Brain tissue binding assay

Regional BTB of the five model compounds was investigated in human post-mortem tissue. Available brain tissue material from 6 AD and 6 control donors, were allocated to BTB experiments as presented in Table ^S4. BTB was investigated in brain tissue homogenates using equilibrium dialysis according to previously published protocol with modifications¹⁶. For each compound, equilibrium dialysis was carried out during three sequential experimental days. Each daily experiment included brain regions from two randomly assigned control donors and two AD patients. A similar setup was used for all compounds. For the validation of inter-day variability, WB homogenate from one rat was included and analyzed in all dialysis experiments.

The BTB of the five compounds was also investigated in rats. FrCx, PrCx, BG, HIP, and CRB were collected from 24 rats and assembled in pools representative of two (paliperidone only) or three animals per pool, in total nine pools. Each drug was studied in 2–3 different pools representing biological replicates. Each experiment also included tissue homogenate from WB.

Independent of tissue origin, the brain tissue homogenates were prepared by diluting the tissue 1:9 (w:v) in 180 mM phosphate buffer saline (PBS), pH 7.4, and homogenizing the tissue using a VCX-130 ultrasonic processor (Sonics, Chemical Instruments AB, Sweden), at an amplitude of 40%. The homogenates were stored in aliquots at −80 °C. On the day of equilibrium dialysis, the homogenate aliquot was thawed and re-sonicated on ice before being spiked with the drug of interest, e.g., paliperidone, donepezil, memantine, indomethacin, or diazepam to a final concentration of 1 µM.

One day prior to the experiment, dialysis membrane strips with a molecular weight cut off of 12–14 kDa (HTDialysis LLC, Gales Ferry, CT, USA) were conditioned in PBS, pH 7.4, for 1 h and thereafter soaked overnight in 20% ethanol in PBS, pH 7.4. At the start of the experiment, the membranes were rinsed with PBS. A 96-well equilibrium dialysis apparatus (HTDialysis LLC, Gales Ferry, CT, USA) was assembled according to the manufacturer’s instructions. One hundred µL of PBS was added to one side of the dialysis membrane (hereafter referred to as buffer side). An equivalent volume of spiked brain homogenate was loaded in triplicates or duplicates to the opposite side of the membrane (hereafter referred to as tissue side). Incubation was carried out at 37 °C and at 200 rpm in a MaxQ 4450 benchtop shaker (Thermo Scientific, Waltham, MA, USA). To prevent pH changes and evaporation, adhesive sealing film was used to cover the samples.

After 6 h of incubation, 50 µL samples were transferred from both buffer and tissue sides to a Corning 0.5 mL, polypropylene round bottom 96-well plate (VWR, Stockholm, Sweden). To assure identical matrix composition for all samples, required for LC-MS/MS analysis of compounds, the buffer side samples were mixed with 50 µL of 1:9 (w:v) blank brain homogenate of the respective region in PBS and tissue side samples were mixed with 50 µL of PBS. The 96-well plate was sealed with aluminum film, vortexed and stored at −20 °C pending bioanalysis. To monitor relative recovery and stability of the compounds during equilibrium dialysis, samples were taken from the spiked brain homogenates at the start of experiment and after the 6 h incubation at 37 °C.