Abstract
In order to achieve a high sample throughput, permeation experiments are often carried out using 96-well sandwich plates. Even though agitation is regarded as important, permeation studies in 96-well format are often carried out without agitation since orbital shaking, the most common agitation method for 96-well plates, has been reported to create difficulties (e.g., well-to-well cross-talk), and high cost and low availability limits the use of other agitation techniques (e.g., magnetic stirring). This study investigates how orbital shaking and magnetic stirring affect the apparent permeability of model compounds with different water-solubilities (methylene blue, carbamazepine, and albendazole) using a novel 96-well sandwich plate comprising a cellulose-hydrate membrane (PermeaPlain® plate). Orbital shaking was found less efficient than magnetic stirring in terms of homogeneously distributing a small volume of dye within the donor compartment. Furthermore, in terms of achieving maximum trans-barrier flux, magnetic stirring was found a more effective agitation method than orbital shaking. Obviously, with orbital shaking the medium in the bottom compartment of the sandwich plates never was mixed in-phase. The impact of insufficient mixing on permeation was found strongest with the most lipophilic compound, which correlates with literature reports that the contribution of the unstirred water layer towards the overall resistance of the barrier is most expressed in case of lipophilic drugs. Finally, it was tested how different liquid volumes in the bottom compartment of the plates affect the well-to-well cross-talk during permeation experiments under orbital shaking. This study revealed that 250–300 µL should be used in the bottom compartment of the sandwich plates to reduce well-to-well cross-talk when using orbital shaking for agitation.
Highlights
Both, regulators and scientists in drug development widely use in vitro permeation tools, such as colon carcinoma cell line (Caco-2), parallel artificial membrane permeability assay (PAMPA), Phospholipid Vesicle Permeation Assay (PVPA) or PermeaPadÒ for biopharmaceutical characterization and classification of drug compounds.[1,2] In drug discovery, the permeability of large compound libraries preferably is screened using 96-well plate-based approaches in high throughput.[3]
When using orbital shaking for agitation, the dipyridamole particles stayed as lumps at the bottom of the wells and did not move
To reduce the risk of well-to-well cross-talk, the current study demonstrated that preferably volumes below 400 mL in the donor compartment of PermeaPlainÒ plates should be applied
Summary
Regulators and scientists in drug development widely use in vitro permeation tools, such as Caco-2, PAMPA, PVPA or PermeaPadÒ for biopharmaceutical characterization and classification of drug compounds.[1,2] In drug discovery, the permeability of large compound libraries preferably is screened using 96-well plate-based approaches in high throughput.[3]. Agitation influences the mass transfer from the solid to the liquid.[10,11] In permeation studies, agitation of the donor compartments often enhances the permeation rate of poorly soluble compounds.[12] This enhancement often is regarded to be caused by a reduction of the thickness of the unstirred water layer (UWL), which is a diffusion barrier of unstirred aqueous buffer adherent to the permeation barrier and which the compound needs to overcome. Agitation of the donor and acceptor compartments are observed to increase the permeation rate of poorly soluble compounds, which is thought to be due to a decrease of the UWL thickness. Effective agitation of 96well permeation devices is regarded a pre-requisite to obtain in vivo relevant permeability predictions[12]
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