Permeability calculation from PAMPA data

Considerations in the Calculation of Permeability from PAMPA Data... 

The equations used to calculate permeability from PAMPA data are derived from Fick s law and assume that equilibrium between the donor and the receiver wells has not been achieved. The equations also assume that the membrane has been fully saturated by the sample upon leaving the donor well before permeation into the receiver well commences. For most compounds, this saturation time is extremely short and does not generally present a problem. Membrane retention also needs to be considered. This will tend to be higher for more lipophilic compounds and has sometimes been seen up to 90%. The consideration, or otherwise, of membrane retention will dictate which set of equations should be used and whether the resulting permeability value is termed the apparent permeability as is used in cell-based determinations or the effective permeability. 

Permeability can be assessed using parallel artiflcial membrane assays or PAMPA (Avdeef 2005). In the PAMPA assay, a sandwich is formed between two 96-well plates such that a donor and receptor cell is created, separated by a 125 pm Alter coated with a dodecane solution of dioleoylphosphatidylcholine. Drug diffusion from the donor to receptor compartments is measured analytically over time using either a UV plate-reader or by LC/MS and effective permeability calculated. Data is then binned as high (a score of 3), medium (a score of 2) or low (a score of 1) based on the following criteria ... 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

HT-solubility/permeability First, solubility is determined at four pH values by comparing the concentration of a saturated compound solution with its dilute, known as the concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support, PAMPA. The two artificial membranes presented here model the GIT and the BBB. Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are (/) reduction of sample usage and preparation time, ( /) elimination of interference from compound precipitation in membrane permeability determination, Hi) maximization of input concentration to permeability assay for improved reproducibility, and (/v) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity. 

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