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.

37 . (new) An assay for screening test compounds, comprising: 
 a) incubating a source containing HERG or a fragment thereof with: 
 i) radiolabeled astemizole of formula (III)  
                     
 ii) the test compound; and  
   b) measuring the effect of the test compound on the amount of reference compound bound to HERG.    
     
     
         38 . (new) An assay for screening test compounds for their ability to induce cardiotoxicity in a subject, comprising: 
 a) incubating a source containing HERG or a fragment thereof with: 
 i) radiolabeled astemizole of formula (III)  
                     
 ii) the test compound; and  
   b) measuring the effect of the test compound on the amount of reference compound bound to HERG.    
     
     
         39 . (new) An assay for screening test compounds for their capability to induce cardiac arrhythmia in a subject, comprising: 
 a) incubating a source containing HERG or a fragment thereof with: 
 i) radiolabeled astemizole of formula (III)  
                     
 ii) the test compound; and  
   b) measuring the effect of the test compound on the amount of reference compound bound to HERG.    
     
     
         40 . (new) An assay according to  claim 37 , wherein the source containing HERG is selected from the group consisting of: 
 i) an isolated and purified nucleic acid which encodes HERG having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or a fragment thereof;    ii) an isolated and purified nucleic acid which encodes HERG comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof;    iii) cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof; or    iv) membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof.    
     
     
         41 . (new) An assay according to  claim 37 , wherein the source containing HERG are membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel consisting of SEQ ID NO:2.  
     
     
         42 . (new) Radiolabeled astemizole of formula (III)  
       
         
           
           
               
               
           
         
       
     
     
         43 . (new) A process for preparing radiolabeled astemizole as in  claim 42  comprising: 
 a) demethylating astemizole of formula (I) using a suitable reagent such as 48% aqueous hydrobromic acid:  
                     
 b) reacting the intermediate of formula (II) with [ 3 H]-methyliodide (CT 3 I) optionally in a reaction inert solvent and in the presence of a base to obtain said radiolabeled astemizole:  
                     
 
     
     
         44 . (new) A high-throughput assay for screening test compounds, the assay comprising: 
 a) contacting membrane preparations of cells expressing on the surface thereof HERG polypeptide channels having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 of fragments thereof, with radiolabeled astemizole as claimed in  claim 42 , for a time sufficient to allow binding of the reference compound with the HERG polypeptide channel;    b) contacting membrane preparations of cells expressing on the surface thereof HERG polypeptide channels having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or fragments thereof, with radiolabeled astemizole of step a) together with the test compound for a time sufficient to allow binding of the reference compound and of the test compound with the HERG polypeptide channel;    c) measuring the amount of radiolabeled astemizole bound to the HERG channel in step a);    d) measuring the amount of radiolabeled astemizole bound to the HERG channel in step b); and    e) compare the amount of radiolabeled astemizole bound to the HERG channel measured in step a) with the amount of radiolabeled astemizole bound to the HERG polypeptide channel measured in step b).    
     
     
         45 . (new) A high-throughput proximity detection assay for screening test compounds the assay comprising: 
 i) HERG labeled with a first label capable of participating in a proximity detection assay;    ii) Radiolabeled astemizole as claimed in claim  6  capable of participating in a proximity detection assay;    iii) contacting HERG of step i) and radiolabeled astemizole of step ii) together with a test compound for a time sufficient to allow binding of the reference compound and of the test compound to HERG; and    iv) detect an interaction between HERG of step i) and radiolabeled astemizole of step ii) by means of proximity of the first label with the second label when HERG and the reference compound interact.    
     
     
         46 . (new) A kit comprising: 
 a) a source containing HERG;    b) radiolabeled astemizole as claimed in  claim 42 .    
     
     
         47 . (new) A kit according to  claim 46  wherein the source containing HERG is being selected from: 
 i) an isolated and purified nucleic acid which encodes HERG having an amino acid sequence that is at least 80% identical to that to SEQ ID NO:2 or a fragment thereof;  
 ii) an isolated and purified nucleic acid which encodes HERG comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof;  
 iii) cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof; or iv) membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof.  
 
     
     
         48 . (new) A kit according to  claim 46  wherein the source containing HERG is an isolated and purified HERG polypeptide channel or a fragment thereof, bound to a solid support.  
     
     
         49 . (new) A kit according to  claim 48  wherein the solid support is a fluorescer comprising solid support.  
     
     
         50 . (new) A kit according to  claim 46  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.  
     
     
         51 . (new) A kit according to  claim 50  wherein said cells are HEK293 cells.  
     
     
         52 . (new) A kit according to  claim 51  optionally comprising means to remove the excess of unbound labeled reference compound from the incubation mixture.  
     
     
         53 . (new) A kit according to  claim 51  wherein the separating means consist of GF/B filtration.  
     
     
         54 . (new) Use of an isolated and purified polynucleotide which encodes HERG comprising the nucleic acid sequence of SEQ ID NO:2 or a fragment thereof in an assay according to  claim 37 .  
     
     
         55 . (new) Use of an isolated an purified polynucleotide which encodes HERG comprising the nucleic acid sequence of SEQ ID NO:1 or a fragment thereof in an assay according to  claim 37 .  
     
     
         56 . (new) Use of cells expressing on the surface thereof the HERG polypeptide channel comprising the amino acid sequence of SEQ ID NO:2 or a fragments thereof in an assay according to  claim 37 .  
     
     
         57 . (new) Use of membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof in an assay according to  claim 37 .  
     
     
         58 . (new) Use of membrane preparations of cells expressing on the surface thereof HERG polypeptide channels encoded by the amino acid sequence consisting of SEQ ID NO:2 in an assay according to  claim 37 .  
     
     
         59 . (new) Use of radiolabeled astemizole as claimed in claim  6  in an assay according to  claim 37.