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. 2016 Mar 13;7(5):482–486. doi: 10.1021/acsmedchemlett.5b00499

Autoradiographic Evaluation of [18F]FECUMI-101, a High Affinity 5-HT1AR Ligand in Human Brain

J S Dileep Kumar †,‡,*, Mark D Underwood †,§, Norman R Simpson †, Suham A Kassir †, Jaya Prabhakaran §, Vattoly J Majo §, Mihran J Bakalian †, Ramin V Parsey ‡, J John Mann †,§, Victoria Arango †,§
PMCID: PMC4867482  PMID: 27190597

Abstract

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[18F]FECUMI-101 ([18F]1) is a 5HT1AR ligand demonstrating specific binding in brain regions corresponding to the distribution of 5-HT1AR in baboons. However, we detected moderate uptake of [18F]1 in baboon thalamus, a brain region lacking 5-HT1AR. We sought to investigate the relative binding of [18F]1 to 5-HT1AR, α1R, and 5-HT7R in vitro. Using autoradiography in human brain sections, specific binding of [18F]1 to 5-HT1AR was confirmed. However, [18F]1 also showed 26% binding to α1R in PFC. The hippocampal formation exhibited 51% and 92% binding of [18F]1 to α1R and 5-HT1AR, respectively. Thalamus and cerebellum showed very little binding. There is no measurable specific binding of [18F]1 to 5-HT7R and no effect of temperature on [18F]1 specific binding to 5-HT1AR or α1R. These results indicate that, while [18F]FECUMI-101 is not a completely selective 5-HT1AR ligand for receptor quantification, it may be useful for occupancy measurements of drugs acting at 5-HT1AR in vivo.

