As for plasma protein binding experiments, 96 well equilibrium dialysis apparatus was used to determine the free fraction in brain tissue homogenate for each compound (HT Dialysis LLC, Gales Ferry, CT). Membranes (12-14 kDA cut-off) were conditioned in deionised water for 60 minutes, followed by conditioning in 80:20 deionised water:ethanol for 20 minutes, and then rinsed in isotonic buffer before use. Frozen brain tissue was thawed and homogenised in artificial CSF at a ratio of 3:1. Homogenate was then spiked with test compound (final concentration 10 ug/mL), and 150 μL aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate. Dialysis vs artificial CSF was carried out for 5 hours in a temperature-controlled incubator at ca. 37ºC (Barworld scientific Ltd, UK) using an orbital microplate shaker at 100 revolutions/minute (Barworld scientific Ltd, UK). 150 μL aliquots of spiked homogenate were stored at 37ºC and 4ºC for the course of the experiment to determine recovery. At the end of the incubation period, 50 μL aliquots of homogenate or buffer were transferred to a 96 deepwell plate (Waters TM ) and the composition in each balanced with control fluid (50 μL), such that the volume of buffer to brain homogenate is the same. Sample extraction was performed by the addition of 200 μL of acetonitrile containing an appropriate internal standard. Samples were allowed to mix for 1 minute and then centrifuged at 3000rpm in 96-well blocks for 15 minutes (Allegra X12-R, Beckman Coulter, USA) after which 150 uL of supernatant was removed to 50 μL of water. All samples were analysed by UPLC-MS/MS. The unbound fraction was determined as the ratio of the peak area in buffer to that in homogenate using the following equation: Where fucr means fraction unbound corrected D is the dilution factor 𝑇𝐵 = (1 𝑓𝑢𝑐𝑟) ⋅ 100 Where TB means Tissue Binding The recovery of the dialysed samples was calculated by comparing the sum of the peak areas (buffer and matrix) of any test compound at 37ᵒC to that of the same compound at 4ᵒC. This is then multiplied by 100% to get the answer in percentage.