Parallel Artificial Membrane Permeability Assay (PAMPA) training video | Pion Inc

Summary

This video provides a comprehensive demonstration of the Pion PAMPA (Parallel Artificial Membrane Permeability Assay) technology, a cost-effective tool for assessing drug permeability and predicting oral absorption. The tutorial covers the selection of plates, preparation of buffers and stock solutions, and step-by-step instructions for setting up the PAMPA sandwich and conducting assays. It also explains the proper use of UV plates for blank, reference, donor, and acceptor measurements, emphasizing careful handling to avoid cross-contamination or membrane disruption. Finally, the video guides users through data collection, refinement, and analysis using the Pamper Explorer software, ensuring reliable and accurate permeability results.

Takeaways

• 😀 PAMPA (Parallel Artificial Membrane Permeability Assay) is used to predict oral absorption and develop structure-permeability relationships in drug discovery.
• 😀 PAMPA is a cost-effective, high-throughput alternative to cellular models, allowing testing with minimal sample (5-10 µL) in 96-well plates.
• 😀 The PAMPA assay is based on a sandwich setup, consisting of a donor plate (bottom) and an acceptor plate (top) with lipid-coated membranes for permeability testing.
• 😀 Key reagents for the assay include ASB (Acceptor Sink Buffer), Prisma HD buffer at three pH values, and GIT lipid, all of which must be prepared and handled carefully.
• 😀 Stock solutions of test compounds are prepared in DMSO, ensuring they are particulate-free before use in the assay.
• 😀 The Excel input file, used by PAMPA Explorer software, must contain essential compound information such as sample names, concentrations, and molecular weights.
• 😀 The software setup involves assigning compounds to wells, defining pH values, and selecting replicate numbers for the assay. The pH values typically used are 5.0, 6.2, and 7.4.
• 😀 UV plates are prepared to collect spectral data before and after the assay, enabling quantification of permeability rates for each compound.
• 😀 The PAMPA sandwich setup is critical; it ensures proper sealing and prevents air gaps between the donor and acceptor plates during the incubation period.
• 😀 The assay involves a 4-hour incubation period in a humidity chamber, after which spectral data is collected from both the donor and acceptor plates to measure permeability.
• 😀 Data refinement in the software provides final permeability constants, which can be reviewed in table or spectral view, enabling detailed analysis of compound performance.

Q & A

What is the primary purpose of the PAMPA assay?

The PAMPA assay is designed to evaluate passive permeability of compounds, helping predict oral absorption and supporting the development of structure-permeability relationships in drug discovery.

How does PAMPA compare to Caco-2 assays?

PAMPA is a cost-effective, high-throughput screening tool that uses small sample volumes, whereas Caco-2 assays are cellular models that may provide more predictive results but are more resource-intensive and slower.

What are the main components of a PAMPA sandwich?

The PAMPA sandwich consists of a top acceptor plate with lipid-coated membranes and a bottom donor plate containing the sample solutions.

How is the ASB (Acceptor Sink Buffer) prepared and stored?

ASB is prepared by submerging it in warm water to reach room temperature and gently inverting to dissolve solids. Shaking should be avoided to prevent bubbles. The buffer is stored refrigerated and should be thawed before use if precipitates are present.

What pH values are used for the Prisma HD buffer in a GIT PAMPA assay?

The Prisma HD buffer is prepared at pH 5.0, 6.2, and 7.4 to simulate different gastrointestinal conditions.

What is the purpose of painting the GIT lipid onto the acceptor plate membrane?

Painting the lipid onto the membrane creates a lipid barrier to mimic biological membranes, which is essential for accurately measuring compound permeability.

How are stock solutions of test compounds prepared?

Stock solutions are prepared at 10 mM concentration by dissolving compounds in DMSO, sonicated for 1–2 minutes if necessary, ensuring they are particulate-free, with a volume of 0.5–1 mL per sample.

What steps are involved in assembling and incubating the PAMPA sandwich?

After filling the donor and acceptor plates, the acceptor plate is placed on top of the donor plate, aligned correctly to avoid air gaps, then the sandwich is placed in a humidity chamber with a wet sponge and incubated for 4 hours.

How is data collected from donor and acceptor plates after incubation?

Aliquots of 150 µL (transferred in two 75 µL steps) are moved from both donor and acceptor plates to fresh UV plates. Bubbles are removed, and UV spectral data is collected using the UV Plate Reader.

How does the PAMPA Explorer software process the assay data?

The software imports compound information from an Excel template, allows setting multi-pH conditions, starts the assay timer, and refines permeability constants. Data can be viewed in table form or spectral form for detailed analysis.

What precautions should be taken when transferring solutions to avoid membrane damage?

During pipetting, tips should not touch the membranes to prevent lipid detachment. Proper angles and careful handling are required, especially when painting lipid or transferring solutions to UV plates.

Why is a blank UV plate necessary in the PAMPA assay?

The blank UV plate measures baseline absorbance of the buffer to correct for background signals, ensuring accurate quantification of compound permeability.