ADME Related Profiling in 96 and 384 Well Plate Format - A Novel and Robust HT-Assay for the Determination of Lipophilicity and Serum Albumin Binding

Abstract

The failure of about half of the drug candidates is associated with poor pharmacokinetic properties leading to a huge loss of time and money [1]. Early profiling of drug like properties provides important information in order to screen out insoluble, poorly absorbed and toxic compounds. Today, large compound libraries have to be screened, and of course the total number of compounds will rise over the next years leading to a growing demand for fully automated assays. A balance between quality, speed, throughput, cost and information content can be accomplished by the careful selection of assays and experimental conditions. Here we describe a novel 384 well format assay for two important ADME related descriptors (lipophilicity and serum protein binding) as input parameters for a precise prediction of fraction absorbed, blood/organ distribution coefficients and permeability, in order to maximize the information about a compound at an early stage of discovery.

Plasma Protein Binding and Volume of Distribution: Determination, Prediction and Use in Early Drug Discovery

“…The speed and simplicity offered by this approach has been somewhat superseded by the increased throughput of standardized in vitro methods utilizing whole plasma as detailed above. A variation on the HPLC methodology, TRANSIL technology, involves immobilization of HSA onto the inert surface of silica beads suspended in PBS buffer [46]. After compound addition and mixing, the beads are separated by low speed centrifugation and the resultant supernatant is analyzed by UV or MS. Validation has been performed on a range of compounds but there are few reported applications in the literature.…”

Adme

“…It is a kinetic parameter related to lipophilicity, which in turn is related to the partition and distribution coefficients. Physicochemical profiling of drug molecules is important in assessing the potential for good oral absorption [11,12]. At the same time, physicochemical properties are not independent of each other, and effective use is in the collective assessment of these parameters.…”

Application of Liposome Based Sensors in High-Throughput Screening Systems

“…TRANSIL ® membranes are stable, unilamellar, ordered and fluid, as shown by DSC and NMR studies, enabling functional incorporation of the integral membrane proteins for receptor binding studies or screening tests [134]. Another advantage of TRANSIL ® based assays over the liposomal ones is the substantial density difference between the TRANSIL ® spheres and the assay medium, which enables a decrease the separation time from several hours for liposome-based tests to minutes for TRANSIL ® assays [135]. However, the method requires an independent concentration determination.…”

“…In addition, the system can be used in two "modes" including a high-throughput (HT) and a high-precision (HP) mode. In the latter, a broader range of lipid concentrations is used, improving the precision of the measurement [135]. It has been shown that the lipid composition of TRANSIL ® spheres can be easily modified which enables different barriers the drug must cross in vivo to be mimicked, for example, by introduction of negatively charged phosphatidylserine (PS) for drugs designed to pass through the blood-brain barrier which is rich in negatively charged compounds [136].…”

“…Human serum albumin (HSA) covered TRANSIL ® beads, branded as TRANSIL ® -HSA, consist of covalently immobilized and randomly oriented HSA on a silica surface [135]. TRAN-SIL ® -HSA based high throughput binding assays enable fast determination of the albumin drug binding profiles which are crucial for the pharmacokinetic modeling and further drug development.…”