Permeability assays

The advent of combinatorial chemistry and high-throughput screening technologies has resulted in a large increase in the number of potential drug candidates being identified for development. Selecting the best compound to take forward into the drug development process is further complicated by development time frames that continue to shrink. Together, these factors have increased the need for an intelligent lead compound selection process that is based not only on biological activities but also on the pharmacological and chemical characteristics of the potential lead compounds. Failure to consider any of these characteristics could lead to significant and costly development problems, delays in getting the product to market, or the failure of the development project altogether.

Cellular permeability is one drug characteristic that is now being considered much earlier in the drug discovery process. Permeability assays using the Caco-2 colon carcinoma cell line are being used throughout the pharmaceutical industry to estimate the ability of potential drug compounds to cross the intestinal epithelium. Traditional permeability assays are very labor-intensive and have a throughput of fewer than 10 compounds per week. Fortunately, however, automation of permeability assays is possible. We have applied a Tecan Genesis 150 workstation configured specifically for cell permeability assays to improve the throughput of potential drug compounds. Using this system, the throughput of permeability assays has been improved 10-fold relative to traditional manual permeability assays and 4-fold relative to streamlined manual permeability screens.

Manual permeability assays

Figure 1. Schematic representation of a Caco-2 permeability assay.

Caco-2 cell permeability studies are performed using Caco-2 cell monolayers grown on microporous membranes in multiwell insert systems. With the inserts suspended in the wells of multiwell plates, test compounds can be added to either the upper (apical) or lower (basolateral) chamber to measure permeability in the absorptive (apical to basolateral) or secretive (basolateral to apical) directions, respectively, as shown in Figure 1. Samples are then taken from the opposite chamber at various time intervals to measure the amount of test compound that has crossed the cell monolayer.

Traditionally, all aspects of permeability assays, including monolayer washing, addition of test compounds, and receiver compartment sampling at multiple time points, have been done manually. The numerous sampling intervals and replicate monolayers used for each transport direction of traditional assays add an even larger burden to performing the assay. As a result, the practical throughput of manual assays has been limited to just a few compounds per week. Throughput can be improved, however, by decreasing the number of replicate monolayers used and the number of sampling intervals examined for each compound. Still, even with such streamlining modifications, the assay remains labor-intensive, with a throughput of only about 10 compounds per day.

The optimum platform for automating the Caco-2 permeability assay can automate the required pipetting operations as well as perform nonpipetting operations. For example, the instrument’s ability to transport assay plates between the work surface and an enclosed incubator is preferable to performing the entire assay on a warmed surface. Likewise, instruments that can remove and replace plate lids allow the assay plates to be covered for extended incubations, thereby minimizing evaporation. The Tecan Genesis workstation is ideal for automating permeability assays because it can perform all of these tasks.

When configured for cell-permeability assays, the Tecan Genesis workstation combines an 8-pipette liquid-handling system, one or more shaking incubators capable of holding four 24-well permeability assay plates, various plate/lid racks, and temperature-controlled carriers and reagent reservoirs.

Unique to this workstation is a robotic manipulator arm (ROMA) that can pick up and move plates, lids, or permeability inserts to and from various locations within its work space. The workstation also includes software that dynamically schedules multiwell plate processing and automatically adjusts the schedule to account for process time variation. Together, the software, liquid handler, and robotic manipulator of the system allow the workstation to perform the entire Caco-2 permeability assay without manual intervention.

Another advantage of this workstation is its flexible pipetting software. With most traditional automation workstations, all pipette tips must perform the same operation simultaneously, and whatever solution is aspirated must be dispensed in the next programming step followed by tip washing. The Genesis workstation aspirates and dispenses using separately controlled tips, reducing cycle times on the platform, and therefore, assay times.

