Combination therapy for treating protein deficiency disorders

Abstract

This application provides methods of improving protein replacement therapy by combining protein replacement therapy with active site-specific chaperones (ASSC) to increase the stability and efficiency of the protein being administered. The application further provides compositions comprising the purified protein and an ASSC, and methods of treatment by administering the compositions.

What is claimed:  
     
         1 . A method for enhancing the stability of a purified protein, which method comprises contacting the protein in a pharmaceutically acceptable carrier with an effective amount of an active site-specific chaperone.  
     
     
         2 . The method of  claim 1 , wherein the purified protein is an enzyme and the active site-specific chaperone is a reversible competitive inhibitor of the enzyme.  
     
     
         3 . The method of  claim 2 , wherein the enzyme is an enzyme associated with a lysosomal storage disorder.  
     
     
         4 . The method of  claim 3 , wherein the enzyme is α-galactosidase A.  
     
     
         5 . The method of  claim 3 , wherein the enzyme is β-glucocerebrosidase.  
     
     
         6 . The method of  claim 4 , wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group  
         R 4  and R 4 ′ independently represent H, OH; and  
         R 7  represents H or OH.  
       
     
     
         7 . The method of  claim 6 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of 1-deoxygalactonojirimycin, α-allo-homonojirimycin, α-galacto-homonojirimycin, α-1-C-butyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , N-methyl-calystegine A 3 , and N-methyl-calystegine B 2 .  
     
     
         8 . The method of  claim 7 , wherein the reversible competitive inhibitor is 1-deoxygalactonojirimycin.  
     
     
         9 . The method of  claim 5 , wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group;  
         R 4  represents OH;  
         R 4 ′ represents H; and  
         R 7  represents OH.  
       
     
     
         10 . The method of  claim 9 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of isofagomine, N-dodecyl-isofagomine, N-nonylisofagomine, N-dodecyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , calystegine B 3  and calystegine C 1 .  
     
     
         11 . The method of  claim 10 , wherein the reversible competitive inhibitor is isofagomine.  
     
     
         12 . The method of  claim 10 , wherein the reversible competitive inhibitor is N-dodecyl-isofagomine.  
     
     
         13 . A method of increasing in vitro the shelf-life of a protein by contacting the protein in a pharmaceutically acceptable carrier with an effective amount of an active site-specific chaperone.  
     
     
         14 . The method of  claim 13 , wherein the protein, pharmaceutically acceptable carrier, and active site-specific chaperone are formulated in a lyophilized powder.  
     
     
         15 . The method of  claim 13 , wherein the protein, pharmaceutically acceptable carrier, and active site-specific chaperone are formulated in a sterile aqueous solution.  
     
     
         16 . The method of  claim 13 , wherein the protein is an enzyme and the active site-specific chaperone is a reversible competitive inhibitor of the enzyme.  
     
     
         17 . The method of  claim 16 , wherein the enzyme is associated with a lysosomal storage disorder.  
     
     
         18 . The method of  claim 17 , wherein the enzyme is α-galactosidase A.  
     
     
         19 . The method of  claim 17 , wherein the enzyme is β-glucocerebrosidase.  
     
     
         20 . The method of  claim 18 , wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group R 4  and R 4 ′ independently represent H, OH; and  
         R 7  represents H or OH.  
       
     
     
         21 . The method of  claim 20 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of 1-deoxygalactonojirimycin, α-allo-homonojirimycin, α-galacto-homonojirimycin, α-1-C-butyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , N-methyl-calystegine A 3 , and N-methyl-calystegine B 2 .  
     
     
         22 . The method of  claim 21 , wherein the reversible competitive inhibitor is 1-deoxygalactonojirimycin.  
     
     
         23 . The method of  claim 19  wherein the reversible competitive inhibitor is represented by a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group;  
         R 4  represents OH;  
         R 4 ′ represents H; and  
         R 7  represents H or OH.  
       
     
     
         24 . The method of  claim 23 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of isofagomine, N-dodecyl-isofagomine, N-nonyl-isofagomine, N-dodecyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , calystegine B 3  and calystegine C 1 .  
     
     
         25 . The method of  claim 24 , wherein the reversible competitive inhibitor is isofagomine.  
     
     
         26 . The method of  claim 24 , wherein the reversible competitive inhibitor is N-dodecyl isofagomine.  
     
     
         27 . A method of extending the half-life and prolonging the activity in vivo of a purified protein in an individual who has been administered the protein in a pharmaceutically acceptable carrier, which method comprises contacting the protein with an effective amount of an active site-specific chaperone in a pharmaceutically acceptable carrier.  
     
     
         28 . The method of  claim 27 , wherein the protein is co-administered with the active site-specific chaperone.  
     
     
         29 . The method of  claim 27 , wherein the protein is an enzyme and the active site-specific chaperone is a reversible competitive inhibitor of the enzyme.  
     
     
         30 . The method of  claim 29 , wherein the enzyme is associated with a lysosomal storage disorder.  
     
     
         31 . The method of  claim 30 , wherein the enzyme is α-galactosidase A.  
     
     
         32 . The method of  claim 30 , wherein the enzyme is β-glucocerebrosidase.  
     
     
         33 . The method of  claim 31 , wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group  
         R 4  and R 4 ′ independently represent H, OH; and  
         R 7  represents H or OH.  
       
     
     
         34 . The method of  claim 33 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of 1-deoxygalactonojirimycin, α-allo-homonojirimycin, α-galacto-homonojirimycin, α-1-C-butyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , N-methyl-calystegine A 3 , and N-methyl-calystegine B 2 .  
     
     
         35 . The method of  claim 34 , wherein the reversible competitive inhibitor is 1-deoxygalactonojirimycin.  
     
