like. Such methods are routine to those skilled in the art.

In a particular embodiment of the invention, the composition is thoroughly combined or mixed with a semi-solid or solid carrier. The mixing can be carried out in any conventional manner, such as milling. Stabilizers may also be added during mixing in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextran, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like,

in a further embodiment, the invention may relate to the use of a pharmaceutical lipid carrier composition comprising NC_Ca-ATPAn inhibitor (antagonist) or related compound of the channel, one or more lipids and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide range of substances characterized as being insoluble in water and extractable with organic solvents. This general class of compounds is well known to those skilled in the art, as the term "lipid" is used herein, and is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are typically biological substances. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether and ester linked fatty acids, and polymerizable lipids, and combinations thereof. Of course, the compositions and methods of the present invention also include compounds other than those specifically described herein that would be understood by one of skill in the art to be lipids.

Those skilled in the art are familiar with a variety of techniques by which the compositions can be dispersed in lipid carriers. For example, NC may be_Ca-ATPThe inhibitor (antagonist) or related compound of the channel is dispersed in a solution containing the lipid, solubilized with the lipid, emulsified with the lipid, mixed with the lipid, combined with the lipid, covalently bound to the lipid, contained as a suspension in the lipid, contained or complexed as a micelle or liposome, or otherwise associated with the lipid or lipid structure by any means known to those skilled in the art. Dispersion may or may not result in the formation of liposomes.

The actual dosage of the compositions of the invention to be administered to an animal patient may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease to be treated, previous or present therapeutic and/or prophylactic intervention, the patient's morbidity and the route of administration. The preferred dose and/or the number of administrations of the effective amount may vary depending on the dose and route of administration, and on the response of the patient. The physician in charge of the administration will in any event determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual patient.

In particular embodiments, the pharmaceutical composition may include, for example, at least about 0.1% active compound. In other embodiments, the active compound may comprise from about 2% to about 75% weight units, or for example, from about 25% to about 60%, and any range derived therefrom. Naturally, the amount of active compound in each therapeutically useful composition is prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Those skilled in the art will appreciate factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other pharmacological considerations in making such pharmaceutical formulations, and thus, various dosages and treatment regimens are desirable.

A. Digestive tract compositions and formulations

In a preferred embodiment of the invention, NC is_Ca-ATPAntagonists of the channels or related compounds are formulated for administration by the digestive tract route. The digestive tract route includes all possible routes of administration where the composition is in direct contact with the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. Thus, these compositions may be formulated with an inert diluent or an assimilable edible carrier, or may be encapsulated in hard-or soft-shell gelatin capsules, or may be compressed into tablets, or may be incorporated directly into the diet.

In particular embodiments, the active compounds may be incorporated into excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, water and the like (Mathiowitz et al, 19971; Hwang et al, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each of which is expressly incorporated by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: binders, such as, for example, gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as, for example, corn starch, potato starch, alginic acid, or a combination thereof; lubricants, such as, for example, magnesium stearate; wetting agents such as, for example, sucrose, lactose, saccharin or combinations thereof; flavoring agents, such as, for example, peppermint, wintergreen oil, cherry flavoring, orange flavoring, and the like. When the unit dosage form is a capsule, it may contain, in addition to materials of the above kind, a lipid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the unit dosage. For example, tablets, pills, or capsules can be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, a carrier, such as a liquid carrier. Gelatin capsules, tablets or pills may be enteric coated. The enteric coating prevents denaturation of the composition in the stomach or upper intestine where the pH is acidic. See, for example, U.S. patent No.5,629,001. Upon reaching the small intestine, the alkaline pH therein dissolves the coating and allows the composition to be released and absorbed by specialized cells such as epithelial digestive tract cells and peyer's patches M cells. A syrup of elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compounds may be incorporated into sustained release formulations and formulations.

For oral administration, the compositions of the present invention may optionally incorporate one or more excipients in the form of a mouthwash, toothpaste, buccal tablet, buccal spray, or sublingual oral dosage formulation. For example, mouthwashes can be prepared by incorporating the active ingredient at the desired level in a suitable solvent, such as a sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be incorporated into an oral solution such as a solution containing sodium borate, glycerin and dipotassium carbonate, or dispersed in a toothpaste, or added in a therapeutically effective amount to a composition comprising water, binders, abrasives, flavoring agents, foaming agents and humectants. Alternatively, the composition may be formulated as a tablet or solution that can be placed under the tongue or otherwise dissolved in the mouth.

Other formulations suitable for other modes of administration to the digestive tract include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, the suppository softens, melts or dissolves in the cavity liquid. Generally, for suppositories, typical carriers may include, for example, polyethylene glycol, triglycerides or combinations thereof. In particular embodiments, suppositories may be formed from mixtures containing, for example, from about 0.5% to about 10%, preferably from about 1% to about 2%, of the active ingredient.

B. Parenteral compositions and formulations

In a further embodiment, NC may be administered by parenteral route_Ca-ATPAntagonists or related compounds of the channel. As used herein, the term "parenteral" includes a route that bypasses the digestive tract. Specifically, the pharmaceutical compositions disclosed herein may be, for example, but not limited to, intravenous, intradermal, intramuscular, intraarterial, intraventricular, intrathecal, subcutaneous, or intraperitoneal, U.S. Pat. nos.6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each of which is incorporated herein by reference in its entirety).