Keywords: 5-HT1AR, α1R, radiotracer, autoradiography, PET, hippocampus

5-HT1AR exists in active and inactive agonist affinity states. The antagonist ligands bind to the active and inactive conformations of 5-HT1AR with equal affinity. Whereas, agonist ligands bind preferentially to the active state of the receptor, thereby providing a more meaningful functional measure of the 5-HT1AR.1 Although, antagonist 5-HT1AR PET tracers can measure the total receptor binding, they cannot detect changes in the active 5-HT1AR binding in disease states or in the context of drug based treatment. Agonist 5-HT1AR radiotracers are likely to be sensitive to measure the changes in the intrasynaptic concentrations of the endogenous 5-HT to the receptor. Additionally 5-HT1AR agonist radiotracers may be useful to monitor desensitization and sensitization of receptors; to provide a better estimate of 5-HT1AR in diseases; measure receptor occupancy of agonist therapeutic agents; and for the efficacy evaluation of SSRI treatment.1−3 The development of specific 5-HT1AR agonist PET tracers has met with limited success despite three decades of effort.1 Most of the agonist radiotracers developed to date have the structural core of amino-tetralin, apomorphine, thiochromine, and arylpiperazine based ligands.1−3 We reported 3,5-dioxo-(2H,4H)-1,2,4-triazine tethered arylpiperazines as promising agonist/partial agonist ligands with high affinity for 5-HT1AR based on GTPγS stimulation studies in CHO cells stably expressing human 5-HT1AR.4−6 Based on structure–affinity relationship (SAR) studies of 3,5-dioxo-(2H,4H)-1,2,4-triazine, [11C]CUMI-101 was identified as a partial agonist radiotracer, and it has been tested in both nonhuman primates and human subjects with PET.5−13 Another study using membrane preparations from CHO cells also observed 5-HT1AR agonist properties of CUMI-101.14 However, the same report did not find stimulation of GTPγS binding by CUMI-101 in rat cortex and rat hippocampal membranes. Recently, brain homogenate assays of CUMI-101 in rat, monkey, and human brain homogenates showed increase of GTPγS stimulation compared to basal expression.15 The dose response curves of the same study were more consistent with that of an antagonist behavior of CUMI-101. In a GTPγS stimulation of rat hippocampal membrane study, buspirone, a partial agonist of 5-HT1AR exhibited very low stimulation to GTPγS binding in comparison to full agonist (R)-8-OH-DPAT.32 The above study indicates that membrane homogenate studies may not provide a definitive answer for 5-HT1AR functional properties of partial agonists.32 Additional findings such as dose–response relationships to behavior and electrophysiological neuron responses in rodents may offer better definitive characterizing of the agonist/antagonist properties of CUMI-101 in vivo. While several 18F-labeled radiotracers based on the CUMI-101 core failed in vivo to image 5-HT1AR, PET studies of [18F]FECUMI-101 ([18F]1, Figure 1), a fluoroethyl analogue of CUMI-101, showed promising imaging characteristics in baboons.16 FECUMI-101 is a high affinity 5-HT1AR ligand (Ki = 0.1 nM) and produced a dose-dependent increase in [35S]GTPγS binding in CHO cells expressing 5-HT1AR with an Emax 77% and EC50 of 0.65 nM.16In vivo pharmacological response following subcutaneous administration of FECUMI-101 induce a moderate lower lip retraction in rats, a characteristic response specific to 5-HT1AR activation. These studies further confirm partial 5-HT1AR agonistic properties of FECUMI-101.33 The highest uptake of [18F]1 radioactivity was found in hippocampus, anterior cingulate cortex, insular cortex, prefrontal cortex, and amygdala.16 Striatum, occipital cortex, and cerebellum showed minimal binding of [18F]1.16 This distribution is consistent with our previous reports of the parent tracer [11C]CUMI-101 in baboon and human.7−11 The thalamus was discrepant showing higher binding of [18F]FECUMI-101 in comparison to [11C]CUMI-101 and [11C]WAY100635, despite the higher selectivity of FECUMI to α1AR.7−13 The binding of [18F]FECUMI-101 to thalamus cannot be solely attributed to 5-HT1AR binding as it is established that thalamus has low 5-HT1AR concentration.1,3,17 Receptor selectivity assays show that the highest affinity of FECUMI-101, apart from 5-HT1AR (Ki = 0.1 nM), is to 5-HT7R (Ki = 17.2 nM), α1AR (Ki = 21.4 nM), and α1BR (26.8 nM).16 Both 5-HT7R and α1R are abundant in the thalamus, and α1R is coexpressed with 5-HT1AR in many brain regions.18−20 Since α1R antagonists are known to produce cardiovascular changes even at 0.01 mg/kg doses in monkeys, it is difficult to perform in vivo blocking experiments to test α1AR selectivity of [18F]1 in nonhuman primates.21,22 The binding affinity measurements of FECUMI-101 were performed at room temperature;16 however, differences in α1R binding at room temperature vs 37 °C (physiological temperature) were recently reported for the parent ligand CUMI-101 in membrane homogenate radioligand binding assays.15

Figure 1.

Figure 1

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Chemical structure of [18F]FECUMI-101.

We sought to determine the binding selectivity of [18F]1 for 5-HT1AR, α1R, and 5-HT7R and test potential temperature effects on 5-HT1AR and α1R binding by performing experiments at room temperature and at 37 °C using in vitro autoradiography in post-mortem human brain sections. Herein, we describe our experiments and results.

The reference standard FECUMI-101 and the corresponding radiolabeling precursor were synthesized using our previously reported procedure.16 Radiosynthesis of [18F]FECUMI-101 was achieved with minor modifications of the reported procedure.16 Briefly, [18F]fluoroethyl tosylate was synthesized and reacted with the corresponding desethyl precursor in DMSO in the presence of K2CO3. The crude product was purified by semipreparative HPLC, followed by elution with C-18 Sep-Pak cartridge, and the radioproduct was reconstructed in ethanol. A portion of the ethanol solution was used for quality control studies and subsequently used for autoradiography studies. [18F]FECUMI-101 was obtained in 30 ± 5% yield with ≥90% radiochemical purities and 1.5 ± 0.5 Ci/μmol specific activity.

