PAMPA Assay | PAMPA Screening

The parallel artificial membrane permeability assay – PAMPA Assay is one of  our in vitro ADME screening services. We deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach. Permeability is an important physicochemical property of a drug, governing the availability of the drug in the systematic circulation. PAMPA measures passive diffusion and is therefore the first port of call when looking into permeability. It also has a higher throughput than Caco-2 or MDCK cell-based permeability assays.

Using GVK BIO’s ADME screening services is the fastest, and most cost-effective way to get permeability predictions for your compounds. Leveraging our unparalleled expertise in absorption measurements will get you reliable and reproducible results.

  • The Parallel Artificial Membrane Permeability Assay (PAMPA Assay) is used as an in vitro model of passive, transcellular permeation.
  • PAMPA Assay avoids the complexities of active transport, allowing test compounds to be ranked based on a simple permeability property alone.
  • The ability of PAMPA assay to evaluate permeability over a large pH range is valuable for an early understanding how new oral compounds might be absorbed across the entire gastrointestinal tract.

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What is PAMPA Assay?

The Parallel Artificial Membrane Permeability Assay (PAMPA Assay) is a method which determines the permeability of a substance from a donor compartment through an artificial phospholipid membrane into an acceptor compartment. Because the membrane has no transporters or efflux systems, only passive permeability is observed. The main objective is the classification of passively transported compounds via a simple and robust in vitro model of passive transcellular permeation.

Drug permeability is one of the most important factors to be considered for predicting drug oral bioavailability. During the early discovery and development phase, drug candidates are screened to select candidates that have better oral-absorption potential. Then a filter is applied to remove poor performers and identify candidates that need to be modified.

PAMPA is one of the most common methods to determine the permeability of substances from a donor compartment into an acceptor compartment through a lipid-infused artificial membrane. A membrane/acceptor compartment is placed on top of a multi-well microtiter plate to form a donor compartment. The test compound is added to the donor compartment at the beginning of the test, with the acceptor compartment being drug-free. After an incubation period which may include stirring, the assembly is separated and the amount of drug in each compartment is measured.

PAMPA Process

The original PAMPA method is first proposed by Kansy et al., this method has been widely used in the drug develop process to predict oral absorption, but it also exists several issues like poor correlation with human absorption and Caco-2 data, mass retention by the membrane, and relatively low stability of the membrane. So after years of development, a new PAMPA membrane (lipid-oil-lipid membrane, which is closer to biological membrane) was designed with advantages including no excessive solvents, structured layers, and short permeation pathway.

Parallel Artificial Membrane Permeability Assay

The PAMPA experimental protocol is as follows:

  1. The donor wells are filled with 180μl of system solution containing the compound (assay DMSO final concentration is 0.5%)
  2. The filter on the bottom of each acceptor well is filled with 200μl of acceptor sink buffer
  3. Assemble the sandwich plate and then incubate for 30 min at room temperature
  4. Disassemble the sandwich and measure the amount of compound present in both donor and acceptor wells
  5. Compare to the spectra obtained from reference standards and effective permeability

PAMPA Assay Description

Working solutions of each compound are prepared from 10 mM stock solution in DMSO diluted to a final concentration of 10 μM in PBS buffer (pH 7.4, 1% DMSO).

1% (w/v) lecithin/dodecane is added to the donor side of the Multi Screen Filter Plate, then 10 μM control or test compound working solution is added. The receiver side of the Multi Screen Filter Plates is filled with PBS buffer containing 1% DMSO. The plates are kept at room temperature for 24 h. Samples are collected from the donor and receiver sides. The donor sides samples are diluted 20-fold with PBS (1% DMSO).  All receiver and diluted donor side samples are mixed with ACN/MeOH (1:1, v/v) containing 25 ng/mL terfenadine and 50 ng/mL tolbutamide as internal standards. Samples are vortexed and then centrifuged at 4 °C. An aliquot of the supernatant is transferred to a 0.65 ml tube for LC-MS/MS analysis.

The MS detection is performed using a Sciex API 4000 instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – Solvent A: water with 0.1% formic acid,  solvent B: ACN with 0.1% formic acid.

Pion PAMPA Models

Gastrointestinal Permeability  The gastrointestinal tract (GIT) has a pH range from pH 1 – 8.  Since the pH of the blood is pH 7.4; there exists a pH gradient between the GIT and the plasma that can affect the transport of ionizable molecules. In an effort to simulate this pH gradient, we have chosen pH 7.4 for the acceptor compartment, and a pH range of 5.0, 6.2, and 7.4 in the donor compartment. To model transport conditions in the blood, the acceptor contains a scavenger binding molecule. Pion’s GIT-0 phospholipid is used for the assay.

Blood-Brain Barrier Permeability  CNS Screening of candidate drug molecules is done using a PAMPA assay to model blood-brain barrier (BBB) permeability. Rodent in vivo and in situ studies of the kinetics of drug uptake across the BBB are valuable tools for assessing factors important to steady-state brain penetration. These are relatively expensive and time consuming assays to do, and are done only sparingly. Animal studies can be augmented by PAMPA assay, which for BBB uses a mixture of phospholipids infused into lipophilic microfilters, with net negative lipid charge, a system mimicking many of the properties of brain lipid membranes. The data is used to predict brain uptake kinetics, as indicated by PS values determined by in situ perfusion methods.

Skin Penetration  Researchers and product developers can now measure drug permeability with a proprietary test system that is highly predictive of the human skin barrier. Unlike conventional skin test methods, Pion’s high throughput method is faster, less expensive and may be conducted manually or on robotic platforms.

The Skin PAMPA Test System excels as a complimentary screen prior to Franz Cell testing due to the lower cost, enhanced usability, higher throughput and reduced sample requirements. Pre-coated with a proprietary formulation, the 96 well plates are disposable, which completely eliminates cross contamination and the additional time and labor required for washing glass cells. Permeability measurements with the artificial skin-mimetic membrane exhibit a high correlation as tested against human skin in Franz cells.

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