A solution of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water, suitably mixed with a surfactant such as hydroxypropyl cellulose. The dispersant may be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. patent No.5,466,468, expressly incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that an easily injectable force is present. Must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, DMSO, polyol (agents, glycerol, ethylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, or by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, mercury ethyl salicylate, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration of aqueous solutions, for example, the solution should be suitably buffered if necessary and the liquid diluent rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used in accordance with the present disclosure are known to those skilled in the art. For example, a dose may be dissolved in 1ml of isotonic NaCl solution and added to 1000ml of subcutaneous perfusion fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15 th edition, p1035-1038 and 1570-. Depending on the condition of the patient to be treated, some variation in the dosage must occur. The person responsible for the administration will in any case decide the appropriate dosage for the individual patient. In addition, for human administration, the formulations should meet sterility, pyrogenicity, general safety and purity standards, as required by FDA office of biological standards.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the technique of lyophilization which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powdered composition is mixed with a liquid carrier, such as, for example, water or a salt solution, with or without a stabilizer.

C. Hybrid pharmaceutical compositions and formulations

In other preferred embodiments of the invention, NC may be formulated_Ca-ATPAntagonists of the channels or related compounds are useful for administration by various miscellaneous routes, e.g., topical (i.e., transdermal), mucosal (intranasal, vaginal, etc.), and/or inhalation.

Pharmaceutical compositions for topical administration may include the active compound formulated for medicated applications such as ointments, pastes, creams or powders. Ointments include all oily, absorbing, emulsifying and water-soluble base compositions for topical application, while creams and lotions are those compositions that include only an emulsifying base. Topically administered drugs may contain permeation enhancers to aid absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerol, alcohols, alkyl methyl sulfoxides, pyrrolidones, and lurocoapram. Possible bases for topical compositions include polyethylene glycols, lanolin, cold creams and petrolatum as well as any other suitable absorbent, emulsifier or water-soluble cream base. Topical formulations may also include emulsifiers, gelling agents, and antimicrobial preservatives as needed to protect the active ingredients and provide a homogeneous mixture. Transdermal administration of the present invention may also include the use of "patches". For example, a patch may provide one or more active substances at a predetermined rate and continuously over a fixed period of time.

In particular embodiments, the pharmaceutical composition may be delivered by eye drops, intranasal spray, inhalation, and/or other aerosol delivery vehicles. Methods of delivering compositions directly to the lungs via nasal aerosol sprays have been described, for example, in U.S. patent nos. 5,756,353 and 5,804,212 (each expressly incorporated herein by reference in its entirety). Likewise, drug delivery using intranasal microparticle resins (Takenaga et al, 1998) and lysophosphatidyl-glycerol compounds (U.S. patent No.5,725,871, each expressly incorporated by reference in its entirety) is also well known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. patent No.5,780,045 (expressly incorporated by reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid particles of a liquid dispersed in a liquefied or pressurized gaseous propellant. A typical aerosol of the invention for inhalation consists of a suspension of the active ingredient in a liquid propellant or a mixture of liquid propellants and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary depending on the pressure requirements of the propellant. The administration of the aerosol will vary according to the age, weight and severity of symptoms and response of the patient.

VIII. diagnosis

Antagonists or related compounds may be useful in diagnosing, monitoring or prognosing spinal cord injury, e.g., monitoring neuronal damage, or monitoring neuronal cells in the edematous region, etc.

A. Genetic diagnosis

One embodiment of the invention includes detecting NC_Ca-ATPMethods for the expression of any part of the channel, e.g. the expression of the regulatory unit SUR1, and/or the expression of pore-forming subunits. This may include determining the level of SUR1 expressed and/or the level of pore-forming subunits expressed. Understood by the invention is NC_Ca-ATPUpregulation or increase in channel expression involves increased SUR1 levels, which are associated with increased neuronal damage, such as edema.

First, a biological sample is obtained from a patient. The biological sample may be a tissue or a liquid. In particular embodiments, the biological sample comprises cells and/or endothelial cells from the spinal cord or microvessels associated with spinal cord or spinal tissue.

The nucleic acids used are isolated from the cells contained in the biological sample according to standard methods (Sambrook et al, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. In the case of RNA, it is desirable to convert the RNA to complementary DNA (cDNA). In one embodiment, the RNA is whole cell RNA; in another embodiment, it is a poly-A RNA. Typically, the nucleic acid is amplified.

Depending on the format, amplification is used directly or followed by a second known nucleic acid to identify a particular target nucleic acid in a sample. The identified product is then detected. In certain applications, detection is performed by visual means (e.g., ethidium bromide staining of the gel). Alternatively, detection may involve indirect identification of the product by chemiluminescence, radiolabelled scintigraphy or fluorescent labelling or even by a system using electrical or thermal pulse signals (Affymax technology; Bellus, 1994).

