Assays that predict passive absorption of orally administered drugs have become increasingly important in the drug discovery process. As previously described by Faller1 and Kansy2 such assays provide rapid, low cost and automation friendly methods to measure a compound’s passive permeability. The HDM-PAMPA (HDM = Hexadecane Method) method is a non-cell based assay designed to predict passive, transcellular permeability of drugs in early drug discovery. The assay is carried out in a 96-well MultiScreen Permeability plate (MPC4NTR10) and measures the ability of a compound to diffuse from a Donor to an Acceptor compartment separated by a hexadecane liquid layer on a polycarbonate membrane support. This protocol note details the steps required to determine compound permeability rates across a hexadecane layer.
To measure the integrity (proper formation) of the hexadecane membrane:
Figure 1.Log Pe can be calculated from the equation as reported by Faller et al. (1)
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 (λmax) 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.
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