Initial characterization of [11C]-GSK1482160 as radioligand for P2X7 receptors

Objectives Neuroinflammation is an essential step in the progression of brain diseases [1], where pro-inflammatory cytokines play a central role [2]. Recent work has shown that the pathogenesis of neuroinflammation is mediated in part by the release of adenosine/uridine derivatives from the damage site, involving a family of ionotropic purinergic receptors including P2X7 with elevated receptor expression [3], microglial proliferation and phagocytosis of the injured site [4, 5]. This mechanism is common amongst a wide array of neurodegenerative inflammatory diseases which include: Alzheimer's, Parkinson's, Huntington’s Disease, frontotemporal dementia, Atrophic Lateral Sclerosis, Multiple Sclerosis, and Traumatic Brain Injury [6-8]. The P2X7 receptors represent a novel molecular target for imaging neuroinflammation via PET. GSK1482160 is an ideal starting point for evaluation of the P2X7 receptors, has high receptor affinity, and ideal blood-brain barrier penetration [9]. Moreover, recent work from our institution has radiolabeled this compound [11C]-GSK1482160 [10] yielding a potential biomarker for neuroinflammation. Therefore, we report the initial physical and biological characterization of this novel ligand.

Methods Radiosynthesis of [11C]-GSK1482160 was according to published methods [10]. To determine receptor density (Bmax), saturation kinetics was employed with using cell membrane homogenate from human embryonic kidney cells (HEK) transfected with human P2X7R gene. Receptor binding potential (Bp) was determined by association kinetics. Secondary confirmation was performed via Radiologic-IHC (RIHC) on facing sections, probed with P2X7R monoclonal antibodies (mAb), and detected via [125I]-Protein-A. Tertiary confirmation was performed via Immuno-fluorescence Assay (IFA) using P2X7 mAb conjugated to NIR-fluorophore. Whole animal biodistribution was performed in 3 mice/time point at 15, 30, and 60min post IV injection. Tissue activity (%ID/g) was determined for brain, blood, heart, lung, liver, spleen, intestines, kidney, and muscle. In addition, in vivo PET/CT (n=3) images were acquired dynamically in listmode, regions segmented manually, and time series analyzed with a 2 compartment 4 parameter tracer kinetic models.

Results Using high specific activity [11C]-GSK1482160, saturation kinetics revealed a Bmax and Kd of 923fmol/mg and 1.1nm in hP2X7R-HEK cells, respectively. Association kinetic determination of kon, koff, and Bp in hP2X7R-HEK cells were 0.231/min[asterisk]nM, 0.272/min, and 0.91, respectively. These results were confirmed via P2X7 binding and IFA in hP2X7R-HEK cell blocks, showing high sensitivity and specificity in this system. Biodistribution of [11C]-GSK1482160 showed a time dependent tissue distribution in all tissues studied, with the liver (36.8±5.0), intestines (16.9±2.6) and kidney (29.1±5.4) showing the highest uptake by 30min. The brain, blood, heart, lung, spleen, and skeletal muscle all had uptake which showed similar temporal patterns, and uptake levels of 2.5±0.27, 8.16±0.90, 11.6±1.25, 12.1±1.5, 8.2±1.1, and 6.9±0.82%ID/g at 30min. To characterize tracer kinetics, in vivo PET/CT [11C]-GSK1482160 was performed in normal mice and time courses modeled for brain, liver, intestines, kidney, muscle and bladder (R2=0.933±0.021). Determination of total volumes of distributions (Vt) for brain, heart, liver, intestine, kidney, and muscle were 0.068±.0008, 0.2±0.003, 0.45±0.02, 0.21±0.02, 0.24±0.02, and 0.11±0.04, respectively.

Conclusions Initial characterization of [11C]-GSK1482160 shows high affinity and favorable association/disassociation kinetics, and were confirmed via two different in vitro assays. In vivo biodistribution indicated that the liver, intestine and kidney uptake were maximal by 30 min, and in vivo PET/CT tracer kinetic modeling indicated that Vt followed expected trends for non-diseased mice.