After testing, the results seen in a given patient can be compared to a statistically significant reference group of normal patients and patients who have been diagnosed with spinal cord injury and or secondary injury associated therewith, and the like.

Further, it is contemplated that chip-based DNA techniques such as those described by Hacia et al (1996) and Shoemaker et al (1996) may be used for diagnosis. Briefly, these techniques involve quantitative methods for rapidly and accurately analyzing a large number of genes. By labeling genes with oligonucleotides and using an immobilized probe array, chip technology can be used to isolate target molecules as a high density array and to screen these molecules based on hybridization. See Pease et al (1994); fodor et al, (1991).

B. Other types of diagnosis

To enhance the utility of molecules, such as compounds and/or proteins and/or antibodies, as diagnostic agents, at least one desired molecule or moiety is typically linked or covalently bound or complexed.

Specific examples of conjugates are those in which a molecule (e.g., a protein, antibody, and/or compound) is linked to a detectable label. "detectable label" is a compound and/or element whose use allows their attached antibodies to be detected, and/or further quantified if desired, due to specific functional and/or chemical characteristics.

It is generally preferred to use the conjugates as diagnostic agents. Diagnostics generally fall into two categories, those used for in vitro diagnostics, such as in various immunoassays, and/or those used in vivo diagnostic protocols, commonly referred to as "molecular-targeted imaging.

Many suitable imaging agents are known in the art, as are methods for their attachment to molecules, such as antibodies (see, e.g., U.S. patent nos. 5,021,236; 4,938,948; and 4,472,509, each of which is incorporated herein by reference). The imaging moiety used may be a paramagnetic ion; a radioactive isotope; a fluorescent dye; an NMR-detectable substance; x-ray imaging.

In the case of paramagnetic ions, mention may be made, by way of example, of ions such as chromium (III), magnesium (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), gadolinium being particularly preferred. Other conditions such as ions useful in X-ray imaging include, but are not limited to, lanthanum (III), gold (III), lead (II), and in particular bismuth (III).

In the case of radioisotopes for therapeutic and/or diagnostic applications, astatine may be mentioned²¹¹、¹¹Carbon, carbon,¹⁴Carbon, carbon,⁵¹Chromium (II),³⁶Chlorine,⁵⁷Cobalt,⁵⁸Cobalt, copper⁶⁷、¹⁵²Eu, Ga⁶⁷、³Hydrogen and iodine¹²³Iodine, iodine¹²⁵Iodine, iodine¹³¹Indium, indium¹¹¹、⁵⁹Iron, iron,³²Phosphorus, rhenium¹⁸⁶Rhenium¹⁸⁸、⁷⁵Selenium,³⁵Sulphur, technetium^99mAnd/or yttrium⁹⁰. It is generally preferred that¹²⁵I is used in particular embodiments due to low energy and useSuitability for long-range detection, technetium^99mAnd/or indium¹¹¹Is generally preferred.

Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine Green, rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and/or Texas Red.

Other types of conjugates contemplated in the present invention are those primarily intended for use in vitro, wherein the molecule is linked to a second binding ligand and/or an enzyme (enzyme label) that will produce a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase or glucose oxidase. Preferred second binding ligands are biotin and/or avidin and streptavidin. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

Various other useful immunoassay procedures have been described in the scientific literature, such as, for example, Nakamura et al (1987). The simplest and most straightforward understanding of immunoassays is the binding assay. Particularly preferred immunoassays are the various types of Radioimmunoassays (RIA) and the immunobead capture assay. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be apparent that detection is not limited to such techniques, and western blots, dot blots, FACS analyses, and the like may also be used in conjunction with the present invention.

The immunological-based detection method used in conjunction with western blotting involves an enzyme-, radiolabel-, or fluorescently-labeled second molecule/anti-NC_Ca-ATPAntibodies to the SUR1 channel or regulatory subunit are considered particularly useful in this regard. Involving the use of such marksU.S. patents include 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated by reference. Of course, other advantages may be found by using a second binding ligand, such as a secondary antibody or biotin/avidin ligand binding arrangement, as is known in the art.

In addition to the above imaging techniques, positron emission techniques, PET imaging or PET scanning are also known to those skilled in the art and may be used as diagnostic examinations. PET scanning involves the acquisition of physiological images based on the detection of radiation from positron emission. Positrons are tiny particles that are emitted from radioactive substances when administered to a patient.

Thus, in particular embodiments of the invention, the antagonist or a related compound thereof is enzymatically-, radiolabeled-, or fluorescently-labeled, as described above and used to diagnose, monitor and/or grade neuronal damage in spinal cord injury, and/or predict or grade secondary injury associated with spinal cord injury. For example, a labeled antagonist or related compound thereof can be used to determine or define a penumbra or area at risk of damage following spinal cord injury.

IX. diagnosis or treatment box

Any of the compositions described herein can be contained in a kit. In a non-limiting example, it is envisioned that a compound that selectively binds or recognizes SUR1 may be included in the diagnostic kit. Such compounds are referred to as "SUR 1 markers" which may include, but are not limited to, antibodies (monoclonal or polyclonal), SUR1 oligonucleotides, SU1 polypeptides, small molecules, or combinations thereof, antagonists, and the like. It is envisioned that any of these SUR1 labels may be linked to radioactive and/or fluorescent and/or enzymatic labels for rapid detection. The kit may also include a lipid and/or other agent, such as a radioactive or enzymatic or fluorescent label, in a suitable container means.

