We provide various in vitro models for assesing the compounds ability to traverse various biological membranes. These models are used to study the bioavailability and distribution in in vitro conditions.
PAMPA membrane permeation
PAMPA (parallel artificial membrane permeation assay) assay provides information on the ability of the compound to traverse through artificial phospholipid based membrane, which is mimicking cell membrane. Use of artificial membrane ensures the transport is done by passive diffusion due to lack of different transporter enzymes. Comparison of the results from PAMPA and cell based models (like Caco-2 and MDCK) are used to estimated the role of transporters in compound permeation and also in efflux.
PAMPA models composed of different lipids are available in addition to a BBB-PAMPA model, which by its lipid composition, is resembling more of the blood-brain-barrier.
|Apparent permeabilities of study and reference compounds across PAMPA membrane at the indicated different pH values. Mean ± SD, n=2|
Conducting PAMPA experiments is fast and as being an artificial membrane, it requires no growth of dense cell layers. We conduct PAMPA experiments at different pH ranges to study the effect of the ionized forms of the test compound. We also include positive and negative permeation controls. Detection of te permeability is done with UPLC-MS or UPLC-UV method.
Cell based permeation studies
Cell based permeation studies involve a transwell cell culture setup, in which the cells are grown on a permeable filter membrane inserts. Dense cell layers polarize on this filter layer to apical and basolateral sides (basolateral side facing to the solid filter growth support). The test compounds can be applied on either side of the cell layer (to the cell insert or the culture plate medium), which allows study of the permeation / transport form apical to basolateral (AB) and basolateral to apical (BA) directions.
Typically the study involves sampling of the test compounds on both sides at given time points, followed by analysis of the test item concentration by UPLC-MS or UPLC-UV method. Based on the measured concentrations, apparent permeation coefficient is calculated (P(app)). We include a set of reference compounds with low and high permeation profiles in cell based permeation studies, to overcome the typical biological variation in cell based permeation measurements. These reference compounds can include lucifer yellow, mannitol, atenolol, sotalol, pindolol, metoprolol, propranolol and quinidine. We also check the cell layer is intact in each well by a TEER (transeptihelial electrical resistance) measurement before the experiment.
The cell based permeation models have some active drug transporter systems, including P-glycoprotein efflux transporter (P-gp, also known as multidrug resistance protein 1, MDR1, a member of the ABC transporters). The involvement of the transporters can be estimated both in the apical-to-basolateral vs basolateral-to-apical permeabilities as well as by using specific transporter inhibitors, like vanadate. In addition, we can also analyze the ability of the compound to inhibit P-gp transporter by inclusion of known P-gp substrates and monitoring their transport +/- the test compounds (see FDA guidance and EMEA guidance).
The cell based permeation models are somewhat also dependent on the size of the test compounds. Small compounds (below ~250 Da) may permeate the cell layer also paracellularly between the cells, whilst the permeation through the cells is the main route for larger compounds.
Caco-2 monolayer permeation
The immortalized human epithelial colorectal adenocarcinoma cell line colon carcinoma (Caco-2) provides a the most common way to measure the compounds ability to be absorbed from small intestine in apical to basolateral (AB) or as an efflux model from basolateral to apical (AB) direction. Caco-2 cells contain the typical epithelial tight junctions and are expressing both influx and eflux transporters. We use commercial Caco-2 plates in our permeation experiments.
|Apparent permeability coefficients (x10-6 cm/s) of studied compounds. The data is presented as average±SD (n=3)|
MDCK cell layer permeation
Madin-Darby canine (dog) kidney epithelial cells (MDCK) provide anoter very popular in vitro cell based permeation model. MDCK cells are also available as stably transfected with different transporters. MDCK cell models can be also used in both AB and BA directions and the roles of the transporters be assessed.
Human skin permeation
Permeation study on human cadaver skin to characterise compounds and formulations intended for topical administration.
Human abdominal cadaver skin from healthy donors is obtained from Kuopio University Hospital under the permission from National Board of Medicolegal Affairs. Epidermis is separated from the underlying dermis by immersion in distilled water at 60 °C. The isolated membrane is dried and stored at –20 °C. We conduct human skin permeation studies in collaboration with the University of Eastern Finland.