Development and Preliminary Evaluation of a ¹²⁵I-Labeled Radioligand ([¹²⁵I]iodotrazoline) for In Vitro Detection of Imidazoline-2 Binding Site in the Brain

Astrocytes exert multiple functions within the brain, including regulating neuroinflammation and maintaining homeostasis, and the reactive astrocytes are implicated in many neurodegenerative disorders. Imidazoline-2 binding site (I₂BS) has been established as a reliable biomarker for precisely quantifying reactive astrocytes. Here, we reported the development of [¹²⁵I]iodotrazoline ([¹²⁵I]<b>8</b>), a novel I₂BS radioligand with high affinity (<i>K</i>_i = 6.8 nM) and exceptional selectivity over α₂-adrenoceptors (>1400 folds). <i>In vitro</i> autoradiography (ARG) using rat brain sections revealed a heterogeneous distribution of [¹²⁵I]<b>8</b>, with high signals in the medulla, midbrain, pons, and hypothalamus. Pretreatment with unlabeled I₂BS-selective ligands, BU224 and FTIMD, reduced the binding by >30%, indicating high <i>in vitro</i> specificity for I₂BS. <i>Ex vivo</i> ARG results confirmed this distribution pattern in the rat brain. Biodistribution results in mice demonstrated a rapid brain uptake of [¹²⁵I]<b>8</b> (3.35% ID/g at 2 min postinjection) with slow washout. Metabolite analysis exhibited the desirable biostability of [¹²⁵I]<b>8</b> in the rat brain. Altogether, this work provides a new ¹²⁵I-labeled radioligand featuring a novel 2-trans-styryl-imidazoline scaffold, which shows significant specificity binding for I₂BS <i>in vitro</i>, serving as a valuable tool for I₂BS detection and astrocyte-related pathology research.