Abstract
Parallel artificial membrane permeability assay (PAMPA) is a screening tool for the evaluation of drug permeability across various biological membrane systems in a microplate format. In PAMPA, a drug candidate is allowed to pass through the lipid layer of a particular well during an incubation period of, typically, 10–16 h. In a second step, the samples of each well are transferred to a UV-Vis–compatible microplate and optically measured (applicable only to analytes with sufficient absorbance) or sampled by mass-spectrometric analysis. The required incubation period, sample transfer, and detection methods jointly limit the scalability of PAMPA to high-throughput screening format. We introduce a modification of the PAMPA method that allows direct fluorescence detection, without sample transfer, in real time (RT-PAMPA). The method employs the use of a fluorescent artificial receptor (FAR), composed of a macrocycle in combination with an encapsulated fluorescent dye, administered in the acceptor chamber of conventional PAMPA microplates. Because the detection principle relies on the molecular recognition of an analyte by the receptor and the associated fluorescence response, concentration changes of any analyte that binds to the receptor can be monitored (molecules with aromatic residues in the present example), regardless of the spectroscopic properties of the analyte itself. Moreover, because the fluorescence of the (upper) acceptor well can be read out directly by fluorescence in a microplate reader, the permeation of the drug through the planar lipid layer can be monitored in real time. Compared with the traditional assay, RT-PAMPA allows not only quantification of the permeability characteristics but also rapid differentiation between fast and slow diffusion events.
Highlights
Drug absorption is a key factor in the evaluation of potential active pharmaceutical ingredients at the early stage of drug development
Most analytes caused a significant decrease of the fluorescence intensity, and changes >10% were considered to be sufficiently strong to be readily followed by RT-Parallel artificial membrane permeability assay (PAMPA), where a lower sensitivity was expected because of the temporal requirement of permeation
The concentrations of the analytes in the fluorescence response experiments were conservatively set as 0.1 mmol/L, below the test concentrations used in the subsequent RT-PAMPA (1 mmol/L), such that already the permeation of a fraction of analyte from the donor to the acceptor well should ensure a fluorescence response in the upper
Summary
Drug absorption is a key factor in the evaluation of potential active pharmaceutical ingredients at the early stage of drug development. Because of its implementation as a single-point assay, the permeability value with PAMPA is obtained after a specific incubation time, typically 10–16 h (Sugano et al, 2001). At this end point, the permeation process is interrupted by separating the acceptor from the donor plate and the samples of the individual wells are transferred to microplates suitable for UV-Vis quantification; for drug candidates with insufficient UV-Vis absorption, liquid chromatography–mass spectrometry (LC/MS) techniques are required for quantitative analysis, adding instrumental–analytical work for each and every well of analyte (Hanlan et al, 2003)
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