Methods for treating neural cell swelling

Abstract

A composition comprising a novel Ca 2+ -activated, [ATP] i -sensitive nonspecific cation (NC Ca-ATP ) channel is described. The channel is found in mammalian neural cells and exhibits a different sensitivity to block by various adenine nucleotides, and is activated by submicromolar [Ca] i . The NC Ca-ATP channel is activated under conditions of ATP depletion, which causes severe cell depolarization, followed by cell swelling. The NC Ca-ATP channel is regulated by a sulfonylurea receptor and is inhibited by sulfonylurea compounds glibenclamide and tolbutamide. Methods employing compositions comprising the NC Ca-ATP channel to screen for compounds that block the channel and the use of such antagonists as therapeutics in preventing brain swelling and damage are described. In addition, methods employing compositions comprising the Kir2.3 channel to screen for compounds that open the channel and the use of such antagonists as therapeutics in preventing brain swelling and damage are described.

1 . A composition comprising a membrane preparation expressing the NCCa-ATP channel. 
     
     
         2 . The composition of  claim 1 , wherein said membrane preparation is derived from neural cells. 
     
     
         3 . The composition of  claim 1 , wherein said membrane preparation is derived from isolated native reactive astrocytes (NRAs). 
     
     
         4 . The composition of  claim 1 , wherein said channel is blocked by antagonists of the type 1 sulfonylurea receptor and opened by SUR1 activators. 
     
     
         5 . The composition of  claim 3 , wherein said astrocytes are freshly isolated native reactive astrocytes. 
     
     
         6 . The composition of  claim 1 , wherein said channel has the following characteristics: (a) it is a 35 pS type channel; (b) it is stimulated by cytoplasmic Ca 2+  in the concentration range from about 10 −8  to about 10 −5  M; (c) it opens when cytoplasmic ATP is less than about 0.8 uM; and (d) it is permeable to the monovalent cations K + , Cs + , Li +  and Na + . 
     
     
         7 . A method of screening for antagonists of the NC Ca-ATP  channel, comprising:
 (a) contacting a test compound with a composition comprising the NC Ca-ATP  channel; and   (b) identifying test compounds that inhibit an activity of said channel by measuring said activity in the presence and absence of said test compound, wherein a test compound that inhibits said activity is identified as an antagonist of the NC Ca-ATP  channel.   
     
     
         8 . The method of  claim 7 , wherein said composition comprises a preparation of neural cells expressing the NC Ca-ATP  channel. 
     
     
         9 . The method of  claim 7 , wherein said composition comprises a membrane preparation expressing the NC Ca-ATP  channel. 
     
     
         10 . The method of  claim 7 , wherein said membrane preparation is derived from isolated native reactive astrocytes (NRAs). 
     
     
         11 . The method of  claim 7 , wherein said antagonist of the NC Ca-ATP  channel is selected from the group consisting of: (a) an NC Ca-ATP  channel inhibitor, an NC Ca-ATP  channel blocker, a SUR1 antagoinist, SUR1 inhibitor, and/or a compound capable of reducing the magnitude of membrane current through the channel. 
     
     
         12 . A method for identifying compounds that inhibit neural cell swelling, comprising:
 (a) contacting a test compound with a composition comprising the NC Ca-ATP  channel, and   (b) determining whether the test compound blocks the NCCa-Ap channel, wherein a test compound that blocks the NC Ca-ATP  channel is identified as a compound for inhibiting neural cell swelling.   
     
     
         13 . A method for identifying compounds that inhibit brain swelling, comprising: (a) contacting a test compound with a composition comprising the NC Ca-ATP  channel, and (b) determining whether the test compound blocks the NC Ca-ATP  channel, wherein a test compound that blocks the NC Ca-ATP  channel is identified as a compound for inhibiting brain swelling. 
     
