Methods for determining plasma free drug concentration by direct measurement of binding affinity of protease inhibitors to plasma proteins

Abstract

Methods for isothermal titration calorimetry analysis of the binding affinity of protease inhibitors to plasma proteins. A method that can quantitatively calculate free drug concentrations of protease inhibitors in human plasma, as well as a method to calculate therapeutic amounts and dosage regimens. Furthermore, the present invention provides a method that can calculate the effect of plasma proteins on the antiviral activity (EC50 values) of protease inhibitors from their binding affinities to plasma proteins. The present invention provides as well a method that can evaluate the in vivo anti-HIV efficacy of PIs in human plasma.

1 - 37 . (canceled)  
   
   
       38 . A method for determining the antiviral activity or EC 50  value of at least one protease inhibitor in the presence of at least one plasma protein, which comprises: 
 i) providing at least one plasma protein;    ii) providing at least one protease inhibitor; and    iii) quantifying a binding affinity between the at least one plasma protein and the at least protease inhibitor through isothermal titration calorimetry measurements;    thereby determining the antiviral activity or EC 50  value of at least one protease inhibitor in the presence of at least one plasma protein.    
   
   
       39 . The method according to  claim 38 , wherein the plasma protein is chosen from albumin, aI-acid glycoprotein, and lipoprotein.  
   
   
       40 . The method according to  claim 38 , wherein the method is a high throughput screening method.  
   
   
       41 . The method according to  claim 38 , wherein the method further comprises providing at least one competitive binding agent.  
   
   
       42 . The method according to  claim 38 , wherein the method further comprises a step of providing one or two additional drugs.