Measurements of plasma protein binding – variety of experimental techniques

Abstract

Drug molecules in vivo may be bound to proteins and lipids in plasma and/or in tissues, or free (unbound) in diffusion among the aqueous environment of the blood and tissues. Data from in vitro plasma protein binding experiments that determine the fraction of protein-bound drug are frequently used in drug discovery [1]. Human plasma proteins contain around 40 % albumin (HSA), 1-acid glycoprotein (AGP) in much lower concentration (1-3 %) and immunoglobulins [2]. Methods used for drug – plasma protein binding (PPB) studies are numerous and can be divided into two main groups: separation methods (enabling the calculation of binding parameters, i.e. the number of binding sites and their respective affinity constants) and non-separation methods (describing predominantly qualitative parameters of the ligand-protein complex) [3]. Sometimes, results of PPB measurements obtained by different techniques are not consistent. High binding affinity to plasma proteins is not necessarily a crucial limiting factor for further delivery of compound to the target organ [1]. As an example, we show the study of the interactions between HSA/AGP and an “in-house” synthesized steroidal derivative that showed remarkable inhibitory potency against BoNT/A holotoxin in mouse embryonic stem cell derived motor neurons [4]. A variety of experimental techniques (ITC, HPLC, spectrofluorimetry, FTIR, and equilibrium dialysis) were used and the results were compared highlighting the advantages and disadvatages of various techniques.