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.

(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.

Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.

In vitro and in vivo evaluation of [18F]-FDEGPECO as a PET tracer for imaging the metabotropic glutamate receptor subtype 5 (mGluR5).

(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-2-(2-(18)F-fluoroethoxy)ethyl oxime, ([(18)F]-FDEGPECO), a novel high affinity radioligand for the metabotropic glutamate receptor subtype 5 (mGluR5) was assessed for its potential as a PET imaging agent. In vitro autoradiography on rat brain slices resulted in a heterogeneous and displaceable binding to mGluR5-rich brain regions. [(18)F]-FDEGPECO showed high stability in rat plasma and brain homogenate as well as in human plasma and microsomes. Good blood-brain barrier passage was predicted from an in vitro transport assay with P-glycoprotein-transfected hMDR1-MDCK cells. In vivo PET imaging on rats revealed specific uptake of radioactivity in the mGluR5-rich brain regions such as hippocampus, striatum and cortex while the cerebellum, a region with low mGluR5-expression, showed negligible uptake. Blockade experiments by co-injection of [(18)F]-FDEGPECO and M-MPEP (6mg/kg), an antagonist for mGluR5, reduced the level of radioactivity in mGluR5-regions to that of the cerebellum, pointing to an effective blockade of specifically bound [(18)F]-FDEGPECO. Postmortem biodistribution studies at 15min p.i. confirmed the distribution pattern observed in PET. HPLC analysis of rat brain extracts indicated that 98.5% and 91% of the total radioactivity were parent compound at 5min and 17min p.i., respectively. Taken together, the high affinity and the high in vivo specificity of [(18)F]-FDEGPECO for mGluR5 in the rat brain as well as the lack of in vivo defluorination make this new [(18)F]-labeled ABP688 derivative a suitable ligand for the preclinical PET imaging of mGluR5. These favorable characteristics warrant further evaluation in humans.