Methods of modulating intracellular degradation rates of toxins

Abstract

The present invention provides for methods of modulating the degradation rate of a toxin in a cell, thereby modulating the half-life of the toxin. Particularly, the invention features methods of modulating the degradation rate of a toxin by modulating fusion between a lysosome and an endosome that carries the toxin in the cell.

1 . A method of modulating the degradation rate of a Clostridial toxin in a cell, the method comprising the step of co-administering to a cell the toxin and a lysosome-endosome fusion modulator.  
   
   
       2 . The method according to  claim 1 , wherein the Clostridial toxin is a beratti toxin, a butyricum toxin, a tetani toxin or a botulinum toxin.  
   
   
       3 . The method according to  claim 2 , wherein the botulinum toxin is a botulinum toxin serotype A, botulinum toxin serotype B, botulinum toxin serotype C1, botulinum toxin serotype D, botulinum toxin serotype E, botulinum toxin serotype F or botulinum toxin serotype G.  
   
   
       4 . The method according to  claim 1 , wherein the lysosome-endosome fusion modulator decreases the degradation rate of the Clostridial toxin.  
   
   
       5 . The method according to  claim 1 , wherein the lysosome-endosome fusion modulator comprises a lysosome-endosome fusion inhibitor.  
   
   
       6 . The method according to  claim 5 , wherein the lysosome-endosome fusion inhibitor is selected from the group consisting of a GTPase inhibitor, ATPase inhibitor, brefeldin A, cytochalasin B, Wortmannin, cytochalasin D, an inhibitor of actin filaments, phorbol12-myristate 13-acetate (PMA), a stimulator of protein kinase C, bafilomycin A, and mixtures of any of the following.  
   
   
       7 . The method according to  claim 6 , wherein the GTPase inhibitor is selected from the group consisting of a Rab GTPase inhibitor, a Rho GTPase inhibitor, and mixtures of any of the following.  
   
   
       8 . The method according to  claim 6 , wherein the ATPase inhibitor comprises an ATPase associated with cellular activities (AAA) type inhibitor.  
   
   
       9 . The method according to  claim 6 , wherein the GTPase inhibitor is selected from the group consisting of a guanine dissociation inhibitor (GDI) protein, an isoprene binding domain of the guanine dissociation inhibitor, a GTPase activating protein (GAP), a fluoroaluminate (AIF 4 ), a guanylyl 5-thiophosphate, a Y-27632 Rho kinase inhibitor, a C3 transferase, a  Clostridium difficile  toxin A, a  Clostridium difficile  toxin B, a  Clostridium sordellii  lethal toxin LT, a  Escherichia coli  cytotoxic necrotizing factor 1 (CNF1), a  Escherichia coli  cytotoxic necrotizing factor 2 (CNF2), a  Bordetella bronchiseptica  dermonecrotizing toxin (DNT), and mixtures of any of the following.  
   
   
       10 . The method according to  claim 1 , wherein the lysosome-endosome fusion modulator increases the degradation rate of the Clostridial toxin.  
   
   
       11 . The method according to  claim 1 , wherein the lysosome-endosome fusion modulator comprises a fusion facilitator.  
   
   
       12 . The method according to  claim 11 , wherein the fusion facilitator comprises a GTPase activator, a type III secreted toxin, or mixtures thereof.  
   
   
       13 . The method according to  claim 12 , wherein the GTPase activator comprises a guanine nucleotide exchange factor (GEF) protein, a GEF protein mimic, or mixtures thereof.  
   
   
       14 . The method according to  claim 12 , wherein the type III secreted toxin is a  Salmonella typhimurium  SopE, a  Salmonella  SptP, a  Yersinia pseudotuberculosis  YopE, a  Yersinia  YopT or a  Pseudomonas aeruginosa  ExoS.  
   
   
       15 . A method of modulating a half-life of a Clostridial toxin in a mammal, the method comprising the step of co-administering to the mammal the Clostridial toxin and a lysosome-endosome fusion modulator.  
   
   
       16 . The method according to  claim 15 , wherein the Clostridial toxin is a beratti toxin, a butyricum toxin, a tetani toxin or a botulinum toxin.  
   
   
       17 . The method according to  claim 16 , wherein the botulinum toxin is a botulinum toxin serotype A, botulinum toxin serotype B, botulinum toxin serotype C1, botulinum toxin serotype D, botulinum toxin serotype E, botulinum toxin serotype F or botulinum toxin serotype G.  
   
   
       18 . The method according to  claim 15 , wherein the lysosome-endosome fusion modulator increases the half-life of the Clostridial toxin.  
   
   
       19 . The method according to  claim 15 , wherein the lysosome-endosome fusion modulator comprises a lysosome-endosome fusion inhibitor.  
   
   
       20 . The method according to  claim 19 , wherein the lysosome-endosome fusion inhibitor is selected from the group consisting of a GTPase inhibitor, an ATPase inhibitor, brefeldin A, cytochalasin B, Wortmannin, cytochalasin D, an inhibitor of actin filaments, phorbol 12-myristate 13-acetate (PMA), a stimulator of protein kinase C, bafilomycin A, and mixtures of any of the following.  
   
   
       21 . The method according to  claim 20 , wherein the GTPase inhibitor is selected from the group consisting of a Rab GTPase inhibitor, a Rho GTPase inhibitor, and mixtures of any of the following.  
   
   
       22 . The method according to  claim 20 , wherein the ATPase inhibitor comprises an ATPase associated with cellular activities (AAA) type inhibitor.  
   
