Utility of protein in the prediction of in vivo effects

A method of evaluating disposition of a candidate compound in an in vitro culture and/or suspension to predict in vivo disposition of the candidate compound, the method comprising:

• (a) providing a cell culture and/or suspension, wherein the cell culture and/or suspension comprises a first sandwich culture of hepatocyte cells and providing a second sandwich culture and/or suspension of hepatocyte cells, each first and second sandwich culture comprising an artificial membrane system and at least one bile canaliculus, wherein the artificial membrane system comprises a cell transfected or knocked-out for transporters so as to be adapted to mimic cells, the first sandwich culture having an intact bile canaliculus and the second sandwich culture having a disrupted bile canaliculus;
• (b) exposing a candidate compound to the first sandwich culture and to the second sandwich culture for a time sufficient to allow uptake of the candidate compound;
• (c) simultaneously exposing the first and second sandwich cultures and/or suspension and the candidate compound to a medium containing proteins that are present extracellularly in liver at physiological concentrations to approximate the in vivo environment of hepatocytes in the liver, wherein the concentration of the proteins contained in the media is a physiologic concentration or a concentration having a characteristic similar to a physiological concentration;
• (d) washing and lysing the first and second cell cultures and/or suspensions;
• and
• (e) determining an amount of the candidate compound taken up in the first and second sandwich cultures and/or suspension, comprising determining an intracellular concentration of the candidate compound, to thereby evaluate disposition of the compound to predict in vivo disposition of the candidate compound without adjusting using protein binding data from a separate experiment.