Synthesis, radiolabelling and in vitro and in vivo evaluation of a novel fluorinated ABP688 derivative for the PET imaging of metabotropic glutamate receptor subtype 5

Abstract

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(2-(3-(18)F-fluoropropoxy)ethyl) oxime ([(18)F]-PSS223) was evaluated in vitro and in vivo to establish its potential as a PET tracer for imaging metabotropic glutamate receptor subtype 5 (mGluR5). [(18)F]-PSS223 was obtained in 20% decay corrected radiochemical yield whereas the non-radioactive PSS223 was accomplished in 70% chemical yield in a S(N)2 reaction of common intermediate mesylate 8 with potassium fluoride. The in vitro binding affinity of [(18)F]-PSS223 was measured directly in a Scatchard assay to give K(d) = 3.34 ± 2.05 nM. [(18)F]-PSS223 was stable in PBS and rat plasma but was significantly metabolized by rat liver microsomal enzymes, but to a lesser extent by human liver microsomes. Within 60 min, 90% and 20% of [(18)F]-PSS223 was metabolized by rat and human microsome enzymes, respectively. In vitro autoradiography on horizontal rat brain slices showed heterogeneous distribution of [(18)F]-PSS223 with the highest accumulation in brain regions where mGluR5 is highly expressed (hippocampus, striatum and cortex). Autoradiography in vitro under blockade conditions with ABP688 confirmed the high specificity of [(18)F]-PSS223 for mGluR5. Under the same blocking conditions but using the mGluR1 antagonist, JNJ16259685, no blockade was observed demonstrating the selectivity of [(18)F]-PSS223 for mGluR5 over mGluR1. Despite favourable in vitro properties of [(18)F]-PSS223, a clear-cut visualization of mGluR5-rich brain regions in vivo in rats was not possible mainly due to a fast clearance from the brain and low metabolic stability of [(18)F]-PSS223.

Keywords: PET imaging; [11C]-ABP688; [18F]-FDEGPECO; [18F]-PSS223; autoradiography; mGluR5; microsome enzymes.

Figure 1

Structures of MPEP and selected PET tracers for imaging of mGluR5

Figure 2

Syntheses of PSS223 and [¹⁸F]-PSS223 from key intermediate 8 via S_N2 reaction.

Figure 3

A. Displacement of [³H]-ABP688 by PSS223. [³H]-ABP688 (total 2 nM) binding (B) as a function of the PSS223 concentration. Symbols represent mean±SD of one experiment performed in triplicate. Non-linear fitting (red line) resulted in IC₅₀ 14 nM and corresponding K_i 6 nM; B. Scatchard plot analysis of the specific binding of [¹⁸F]-PSS223 to rat brain membranes. Symbols, specific binding (total binding minus binding in the presence of 100 μM MMPEP) of one representative experiment; red line, linear regression.

Figure 4

In vitro autoradiography with rat brain slices. A. Incubation with 1 nM [¹⁸F]-PSS223 showing heterogeneous distribution with highest expression in striatum, hippocampus and cortex; B. Brain slices incubated with 1 nM [¹⁸F]-PSS223 and 100 nM ABP688; C. Brain slices incubated with 1 nM [¹⁸F]-PSS223 and 100 nM mGluR1 antagonist JNJ16259685. Blue, green, orange, red, dark red indicate radioactivity from low to high. Columns represent same experiment performed in duplicate.

Figure 5

A. Metabolism of [¹⁸F]-PSS223 by rat liver microsomes. Green symbols, [¹⁸F]-PSS223; blue symbols, polar ¹⁸F-radiometabolite; lines, fitted exponential functions; B. Metabolism of [¹⁸F]-PSS223 by human liver microsomes. Green symbols, [¹⁸F]-PSS223; blue and red symbols, polar ¹⁸F-radiometabolites.

Figure 6

A. PET dynamic scan of the head of a male Wistar rat injected with [¹⁸F]-PSS223. Transverse planes through the head summarized for the indicated time windows. White, CT; B. Time activity curves of [¹⁸F]-PSS223 uptake in a male Wistar rat in different brain regions showing increasing uptake in jaw due to rapid defluorination.

Figure 7

Rat brain PET images of [¹¹C]-ABP688, [¹⁸F]-FDEGPECO and [¹⁸F]-PSS223. Planes (crosshairs) and maximal intensity projections (MIP) averaged from 2 to 45 min after tracer injection.

Figure 8

Likely mechanism of defluorination of [¹⁸F]-PSS223 involves cytochrome P450-catalyzed C-oxygenation. The aldehyde 9 is oxidized or reduced to the respective carboxylic acid or alcohol while [¹⁸F]-fluoride is accumulated in bone.