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

A method for determining the concentration of at least one protease inhibitor present in free form within a test medium, said test medium also containing a bound form of said protease inhibitor with at least one plasma protein in equilibrium with said free form, the method comprising:

• i) providing at least one plasma protein;
• ii) providing at least one protease inhibitor;
• iii) quantifying the concentration of protease inhibitor in free form through isothermal titration calorimetry measurements.

A kit for determining the binding affinity of at least one protease inhibitor to at least one plasma protein comprising:

• i) at least one plasma protein, and variants thereof;
• ii) at least one protease inhibitor.

A kit for determining the concentration of at least one protease inhibitor present in free form within a test medium, said test medium also containing a bound form of said protease inhibitor with at least one plasma protein in equilibrium with said free form, the kit comprising:

• i) at least one plasma protein, and variants thereof;
• ii) at least one protease inhibitor.

A kit for determining the antiviral activity or EC50 value of at least one protease inhibitor in the presence of at least one plasma protein, the kit comprising:

• i) at least one plasma protein, and variants thereof;
• ii) at least one protease inhibitor.

A kit for determining the in vivo efficacy of at least one protease inhibitor in human plasma, the kit comprising:

• i) at least one plasma protein, and variants thereof;
• ii) at least one protease inhibitor.