Kits may include appropriate aliquots of SUR 1-labeled, lipid and/or other reagent compositions of the invention, whether labeled or unlabeled, as may be used to prepare standard curves for detection assays. The components of the kit may be packaged in aqueous media or in lyophilized form. The container means of the kit generally comprises at least one vial, test tube, flask, bottle, syringe or other container means into which the ingredients are placed, preferably in appropriate aliquots. Where more than one component is present in the kit, the kit will typically also contain a second, third or other additional container into which the other components may be separately placed. However, various combinations of ingredients may be included in the vial. The kits of the present invention also typically include a device for commercially selling closed structures containing the SUR1 label, the lipid, the other reagent, and any other reagent containers. Such containers may include injection or blow molded plastic containers in which the desired vials are stored.

Therapeutic kits of the invention are kits comprising an antagonist or a compound related thereto. Thus, the kit may include a SUR1 antagonist or a related compound to block and/or inhibit NC_Ca-ATPA channel. Such kits typically contain a pharmaceutically acceptable formulation of the SUR1 antagonist or related compound in a suitable container means. The kit may have a single container means and/or may have different container means for each compound.

Where the components of the kit are provided as one and/or more liquid solutions, the liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred. SUR1 antagonists or related compounds may also be formulated into injectable compositions. In this case, the container means may itself be a syringe, pipette and/or other such similar device from which the formulation is applied to an affected site of the body, injected into an animal, and/or even applied to and/or mixed with other components of the cartridge.

Examples of aqueous solutions include, but are not limited to, ethanol, DMSO, and/or ringer's solution. In particular embodiments, DMSO or ethanol is used at a concentration of no more than 0.1% or (1 ml/1000L).

However, the components of the kit may be provided as a dry powder. When the reagents and/or ingredients are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another containment device.

The container means typically comprises at least one vial, test tube, flask, bottle, syringe, and/or other container means, wherein the SUR1 antagonist or related compound thereof is suitably dispensed. The kit may further comprise a second container means for holding sterile, pharmaceutically acceptable buffers and/or other diluents.

The kits of the present invention also typically include a device for containing the vials in a commercially available closed configuration, such as, for example, injection and/or blow molded plastic containers, in which the desired vials are stored.

Regardless of the number and/or type of containers, kits of the invention may further comprise a device for assisting in the injection/administration and/or placement of a SUR1 antagonist or related compound thereof into an animal, and/or packaging of a kit of the invention with such a device. Such a device may be a syringe, pipette, forceps and/or any medically approved delivery vehicle.

In addition to the SUR1 antagonist or related compound, the kit can also include a second active ingredient. Examples of the second active ingredient include substances for preventing hypoglycemia (e.g., glucose, D5W, glucagon, etc.), steroids (e.g., methylprednisolone), and the like. These second active ingredients may be combined in the same vial with the SUR1 antagonist or related compound, or they may be contained in separate vials.

Still further, kits of the invention may also include a glucose test kit. Thus, the glucose of a patient is measured using a glucose test kit, and then a SUR1 antagonist or its related compound may be administered to the patient, followed by measuring the patient's glucose.

In addition to the above-described kits, the therapeutic kit of the present invention may be assembled such that the IV bag includes a septum or chamber that can be opened or broken to release the compound into the IV bag. Another kit may include a bolus kit, where the bolus kit may include a pre-filled syringe or similar easy-to-use rapid drug delivery device. An infusion kit may include a vial or ampoule and an IV solution (e.g., ringer's solution) for adding the vial or ampoule thereto prior to infusion. The infusion kit may also include a bolus kit for administering a bolus/loading dose to a patient before, during, or after infusion.

X example

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

To NC_Ca-ATPAdjustment of channels

Through Na⁺When the cell depolarizes due to a large influx, H is due to the osmotic gradient₂O enters the cell. H₂The influx of O results in cell blebbing, i.e., cytotoxic edema. Scanning Electron Microscopy (SEM) and phase contrast microscopy were used to examine this phenomenon for R1 astrocytes. Freshly isolated cells examined by SEM showed a complex surface decorated with numerous fine protrusions (fig. 1A). Shortly after exposure to sodium azide, but after expected depolarization, the complex cell surface begins to be replaced by surface bubbles, with a flattening of the membrane (fig. 1B). After that, the film appearance was mainly bubbles, which completely lost the fine protrusions observed in the control (fig. 1C).

In the absence of ATP depletion, NC was opened simply by diazoxide_Ca-ATPChanneling, reproducing the bubbling (figure 2). In contrast, the foaming observed with normally sodium azide-induced depletion of APT was completely prevented by glibenclamide (figure 2). Blistering and cytotoxic edemaIndicating necrotic cell death.

