Calculation of Permeability from PAMPA Data

3 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 is a kinetic process, and is quoted as a rate. In cell-based assays such as Caco-2, a number of time points are generally taken from both the donor (apical) wells and the receiver (basolateral) wells. Because of this, retention on the membranes is not determined and the permeability values quoted are apparent, Pa. The main benefit of PAM PA type assays is their usefulness as high-throughput tools and therefore taking time point measurements will create a bottleneck. Measurements of the donor and receiver wells are made at the end of the incubation period and referenced back to the starting concentration of the solute in the donor wells. 

Membrane retention values can be determined using mass balance and inclusion of this gives the effective permeability, Pe. These are shown in Equations 6.4—6.6  

Effective permeability (Pe) and apparent permeability (Pa) can be determined using Equations 6.4 and 6.5 and Equation 6.6, respectively, where A is the membrane area (cm2), t is the incubation time (s), VR is the volume of the receiver well (cm3), VD is the volume of the donor well (cm3), CR is the concentration of solute in the receiver well at time t (mol/cm3), CD is the concentration of the solute in the donor well at time t = 0 (mol/cm3), and Ce is the concentration of the solute at equilibrium (mol/cm3). 

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