     
         14 . A method for identifying compounds that inhibit brain swelling, comprising: (a) contacting a test compound with a composition comprising the NC Ca-ATP  channel, and (b) determining whether the test compound inhibits neural cell swelling, wherein a test compound that inhibits neural cell swelling is identified as a compound for inhibiting brain swelling. 
     
     
         15 . A method for identifying compounds that inhibit neural cell swelling in an animal, comprising: (a) contacting a test compound with a composition comprising the NC Ca-ATP  channel and determining whether the test compound blocks the channel, and (b) administering the test compound to an animal having a brain injury or cerebral ischemia, and determining whether the test compound inhibits brain swelling of the treated animal, wherein test compounds that inhibit brain swelling are identified as compounds that inhibit neural cell swelling in an animal. 
     
     
         16 . A method for identifying compounds that inhibit brain swelling, comprising:
 (a) contacting a test compound with a composition comprising the NC Ca-ATP  channel, and determining whether the test compound blocks the channel, and   (b) administering the test compound to an animal having a brain injury or cerebral ischemia, and determining whether the test compound inhibits brain swelling of the treated animal, wherein test compounds that block the NC Ca-ATP  channel are identified as compounds that inhibit brain swelling.   
     
     
         17 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said composition comprises a preparation of neural cells expressing the NC Ca-ATP  channel. 
     
     
         18 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said composition comprises a membrane preparation expressing the NC Ca-ATP  channel. 
     
     
         19 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said composition comprises a membrane preparation expressing the NC Ca-ATP  channel and further wherein said membrane preparation is derived from isolated native reactive astrocytes (NRAs). 
     
     
         20 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said compound is an antagonist of a type 1 sulfonylurea receptor. 
     
     
         21 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said compound is an antagonist of a type 1 sulfonylurea receptor, and further wherein said antagonist is a sulfonylurea compound. 
     
     
         22 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said determining step is selected from the group consisting of:
 (a) measuring channel currents; 
 (b) measuring membrane potential; 
 (c) detecting expression of annexin V; 
 (d) detecting expression of propidium iodide; 
 (e) in vitro binding assays; 
 (f) identifying appearance of cell blebbing and/or cell swelling; and 
 (g) combinations thereof. 
 
     
     
         23 . The method according to any one of  claims 12 ,  13 ,  14 ,  15  and  16 , wherein said composition comprises a preparation of neural cells expressing the NC Ca-ATP  channel, and further wherein said cell is a native reactive astrocyte. 
     
     
         24 . A method of preventing neural cell swelling in the brain of a subject, said method comprising administering to the subject a formulation containing an effective amount of a compound that blocks the NC Ca-ATP  channel and a pharmaceutically acceptable carrier. 
     
     
         25 . A method of alleviating the negative effects of traumatic brain injury or cerebral ischemia stemming from neural cell swelling in a subject, comprising administering to the subject a formulation comprising an effective amount of a compound that blocks the NC Ca-ATP  channel and a pharmaceutically acceptable carrier. 
     
     
         26 . The method according to any one of  claims 24  and  25 , wherein said administration is delivery into the brain. 
     
     
         27 . The method according to any one of  claims 24  and  25 , wherein said compound is an antagonist of a type 1 sulfonylurea receptor. 
     
     
         28 . The method according to any one of  claims 24  and  25 , wherein said compound is an antagonist of a type 1 sulfonylurea receptor, and further wherein said antagonist is a sulfonylurea compound. 
     
     
         29 . The method according to any one of  claims 24  and  25 , wherein said compound is an antagonist of a type 1 sulfonylurea receptor, and further wherein said antagonist is selected from the group consisting of glibenclamide and tolbutamide. 
     
     
         30 . The method according to any one of  claims 24  and  25 , wherein said compound is an antagonist of a type 1 sulfonylurea receptor, and further wherein said antagonist is selected from the group consisting of repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382, gliclazide, glimepiride, MgADP, and combinations thereof. 
     