   
       23 . The method according to  claim 20 , wherein the GTPase inhibitor is selected from the group consisting of a guanine dissociation inhibitor (GDI) protein, an isoprene binding domain of the guanine dissociation inhibitor, a GTPase activating protein (GAP), a fluoroaluminate (AIF 4 ), a guanylyl 5-thiophosphate, a Y-27632 Rho kinase inhibitor, a C3 transferase, a  Clostridium difficile  toxin A, a  Clostridium difficile  toxin B, a  Clostridium. sordellii  lethal toxin LT, a  Escherichia coli  cytotoxic necrotizing factor 1 (CNF1), a  Escherichia coli  cytotoxic necrotizing factor 2 (CNF2), a  Bordetella bronchiseptica  dermonecrotizing toxin (DNT), and mixtures of any of the following  
   
   
       24 . The method according to  claim 15 , wherein the lysosome-endosome fusion modulator decreases the half-life of the Clostridial toxin.  
   
   
       25 . The method according to  claim 15 , wherein the lysosome-endosome fusion modulator comprises a lysosome-endosome facilitator.  
   
   
       26 . The method according to  claim 25 , wherein the lysosome-endosome facilitator comprises a GTPase activator, a type III secreted toxin, or a mixture thereof.  
   
   
       27 . The method according to  claim 26 , wherein the GTPase activator comprises a guanine nucleotide exchange factor (GEF) protein, a GEF protein mimic, or mixtures thereof.  
   
   
       28 . The method according to  claim 26 , wherein the type III secreted toxin is a  Salmonella typhimurium  SopE, a  Salmonella  SptP, a  Yersinia pseudotuberculosis  YopE, a  Yersinia  YopT or a  Pseudomonas aeruginosa  ExoS.  
   
   
       29 . A method of treating a biological disorder in a patient, the method comprising the step of co-administering to a patient in need thereof a Clostridial toxin and a lysosome-endosome fusion inhibitor modulator.  
   
   
       30 . The method according to  claim 29 , wherein the biological disorder comprises at least one of a neuromuscular disorder, an autonomic disorder and pain.  
   
   
       31 . The method according to  claim 29 , wherein the Clostridial toxin is a beratti toxin, a butyricum toxin, a tetani toxin or a botulinum toxin.  
   
   
       32 . The method according to  claim 31 , wherein the botulinum toxin is a botulinum toxin serotype A, botulinum toxin serotype B, botulinum toxin serotype C1, botulinum toxin serotype D, botulinum toxin serotype E, botulinum toxin serotype F or botulinum toxin serotype G.  
   
   
       33 . The method according to  claim 29 , wherein the lysosome-endosome fusion modulator decreases the degradation rate of the Clostridial toxin.  
   
   
       34 . The method according to  claim 29 , wherein the lysosome-endosome fusion modulator increases the half-life of the Clostridial toxin.  
   
   
       35 . The method according to  claim 29 , wherein the lysosome-endosome fusion modulator comprises a lysosome-endosome fusion inhibitor.  
   
   
       36 . The method according to  claim 35 , wherein the lysosome-endosome fusion inhibitor is selected from the group consisting of a GTPase inhibitor, ATPase inhibitor, brefeldin A, cytochalasin B, Wortmannin, cytochalasin D, an inhibitor of actin filaments, phorbol12-myristate 13-acetate (PMA), a stimulator of protein kinase C, bafilomycin A, and mixtures of any of the following.  
   
   
       37 . The method according to  claim 36 , wherein the GTPase inhibitor is selected from the group consisting of a Rab GTPase inhibitor, a Rho GTPase inhibitor, and mixtures of any of the following.  
   
   
       38 . The method according to  claim 36 , wherein the ATPase inhibitor comprises an ATPase associated with cellular activities (AAA) type inhibitor.  
   
   
       39 . The method according to  claim 36 , wherein the GTPase inhibitor is selected from the group consisting of a guanine dissociation inhibitor (GDI) protein, an isoprene binding domain of the guanine dissociation inhibitor, a GTPase activating protein (GAP), a fluoroaluminate (AIF 4 ), a guanylyl 5-thiophosphate, a Y-27632 Rho kinase inhibitor, a C3 transferase, a  Clostridium difficile  toxin A, a  Clostridium difficile  toxin B, a  Clostridium sordellii  lethal toxin LT, a  Escherichia coli  cytotoxic necrotizing factor 1 (CNF1), a  Escherichia coli  cytotoxic necrotizing factor 2 (CNF2), a  Bordetella bronchiseptica  dermonecrotizing toxin (DNT), and mixtures of any of the following  
   
   
       40 . The method according to  claim 29 , wherein the lysosome-endosome fusion modulator increases the degradation rate of the Clostridial toxin.  
   
   
       41 . The method according to  claim 29 , wherein the lysosome-endosome fusion modulator decreases the half-life of the Clostridial toxin.  
   
   
       42 . The method according to  claim 29 , wherein the lysosome-endosome fusion modulator comprises a fusion facilitator.  
   
   
       43 . The method according to  claim 42 , wherein the fusion facilitator comprises a GTPase activator, a type III secreted toxin, or mixtures thereof.  
   
   
       44 . The method according to  claim 43 , wherein the GTPase activator comprises a guanine nucleotide exchange factor (GEF) protein, a GEF protein mimic, or mixtures thereof.  
   
   
       45 . The method according to  claim 43 , wherein the type III secreted toxin is a  Salmonella typhimurium  SopE, a  Salmonella  SptP, a  Yersinia pseudotuberculosis  YopE, a  Yersinia  YopT or a  Pseudomonas aeruginosa  ExoS.  
   
   
       46 . (canceled)