Test compound (0.5µM) was incubated with female CD1 mouse, male rat Han Wistar and pooled human liver microsomes (Xenotech TM ; 0.5mg/mL 50mM potassium phosphate buffer, pH7.4) and the reaction started with addition of excess NADPH (8mg/mL 50mM potassium phosphate buffer, pH7.4). Immediately, at time zero, then at 3, 6, 9, 15 and 30 minutes an aliquot (50uL) of the incubation mixture was removed and mixed with acetonitrile (100uL) to stop the reaction. Internal standard was added to all samples, the samples centrifuged to sediment precipitated protein and the plates then sealed prior to UPLC-MSMS analysis (Xevo TQ-S Micro, Waters TM ). XLfit (IDBS, UK) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each timepoint. The rate of intrinsic clearance (CLi) of each test compound was then calculated using the following calculation: CLi(mL/min/g liver) = k x V x Microsomal protein yield Where V (mL/mg protein) is the incubation volume/mg protein added and microsomal protein yield is taken as 48, 46 and 40 mg protein/ g liver for mouse, rat and human respectively. Verapamil (0.5 µM) was used as a positive control to confirm acceptable assay performance. RealSOL Solubility Solubility was assessed using an "in-house" developed method known as "RealSOL". This solubility method measures the solubility in physiological strength phosphate buffered saline starting from 10 mM DMSO solutions of the test compounds. Although a "kinetic" solubility method, the long shaking times appears to give solubility values that reflect more closely those made from solid samples when compared to a previous nephelometric method (unpublished in-house data). Test compounds were dissolved in DMSO to give 10 mM solutions. Solubility test samples were prepared by adding a volume (5 µL) of the 10 mM solution to a volume (195 µL) of phosphate buffered saline, pH 7.4 (Sigma-Aldrich, Cat no. P4417, made as per manufacturer's instructions). This solution was then mixed for 24 hours (rotary mixing, 900 rpm, 25°C) excluding light. After mixing, the solubility test samples were filtered to remove any undissolved material using a proprietary filter (Millipore Multiscreen HTS filter, 96-well format). Samples were drawn through the filter using vacuum. The filtrate from the above was analysed for dissolved drug compound using a truncated UHPLC methodology. A Shimadzu Nexera X2 UHPLC system was used, with a reversed-phase column and a simple formic acid gradient elution. 4 nm A calibration solution was prepared in the following way: The same 10 mM solution used to prepare the solubility test sample was diluted in DMSO to give a 500µM solution. This solution was then again diluted with 50:50 acetonitrile: water to give a 50µM solution. Aliquots (0.2, 2.0 and 5.0 µL) of this 50 µM solution were then injected onto the UHPLC system and the areas of the resultant peaks integrated to produce a calibration line. Aliquots of the test sample filtrate (0.4 and 5.0 µL) were then injected onto the UHPLC system and the resultant peak areas for any peaks corresponding to the test compound determined and quantified using the calibration line (the injection volume that gave a peak area closest to the calibrated range was used for determining solubility).