Selective binding of 2-[125I]iodo-nisoxetine to norepinephrine transporters in the brain

A radioiodinated ligand, (R)-N-methyl-(2-[I-125]iodo-phenoxy)-3-phenylpropylamine, [I-125]2-INXT, targeting norepinephrine transporters (NET), was successfully prepared. A no-carrier-added product, [I-125]2-INXT, displayed a saturable binding with a high affinity (K-d=0.06 nM) in the homogenates prepared from rat cortical tissues as well as from LLC-PK1 cells expressing NET. A relatively low number of binding sties (B-max=55 fmol/mg protein) measured with [I-125]2-INXT in rat cortical homogenates is consistent with the value reported for a known NET ligand, [3 H]nisoxetine. Competition studies with various compounds on [I-125]2-INXT binding clearly confirmed the pharmacological specificity and selectivity for NET binding sites. Following a tail-vein injection of [I-125]2-INXT in rats, a good initial brain uptake was observed (0.56% dose at 2 min) followed by a slow washout from the brain (0.2% remained at 3 hours post-injection). The hypothalamus (a NET-rich region) to striatum (a region devoid of NET) ratio was 1.5 at 3 hours post-i.v. injection. Pretreatment of rats with nisoxetine significantly inhibited the uptake of [I-125]2-INXT (70-100% inhibition) in locus coeruleus, hypothalamus and raphe nuclei, regions known to have a high density of NET; whereas escitalopram, a serotonin transporter ligand, did not show a similar effect. Ex vivo autoradiography of rat brain sections of [I-125]2-INXT (at 3 hours after an i.v. injection) displayed an excellent regional brain localization pattern corroborated to the specific NET distribution in the brain. The specific brain localization was significantly reduced by a dose of nisoxetine pretreatment. Taken together, the data suggest that [I-123]2-INXT may be useful for mapping NET binding sites in the brain. (C) 2004 Elsevier Inc. All rights reserved.