Example 2

Regulation by stimulants

K_ATPA characteristic feature of the channels (Kir6.1, Kir6.2) is the phosphatidylinositol 4, 5-bisphosphate (PIP) passage through the membrane lipid₂) To modulate channel affinity for ATP. PIP (picture in picture)₂Application to the cytosolic side of the membrane to increase K_ATPOpen state stability of the channel (Ashcroft, 1998; Baukrowitz et al, 1998; Rohacs et al, 1999). The improvement in open state stability appears to be an increase in the likelihood of channel opening in the absence of ATP and a corresponding decrease in sensitivity to ATP inhibition (Enkvertchaul et al, 2000; Haruna et al, 2000; Koster et al, 1999; and Larsson et al, 2000).

Known as K_ATPChannel and NC_Ca-ATPNumerous similarities between channels, the inventors assume NC_Ca-ATPThe ATP-sensitivity of the channel will respond in the same way to PIP₂. By investigating NC in an inner-facing diaphragm_Ca-ATPChannels to test this, using Cs⁺As charge carrier and using 1 μ M Ca in the bath²⁺And 10 μ M ATP, the latter being expected to completely block the channel. Under these conditions, only NC was recorded in R1 astrocytes_Ca-ATPA channel. PIP (picture in picture)₂When added (50 μ M) to the bath, channel activity became apparent (FIG. 3), as by PIP₂To K_ATPThe analogy of the channel effect is predicted. The identification of the channel was confirmed by the blocking of this channel activity by glibenclamide.

To determine whether receptor-mediated mechanisms involved NC_Ca-ATPModulation of channel activity using the well-known phospholipase C (PLC) to investigate whether PLC activation leads to PIP₂And thus increase the affinity for ATP, e.g., decrease channel opening. Estrogen is a well-known PLC activator in the brain and elsewhere (Beyer et al, 2002; Le Mellay et al, 1999; Qui et al, 2003). For this experiment, the patch of cell attachment was studied to prevent modification of the intracellular signaling mechanismAnd (6) changing. Generation of NC by depletion of cellular ATP by exposure to sodium azide_Ca-ATPChannel activity (figure 4, first part of the recording).

When estrogen (E2; 10nM) was applied to the bath, the passage from the NC to the bath was quickly terminated_Ca-ATPChannel induced activity (figure 4). This indicates that estrogen exerts its effect on NC_Ca-ATPRegulatory control of the channels and suggests the presence of estrogen receptors on these cells that are capable of rapid (non-genomic) activation of signaling cascades.

Second, to determine whether estrogen receptors could be detected in R1 astrocytes from males and females. Gelatin sponge implants were collected 7 days after implantation in one group of 3 female rats (F) and another group of 3 male rats (M). Proteins pooled from each group were analyzed by western blotting at 2 dilutions (4x 50 μ g total protein; 1x 12.5 μ g total protein) and proteins from uterus were used as controls (fig. 5A). Membranes were blotted with antibodies recognizing alpha and beta estrogen receptors. Males and females showed distinct bands at the appropriate molecular weights for the alpha (66kDa) and beta (55kDa) receptors (FIG. 5) (Hiroi et al, 1999). The same protein samples from males and females were also used to confirm the presence of SUR1, and protein from pancreas was used as a positive control (fig. 5B). Notably, estrogen receptors from male and female astrocytes have been previously reported (Choi et al, 2001). In the cerebral cortex, a greater beta isoform content was reported (Guo et al, 2001), as indicated by western blotting.

Next, the electrophysiological experiment of fig. 4 was repeated using R1 astrocytes collected from male rats. As above, cell-attached patches were studied in which NC was activated by depletion of intracellular ATP following exposure to sodium azide_Ca-ATPChannel activity (figure 6A). Examining the higher time-resolved recordings confirms that for NC_Ca-ATPThe proper conductance of the channel adequately defines the activity of the channel (FIG. 6B). When estrogen was administered to the bath (FIG. 6, E2, 10nM, arrow), rapid termination by NC was observed_Ca-ATPChannel induced activity (figure 6). These data provide more estrogen pairs NC_Ca-ATPChannel exertionEvidence for regulatory control has also shown that this response is equally strong in R1 astrocytes from both males and females.

By analogy to the estrogenic effects, depletion of PIP is expected₂Including other receptor-mediated mechanisms and more direct PLC activators such as C-protein, etc., to the NC_Ca-ATPThe activity of the channel has a similar inhibitory effect and therefore exerts a protective effect.

Example 3

NC_Ca-ATPPassage and necrotic death

Applicants have discovered a novel mechanism for necrotic death of reactive astrocytes in brain injury and stroke, which play an important role in spinal cord injury. Blebbing and cytotoxic edema are indicative of necrotic cell death. Freshly isolated reactive astrocytes were labeled with propidium iodide, which is a marker of necrotic death, and annexin V, which is a marker of apoptotic death. Cells exposed to sodium azide showed a significant increase in necrotic but not apoptotic death (figure 7). However, sodium azide-induced necrotic cell death was significantly reduced in the presence of glibenclamide (figure 7). These in vitro data indicate NC_Ca-ATPThe important role of the channel in the necrotic death of reactive astrocytes and suggests that antagonists of SUR1, such as glibenclamide, are useful in preventing cytotoxic edema and necrotic death in vivo.

