Methods and compositions for overcoming resistance to biologic and chemotherapy

Abstract

This invention provides a method for identifying potential therapeutic agents by contacting a target cell with a candidate therapeutic agent which is a selective substrate for an endogenous, intracellular enzyme in the cell which is enhanced in its expression as a result of selection by biologic or chemotherapy. This invention also provides methods and examples of molecules for selectively killing a pathological cell by contacting the cell with prodrug that is a selective substrate for an endogenous, intracellular enzyme. The prodrug is subsequently converted to a cellular toxin. Further provided by this invention is a method for treating a pathology characterized by pathological, hyperproliferative cells in a subject by administering to the subject a prodrug that is a selective substrate for an endogenous, overexpressed, intracellular enzyme, and converted by the enzyme to a cellular toxin in the hyperproliferative cell.

1 - 36 . (canceled)  
     
     
         37 . A method for identifying potential therapeutic agents, comprising: 
 contacting a target cell with a candidate therapeutic agent that is a selective substrate for a target enzyme, under conditions that favor the incorporation of the agent into the intracellular compartment of the target cell; and    assaying the target cell for inhibition of cellular proliferation or cell killing.    
     
     
         38 . A method for selecting a uridine analogue for reducing or inhibiting the replication or spread of tumor cells, comprising the steps of: 
 providing a uridine analog unsubstituted in the 5-position;    testing said uridine analog for activation by thymidylate synthase; and    selecting said uridine analog when found to be activated by thymidylate synthase.    
     
     
         39 . The method according to  claim 38 , wherein the testing step comprises: 
 measuring cytotoxicity of said uridine analogue with respect to at least one cell line with a high expression of thymidylate synthase enzyme and at least one cell line with a low expression of thymidylate synthase enzyme; and    the selecting step comprises:    selecting said uridine analogue when the cytotoxicity measured with respect to the at least one cell line with a high expression of thymidylate synthase enzyme is greater than the cytotoxicity measured with respect to the at least one cell line with a low expression of thymidylate synthase enzyme.    
     
     
         40 . The method according to  claim 38 , wherein the uridine analog contains a radioisotope.  
     
     
         41 . The method of  claim 38  wherein the uridine analogue is a compound of the following general formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A=N, C;  
 B=H, hydroxyl, halogen, acyl (C 1 -C 6 ), alkyl (C 1 -C 6 ), alkoxy (C 1 -C 6 );  
 D=O, S, NH 2 ; and  
 G=substituted or unsubstituted cyclic sugar, substituted or unsubstituted acyclic sugar, substituted or unsubstituted mono, di, or tri-phospho-cyclic-sugar phosphate; substituted or unsubstituted mono, di, or tri-phospho-acyclic-sugar phosphate; substituted or unsubstituted mono, di, or tri-phospho-cyclic sugar analogues; substituted or unsubstituted mono, di, or tri-phospho-acyclic sugar analogues wherein the substituents are alkyl (C 1 -C 6 ), alkoxy (C 1 -C 6 )  or halogen.    
 
     
     
         42 . The method according to  claim 41 , wherein the compound contains a radioisotope.  
     
     
         43 . A method of imaging an organism, comprising the steps of: contacting the organism to be imaged with a compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A=N, C;  
 B=H, hydroxyl, halogen, acyl (C 1 -C 6 ), alkyl, alkoxy (C 1 -C 6 );  
 D=O, S,NH 2 ;  
 E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, or any substituent which is readily cleaved in the body to generate H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, or halogen;  
 at least one of W, X, Y, Z is a label or a label containing moiety having sufficient isotopic activity for imaging and the remainder of W, X, Y, Z=H, hydroxyl, halogen, alkyl (C 1 -C 6 ), substituted alkyl (C 1 -C 6 ), alkoxy (C 1 -C 6 ), substituted alkoxy (C 1 -C 6 );  
 J=C, S; and  
 K=O, C; and imaging the organism.  
 
