Improved Capabilities of Noncell-Based Permeability Assays

Assays that predict passive absorption of orally administered drugs have become increasingly important in the early drug discovery process. A molecule’s ability to be orally absorbed is one of the most important aspects in deciding whether it is a potential lead candidate. The cell-based assays, like those using Caco-2 cells, are common functional models for drug absorption; however, this labor-intensive and expensive technique is often pursued late in the drug discovery process. Assays described by Kansy1 and Faller2 have addressed these issues by providing rapid, low-cost, and automation-friendly methods to measure a compound’s passive permeability. These in vitro assays have become an integral part of early drug discovery because of their simplicity.3,4 In vitro permeability assays help predict the potential bioavailability or in vivo absorption of a molecule. This paper discusses the benefits of enhanced throughput, automation, and robustness of in vitro permeability assays with progressive plate technology.

Figure 1 - PAMPA passive diffusion process.

The parallel artificial membrane permeability assay (PAMPA) is a noncell-based in vitro technique used to predict the passive, transcellular membrane permeability of potential new pharmaceuticals. Microplate filters impregnated with mixtures of phospholipid/organic solvent fit precisely into discrete receiver wells to form 96 microdiffusion chambers. Within the phospholipid barrier, a concentration gradient drives the passive diffusion of molecules, which is the principal mechanism of drug transport through biological barriers (see Figure 1). Data produced by these assays result in a permeability estimate (effective permeability, Pe). Fast, flexible, and cost-effective compared to alternative cell-based models, PAMPA also enables the evaluation of different in vivo absorption models by varying pH or lipid composition.

Challenges

 Traditional products for PAMPA had limited handling capabilities with standard laboratory robotics. Automated assembly introduced trapped air between the filter and buffer compartments, reducing diffusion surface area; receiver well shape limited shaking or stirring capability; and inconsistent evaporation necessitated the need for controlled, humidified environments due to inconsistent evaporation.

Figure 2 - PAMPA transport receiver design features.

A newly designed transport receiver plate (Millipore Corp., Danvers, MA) offers high data quality and throughput for PAMPA. The molded receiver plate has such built-in design features (Figure 2) as increased diffusion space (1), recessed side walls (2), rib locaters (3), and a continuous flat top surface (4) to help overcome many of the challenges mentioned above.

Experiments

Handling consistency, assay flexibility, and data reproducibility were evaluated with standard automation. Eighteen molded receiver plates were tested for automated assembly/disassembly and associated cross-talk, evaporation, and occurrence of trapped air. Tests were run on three consecutive days with three different automated liquid handlers (Packard® MultiPROBE® II [PerkinElmer, Boston, MA], Tecan® Genesis [Zurich, Switzerland], and Beckman® BioMek® FX [Fullerton, CA]). Three machined Teflon® (DuPont, Wilmington, DE) receiver plates were used as controls. Plate performance was evaluated before, during, and after automated handling and after overnight incubation on the robot deck.

Cross-talk and trapped air

Cross-talk (cross-contamination) between discrete adjacent wells was measured fluorescently with a Spectrafluorplate reader (Tecan). Trapped air was checked visually on a light box, and well-to-well evaporation was measured using pathcheck on the SpectraMax® Plus plate reader (Molecular Devices, Sunnyvale, CA). Fluorescent test solution or buffer (300 μL/well) was dispensed in wells in a criss-cross pattern, and cross-talk was calculated by dividing relative fluorescence units (RFU) of blank wells by RFU of adjacent fluorescent wells (485-nm excitation/535-nm emission). Molded plates had no cross-talk and controls averaged 22 wells per plate. Molded plates averaged one trapped air bubble, while controls averaged 12 per plate.

Evaporation

Final permeate absorbance is required to calculate passive transport. If permeate concentration and absorbance increase due to evaporative volume loss, the resulting calculation leads to artificially increased permeability rates. Therefore, minimal and uniform evaporation are critical for reproducible and reliable data.

PAMPA incubations at ambient humidity and temperature were evaluated for overnight evaporative volume loss. Based on a 300-μL starting volume, outer perimeter and inner well evaporation for the molded plates was 4.5% ± 0.8% and 4.5% ± 0.6%, respectively (n = 18). Machined Teflon control loss averaged 8.9% ± 3.8% and 4.6% ± 3.2% (n = 3), a twofold and fivefold increase in volume loss and standard deviation, respectively.

Shaking and stirring

The diffusion rate of molecules through a phospholipid barrier is limited by the barrier and/or by adjacent aqueous boundary layers (ABL).5 Nonagitated in vitro ABLs can be 10–100 times greater than in vivo measurements. When the ABL is thicker than the phospholipid barrier, it becomes the rate-limiting factor for the diffusion of nonpolar, lipophilic molecules. Therefore, agitation of the bulk solution by shaking or stirring can significantly reduce the ABL, resulting in more accurate in vitro permeability predictions.

  1. Shaking. Six molded plates were assembled with the BioMek FX liquid handler and evaluated for cross-talk after overnight shaking on the MicroMix5® (DPC, Randolph, NJ) orbital shaker at 1200–1500 rpm (form 22, amplitude 5). One control plate was static tested. Fluorescence was measured with the Spectrafluor plate reader before and after assembly as well as after overnight shaking. Molded plates had no cross-talk, and the control had seven wells. Molded plates averaged 2.5 trapped air bubbles, and the control had 22.
  2. Stirring. Six molded receiver plates were assembled manually with super-tumble stir disks (catalogue no. VP 721F-1, V&P Scientific, Inc., San Diego, CA) in every well. Cross-talk was evaluated after stirring for 1 hr (750 rpm) on a VP 710C1 rotary tumble stirrer (V&P Scientific). No cross-talk was observed.

An integrity test was also performed for potential stirring-related filter damage. A lucifer yellow-buffer test solution (150 μL) was dispensed to filter plates. After 30 min of stirring (300 rpm), permeate fluorescence was measured with a Victorspectrofluorometer (PerkinElmer) at 485/535 nm. Fluorescence of an equilibrium plate (135 μL/well buffer + 65 μL/well test solution) and a blank buffer plate were required to calculate integrity. 1-(sample RFU-mean blank RFU)/(mean equilibrium RFU-mean blank RFU) = % integrity. All stirred assemblies had ≥99% filter integrity.

Conclusion

As a result of design enhancements, PAMPA can be a highly effective way to characterize and optimize lead candidates early in the drug discovery pipeline. Being flexible, fast, low cost, and automation-friendly, PAMPA has become the emerging method to complement cellular based absorption models.

References

  1. Kansy, M.; Senner, F.; Gubernator, K. Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J. Med. Chem. 1998, 41, 1007–10.
  2. Wohnsland, F.; Faller, B. Highthroughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J. Med. Chem. 2001, 44, 923–30.
  3. Di, L.; Kerns, E. Profiling drug-like properties in discovery research. Curr. Opin. Chem. Biol.2003, 7, 402–8.
  4. Brennan, M.B. Drug discovery (filtering out failures early in the game). Chem. Eng. News 2000, 78, 63.
  5. Kansy, M.; Avdeef, A.; Fischer, H. Advances in screening for membrane permeability: high resolution PAMPA for medicinal chemists. Drug Discov. Today2004, 1, 349–55.

    Mr. Kazan is a Biochemical Scientist, Bioscience Div., Millipore Corp., 17 Cherry Hill Dr., Danvers, MA 01923, U.S.A.; tel.: 978-762- 5197; fax: 978-762-5386; e-mail: [email protected].

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