A B Ratio AUC0-inf Ratio AUC0-inf = 5.2 Modeling of the PK and theoretical enzyme occupancy (Equation ) after a single 100 mg/kg dose identified 37 to be the optimal compound to progress into a PK/PD study with a 150 mg/kg BID, Q4hr dosing regimen. This study design, in conjunction with the measured mouse cell IC50, the concentration of 37 in the cerebellum and the corresponding free fraction in this tissue was predicted to provide enzyme occupancy and coverage commensurate with an in vivo biomarker response (see Figure ). Projected tissue concentrations at 15, 50 and 150 mg/kg were calculated following a linear extrapolation of the measured values obtained from the 100 mg/kg dosing cohort illustrated in Figure . These data were used in Equation 1 to generate the %tEO profiles in Figure . Both 37 and 42 have been extensively screened in vitro for potential off-targets. In the Eurofins Safety/Diversity panel (Table ), COX2 is the only off-target for both 37 and 42, representing about 93-fold in vitro selectivity for 37 and 132-fold for 42. In addition, the compounds have also been screened against six additional CNS targets at Eurofins, and none of them showed significant activity at 10 µM on the six off-targets. No significant inhibition of the major human CYP enzymes (<40%, 3A4, 2D6, 2C9, 2C19, 2C8, 1A2) was observed for either compound at 10 µM. In addition, there was a complete absence of any cytotoxicity signal for either compound when they were tested at 100 µM in a HepG2 assay that measured 72-hour ATP production and 24-hour Glu/Gal mitotoxicity. Following ethical review and approval of the study protocol, the PK/PD assessment was undertaken to measure the modulation of D-serine levels in the cerebella of mice following administration of the test compound at one dose using the regimen described above. Two cohorts of animals were tested (compound and vehicle) using 33 animals in total (n=8/group for 37 and n=3/timepoint for vehicle). In both cases plasma and cerebella samples were collected following animal "take-down" at 4-hour, prior to 2nd dosing, 6-hour, and 10-hour after the initial dosing. The levels of D-serine in plasma and brain tissue were quantitatively determined using a chiral LC-MS/MS method, ensuring both adequate sensitivity and selectivity. In addition, CSF was sampled from the animals at the 10-hour timepoint to determine the free, unbound levels of 37.