Assessment of the Target Engagement and D-Serine Biomarker Profiles of the D-Amino Acid Oxidase Inhibitors Sodium Benzoate and PGM030756.

Irregular N-methyl-D-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of schizophrenia. Although direct agonism of the NMDAR has not yielded promising therapeutics, advances have been made by modulating the NMDAR co-agonist site which is activated by glycine and D-serine. One approach to activate the co-agonist site is to increase synaptic D-serine levels through inhibition of D-amino acid oxidase (DAO), the major catabolic clearance pathway for this and other D-amino acids. A number of DAO inhibitors have been developed but most have not entered clinical trials. One exception to this is sodium benzoate which has demonstrated efficacy in small trials of schizophrenia and Alzheimer's disease. Herein we provide data on the effect of sodium benzoate and an optimised Takeda compound, PGM030756 on ex vivo DAO enzyme occupancy and cerebellar D-serine levels in mice. Both compounds achieve high levels of enzyme occupancy; although lower doses of PGM030756 (1, 3 and 10 mg/kg) were required to achieve this compared to sodium benzoate (300, 1000 mg/kg). Cerebellar D-serine levels were increased by both agents with a delay of approximately 6 h after dosing before the peak effect was achieved. Our data and methods may be useful in understanding the effects of sodium benzoate that have been seen in clinical trials of schizophrenia and Alzheimer's disease and to support the potential clinical assessment of other DAO inhibitors, such as PGM030756, which demonstrate good enzyme occupancy and D-serine increases following administration of low oral doses.

Evaluation of monoamine oxidase A and B type enzyme occupancy using non-radiolabelled tracers in rat brain.

Monoamine oxidase (MAO) enzymes, type A and B metabolise the amine neurotransmitters of the body. Selective inhibition of either enzyme is an approach for treating neurodegenerative and stress-induced disorders, and inhibition of an enzyme is proportional to the binding of the MAO inhibitor. Conventionally, the binding of test compounds to enzymes is assessed by radiolabelled ligands in ex vivo and in vivo occupancy assays. Regulatory restrictions and turnaround time are the limitations of the methods that use radiolabelled ligands. But the use of non-radiolabelled tracers and sensitive mass spectrometry (LC-MS/MS) based assays accelerated the determination of target occupancy in pre-clinical species. A report on use of non-radiolabelled ligand in in vivo MAO occupancy assay is not available. The objectives of the present study were to optimise non-radiolabelled harmine and deprenyl as selective tracers in MAO-A and MAO-B occupancy assays and evaluate MAO occupancy of test compounds in rat brain. Tracer optimisation resulted in a detectable, stable, and low ratio (<3.0) of tracer concentrations between any two brain tissues. In occupancy assay, tracer was intravenously administered (10 µg/kg, harmine or 60 µg/kg, L-deprenyl) after the treatment with test compound (clorgyline or tranylcypromine or pargyline or phenelzine or thioperamide). Specific brain tissues were isolated at a defined interval and tracer concentrations were quantified using LC-MS/MS method. Pre-treatment with MAO inhibitors resulted in a decrease (maximum, 80-85%) in harmine or an increase (maximum, 85-300%) in L-deprenyl concentrations. But we considered the change in tracer concentration, relative to the vehicle and positive control groups to calculate MAO occupancy. The observed selectivity and ratio of occupancies (ED50) of test compound towards MAO-A and MAO-B are comparable with the results from in vitro radiolabelled ligand-based inhibition assay. The results demonstrated the application of these non-radiolabelled tracers as suitable pre-clinical tools to determine MAO occupancy.

In vivo measurement of PDE10A enzyme occupancy by positron emission tomography (PET) following single oral dose administration of PF-02545920 in healthy male subjects.

Phosphodiesterase 10A (PDE10A) is an enzyme highly enriched in the striatal medium spiny neurons. It is involved in the regulation of cytoplasmic levels of cAMP and cGMP and signaling within the basal ganglia. This study with PDE10A radioligand [18F]MNI-659 was designed to measure the enzyme occupancy of PF-02545920 in 8 healthy male volunteers (48 ± 4 years) after a single oral dose (10 mg or 20 mg) and to evaluate safety and tolerability. Arterial blood sampling was performed to obtain a metabolite-corrected plasma input function for the quantification of [18F]MNI-659 binding to PDE10A. The occupancy of PF-02545920 was calculated with two different methods: In Method 1, [18F]MNI-659 enzyme occupancy was calculated from the estimates of binding potential, using the cerebellum as a reference region; in Method 2, occupancy was estimated from the slope of the revised Lassen's plot. Serum concentrations of PF-02545920 were measured to determine the relationship between concentration and occupancy. Based on Method 1, striatal PDE10A occupancy increased with increasing PF-02545920 dose: 14-27% at 10 mg dose (N = 4) and 45-63% at 20 mg dose (N = 3). Comparable occupancies were observed using Lassen's plot Method 2: 10 mg: 14-37%; 20 mg: 46-55%. The relationship between exposure and occupancy was best described using an Emax model. The serum concentration associated with 50% occupancy was estimated to be 93.2 ng/mL. Single oral doses of 10 mg or 20 mg of PF-02545920 were safe and well tolerated in healthy male volunteers [NCT# 01918202].