Investigation of Drug-Drug Interaction with Human Serum Albumin by Using Equilibrium Sampling Through Membrane and Biophysical Models

Competitive and displacement binding interactions of drugs with human serum albumin (HSA) take place when two or more drugs binding to the same binding site on HSA are co-administered into the blood stream. When two drugs simultaneously binding to two different neighboring binding sites on HSA, binding one of the two drugs to one site would influence binding the other drug to the other site, which is called allosteric drug-drug interaction that is not always straight forward to evaluate. We hypothesize that allosteric drug-drug interactions with HSA (compared to single drug interactions) may result in differences related to drug binding association constant as well as the number of drug molecules bound to each binding site on HSA. To test our hypothesis, we want to develop an integrated method that will enable us to measure only unbound (free) drug fraction in the drug-protein sample (Objective 1). From the free drug fraction determination, biophysical models, such as Bjerrum and Scatchard modeling (usually used in medical and pharmaceutical sciences), will be employed to quantitatively evaluate drug-drug interaction with HSA at molecular level; in terms of binding association constants and number of drug molecules binding on each of the two binding sites on HSA (Objective 2). Our approach is based on using a short piece of a miniaturized porous polymeric hollow-fiber membrane; the pores of which are impregnated with a tinny amount of an organic solvent and the fibers lumen is filled with a proper volume of buffer solution. This tinny sampling device is then put in the drug-protein solution until a thermodynamic equilibrium is reached (hence the approach is called Equilibrium Sampling Through Membrane, ESTM) at finely tuned nonedepleting drug extraction conditions. At these conditions, the free drug fraction in drug-protein solution is measured (Satisfying Objective 1), which will be used in Bjerrum and Scatchard models to further characterize drug-drug interaction with HSA (Satisfying Objective 2). New data and knowledge from studying two model drugs interacting with HSA will be the major outcome of this work. The approach used in this project is expected to be adapted for other applications concerning small molecule and large biomolecule interactions.