Post-mortem sections (20 μm) from four nonpsychiatric, medication-free normal human brains that included hippocampus, prefrontal cortex (BA9), thalamus, and cerebellum were used for the autoradiography studies.23−25 Initially we examined the distribution of [18F]1 in slide-mounted post-mortem human brain sections at 37 °C (Figure 2). The binding data obtained were averaged from two experiments for hippocampus, BA9, and thalamus, four experiments for white matters, and one experiment for cerebellum with measures in triplicate. The highest concentration of [18F]1 binding was found in hippocampus, with less binding in other structures (rank order relative to adjacent white matter: hippocampus (28:1) > prefrontal cortex (7.1:1) > thalamus (2.8:1) > cerebellum (2.2:1) > white matter (1:1)). The data for individual regions are corresponding to binding in gray matter relative to adjacent white matter in the same tissue section. The distribution of [18F]FECUMI-101 was heterogeneous and generally consistent with the known distribution of the 5-HT1AR receptor (Figure 2).

Figure 2.

Figure 2

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Regional distribution of [18F]FECUMI-101 in post-mortem human brain sections at 37 °C.

We determined [18F]1 binding selectivity in BA9, hippocampus, thalamus, and cerebellum by displacing [18F]FECUMI-101 uptake with specific blockers; WAY100635 for 5-HT1AR, prazosin for α1R, and SB269970 for 5-HT7R at 37 °C (Figure 3). Percentages of binding represented are based on the corresponding specific binding of 5-HT1AR, α1R, and 5-HT7R ligands with respect to the total binding of [18F]FECUMI-101. In BA9, prazosin displaced approximately 26% of [18F]FECUMI-101 binding, while WAY100635 displaced 98% of binding suggesting that [18F]FECUMI-101 has high affinity for 5-HT1AR but also modest measurable binding to α1R. The hippocampal sections for this experiment were from the most anterior level where only cornu ammonis1 (CA1) is present as dentate gyrus (DG) begins 4 mm posterior to the CA1. WAY100635 displaced all the binding of [18F]FECUMI-101 in the CA1 area, and we did not find any measurable binding for α1R as evident from prazosin displacement (Figure 3). In a subsequent experiment, with sections of hippocampus containing both the CA and DG fields, [18F]FECUMI-101 was seen to label both the CA and DG (Figure 4). When the entire hippocampus was sampled, prazosin and WAY100635 displaced 51% and 92% of [18F]FECUMI-101 binding, respectively. In the DG, prazosin and WAY100635 provided comparable displacement of [18F]FECUMI-101 binding (84% and 89% respectively); while in CA1, WAY100635 displaced all [18F]FECUMI-101 binding (99%) in CA1 compared to prazosin (13%, Figure 4). In contrast, in vivo PET imaging in baboon did not distinguish CA1 and DG subfields and only measured whole hippocampus.16

Figure 3.

Figure 3

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In vitro selectivity of [18F]FECUMI-101 in slide mounted post-mortem human brain sections at 37 °C.

Figure 4.

Figure 4

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Autoradiograms of [18F]FECUMI-101 in human brain sections at 37 °C. (A) Total binding of [18F]FECUMI-101. (B) Displacement with 1 μM WAY100635. (C) Displacement with 1 μM prazosin.

In the thalamus and cerebellum, there is comparatively little [18F]FECUMI-101 binding and 69% of that small amount of binding was displaced by prazosin and 87% by WAY100635 in the thalamus and 85% and 87% in cerebellum, respectively (Figure 3). Specificity of cerebellar and thalamus binding of [18F]FECUMI-101 are consistent with in vivo baboon PET studies.16 WAY100635 displaces 42% and 55% binding of [18F]FECUMI-101 in vivo in baboon cerebellum and thalamus, respectively.16 These findings are in contrast to the parent ligand [3H]CUMI-101 (5-HT1AR Ki = 0.15 nM; α1R Ki = 6.5 nM), which shows high amounts of specific binding in 5-HT1AR rich brain regions and no significant binding to α1R except for a small level of binding in DG.13 The reason for why there is more binding of [18F]FECUMI-101 to α1R in prefrontal cortex and hippocampal tissues comparison to [3H]CUMI-101 is not clear, especially given its lower α1R affinity (Ki = 21.4 nM vs 6.5 nM).

