Intestine

Intestinal barrier function regulates transport and host defense mechanisms at the mucosal interface with the outside world. Transcellular and paracellular fluxes are tightly controlled by membrane pumps, ion channels and tight junctions, adapting permeability to physiological needs. Permeability greatly defines the absorbance of compounds in the intestine. In case of high permeability compounds it is unlikely that the absorption will be modified by transporters, yet the test compound might be involved in transporter mediated drug-drug interactions. The absorption of medium and low permeability compounds can be affected by membrane transporters located at the endothelial cells of the intestinal barrier.

In the intestine transporters are localized in the brush border membrane and on the basolateral side of intestinal cells. Four major ABC efflux transporters have been shown to localize at the apical/luminal membrane of enterocytes, and thus are thought to form a barrier to intestinal absorption of substrate drugs: P-gp, BCRP, MRP2 and MRP4. Their expression level varies between different segments of the intestine. In general, BCRP, MRP2 and P-gp are expressed at high level in the small intestine, considered by many in the field as the rate limiting barrier to oral drug absorption.
Several different uptake transporters are localized on the apical (brush border) membrane of enterocytes that facilitate the entry of non-membrane-permeable molecules into the blood circulation. The most well known of these is the peptide transporter 1 (PEPT1) responsible for the uptake of di- and tripeptides formed during the digestion of proteins. This transporter has also been shown to transport drugs coupled to amino acids (like valacyclovir) or peptide like drugs. There are a number of other uptake transporters expressed in enterocytes (OATPs OATs, OCTs,). These transporters also have significant effect on the absorption of drugs.

Localization of efflux and uptake transporters in enterocytes.

Available assays within the intestinal package

Membrane based HTS efflux transporter assays

Whole cell based HTS transporter assays

Monolayer assays:

Screening strategy

Penetration package: Start with a monolayer permeability assay on Caco-2, follow with membrane assays (ATPase and vesicular transport) and/or Caco-2 assays with specific inhibitors.

Specific transporter interaction package: Start with high throughput, low-cost methods (ATPase, vesicular transport and Calcein assay) and inhibitory uptake transporter assays. Based on the results transfected cell monolayer efflux assays can follow or Caco-2 assays in the presence of specific inhibitors.

Drug-drug interaction package: Start with single transporter inhibitory assays (vesicular transport, Calcein dye efflux and uptake transporter assays), carry out Caco-2 inhibition assays, (e.g. digoxin, chlorothiazide) to study whether the drug inhibits the transport of other drugs. Caco-2 permeability with specific inhibitors can be performed to study whether the transport of the drug can be inhibited by other compounds.

P-gp toolbox: Specifically for P-gp interaction assessment (substrate or inhibitor) SOLVO recommends to follow the procedure and decision tree in compliance with the FDA draft guidance.