Parallel Artificial Membrane Permeability Assay

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.

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.

Key Features

  1. Improvement of correlation with human absorption and Caco-2
  2. Lower mass retention
  3. High stability and reproducibility
  4. Compatible with buffers containing organic solvents
  5. Professional technical support and consultation

PAMPA Methods

I.Original PAMPA
II.DOPC-PAMPA
III.HDM-PAMPA
IV.Bio-mimetic PAMPA (BM-PAMPA)
V.Double-Sink™ PAMPA (DS-PAMPA)
VI.BD Gentest™ Pre-coated PAMPA Plate

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.

The schematic diagram of PAMPA model

Fig. The schematic diagram of PAMPA model


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.

Creative Bioarray offers a cost effective method with high reliability and accuracy to predict drug oral bioavailability. With multiple specialists in this area and years of experience, we choose BD Gentest™ Pre-coated PAMPA Plate to perform PAMPA, and we guarantee you quality data and customized results to meet your project requirements. If you have any additional requirements or questions, don’t hesitate to contact us. Creative Bioarray will be your best partner.

Case Study

1. Preparation of BBB PAMPA Assay

150 µL of 10 µM compound donor solutions were added to each well of the donor plate, whose PVDF membrane was precoated with 5 µL of 1% brain ploar lipid extract (Porcine) /dodecane mixture. 300 µL of PBS was added to each well of the PTFE acceptor plate. The donor plate and acceptor plate were combined together and incubated for 4h.

Parallel Artificial Membrane Permeability AssayFigure 1. PAMPA Model.

2. Method Validation

Apparent permeability and recovery of the test compounds was determined in duplicate. Compounds were quantified by LC-MS/MS analysis based on the peak area.

Table 1. Papp and Efflux Ratio Determination in BBB PAMPA model
Compound IDMean Papp (10-6 cm/s)Mean Recovery %Rank
Atenolol<0.015<103.7Low
Propranolol66.88640.8High
Sample#126.25940.7High

3. Data Analysis and criteria

  • Papp = (dQ/dt) × (1/C0) × (1/A)
  • %Recovery = (Total compound mass in donor and receiver compartments at the end of the incubation /Initial compound mass in the donor compartment) x 100

Note: dQ/dt is the permeability rate, C0 is the initial concentration in the donor compartment, and A is the surface area of the membrane.

Criteria:

  • Low permeability, Papp < 1;
  • Moderate permeability, 1 < Papp < 10;
  • High permeability, Papp > 10;

For research use only. Not for any other purpose.