ATPase assays

The ATPase assay is an in vitro membrane assay, designed to indicate the nature of the interaction between the compound and the transporter.
By measuring ATPase activity, both 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 with a colorimetric method.

To assess activation, ABC transporter-rich membranes are incubated with various (typically in 8) 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. The activation and inhibition tests are complementary assays.

Stimulation detected in the activation assay indicate that the compound is a transported substrate of the transporter, while interactions detected in the inhibition test indicate interaction of the test compounds with the transporter, but do not give information on the nature (substrate or inhibitor) of the interaction.
In some cases inhibitors or slowly transported compounds may inhibit the baseline transporter ATPase activity as well.

Slowly transported substrates often do not stimulate the ATPase activity in a detectable extent; however the existing interaction can be identified in the inhibition assay.

Vesicular transport assay

"Inside-out" membrane vesicles containing ABC transporters are applied in the vesicular transport assay to determine the IC50 of the compound. After incubating the test article with the inverted membranes, the membranes are then 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 new PREDIVEZ Vesicular Transport Kits were developed for fluorescent reporter substrates).

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 (typically in 7 concentrations) and IC50 is defined as the concentration inhibiting the transport of the reporter substrate by 50%.

Should radiolabeled form of the investigated compound or adequate analytical methods (LC/MS, HPLC) be available, the vesicular transport assay may be performed in a direct format without the reporter substrate and may identify substrate nature of the test article. Direct vesicular transport assay is low throughput assay. It is suitable for low permeability test compounds as high permeability compounds may escape from the vesicles through the lipid bilayer. Standard assays are always recommended before running the direct measurements with the test compound.

The project based on the Direct Vesicular Transport assay is organized into two basic Parts (Part 1 and 2), and one optional Part (Part 3) to evaluate potential drug-drug interactions. The launch of the given Part(s) depend(s) on the success of the previous Part(s).

Part 1 - Assay Development: pilot experiments to determine feasibility of testing -The vesicular transport of the test compound is determined using membranes containing the selected efflux transporter and using control membranes at two incubation time points and at two concentrations of the test drug. The expected outcome of Part 1 is the determination of whether or not the effective efflux transporter mediated vesicular uptake of the isotope-labeled compound into the inside out vesicles can be measured.

Part 2 - Detailed characterisation of the transport: Part 2 will be started if the effective transport of the test drug in Part 1 can be detected. The aim of the Part 2 is to optimize the incubation parameters. Time dependence of transport at 8 time points will determine the length of the first, linear phase of transport. Concentration dependence will be measured to determine the KM and Vmax. The corresponding reporter substrates and reference inhibitors of the standard assays (Table 1) are used to confirm the transporter-test drug interaction.

Part 3 - Optional Part for characterizing potential drug-drug interactions: The IC50s of selected known transporter-interacting drugs will be determined in incubations of TA with transporter expressing membranes as established in Part 2.

Calcein Assay

SOLVO patented Calcein Assay® provides information on the interaction between MDR1/P-gp (ABCB1) or MRP1 (ABCC1) transporters and the test compound. Non-fluorescent, hydrophobic calcein AM, dissolved in the lipid bilayer is pumped out of the cell by MDR1/P-gp (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 (typically in 8 concentrations), negative control and positive control, IC50 can be obtained, defined as the concentration required to inhibit the transport of the reporter substrate by 50%.

Hoechst Assay is also a whole-cell based dye transport assay that addresses BCRP membrane transporter in a similar fashion to Calcein Assay.
A similar dye transport-based assay, utilizing Hoechst 33342 has been worked out to measure the activity of BCRP (ABCG2/MXR). Hoechst 33342 is a non-fluorescent substrate of both MDR1 and BCRP, and the dye becomes fluorescent after entering the cell and binding to DNA.

Uptake transporter assays

Uptake transporter assays are indirect inhibitory whole-cell assays performed on cells lines expressing the uptake transporter studied. These inhibitory assays provide information on any interaction between the uptake transporter and the test drug, however they do not give information on the nature of the interaction (transported substrate or inhibitor.)
Uptake transporters carry substrates into the cells from the outside and use the concentration gradient of another substrate (usually an ion) as energy source. Standard high throughput uptake transporter assays use cold test compounds and measure the inhibitory potential of the test drug (typically in 8 concentrations) on the transport of a known, radiolabeled or fluorescent substrate. IC50 is defined as the concentration required to inhibit the transport of the reporter substrate by 50%.
Should radiolabeled form of the investigated compound or adequate analytical methods (LC/MS, HPLC) be available, the uptake transporter assay may be performed in a direct format without the reporter substrate and may identify substrate nature of the test article. Direct assays are low throughput assays are suitable for low (and medium) permeability test compounds as high permeability compounds may escape from the cells.
Standard assays are always recommended before running the direct measurements with the test compound.
Uptake transporter assays are available as screening services.

Caco-2 Monolayers assays

Monolayers based on Caco-2 cells (a human colon carcinoma cell line) express a wide range of transporter proteins on its cell membranes similar to those of intestinal endothelium cells (Siissalo S et al. 2007, Calcagno AM et al. 2006), thus this cell line is ideal for intestinal absorption simulations. In fact, in the last decade, the utilization of Caco-2 cells has become an industry standard for the investigation of intestinal absorption, permeability and drug-drug interactions (DDIs) (Oh DM et al. 2002.).

SOLVO Biotechnology offers Caco-2 studies as part of its PrediScreen™ service solutions. Three types of studies are available:

1. Passive permeability studies
2. Specific transporter studies
3. Drug-Drug Interaction studies

Caco-2 studies can be performed in different setups. SOLVO offers a number of standard setups, which differ in the amount of information derived and cost. These standard setups serve as starting points to define the optimal final study parameters.

The following standard setups are available:
1. Basic - Studies at one concentration
2. Extended - Studies at several concentrations, more controls
3. FDA Guidelines - Studies according to FDA draft guidance (2006)