Evaluation of serotonin 5-HT(1A) receptors in rodent models using [¹⁸F]mefway PET

Abstract

Introduction: Serotonin 5-HT(1A) receptors have been investigated in various CNS disorders, including epilepsy, mood disorders, and neurodegeneration. [¹⁸F]Mefway (N-{2-[4-(2'-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(cis/trans-4'-[¹⁸F]fluoromethylcyclohexane)-carboxamide) has been developed as a suitable positron emission tomography (PET) imaging agent for these receptors. We have now evaluated the suitability of [¹⁸F]trans-mefway in rat and mouse models using PET and computerized tomography (CT) imaging and corroborated with ex vivo and in vitro autoradiographic studies.

Methods: Normal Sprague-Dawley rats and Balb/C mice were used for PET/CT imaging using intravenously injected [¹⁸F]trans-mefway. Brain PET data were coregistered with rat and mouse magnetic resonance imaging template and regional distribution of radioactivity was quantitated. Selected animals were used for ex vivo autoradiographic studies to confirm regional brain distribution and quantitative measures of binding, using brain region to cerebellum ratios. Binding affinity of trans-mefway and WAY-100635 was measured in rat brain homogenates. Distribution of [¹⁸F]trans-4-fluoromethylcyclohexane carboxylate ([¹⁸F]FMCHA), a major metabolite of [¹⁸F] trans-mefway, was assessed in the rat by PET/CT.

Results: The inhibition constant, K(i) for trans-mefway was 0.84 nM and that for WAY-100635 was 1.07 nM. Rapid brain uptake of [¹⁸F]trans-mefway was observed in all rat brain regions and clearance from cerebellum was fast and was used as a reference region in all studies. Distribution of [¹⁸F]trans-mefway in various brain regions was consistent in PET and in vitro studies. The dorsal raphe was visualized and quantified in the rat PET but identification in the mouse was difficult. The rank order of binding to the various brain regions was hippocampus > frontal cortex > anterior cingulate cortex > lateral septal nuclei > dorsal raphe nuclei.

Conclusion: [¹⁸F]trans-Mefway appears to be an effective 5-HT(1A) receptor imaging agent in rodents for studies of various disease models.

Keywords: 5-HT1A receptors; WAY-100635; mefway; microPET; serotonin.

Figure 1

Chemical structure of [¹⁸F]trans-mefway and [¹⁸F]cis-mefway.

Figure 2

Displacement curves of mefway and WAY-100635 measured in rat brain homogenates using [³H]WAY-100635 for the serotonin 5-HT_1A receptor sites.

Figure 3

PET/CT imaging of rats showing two different orthogonal planes. PET images were summed from 60 to 90 min, postinjection: (A). MR template images of the rat brain; (B). [¹⁸F]Mefway PET/MR images at two different planes; through the dorsal hippocampus and through the ventral hippocampus and dorsal raphe. (C). CT images showing brain and skull; (D). Coregistered CT images of mefway PET. Asterisks indicate radioactivity containing areas outside the brain confirmed by coregistered CT.

Figure 4

(A). Time-activity curve of [¹⁸F]mefway in the rat brain showing hippocampus (HP), lateral septum (LSN), frontal cortex (FC), anterior cingulate cortex (ACC), dorsal raphe nucleus (DRN) and cerebellum (CB). (B). Averaged in vivo brain activity of rats (n=4) in the different brain regions measured by PET and brain region to cerebellum ratios.

Figure 5

(A). In vivo PET rat brain slice of [¹⁸F]mefway coregistered to MR template after injection of 24.6 ± 11.3 MBq dose of [¹⁸F]mefway. (B). Ex vivo PET of [¹⁸F]mefway taken immediately after completion of the in vivo PET scan. (C). After the ex vivo scan is completed, the brain is cut into 40micron sections and exposed to phosphor screens. (D). Correlation of [¹⁸F]mefway binding in vivo (A) with the ex vivo autoradiographs (C). Correlation is high when the frontal cortex and dorsal raphe nucleus are excluded. (E). Correlation of [¹⁸F]mefway binding ratio with reported binding of [³H]8-OH-DPAT in different brain regions.

Figure 6

(A-D). Ex vivo horizontal brain slices of rodent brain showing binding of [¹⁸F]mefway (red = highest binding and white = lowest binding; OL=outer layer of cortex; ML= middle layer of cortex; IL=inner layer of cortex; ACC=anterior cingulated cortex; DG= dentate gyrus; iEC= inner enthorinal cortex; oEC= outer enthorinal cortex; LS= lateral septal nuclei; DRN= dorsal raphe nucleus; CA1, CA2, CA3= hippocampal regions). (E). Quantitation of [¹⁸F]mefway binding in hippocampal regions, dorsal raphe and septal nuclei in ex vivo autradiographs and ratio with respect to the cerebellum; (F). Quantitation of [¹⁸F]mefway binding in cortical layers in ex vivo autradiographs and ratio with respect to cerebellum.

Figure 7

[¹⁸F]Mefway PET imaging of mice: (A). Horizontal mouse brain section showing [¹⁸F]mefway binding, ACC= anterior cinglate cortex; LS= lateral septal nuclei; HP= hippocampus; DRN= dorsal raphe nucleus; EC= enthorinal cortex); (B). Time-activity curves of [¹⁸F]mefway in the mouse brain; (C). PET images were summed from 60 to 90 min, postinjection and coregistered with mouse brain MR template to show [¹⁸F]Mefway PET/MR images of mice through the dorsal hippocampus. (D). Quantitation of [¹⁸F]mefway in the mouse brain regions and ratios with respect to cerebellum.

Figure 8

(A). Schematic showing potential breakdown of [¹⁸F]mefway at the amide linkage to provide the [¹⁸F]trans-4-fluoromethylcyclohxane carboxylate ([¹⁸F]FMCHA) and WAY-100634. Decompostion of [¹⁸F]FMCHA and [¹⁸F]mefway may further occur to give [¹⁸F]fluoride ion. (B). Rat summed of [¹⁸F]FMCHA showing upper body including head and liver of [¹⁸F]FMCHA distribution and time activity curves of various regions of the body; (C). Brain slices showing little [¹⁸F]FMCHA.