Applicants have studied NC in rodent models of stroke_Ca-ATPA channel. In the penumbra, the SUR1 marker was found in stellate cells that were also GFAP-positive (fig. 8A). In the middle of stroke, no stellate cells were present, but the SUR1 marker was found in round cells that also were GFAP-positive and presented a blister-like appearance (fig. 8B, C). Round cells that bleb in situ resemble reactive astrocytes that undergo necrotic death after in vitro exposure to sodium azide. The effect of glibenclamide compared to saline was determined. Administration (300. mu.M, 0.5. mu.l @) by an osmotic mini-pump implanted subcutaneouslyhr). In saline-treated rats, the 3-day mortality rate after stroke was 68%, while in glibenclamide-treated rats, the 3-day mortality rate decreased to 28% (n-29 per group; by x)²P < 0.001). In separate animals, stroke hemispheres in glibenclamide-treated rats were found to contain as much excess water as half of saline-treated rats (n-5 per group; p < 0.01 by t-test), confirming that NC was_Ca-ATPThe important role of the channel in edema formation.

SUR1 was also studied in a traumatic rodent model. The effect of direct infusion of drugs into the site of trauma using an implanted osmotic mini-pump was examined. The channel inhibitor glibenclamide was used to reduce the death of reactive astrocytes, and the channel activator diazoxide was used to promote astrocyte death. In short, it was found that glibenclamide infusion reduced the overall injury response, stabilized the glial capsule surrounding the foreign implant and minimized the inflammatory response compared to the control.

In contrast, diazoxide substantially destroyed the glial membrane sac and elicited a massive inflammatory response characterized by massive influx of PMNs (fig. 9A, B). These data indicate NC_Ca-ATPChannels play a key role in the response to injury, they strongly support inflammation and NC_Ca-ATPThe activity of the channel and the necrotic death of the reactive astrocytes are closely related hypotheses.

Example 4

Functional NC in spinal cord contusion_Ca-ATPExpression of the channel

SUR1 was identified in rodent models of spinal cord contusion. The immune-labeled spinal cord sections showed a large increase in SUR1 expression in the injury region (fig. 10B) compared to the control (fig. 10A). SUR1 co-localized with GFAP (fig. 10C), confirming the involvement of reactive astrocytes. Examination of the cells at high magnification confirmed that SUR 1-positive cells were star-shaped (FIG. 10D) GFAP-positive cells, expressing NC with reactive astrocytes in spinal cord injury_Ca-ATPThe assumptions of the channels are consistent.

Further characterization of reactive astrocytes was performed by isolating reactive astrocytes from the contused spinal cord 3-5 days post injury using Fluorescence Assisted Cell Sorting (FACS). Freshly isolated cells were patch-clamped to demonstrate channels with the expected physiological and pharmacological properties. The Spinal Cord Injury (SCI) model was used and included the use of NYU-type impactor (Yu et al, 2001). Reactive astrocytes were isolated from zymolyzed spinal cord tissue using anti-SUR 1 antibody and FACS. FACS was used to isolate another subtype of reactive astrocytes in brain injury (Dalton et al, 2003). The patch clamp method was used to measure single channel conductance, sensitivity to ATP, and sensitivity to glibenclamide and diazoxide as described for astrocytes isolated from brain lesions (Chen et al, 2001; Chen and Simond, 2003).

Example 5

Blockade of SUR1 prevents delayed bleeding conversion

Injury in spinal cord contusion is caused not only by physical trauma to the tissue, but also by secondary injury that causes the initial injury to propagate and exacerbate the neurological compromise. The mechanisms of secondary injury are generally attributed to the occurrence of ischemia, edema, release of stimulatory amino acids, oxidative damage, and inflammation. Applicants have discovered that bleeding is also a critical component of this secondary injury process. The expansion of bleeding following injury due to the progressive pathological involvement of the capillaries is a phenomenon known as "bleeding conversion".

To study SUR 1-adjusted NC_Ca-ATPThe role of the channel in SCI, a model of semi-cervical spinal cord contusion was used. For this model, a weight of 10 grams was dropped 2.5cm onto the left half of the exposed dura mater at C4-5 in adult female Long-Evans rats. Histopathological studies 24 hours after injury showed a large upregulation of SUR1 in capillaries surrounding the injury site, but not in the control (fig. 11). In addition, capillaries showing SUR1 upregulation in the site of injury were also found to express vimentin (fig. 12), an intermediate filamentous protein commonly associated with astrocytes, but not normally associated with astrocytesAlso expressed by damaged capillary endothelial cells in the brain and spinal cord.

To provide further molecular evidence of the involvement of SUR1, the transcription factor SP1, a major transcription factor known to regulate the expression of SUR1, in tissues in spinal cord injury was also examined. Compared to the control (fig. 13A), the immunolabeling of the tissue in the damaged area showed a significant up-regulation of SP1 (fig. 13B).