     
         31 . The method according to any one of  claims 24  and  25 , wherein said pharmaceutically acceptable carrier renders said formulation suitable for administration, said administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intracutaneous, intragastric and oral administration. 
     
     
         32 . A formulation for preventing or inhibiting neural cell swelling in the brain of a subject, wherein said formulation comprises a compound that blocks the NC Ca-ATP  channel and a pharmaceutically acceptable carrier, wherein the quantity of said compound is at least 10 times greater than the quantity of said compound in formulations for treating diabetes. 
     
     
         33 . A method for identifying compounds that inhibit neural cell swelling, comprising:
 (a) contacting a test compound with a composition comprising the Kir2.3 channel,   (b) determining whether the test compound opens the Kir2.3 channel, wherein a test compound that opens the Kir2.3 channel is identified as a compound for inhibiting neural cell swelling.   
     
     
         34 . A method for identifying compounds that inhibit brain swelling, comprising: (a) contacting a test compound with a composition comprising the Kir2.3 channel, and (b) determining whether the test compound opens the Kir2.3 channel, wherein a test compound that opens the Kir2.3 channel is identified as a compound for inhibiting brain swelling. 
     
     
         35 . A method for identifying compounds that inhibit neural cell swelling and/or brain swelling in an animal, comprising: (a) contacting a test compound with a composition comprising the Kir2.3 channel, and (b) determining whether the test compound opens the Kir2.3 channel, wherein a test compound that opens the Kir2.3 channel is identified as a compound for inhibiting neural cell swelling and/or brain swelling in an animal. 
     
     
         36 . A method for identifying compounds that prevent, inhibit and/or alleviate brain swelling in a subject, comprising:
 (a) contacting a test compound with a composition comprising the Kir2.3 channel, and determining whether the test compound opens the Kir2.3 channel, and   (b) administering the test compound to a subject having a brain injury or cerebral ischemia, and determining whether the test compound prevents, inhibits and/or alleviates brain swelling in the subject, wherein test compounds that open the Kir2.3 channel are identified as compounds that inhibit brain swelling.   
     
     
         37 . A method for identifying compounds that inhibit neural cell swelling in an animal, comprising:
 (a) contacting a test compound with a composition comprising the Kir2.3 channel, and determining whether the test compound opens the Kir2.3 channel, and   (b) administering the test compound to an animal having a brain injury or cerebral ischemia, and determining whether the test compound inhibits brain swelling of the treated animal, wherein test compounds that inhibit brain swelling are identified as compounds that inhibit neural cell swelling in an animal.   
     
     
         38 . A method of preventing neural cell swelling in the brain of a subject, said method comprising administering to the subject a formulation containing an effective amount of a compound that opens the Kir2.3 channel and a pharmaceutically acceptable carrier. 
     
     
         39 . A method of alleviating the negative effects of traumatic brain injury or cerebral ischemia stemming from neural cell swelling in a subject, comprising administering to the subject a formulation comprising an effective amount of a compound that opens the Kir2.3 channel and a pharmaceutically acceptable carrier. 
     
     
         40 . The method according to any one of  claims 33 ,  34 ,  35 ,  36 ,  37 ,  38  and  39 , wherein said compound is Tenidap. 
     
     
         41 . The method according to any one of  claims 33 ,  34 ,  35 ,  36 ,  37 ,  38  and  39 , wherein said determining step is selected from the group consisting of:
 (a) measuring channel currents; 
 (b) measuring membrane potential; 
 (c) detecting expression of annexin V; 
 (d) detecting expression of propidium iodide; 
 (e) in vitro binding assays; 
 (f) identifying appearance of cell blebbing and/or cell swelling; and 
 (g) combinations thereof. 
 
     
     
         42 . A composition comprising freshly isolated native reactive astrocytes, wherein said astrocytes express the NC Ca-ATP  channel. 
     
     
         43 . The composition of  claim 42 , wherein at least about 80% to about 100% of said astrocytes express the NC Ca-ATP  channel.