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	OVERVIEW
	ENZYME INHIBITION
	ENZYME INDUCTION
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	DRUG TRANSPORTERS
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Drug Transporters

Both inhibition and induction of transporters have been implicated as one of the mechanisms governing certain drug-drug interactions, and the FDA’s Sept ’06 Guidance document (PDF) includes specific mention of P-glycoprotein (PgP) as a ubiquitous transporter for specific study. Many drugs are substrates and/or inhibitors of numerous ABC transporters and can have a significant effect on the absorption, distribution, metabolism and elimination (ADME) of these drugs, their toxicity and drug-drug interactions.

XenoTech has a multi-pronged study approach to provide information about the drug candidate’s interaction with a variety of transporters. Permeability information about a compound is helpful to determine the best methods of assaying for transporter interaction and will be used to design the study.

The contract services provided by XenoTech’s Drug Transport Group include:

ATPase assays for ABC transporters
Activation and inhibition of transporters can be investigated using membranes from baculovirus-infected insect cells or mammalian cell membranes containing high levels of human or rat wild-type* transporters. ABC transporters mediate the transport of substrates against a concentration gradient using energy derived from ATP hydrolysis, which is proportional to the transporter activity and easily detected. To assess activation, ABC transporter-rich membranes are incubated with various concentrations of the test article and the effect on basal ATPase activity is measured. Compounds that stimulate ATPase are generally considered substrates for the transporter. For inhibition, a test article is examined for its ability to modify the activity of a given ABC transporter stimulated with its prototypical substrates.

* unless otherwise stated.

For a complete list of membranes available for ATPase assays, see the Membrane Preparations for ATPase Assays on the products page (click here).
Vesicular transport assays for ABC transporters
“Inside-out” membrane vesicles containing ABC transporters are utilized in the vesicular transport assay to determine the IC50 of the compound. After incubating the test article with the inverted membranes, the membranes are separated from the drug-containing buffer and washed before measuring their contents for transported compound. Radiolabeled reporter substrates are used for measuring transported substrate by liquid scintillation counting. The standard vesicular transport assay is an indirect inhibitory assay, performed with cold test articles. This assay provides information on any interaction between the ABC transporter and the test article. The transport of the reporter substrate is measured in the presence of the test article and IC50 is defined as the concentration inhibiting the transport of the reporter substrate by 50%.

For a complete list of membranes available for Vesicular transport assays, see the Membrane Preparations for Vesicular transport assays on the products page (click here).
Calcein Assay
This is an indirect inhibitory whole-cell assay that provides information on any interaction between the ABC transporter* and the test drug by measuring the change in the transport of Calcein AM. Non-fluorescent, hydrophobic Calcein AM, dissolved in the lipid bilayer, is pumped out of the cell by Pgp/MDR1 (ABCB1) or MRP1 (ABCC1), thus keeping the intracellular concentration of Calcein AM low. Modulators of the transporter activity reduce the rate of Calcein AM efflux, leading to increased intracellular Calcein AM, which is then hydrolyzed by intracellular esterases, making it fluorescent. Free Calcein is hydrophobic and thus retained inside the cells. With no inhibitors, the rate of Calcein accumulation (and fluorescence) is slow; however, the addition of inhibitors of the transporter results in a faster rate of Calcein accumulation. By measuring the reporter compound in the presence of the test article, negative control and positive control, IC50 can be obtained, defined as the concentration required to inhibit the transport of the reporter substrate by 50%.

*Pgp/MDR1 (ABCB1) and MRP1 (ABCC1). BCRP (MXR) is also available using Hoechst 33342 dye.
Bi-directional permeability using CaCo-2
This type of study delivers apical-to-basolateral (A-B) or basolateral-to-apical (B-A) transport information that can be used to indicate the presence of an active transport process having significant contribution to the overall net flux of the compound. Measure uni- and bi-directional permeability in polarized monolayers of human epithelial cells to assess intestinal absorption and/or map transporter interactions.
Uptake transporter assays
Uptake transporters in the human body have a variety of native substrates and perform such functions and nutrient uptake, transmitter/cholesterol recycling, kidney re-absorption, redox/pH homeostasis and vitamin/cofactor uptake. Interference with some uptake transporters are known to cause drug-drug interactions that can influence the PK of certain drugs or induce toxicity, causing adverse reactions and/or death. These assays are designed to simulate intestinal absorption and/or map transporter interactions to help predict such interactions.
Assays available:

Some services listed here may be contracted through XenoTech and performed in conjunction with our partner, SOLVO Biotechnology. If you have any questions about our transporter contract studies, please write us at Transporters@xenotechllc.com.

CONTRACT SERVICES

Drug Transporters