Rapid Identification of a Novel Small Molecule Phosphodiesterase 10A (PDE10A) Tracer

Abstract

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

AMG 580: A Novel Small Molecule Phosphodiesterase 10A (PDE10A) Positron Emission Tomography Tracer

“…In the first set of experiments, chemically distinct, nonlabeled, and selective PDE10 inhibitors, AMG 580 (IC 50 5 0.13 nM), AMG 579 (IC 50 5 0.19 nM) (ketobenzamidazole series) , AMG 0074 (IC 50 5 1.0 nM) (quinolone series) (Verhoest et al, 2009), and AMG 7980 (IC 50 5 1.9 nM) (cinnoline series) (Hu et al, 2012) were used to compete for [ 3 H]AMG 580 binding to rat, baboon, and human striatal homogenates ( Fig. 6; Table 4).…”

“…Previously, we reported a high-affinity, selective PDE10A tracer AMG 7980 (Hu et al, 2012;Hwang et al, 2014). AMG 7980 has a fast off-rate, limiting its utility for PET imaging in higher species.…”

“…AMG 7980 has a fast off-rate, limiting its utility for PET imaging in higher species. To identify PDE10A tracer candidates with suitable retention time on the target, SPR spectroscopy was used for the on-and off-rate assessment of the compounds using the purified human PDE10A catalytic domain described previously (Hu et al, 2012). This allowed us to eliminate compounds with fast off-rate (cutoff criterion: approximately 10-fold slower than AMG 7980).…”

“…Therefore, occupancy of AMG 580 at 10 and 30 mg/kg in the alternative formulation was performed and data were combined with the original formulation. STR, with the highest PDE10A expression, was used as the target tissue and the thalamus (THA) was treated as the reference tissue (Hu et al, 2012). Occupancy was calculated based on the reference tissue model determined using the following equations:…”

“…Brain homogenates were prepared from rat, baboon, and human STR using a Covaris E110 Acoustic Homogenizer, as described previously (Hu et al, 2012). For saturation binding experiments, rat STR homogenate (10 mg/well) or baboon and human STR homogenate (25 mg/well) were incubated with [ 3 H]AMG 580 in a 96-well plate with or without nonradiolabeled AMG 580 in assay buffer (50 mM Tris, pH 7.5, 5 mM MgCl 2 , 1Â Roche complete protease inhibitor tablet (Indianapolis, IN), EDTA free) for 1 hour at room temperature.…”