Background:  Fraction unbound (fu) of tissue is an essential parameter for the calculation of in vivo free drug concentrations in the tissues (free drug concentration = total drug concentration x fu).

Service Details: We determine binding capabilities of drug candidates to tissues by spiking test compounds into tissue homogenate in DPBS (1:4, w/v) and dialyzing against buffer until equilibrium is achieved (Riccardi K. et al., Drug Metabolism and Disposition, April 2018, 46 (4), 415-421)). The assay is performed in a multiple-use 96-well dialysis unit HTD96b dialyser (HTDialysis). Each individual well unit consists of 2 chambers separated by a vertically aligned dialysis membrane of predetermined pore size (MWCO 12-14 kDa). Tissue homogenate spiked with the compound of interest is added to one chamber and the dialysis buffer to the other chamber. Free compound diffuses from the tissue chamber to the buffer chamber until equilibrium is reached. Concentrations of the compounds in homogenate and buffer are determined by LC-MS, and the fraction unbound is calculated. All incubations are performed in duplicates. This binding test can be performed for various tissues, including brain, liver, kidney, or lungs.

Deliverable: Fraction unbound (fu) and recovery are calculated based on the LC/MS measurements of the compound concentrations in homogenate and buffer solutions.

Sample Submission: A minimal weighted amount of dry compound (~1 mg) or 100 μL of 10 mM stock DMSO solution is required for this assay. We do not need to know structures of the molecules for ADME testing. However, we ask our customers to provide brutto formulas, if at all possible, for all studies involving MS detection.