Automated permeability screening
The automated workstation used at Pharmacia for performing permeability assays is configured to process four 24-well permeability inserts per assay. Typical assays use one insert to measure transport across Caco-2 cells in the absorptive direction and one for measurements in the secretory direction. This approach yields a throughput of 48 bidirectional analyses per assay, which translates into a practical throughput of approximately 200 bidirectional analyses per week. Therefore, the automated permeability assay increases throughput approximately 20-fold relative to traditional permeability assays and approximately 4-fold relative to streamlined manual assays.

Researchers perform an automated permeability assay by configuring the workstation with appropriate physiological buffers, lab ware, and test compounds. Multiwell insert systems containing Caco-2 cells are placed on the workstation and loaded into the temperature-controlled shaking incubator using the ROMA. Individually, the plates containing the inserts are brought to a prewarmed carrier on the workstation, where the plate lid is removed and the insert containing the Caco-2 cell monolayers is transferred to a new 24-well base plate. The growth medium is then removed from the insert chambers, and the cell monolayers are washed with assay buffer. Test compounds and assay buffers are then added to the donor and receiver compartments of the transport inserts, respectively, and a small sample is taken from the donor chamber and automatically diluted for analysis to verify the concentration of the test compound. The ROMA then retrieves the assay plate lid and returns the plate to the incubator for two hours. Following incubation, samples are taken from each chamber. The conical shape of the permeability inserts used at Pharmacia allows the liquid-handling arm to sample from either of the two chambers created by the placement of the insert. The final step involves adding a fluorescent marker compound to every well, performing another incubation step, and removing a sample to determine if the cell monolayer has been perturbed. This procedure, which was not part of the manual assay due to the increase in assay time, provides an important validation step in every assay that is performed.

Scheduling software
The Tecan Genesis system software controls and schedules processes that include setting up the initial work space layout and the individual test specifications, combining individual tests into a profile, executing the assay, and interfacing with other systems. Researchers define and organize the initial configuration on a work space setup screen, which shows the arrangement of samples, microplates, and reagents and allows items to be dragged and dropped into the appropriate positions. The dynamic scheduling software overcomes the problems commonly experienced with scheduling automated assay systems. For example, if dispensing takes less time than expected, the scheduler recalculates and adjusts future process steps to account for the actual time used by completed steps. The scheduler will continually reevaluate the schedule to optimize throughput.

Validation

TABLE 1

Comparison of apparent permeability coefficients (Papp) obtained using the automated permeability screening assay and a traditional manual method

Test compound Papp (10–6 cm/s) Manual assay Automated assay A>B B>A A>B B>A 1 >40 >40 62 54 2 >40 >40 53 48 3 35 36 26 24 4 16 14 11 12 5 2 20 1.8 14 6 1 6 1.0 5.8 7 0.8 3 0.7 2.8 8 0.1 1 NTD 2.3

A>B = Apical to basolateral transport B>A = Basolateral to apical transport. NTD = No transport detected

The automated assay was validated by comparing the apparent permeability coefficients Papp obtained for a set of proprietary test compounds known to represent a wide range of apparent permeabilities. Accuracy of the screening assay is excellent for P_app values below 10 × 10^–6 cm/s. P_app values were slightly underestimated for highly permeable compounds (P_app > 10 × 10^–6), but the rank order was maintained relative to values obtained using traditional assays (Table 1). The precision of the automated screening assay was excellent, with an interassay RSD of approximately 10%.

The new automated Caco-2 cell permeability assay provides a throughput of 48 compounds per assay, four times the amount that could be produced by manual screening. The assay runs in about 6.5 h without manual intervention, saving approximately 2 h of manual labor per assay. The assay can now be performed much earlier in the drug discovery process (immediately after confirmation of activity through high-throughput and secondary screening) due to the increased throughput from automation. As a result, researchers can now determine permeability coefficients and flag poorly permeable compounds before preparing analogues and developing cell and animal models. The primary payback will be an acceleration of the drug discovery process, part of Pharmacia’s multimillion-dollar effort to move critical assays further up the drug discovery pipeline.