Parallel Artificial Membrane Permeability Assay (PAMPA)
Creative Bioarray is a reliable Parallel Artificial Membrane Permeability Assay (PAMPA) provider. Over the past years, Creative Bioarray has developed expertise in PAMPA. By working with a variety of customers, we can perfectly meet your project requirements and budgets.
- Diffusion and absorption of drugs
Drug permeability is one of the most important factors to be considered for predicting oral drug bioavailability. Permeation mechanisms through biological barriers include active transport, passive diffusion, paracellular, and efflux. The body absorbs nearly 80~95% of the recorded commercial drugs through passive diffusion.
For permeability screening, the Caco-2 cell monolayer permeation method has been widely and successfully used in drug discovery and early development. With the need for reduced cost, increased predictability, and higher throughput in drug discovery, other methods are needed to assist Caco-2 assay.
- Why PAMPA?
PAMPA uses two aqueous buffer solution holes separated by an artificial membrane. The artificial membrane is composed of a lipid-oil-lipid sandwich structure in an organic diluent supported by a porous filter plate matrix. And then, the test compound diluted in the buffer is placed in the donor well. The compound enters the artificial membrane from the donor pore, entering the acceptor pore by passive diffusion. The compound's effective permeability (Pe) is utilized to determine the rate of permeation. Compared with cell-monolayer methods, the time required for the experiment is greatly reduced. Only passive diffusion is tested, and there is no metabolism, no transporter proteins, so there is no need to worry about saturation.
- Improvement of correlation with human absorption and Caco-2
- Lower mass retention
- High stability and reproducibility
- Compatible with buffers containing organic solvents
- High throughput
- No metabolism
- Rapid quantitation
- Low cost
The PAMPA permeability test is based on the passive diffusion of the target compound through the artificial membrane. PAMPA synthetic membrane has a lipid-oil-lipid sandwich structure built into the pores of the porous filter. The middle oil layer maintains a strong and stable PAMPA membrane, ultra-thin to minimize compound retention and interference with compound penetration (Avdeef, 2005; Kerns et al., 2004).
Figure 1. The schematic diagram of the PAMPA model
- Preparation of PAMPA Assay
Compound donor solutions were added to each well of the donor plate, whose PVDF membrane was precoated with 5 µL of 1% lecithin/dodecane mixture. 300 µL of PBS was added to each well of the PTFE acceptor plate. The donor plate and acceptor plate were combined and incubated for 4h.
Figure 2. PAMPA Model.
- Method Validation
The apparent permeability and recovery of the test compounds were determined in duplicate. Compounds were quantified by LC-MS/MS analysis based on the peak area.
|PAMPA Test Results|
|Compound||Test Conc. (µM)||Incubation Time (hour)||Mean Pe (nm/s)||Mean %Recovery||Permeability|
VD = 0.15 mL; VA = 0.30 mL; Area = 0.28 cm2; time = 14400 s.
[drug]acceptor = (Aa/Ai×DF)acceptor; [drug]donor= (Aa/Ai*DF)donor;
Aa/Ai: Peak area ratio of analyte and internal standard; DF: Dilution factor.
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- Avdeef, A. The rise of PAMPA. Expert Opinion on Drug Metabolism & Toxicology, (2005), 1(2), 325-342.
- Kerns, E. H.; et al. Combined Application of Parallel Artificial Membrane Permeability Assay and Caco-2 Permeability Assays in Drug Discovery. Journal of Pharmaceutical Sciences, (2004), 93(6), 1440-1453.
For research use only. Not for any other purpose.