HDM-PAMPA to predict gastrointestinal absorption, binding percentage, equilibrium and kinetics constants with human serum albumin and using 2 end-point measurements

Abstract

The parallel artificial membrane permeability assay (PAMPA) is a high-throughput screening (HTS) technique developed to predict passive permeability through numerous different biological membranes, such as the gastrointestinal tract (GIT), the blood brain barrier (BBB), and the dermal layer. PAMPA is based on an artificial membrane, such as hexadecane (HDM), which separates two compartments (i.e., a donor and an acceptor compartment). In the present study, an HDM-PAMPA method was developed with human serum albumin (HSA) under iso-pH and gradient-pH conditions to predict the percentage of binding, dissociation/association constants (K_d and K_a, respectively) and dissociation/association kinetic rates (k_off and k_on, respectively) between a given drug and HSA. Thanks to the kinetic properties of PAMPA, a two end-point assay was implemented to obtain all three properties. The assay was used to measure basic, acidic, and amphoteric compounds. The protein was free in solution, allowing a direct comparison between this assay and equilibrium dialysis (ED). The developed PAMPA enabled screening of up to 96 compounds in a single run, generating valuable information on absorption and distribution in a high-throughput and high-repeatable manner.

Keywords: Binding percentage; Dissociation/association constants; Dissociation/association microconstants; Gastrointestinal tract absorption; Human serum albumin; PAMPA; Passive permeability.