ABSORPTION: PASSIVE AND ACTIVE TRANSPORT (PERMEABILITY) -2

Parallel Artificial Membrane Permeation Assay (PAMPA):   

As the name suggests, this is a cell-free assay to predict passive, transcellular permeability of the drugs in early drug discovery.  These artificial membranes have no active transport systems and  no metabolizing enzymes, moreover this assay would not provide us the information regarding actively transported molecules.  This gives purely passive diffusion of unchanged compound.

The ability of this 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.

·         The artificial membrane is first built-up by pipetting a solution of lipids on a supporting filter material in micro titer plates ( say 96-well plates)

·         There should also be a control well where lipid layer is absent.

·         Test compounds are added to the donor compartment.

·         Permeation occurs through the artificial membrane into the acceptor compartment

·         Now compound permeated to the acceptor compartment is measured photometrically or by LCMS.

In few protocols, 96-well microtitre plate and a 96-well filter plate is sandwiched such that each composite well is divided into two chambers: donor at the bottom and acceptor at the top.

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 mimic this pH gradient in vitro, alternative assays with pH 7.4 for the acceptor compartment and pH values 5.0, 6.2, and 7.4 in the donor compartment are used.

Permeability values are reported in (10^-6 cm/s)

Compounds which have a

PAMPA: Papp < 10 x 10-6cm/s are classified as low permeability and 

               Papp > 10 x 10-6cm/s are classified as high permeability

___________________________

Caco-2: 

The Caco-2 cell line is derived from a human colon (large intestine) carcinoma.

The cells have characteristics that resemble intestinal epithelial cells such as the formation of a polarized monolayer, well-defined brush border on the apical surface and intercellular junctions.

Caco-2 cells express tight junctions, microvilli, and a number of enzymes and transporters that are characteristic of such enterocytes lining the small intestine.

     ·         Caco-2 human colon adenocarcinoma cells are grown to confluence (to form a monolayer covering the surface of the filter). 

·         Test compounds are added to apical side of the compartment.

·         Permeation occurs through the top side of the monolayer,

·         Now compound permeated to the basolateral compartment is measured photometrically or by LCMS.

·         Apparent permeability coefficient (P_app in cm/s) is determined. 

Rule of thumb numbers for Caco-2 permeability 

Low Caco-2 permeability (<5 x 10-6 cm/s) ; high Caco-2 permeability (>20 x 10-6 cm/s)

Caco-2 assay allows us to assess transport of the drug in both directions i.e. apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer. (known as bi-directional Caco-2 permeability assay)

Thus enabling us to determine an efflux ratio which provides an indicator as to whether a compound undergoes active efflux.

The result is typically reported as an efflux ratio i.e. Papp(B-A)/Papp(A-B). If the efflux ratio is greater than two (Papp(B-A)/Papp(A-B) > 2) then this indicates drug efflux is occurring.

PAMPA is a pre-screening tool in early drug discovery.  Molecules/compounds which behaved well in PAMPA assay (permeated well) are further test with Caco-2 model.

PAMPA allows high throughput screening of molecules (compared to Caco-2)

Caco-2 model is more descriptive – as it gives passive as well as active transport. 

Madin-Darby Canine Kidney (MDCK) cells are also used in place of Caco-2 cells for permeability assays.  These cells can also be grown as a monolayer with tight junctions.  However, MDCK cells are from canine source (dog or dog-like mammals).  Moreover, these are kidney-derived, not intestine-derived as Caco-2 cells.  Therefore, given a choice Caco-2 is more desirable than MDCK.

___________________________

Ø  MDR1-MDCK cells are common choice for evaluating P-gp substrates and inhibitors.  Cell lines are transfected with transporters of interest.  MDR1 is the gene encoding for the efflux protein, P-glycoprotein (P-gp)

Ø  P-gp is one of the mosot well-recognized efflux transporters in many tissues including the brain, kidney and intestine.

Ø  Therefore, MDR1-MDCK has been found to be useful predictor of blood brain barrier permeability.

MDR1-MDCK P_app (apical to basolateral) → gives an indication of the extent of permeation across cells which express P-gp (e.g. gastrointestinal tract and blood brain barrier)

Efflux ratio → gives the extent of drug efflux by P-gp

Even Caco-2 study will give us Efflux ratio [by performing bidirectional assay].  Exactly same as above, by assessing transport in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer enables an efflux ratio to be determined.  Thus, we can know if the compound undergoes active efflux.

Add a P-gp inhibitor to suppress the efflux – the result obtained confirms the role of P-gp in the efflux

Add a known P-gp substrate A – then add the test compound B → If the test compound is P-gp inhibitor then substrate A becomes permeable.  Thus test compound B’s P-gp inhibitory effect can be evaluated.

NOTE: 

Caco-2 experiments also do not predict paracellular absorption well because the paracellular route is tighter in Caco-2 cells than that in the small intestine in vivo. While the average pore radius of the tight junctions in the human small intestine is around 8–13Å, the corresponding radius in Caco-2 cells is about 4Å. When the paracellular pathway is narrower, the intrinsic permeability will be lower than in the in vivo situation.

___________________________