HDM-PAMPA with the MultiScreen Permeability Filter Plate

Figure 1. Log P_e can be calculated from the equation as reported by Faller et al. (1)

Table 1 Description of variables used to calculate Log P_e

Term	Definition	Notes
VD	Volume of donor compartment	Expressed in cm3, 150 µL = 0.15 cm3
VA	Volume of acceptor compartment	Expressed in cm3, 300 µL = 0.30 cm3
Area	Active surface area of membrane	Defined as membrane area x porosity. For the membrane in MultiScreen Permeability Filter Plate, area = .24 cm2 x 20%; or .048 cm2
Time	Incubation time for the assay	Expressed in seconds, 1 hr = 3600 s
[drug]acceptor	Absorbance units of compound in acceptor compartment at the completion of the assay	The absorbance of the sample as recorded by the SoftMax Pro Software
[drug]equilibrium	Absorbance of the compound at theoretical equilibrium from step 9 above	The absorbance of the equilibrium sample as recorded by the SoftMax Pro Software

Note 1:  For consistent and optimal performance, it is important to allow for complete evaporation of hexane.  Once evaporation is complete the hexadecane membrane is stable for weeks.

Note 2:  To ensure the hexadecane layer is intact, electrical resistance measurements can be made both before and after permeability assays are conducted. Intact hexadecane layers exhibit extremely high electrical resistance (normally exceeding 25 kΩ.). Data from wells with electrical resistance measurements below 5 kΩ should be excluded.

Note 3:  An acceptor plate is included in the packaging of the permeability plate. Alternatively, the PTFE Acceptor plate (# MSSACCEPT0R) is recommended in place of the packaged acceptor plate.

Note 4:  To avoid evaporation, the plate should be placed in humidity controlled environment such as a sealed container with wet paper towels during incubation.

Note 5:  Generally sample analysis using a 96 well UV/Vis plate reader is recommended. Sample quantification techniques such as scanning over a broad absorbance range (e.g. 250-500 nm), a single wavelength (λ_max) or a summation of pre-selected fixed wavelengths are all acceptable for analysis. HPLC-UV or LC-MS/MS are also alternative means of detection.

References

1. Wohnsland, F.; Faller, B. High-throughput Permeability pH Profile and High-throughput Alkane/Water Log P With Artificial Membranes, J. Med. Chem., 2001; 44, p. 923–930.

2. 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, p. 1007–1010.