Cardiovascular safety assay

Abstract

The present invention provides assays and kits for the screening of test compounds for their capability to induce cardiotoxicity in a subject. Said assays and kits are based on the finding that the interaction of astemizole with the HERG potassium channel can be exploited to predict cardiotoxicity of compounds during the development of new therapeutics and other agents.

1 - 36 . (canceled) 
     
     
         37 . A kit comprising:
 a) a source containing a HERG polypeptide or functional fragment thereof;   b) radiolabeled astemizole of formula III:   
       
         
           
           
               
               
           
         
       
     
     
         38 . A kit according to  claim 37  wherein the source containing said HERG polypeptide or functional equivalent thereof is selected from:
 i) an isolated and purified nucleic acid which encodes a HERG polypeptide having an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 2 or a functional fragment thereof; 
 ii) an isolated and purified nucleic acid which encodes a HERG polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof; 
 iii) cells expressing on the surface thereof a HERG polypeptide having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or a functional fragment thereof; 
 or 
 iv) membrane preparations of cells expressing on the surface thereof a HERG polypeptide having an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 2 or a functional fragment thereof. 
 
     
     
         39 . A kit according to  claim 37  wherein the source containing HERG is an isolated and purified HERG polypeptide having an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 2 or a functional fragment thereof, bound to a solid support. 
     
     
         40 . A kit according to  claim 39  wherein the solid support is a fluorescer comprising solid support. 
     
     
         41 . A kit according to  claim 39  wherein the source containing HERG consists of membrane preparations of cells expressing on the surface thereof HERG polypeptide channels encoded by the nucleic acid sequence consisting of SEQ ID NO: 2. 
     
     
         42 . A kit according to  claim 41  wherein the cells are HEK293 cells. 
     
     
         43 . A kit according to  claim 42  optionally comprising means to remove the excess of unbound labeled reference compound from the incubation mixture. 
     
     
         44 . A kit according to  claim 42  wherein the separating means consist of GF/B filtration. 
     
     
         45 . An assay for screening test compounds, comprising:
 a) incubating an isolated and purified polynucleotide which encodes HERG comprising the nucleic acid sequence of SEQ ID NO: 2 or a functional fragment thereof with:
 i) radiolabeled astemizole of formula III 
   
       
         
           
           
               
               
           
         
         
           ii) the test compound; and 
         
         b) measuring the effect. 
       
     
     
         46 . An assay for screening test compounds, comprising:
 a) incubating an isolated and purified polynucleotide which encodes a HERG polypeptide having an amino sequence that is at least 80% identical to that of SEQ ID NO: 2; said nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 1 or a functional fragment thereof with:
 i) radiolabeled astemizole of formula III 
   
       
         
           
           
               
               
           
         
         
           ii) the test compound; and 
         
         b) measuring the effect. 
       
     
     
         47 . An assay for screening test compounds, comprising:
 a) incubating membrane preparations of cells expressing on the surface thereof a HERG polypeptide encoded by nucleic acid sequence consisting of SEQ ID NO: 1 with:
 i) radiolabeled astemizole of formula III 
   
       
         
           
           
               
               
           
         
         
           ii) the test compound; and 
         
         b) measuring the effect.