     
         36 . The method of  claim 32  wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group;  
         R 4  represents OH;  
         R 4 ′ represents H; and  
         R 7  represents H or OH.  
       
     
     
         37 . The method of  claim 36 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of isofagomine, N-dodecyl-isofagomine, N-nonyl-isofagomine, N-dodecyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , calystegine B 3  and calystegine C 1 .  
     
     
         38 . The method of  claim 37 , wherein the reversible competitive inhibitor is isofagomine.  
     
     
         39 . The method of  claim 37 , wherein the reversible competitive inhibitor is N-dodecyl-isofagomine.  
     
     
         40 . A method for increasing the production of a recombinant protein by a non-mammalian host cell, wherein the host cell comprises an expression vector comprising a nucleic acid sequence which encode the recombinant protein, which method comprises culturing the host cell in a medium comprising an active site-specific chaperone for the protein.  
     
     
         41 . The method of  claim 40 , wherein the protein is an enzyme and the active site-specific chaperone is a reversible competitive inhibitor of the enzyme.  
     
     
         42 . The method of  claim 41 , wherein the enzyme is associated with a lysosomal storage disorder.  
     
     
         43 . The method of  claim 42 , wherein the enzyme is α-galactosidase A.  
     
     
         44 . The method of  claim 42 , wherein the enzyme is β-glucocerebrosidase.  
     
     
         45 . The method of  claim 43 , wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group R 4  and R 4 ′ independently represent H, OH; and  
         R 7  represents H or OH.  
       
     
     
         46 . The method of  claim 45 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of 1-deoxygalactonojirimycin, α-allo-homonojirimycin, α-galacto-homonojirimycin, α-1-C-butyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , N-methyl-calystegine A 3 , and N-methyl-calystegine B 2 .  
     
     
         47 . The method of  claim 46 , wherein the reversible competitive inhibitor is 1-deoxygalactonojirimycin.  
     
     
         48 . The method of  claim 44 , wherein the reversible competitive inhibitor is a compound of the following formula:  
       
         
           
           
               
               
           
         
         wherein R 0  represents H or a C 1 -C 12  alkyl chain;  
         R 0 ′ represents H, a straight chain or branched saturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group;  
         R 1  and R 1 ′ independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;  
         R 2  and R 2 ′ independently represent H, OH or a C 1 -C 12  alkyl group;  
         R 4  represents OH;  
         R 4 ′ represents H; and  
         R 7  represents H or OH.  
       
     
     
         49 . The method of  claim 48 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of isofagomine, N-dodecyl-isofagomine, N-nonyl-isofagomine, N-dodecyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , calystegine B 3  and calystegine C 1 .  
     
     
         50 . The method of  claim 49 , wherein the reversible competitive inhibitor is isofagomine.  
     
     
         51 . The method of  claim 49 , wherein the reversible competitive inhibitor is N-dodecyl-isofagomine.  
     
     
         52 . A pharmaceutical composition comprising a purified protein and a active site-specific chaperone for the protein in a pharmaceutically acceptable carrier.  
     
     
         53 . The composition of  claim 52 , wherein the protein is an enzyme and the active site-specific chaperone is a reversible competitive inhibitor of the enzyme.  
     
     
         54 . The composition of  claim 53 , wherein the enzyme is associated with a lysosomal storage disorder.  
     
     
         55 . The composition of  claim 54 , wherein the enzyme is α-galactosidase A.  
     
     
         56 . The composition of  claim 54 , wherein the enzyme is β-glucocerebrosidase.  
     
     
         57 . The composition of  claim 55 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of 1-deoxygalactonojirimycin, α-allo-homonojirimycin, α-galacto-homonojirimycin, α-1-C-butyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , N— methyl-calystegine A 3 , and N-methyl-calystegine B 2 .  
     
     
         58 . The composition of  claim 57 , wherein the reversible competitive inhibitor is 1-deoxygalactonojirimycin.  
     
     
         59 . The composition of  claim 56 , wherein the reversible competitive inhibitor is a compound selected from the group consisting of isofagomine, N-dodecyl-isofagomine, N-nonyl-isofagomine, N-dodecyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , calystegine B 3  and calystegine C 1 .  
     
     
         60 . The composition of  claim 59 , wherein the reversible competitive inhibitor is isofagomine.  
     
     
         61 . The composition of  claim 59 , wherein the reversible competitive inhibitor is N-dodecyl-isofagomine.  
     
     
         62 . A method of treating an individual having a disorder requiring protein replacement, comprising administering to the individual a composition comprising an effective amount of purified, wild-type replacement protein and an effective amount of an active site-specific chaperone for the replacement protein.  
     
     
         63 . The method of  claim 62 , wherein the disorder is Fabry disease, the replacement protein is α-galactosidase A, and the active site-specific chaperone is selected from the group consisting of 1-deoxygalactonojirimycin, α-allo-homonojirimycin, α-galacto-homonojirimycin, α-1-C-butyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , N— methyl-calystegine A 3 , and N-methyl-calystegine B 2 .  
     
     
         64 . The method of  claim 63  wherein the active site-specific chaperone is 1-deoxygalactonojirimycin.  
     
     
         65 . The method of  claim 62 , wherein the disorder is Gaucher disease, the replacement protein is β-glucocerebrosidase and the active site-specific chaperone is selected from the group consisting of isofagomine, N-dodecyl-isofagomine, N-nonyl-isofagomine, N-dodecyl-deoxynojirimycin, calystegine A 3 , calystegine B 2 , calystegine B 3  and calystegine C 1 .  
     
     
         66 . The method of  claim 65  wherein the active site-specific chaperone is isofagomine.  
     
     
         67 . The method of  claim 65  wherein the active site-specific chaperone is N-dodecyl-isofagomine.