Parallel Artificial Membrane Permeability Assay (PAMPA)

Drug absorption assessment is crucial in investigating ADME of novel pharmaceuticals. Creative Biolabs provides parallel artificial membrane permeability assay (PAMPA) to evaluatedrug absorption.

Figure 1. Schematic of the PAMPA model (Yu et al. 2015)

Passive diffusion is an important way for drug absorption. It influences drugs pass through the intestinal epithelium, the blood brain barrier or transport across cell membranes. The PAMPA model is an artificial in vitro model suitable for the passive diffusion determination (passive permeability) of a compound. In PAMPA model, a lipid-infused artificial membrane is generated and acts as a supplement for cell-based assays. Test compound is added to multi-well microtitre plate in the donor compartment and permeating an artificial hexadecane membrane to the acceptor compartment. The result is quantified by LC-MS/MS. Lucifer yellow is used to assess membrane integrity.

PAMPA allows permeability evaluation of a compound over a wide pH range and is valuable for an early understanding of its site of absorption along the gastrointestinal tract.

The PAMPA model allows assessment of:

• Contribution of passive diffusion in the drug absorption
• Influence of the pH on permeability
• Screening of compounds of interest
• Comparison of oral absorption efficiency for various pharmaceutical formulations
• Determination of absorption kinetic parameters

PAMPA Process

Permeation experiments were carried out in hydrophobic PVDF 96-well microtiter filter plates. Each well was coated with 17 µL of 35% (v/v) liquid membrane dissolved in hexane for 20 min to completely evaporate the solvent.

The typical PAMPA experimental protocol is as follows:

1. The donor plate was placed on a Teflon acceptor plate that had been prefilled with 280 µL of buffer containing 5% DMSO.
2. The filter on the bottom of each acceptor well is filled with 200 μL of acceptor sink buffer.
3. The resulting sandwich was incubated at room temperature under constant shaking (150 rpm).
4. The sandwich was disassembled and both donor and acceptor compartments were transferred to a UV quartz plate.
5. Compare to the spectra obtained from reference standards and effective permeability.
6. Each compound was measured in triplicate or quadruplicate.
7. Lucifer yellow is used to assess membrane integrity on the filter plate at the end of the incubation time.

Correlation with the Caco-2 Model

The PAMPA model determines the permeability by passive diffusion alone whereas Caco-2 permeability assay assesses the permeability by passive diffusion, by active uptake/efflux and by paracellular active transport. Therefore, if the compound crosses through the membranes only by passive diffusion, a good correlation is observed between the Caco-2 permeability assay and PAMPA. If the compound is a substrate for active efflux, the permeability will be overestimated by PAMPA. Similarly, if the compound undergoes active uptake or paracellular, the permeability will be underestimated by PAMPA.

Creative Biolabs offers a cost effective method with high reliability and accuracy to predict drug absorption. For more detailed information, please feel free to contact us or directly sent us an inquiry.

References

1. Yu H, Wang Q, Sun Y, et al. (2015) “A New PAMPA Model Proposed on the Basis of a Synthetic Phospholipid Membrane.” PLOS ONE 10(2): e0116502. doi: 10.1371/journal.pone.0116502
2. Ottaviani G, Martel S and Carrupt P A (2006). “Parallel Artificial Membrane Permeability Assay:  A New Membrane for the Fast Prediction of Passive Human Skin Permeability” J Med Chem 49(13): 3948–3954

For Research Use Only.