Characterization of the relationship between target occupancy/modulation and preclinical/clinical responses for the glycine transporter 1 (GlyT1) inhibitor Org 25935

Abstract

Background: Selective inhibitors of the glycine transporter I (GlyT1), eg Org 25935, may represent a novel approach for the effective treatment of schizophrenia. Here we describe the relationship between GlyT1 target occupancy, cerebrospinal fluid (CSF) glycine elevation and activity in preclinical behavioural models for Org 25935. The translation of pre-clinical to human glycinergic signaling was an important consideration in the assessment of compound viability. Org 25935 glycinergic responses as well as proof of reserve capacity (and lack of tachyphylaxis) in healthy male volunteers is also presented. These data play a key role in guiding the selection of dosing regimens for future proof of concept studies. Methods: Rat GlyT1 occupancy was determined by measuring specific binding of the selective GlyT1 radiotracer, [3H]CPyBP, to whole brain homogenates prepared from male Sprague Dawley rats treated, in-life, with Org 25935. Org 25935 receptor occupancy was measured in Rhesus monkeys by PET imaging with the GlyT1 tracer [18F] MK-6577. Org 25935-mediated changes in glycine levels were determined in rhesus monkeys and rat CSF, and in the medial prefrontal cortex (mPFC) of conscious rats by in vivo microdialysis. Conditioned avoidance response (CAR) was determined in rats treated with Org 25935 (1-10 mg/kg ip). The effect of Org 25935 on a prefrontal cortex mediated cognition task was evaluated using an object retrieval task in scopolamine-impaired rhesus monkeys. Blood plasma samples were drawn in all studies in order to measure drug levels. Healthy male volunteers received multiple oral doses of placebo, 4, 8 or 16 mg Org 25935 BID for 13 days and a next higher single dose on day 14 (8, 16 or 32 mg respectively). CSF was collected continuously in fractions of 30 minutes each for 36 hours following the first dose on Day 1 and for 48 hours following the A.M. dose of Day 13 to measure glycine responses and Org 25935. Simultaneously, Org 25935 was measured in plasma at selected time points. Results: Preclinical receptor occupancy (Rocc) studies estimated plasma Occ50 for Org 25935 to be 216 nM (Hill coefficient 1.65) in monkeys and 573 nM (Hill coefficient 1.09) in rats. Significant increases in mPFC glycine levels (rats) and in CSF glycine levels (rats and monkeys) were observed at Org 25935 plasma concentrations corresponding to Rocc of >30%. Maximal increases in glycine levels (rat mPFC and rat and monkey CSF) were observed at GlyT1 Rocc 60-70%. In healthy volunteers, the plasma and CSF kinetics of Org 25935 show dose proportionality with a Tmax delay in CSF of about 4 hours. Maximum Org 25935 concentrations in CSF and plasma ranged from 4.06 to 24.3 ng/mL and 127-648 ng/mL,respectively. On Day 1, CSF glycine concentrations increased with a Cmax observed at about 5-6 hours after dosing. Following the single dose on Day 1, the baseline corrected Cav values during the first 12 hours (one dosing interval) were 1.57, 1.73 and 2.05 íg/mL upon 4, 8 and 16 mg Org 25935 respectively. Cav during a dosing interval of 12 hours at steady state following the A.M. dose on day 13 and 14 (next higher dose) were 2.12/2.74, 2.46/3.01 and 2.46/3.06 íg/mL respectively. All doses were well tolerated in volunteers. In the rat CAR model, Org 25935 treatment resulted in a modest but significant disruption in conditioned responding at plasma levels corresponding to Rocc> 80%. At these plasma levels, Org 25935 did not produce any escape failures. Scopolamine treatment produced a significant deficit in the accuracy of the object retrieval task measured in monkeys. Reversal of this deficit was observed at doses of Org 25935 (0.1, 0.3 mg/kg po) which achieved GlyT1 Rocc in the range of 15-60%. However, a higher dose (3 mg/kg po, Rocc 98%) Org 25935 failed to significantly reverse the scopolamine deficit. Discussion: These data show that Org 25935 can effectively elevate glycine levels in the CSF of healthy volunteers and preclinical species. In healthy volunteers, there were no indications for desensitization upon multiple doses. Preclinical behavioral data support the hypothesis that GlyT1 inhibitors may prove to be effective in the treatment of symptom domains associated with schizophrenia. However, these data also suggest a complex relationship between GlyT1 receptor occupancy, CSF glycine changes and behavioral efficacy. As such, careful consideration of GlyT1 receptor occupancies and CSF glycine changes is required in order to guide the selection of dosing regimens for future proof of concept studies in schizophrenia patients.