Parallel artificial membrane permeability assay
Evaluating compound permeability through cell a monolayer is a good indication of intestinal permeability and oral bioavailability. The parallel artificial membrane permeability assay (PAMPA) provides a high throughput, non-cell based method for predicting passive, transcellular intestinal absorption, the process by which the majority drugs enter circulation. In the PAMPA method, an artificial more ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, Lake Nona, FL)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number:
Assay Provider: Dr. Layton Smith, Sanford-Burnham Medical Research Institute
Evaluating compound permeability through cell a monolayer is a good indication of intestinal permeability and oral bioavailability. The parallel artificial membrane permeability assay (PAMPA) provides a high throughput, non-cell based method for predicting passive, transcellular intestinal absorption, the process by which the majority drugs enter circulation. In the PAMPA method, an artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. The drug is introduced in the donor compartment. Following the permeation period, the donor and acceptor compartments are quantified using LC/MS/MS.
The gastrointestinal tract (GT) has a pH range from pH 1-8. The pH of the blood is constant at pH 7.4; therefore it is possible for a pH gradient to exist between the GT and the plasma that can affect the transport of ionizable molecules. In an effort to create such gradient pH conditions, we offer an alternative assay with pH 7.4 for the acceptor compartment and pHs 5.0, 6.2, and 7.4 in the donor compartment.
PAMPA is a well established predictive assay that models the absorption of drugs in the gut. However, PAMPA is an artificial system that may provide inaccurate and potentially misleading results. Despite these limitations, PAMPA can be a useful tool to prioritize lead compounds in early stages of development. The caco-2 cell permeability assay is the industry standard for in vitro prediction of intestinal absorption of drugs, but it too has its limitations. Caco-2 cells require extensive culturing (20+days), and often fail to form the cohesive monolayer necessary for uniform transport of compounds across the cell layer. The assay requires a significant amount of compound to perform the assay (typically ~20mg). Together, the limitations of time and compound consumption decrease the value of the results obtained by caco-2 at the early stages of drug development.
1) Aqueous buffer solution (System Solution, pION Inc) pH 5.0, 6.2, and 7.4.
2) 20% acetonitrile/aqueous buffer solution (Cosolvent System Solution, pION Inc) pH 5.0, 6.2, and 7.4 (acetonitrile is used to render the compounds more soluble during the assay).
3) A "sandwich" plate (pION Inc) consisting of a donor bottom plate with stir bars and an acceptor filter plate.
4) GIT-0 lipid (pION Inc).
5) Acceptor Sink Buffer, pH 7.4 (pION Inc) containing a surfactant to mimic the function of serum proteins
6) Gut-Box(tm) (pION, Inc) for moderate stirring (40 um Aqueous Boundary Layer thickness).
7) Control compounds: Verapamil HCl (highly permeable), Metoprolol (moderately permeably), and Ranitidine (poorly permeable), final concentration in assay: 50 uM.
8) Test compounds, final concentration in assay: desirably as high as the control compounds.
9) The Tecan Freedom Evo 150 automated liquid handling instrument (Tecan US) is used in this assay.
1) Permeability was assessed using the Parallel Artificial Membrane Permeability Assay, PAMPA, in a 96-well format, in duplicate.
2) The donor wells are filled with 180 ul of system solution (and/or 190 uL of cosolvent system solution) containing the compound (assay DMSO final concentration: 0.5%).
3) The filter on the bottom of each acceptor well is coated with GIT-0 lipid and filled with 200 ul of Acceptor Sink Buffer.
4) The sandwich plate is assembled and incubated for 30 min in the Gut-Box(tm).
5) After the permeation time, the sandwich is disassembled and the amount of compound present in both the donor and acceptor wells is measured (by UV absorbance, 250-498 nm, using the Infinite M200, Tecan US).
6) The results are compared to the spectra obtained from reference standards and the effective permeability, Pe, is calculated using the software PAMPA Evolution Plus, version 3.2 (pION Inc). Mass balance is used to determine the amount of material embedded in the membrane filter.
Active compounds in PAMPA will have a log Pe at pH 7.4 of <= - 3.5
Scoring for single-concentration screening:
Activity scoring rules developed at San Diego Center for Chemical Genomics employs a 3-tiered system:
1) The first tier (0-40 range) is reserved for primary or single-concentration screening data and is not applicable to this assay.
2) The second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits that are cherry picked from the HTS mother plates and is not applicable to this assay.
3) The third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues.
Compounds that are inactive in this assay receive a score of 81.
Compounds that are active in this assay receive a score of 90.
** Test Concentration.
Data Table (Concise)