     
     
         44 . A method of determining the proliferation rate of a tissue, comprising the steps of: contacting the tissue with a compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A=N, C;  
 B=H, hydroxyl, halogen, acyl (C 1 -C 6 ), alkyl, alkoxy (C 1 -C 6 );  
 D=O, S,NH 2 ;  
 E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, or any substituent which is readily cleaved in the body to generate H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, or halogen;  
 at least one of W, X, Y, Z is a label or a label containing moiety having sufficient isotopic activity for imaging and the remainder of W, X, Y, Z=H, hydroxyl, halogen, alkyl (Cl-C 6 ), substituted alkyl (Cl-C 6 ), alkoxy (Cl-C 6 ), substituted alkoxy (C 1 -C 6 );  
 J=C, S; and  
 K=O, C;  
 imaging the tissue; and  
 determining the amount of the compound incorporated into the tissue, wherein the amount of the compound incorporated into the tissue correlates to the proliferation rate of the tissue.  
 
     
     
         45 . The method of  claim 44 , wherein the compound is a labeled uridine analog.  
     
     
         46 . A method of assessing the response of tumors to treatment with a thymidylate synthase inhibitor, comprising the steps of: 
 (a)  administering a uridine analogue selected by a method comprising the steps of: providing a uridine analogue unsubstituted in the  5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase; wherein said uridine analogue is labeled with a positron emitter; and    (b)  determining an extent of maximum thymidylate synthase inhibition and persistence of thymidylate synthase inhibition over time between doses by external imaging.      
     
     
         47 . A method for assessing the response of tumors to treatment with a combination of thymidylate synthase inhibitor and at least one additional compound with antitumor activity, comprising the steps of: 
 (a)  administering a combination of a uridine analogue and at least one additional compound with anti-tumor activity, wherein said uridine analogue is selected by a method comprising the steps of:  
 providing a uridine analogue unsubstituted in the 5-position;  
 testing said uridine analog for activation by thymidylate synthase; and  
 selecting said uridine analog when found to be activated by thymidylate synthase;  
 wherein said uridine analogue is labeled with a positron emitter; and  
   (b)  determining an extent of maximum thymidylate synthase inhibition and persistence of thymidylate synthase inhibition over time between doses by external imaging.      
     
     
         48 . The method of  claim 47 , wherein said at least one additional compound with anti- tumor activity comprises a uridine analog.  
     
     
         49 . A compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A=N, C;  
 B=H, hydroxyl, halogen, acyl (C 1 -C 6 ), alkyl, alkoxy (C 1 -C 6 );  
 D=O, S, NH 2 ;  
 E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, or any substituent which is readily cleaved in the body to generate H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, or halogen;  
 at least one of W, X, Y, Z is a label or a label containing moiety having sufficient isotopic activity for imaging and the remainder of W, X, Y, Z=H, hydroxyl, halogen, alkyl (Cl-C 6 ), substituted alkyl (Cl-C 6 ), alkoxy (Cl-C 6 ), substituted alkoxy (C 1 -C 6 );  
 J=C, S; and  
 K=O, C.  
 
     
     
         50 . A compound according to  claim 49 , wherein E is selected from H, methyl and iodine.  
     
     
         51 . A method of diagnosing tumors which are resistant to thymidylate synthase inhibitors, comprising the steps of: 
 (a)  administering a uridine analogue selected by a method comprising providing a uridine analogue unsubstituted in the  5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase prior to obtaining biopsy specimens;    (b)  obtaining biopsy specimens; and      (c)  analyzing DNA from the biopsy specimens for extent of analogue incorporation.      
     
     
         52 . A method of diagnosing tumors which are resistant to thymidylate synthase inhibitors, comprising the steps of: 
 (a)  administering to said tumors a uridine analogue labeled with a positron emitting isotope selected by a method comprising providing a uridine analogue unsubstituted in the  5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase; and    (b)  analyzing DNA incorporation in said tumor by external imaging.      
     
     
         53 . A method of treating cancer, comprising: 
 administering a uridine analog in an amount effective to inhibit the replication or spread of cancer cells, said uridine analog selected by a method comprising the steps of:    providing a uridine analogue unsubstituted in the 5-position;    testing said uridine analog for activation by thymidylate synthase; and    selecting said uridine analog when found to be activated by thymidylate synthase.