To evaluate the role of the newly expressed SUR1 in SCI, 2 groups of animals, a control group and a treatment group, both groups received a mini osmotic pump delivering saline or a selective SUR1 blocking agent subcutaneously following injury to the hemicervical spinal cord contusion (blinded), with the SUR1 blocking agent being a low dose of glibenclamide (300 μ M solution delivered at 0.5 μ l/hr s.q.). Studies at 24 hours post injury showed that glibenclamide treated animals had significantly less blood at the site of contusion compared to controls (figure 14A, B). In addition, homogenates of spinal cord tissue showed significantly less staining from hemoglobin/ferrihemoglobin (fig. 14C). Quantitative studies of hemoglobin concentration as a function of time post spinal cord contusion showed a progressive increase in saline treated animals over the first 6 hours post injury, which was significantly alleviated by glibenclamide treatment (fig. 15).

Next, lesions in both groups of animals were assessed using GFAP to label reactive astrocytes and eriochrome cyanine-R to label myelin. Studies at 24 hours post-injury showed that glibenclamide treated animals had significantly less injury, significantly reduced GFAP expression, and significantly better preservation of the contralateral long tract compared to controls (figure 16).

Still further, behavioral assessments in both groups of animals were performed. Animals were videotaped and behaviours were quantitatively probed vertically in an environment to which the animals had not been previously exposed. Studies at 24 hours post-injury showed that glibenclamide treated animals exhibited significantly improved vertical probing behavior compared to controls (figure 17).

It is recognized that delayed hemorrhagic transformation in spinal cord contusion provides a particular opportunity to mitigate secondary injury. As is well known, blood is extremely toxic to CNS tissues and causes the formation of edema, the production of reactive oxygen species and the initiation of inflammation. The concept of delayed bleeding conversion is a new concept in SCI, and the discovery that glyburide can be used to alleviate this condition provides unprecedented opportunities to improve outcomes by mitigating secondary injury.

Example 6

NC_Ca-ATPChannel necrosis in cytotoxic edema and astrocytes

And in the release of bioactive molecules that promote tissue inflammation

The identification of candidate intracellular molecules released by cell membrane lysis during necrotic cell death that play a role in initiating the inflammatory response in spinal cord injury is also contemplated in the present invention. FACS was used to isolate reactive astrocytes from the contused spinal cord 3-5 days after injury. The effect of sodium azide (1mM) vs. sodium azide plus glibenclamide (1 μ M) on necrotic death vs. apoptotic death during the first 3 hours post-intoxication was evaluated using freshly isolated cells by the following method: performing morphological studies using phase contrast microscopy, scanning electron microscopy and transmission electron microscopy; labeling propidium iodide vs. annexin V; and assessing DNA degradation using TUNEL labeling and DNA fragmentation ladders.

Using freshly isolated cells, the release of HSP-32 and HSP-70 following sodium azide-induced astrocytic necrotic death was measured using ELISA. Furthermore, using the same experimental model, we will evaluate the protective effect of glibenclamide on sodium azide-induced release of HSP-32 and HSP-70. Standard FACS methods are used, as well as scanning and transmission electron microscopy and phase contrast microscopy, the latter allowing continuous tracking of individual cells during the foaming process. Immunofluorescence is also used, as described herein.

Example 7

NC_Ca-ATPAntagonists of the channel inAbility to reduce inflammatory response in spinal cord contusion in vivo

The tissues were studied about 3 days after injury. Quantitative immunofluorescent markers for activated microglia (OX-42), macrophages (MAC-387, Novus), PMNs (MMP-8, Chemicon), and iNOS were used to assess in situ inflammatory responses in spinal cord contusion rats treated with saline or glibenclamide. For these experiments, fresh-frozen sections of the spinal cord in or near the contusion area were studied. Quantitative FACS analysis of macrophages (MAC-387) and PMNs (MMP-8) was performed. For these experiments, 15-mm spinal segments containing the bruised area were obtained and subjected to enzymatic dispersion for FACS analysis. Quantitative western blotting for SUR1 and iNOS, for these experiments, 15-mm spinal segments containing contusion areas were obtained and homogenized for western blotting. Standard methods and materials were used, as described above, including FACS analysis, western blot and immunofluorescence imaging.

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Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims (43)

1. A method of treating a patient having a spinal cord injury comprising effectively inhibiting NC in neuronal cells, glial cells, neuroendothelial cells, or a combination thereof_Ca-ATPThe compound of the channel is administered to the patient.

2. The method of claim 1, wherein the compound reduces cell death.

3. The method of claim 1, wherein the compound reduces an inflammatory response.

4. The method of claim 1, wherein the compound reduces hemorrhagic transformation.

5. The method of claim 1, wherein the compound reduces secondary injury associated with spinal cord injury.

6. The method of claim 1, wherein the compound is a type 1 sulfonylurea receptor (SUR1) antagonist.

7. The method of claim 6, wherein the SUR1 antagonist is selected from the group consisting of glyburide, tolbutamide, repaglinide, nateglinide, meglitinide, imiglizole, LY 39364, LY389382, glyclazide, glimepiride, estrogen, and estrogen-related compounds.

8. The method of claim 6, wherein the amount of SUR1 antagonist administered to the patient is from about 0.0001 μ g/kg/day to about 20 mg/kg/day.

