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Please provide an overview of the Cloe Screen PAMPA service.
PAMPA (parallel artificial membrane permeation assay) is an
in vitro
model of passive diffusion.
The test compound is added to the donor compartment of a 96 well plate. The permeation of compound across an artificial hexadecane membrane is quantified by LC-MS/MS after a five hour incubation at room temperature. Lucifer yellow is used to assess membrane integrity.
The apparent permeability for each compound (Papp) is calculated from the following equation:
Where VD and VA are the volumes of the donor and acceptor compartments, respectively, area is surface area of the membrane multiplied by the porosity and the equilibrium drug concentration is the concentration of test compound in the total volume of the donor and acceptor compartments.
How do I interpret the data from the PAMPA assay?
The PAMPA assay measures permeability across an artificial membrane. This
method provides an in vitro model for passive diffusion. Passive
diffusion is an important factor in determining transport through the
gastrointestinal tract, penetration of the blood brain barrier, as well
as transport across cell membranes. Permeability can also be influenced
by several other mechanisms including paracellular transport and active
uptake or efflux which are not assessed in PAMPA. Therefore, PAMPA provides
a simplistic approach to permeability by only measuring a single mechanism.
This avoids the complexities of active transport/efflux and enables the
compounds to be ranked on a single permeability property.
The compounds can be categorised into low and high permeability. Generally,
compounds which have a
Papp < 10 x 10-6cm/s
are classified as low permeability and compounds with a
Papp >10 x 10-6cm/s
are classified as high permeability.
What is a typical strategy for assessing the permeability of a compound?
Often, PAMPA is used as a primary permeability screen where a simple measure of passive
diffusion is required. However, using PAMPA results in isolation can be misleading in the understanding of true
in vivo permeability. Cell based assays, such as Caco-2, assess permeability by transcellular passive
diffusion as well as active and paracellular transport. Therefore, the Caco-2 permeability screen provides more
detailed mechanistic information. The PAMPA and Caco-2 assays can be valuable when used in conjunction to identify
the cause of poor absorption (Figure 4).
What is the relationship between Caco-2 and PAMPA?
PAMPA only measures permeability by passive diffusion whereas the Caco-2 permeability
assay also assesses active uptake/efflux and paracellular transport. Therefore,
a good correlation is observed between the Caco-2 permeability assay and PAMPA
if the compound crosses the membrane by passive diffusion alone. If the compound
is a substrate for active efflux then the PAMPA overestimates the permeability
and if the compound undergoes active uptake or paracellular then the PAMPA underestimates
the permeability. The relationship between Caco-2 permeability and PAMPA permeability
can be useful in diagnosing the mechanism of permeation. This is illustrated in Figure 4.
Figure 4
Illustration showing the typical relationship between Caco-2 permeability and PAMPA permeability3.
What pH do you use for PAMPA?
Typically, a pH of 7 is used. However, a number of different pH values can be investigated in PAMPA. Therefore, passive diffusion can be determined at the pH values observed in the gastrointestinal tract.
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