9. The method of claim 6, wherein the amount of SUR1 antagonist administered to the patient is from about 0.01 μ g/kg/day to about 100 μ g/kg/day.

10. The method of claim 6, wherein the amount of SUR1 antagonist administered to the patient is from about 100 μ g/kg/day to about 20 mg/kg/day.

11. The method of claim 6, wherein the SUR1 antagonist is administered as a bolus injection.

12. The method of claim 6, wherein the SUR1 antagonist is administered as an infusion.

13. The method of claim 6, wherein the SUR1 antagonist is administered as a bolus infusion in combination with an infusion.

14. The method of claim 6, wherein the amount of SUR1 antagonist administered to the patient is from about 0.0001 μ g/kg/treatment to about 20 mg/kg/treatment.

15. The method of claim 6, wherein the amount of SUR1 antagonist administered to the patient is from about 0.01 μ g/kg/treatment to about 100 μ g/kg/treatment.

16. The method of claim 6, wherein the amount of SUR1 antagonist administered to the patient is from about 100 μ g/kg/treatment to about 20 mg/kg/treatment.

17. The method of claim 6, wherein the SUR1 antagonist blocks Na + influx into the cell, thereby preventing depolarization of the cell.

18. The method of claim 6, wherein the SUR1 antagonist blocks Na + influx into the cell, thereby preventing cytotoxic edema.

19. The method of claim 1, wherein the compound is administered via the digestive tract or parenterally.

20. The method of claim 19, wherein the gastrointestinal administration comprises oral, buccal, rectal, or sublingual administration.

21. The method of claim 19, wherein parenteral administration comprises intravenous, intradermal, intramuscular, intraarterial, intrathecal, subcutaneous, intraperitoneal or intraventricular administration.

22. The method of claim 1, wherein the compound is administered mucosally.

23. The method of claim 22, wherein mucosal administration comprises intranasal administration.

24. The method of claim 1, wherein for NC_Ca-ATPInhibition of the channel results in a reduction in patient morbidity.

25. The method of claim 1, wherein for NC_Ca-ATPInhibition of the channel results in a reduction of extravasated blood near the site of contusion in the patient.

26. The method of claim 1, wherein for NC_Ca-ATPInhibition of the channel reduces the size of the spinal cord injury.

27. The method of claim 24, wherein the reduction in lesion size reduces involvement on the contralateral side of the spinal cord.

28. The method of claim 1, wherein for NC_Ca-ATPChannel suppression reduces GFAP up-regulation.

29. The method of claim 1, wherein the NC is present in neuronal cells, glial cells, endothelial cells, or a combination thereof_Ca-ATPInhibition of the channel preserves the myelinated long bundle.

30. The method of claim 1, wherein for NC_Ca-ATPThe inhibition of the channel improves the patient's movement or sensation.

31. A method for reducing edema in a spinal cord injury penumbra in a patient, comprising inhibiting NC in neuronal cells, glial cells, neuroendothelial cells, or a combination thereof_Ca-ATPThe compound of the channel is administered to the patient.

32. A method of treating a patient at risk of developing a spinal cord injury comprising effectively inhibiting NC in neuronal cells, glial cells, neuroendothelial cells, or a combination thereof_Ca-ATPOf passagesThe compound is administered to a patient.

33. The method of claim 32, wherein the patient is undergoing surgical treatment.

34. The method of claim 32, wherein the patient is undergoing radiation therapy.

35. A method of reducing extravasation of blood from a spinal cord injury comprising NC effective to inhibit neuronal cells, glial cells, neuroendothelial cells, or a combination thereof_CA-ATPThe compound of the channel is administered to the patient.

36. The method of claim 35, wherein the compound is a type 1 sulfonylurea receptor (SUR1) antagonist.

37. The method of claim 36, wherein the SUR1 antagonist is selected from the group consisting of glyburide, tolbutamide, repaglinide, nateglinide, meglitinide, imiglizole, LY 39364, LY389382, glyclazide, glimepiride, estrogen, and estrogen-related compounds.

38. The method of claim 35, wherein the patient is at risk of spinal cord injury.

39. The method of claim 38, wherein the compound is administered before, during or after surgical treatment or radiation therapy.

40. A method of diagnosing neuronal cell edema and/or cytotoxic injury in a spinal cord of a patient, comprising:

an antagonist labeled SUR 1;

administering a labeled SUR1 antagonist to the patient;

measuring the level of the labeled SUR1 antagonist in the spinal cord of the patient, wherein the presence of the labeled SUR1 antagonist in the spinal cord of the patient indicates neuronal cell edema and/or cytotoxic injury in the spinal cord.

41. A method of determining the posterior penumbra of spinal cord injury in a patient, comprising:

an antagonist labeled SUR 1;

administering a labeled SUR1 antagonist to the patient;

visualizing a labeled SUR1 antagonist in the spinal cord of the patient, wherein the presence of the labeled SUR1 antagonist indicates a penumbra after spinal cord injury in the patient.

42. The method of claim 41, wherein determining the penumbra indicates the location of neuronal damage.

43. The method of claim 41, wherein determining the penumbra monitors the progression of the disease.