The 5-HT7R ligand, SB269970, did not displace [18F]FECUMI-101 binding in any of the regions measured (Figure 3). This suggests that the [18F]FECUMI-101 has no appreciable binding to the 5-HT7R or that the density of 5-HT7R is low in these regions in human brain and hence are not contributing significantly to the total binding.

To determine whether there is a difference in the relative amount of binding to the 5-HT1AR compared to the α1R under physiological temperatures, we examined [18F]FECUMI-101 binding displacement by WAY100635 and prazosin at room temperature and 37 °C. At both temperatures, [18F]FECUMI-101 displayed comparable specific binding in hippocampus, BA9, and thalamus (Figure 5). The binding data obtained are from one experiment in triplicate measurements for hippocampus, BA9, and duplicate measurements for thalamus.

Figure 5.

Figure 5

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Binding of [18F]FECUMI-101 in post-mortem human brain tissues at 37 °C and room temperature.

In summary, autoradiography studies of [18F]FECUMI-101 in post-mortem human brain sections show 5-HT1AR is the major binding site. The typical cortical lamination of the 5-HT1AR obtained in vitro with [3H]8-OH-DPAT is seen with [18F]FECUMI-101 here.26,27 The anatomical distribution of 5-HT1AR binding is consistent with reported in vivo and in vitro binding of [18F]FECUMI-101 in baboon by PET.16 While there was measurable specific binding of the radiotracer to α1R in hippocampus (mainly DG) and prefrontal cortex, [18F]1 binding was effectively completely displaced by WAY100635 in the prefrontal cortex and CA1 subfield of the hippocampus. The thalamus and cerebellum had little [18F]1 binding, but the binding in these regions was blocked by WAY100635 and prazosin. Because there is displaceable [18F]1 binding in cerebellum it should not be used as a reference region for in vivo PET applications. Despite the known high concentration of α1R in thalamus and cerebellum, we did not obtain a proportionately high amount of binding of [18F]1 in these regions.28−31 Similarly, the in vitro binding we observed in human thalamus was much lower than what we measured in vivo in baboon.16 A possible explanation for this discrepancy may be the difference in α1R concentration in the thalamus of baboon and human or it could be due to the binding of [18F]FECUMI-101 to an unknown target in thalamus. There is no detectable binding of [18F]FECUMI-101 to 5-HT7R sites in the tested brain regions. Similarly, we found no significant change of specific binding to 5-HT1AR and α1R at room temperature and 37 °C. The sensitivity of in vitro autoradiography is much higher than in vivo PET; hence, further in vivo blocking studies with α1R are required to establish in vivo α1R selectivity of [18F]FECUMI-101. In conclusion, our experiments suggest that [18F]FECUMI-101 is a relatively selective 5-HT1AR tracer that holds promise for occupancy measurement of 5-HT1AR and α1R dual or selective drugs in vivo with PET.

Experimental Procedures

Author: [18F]FECUMI-101 was synthesized as described before.16In vitro autoradiography experiments were performed using four nonpsychiatric medication-free normal human brain sections. Brain sections from BA9 and hippocampus came from one subject, whereas cerebellum and thalamus sections were obtained from two other subjects. Three sections (20 μm) each for total and nonspecific binding per brain region were assayed for distribution and specificity studies. Three brain sections (20 μm) for hippocampus and BA9 brain regions and two brain sections for thalamus were assayed for effect of temperature for total and nonspecific binding. Slide-mounted sections of frontal cortex, hippocampus, thalamus, and cerebellum were brought to room temperature and incubated in 170 mM Tris HCl (pH 7.6) containing 4 mM CaCl2 and 138 mM sucrose and 5–10 nM of radioligand (1–2.3 mCi in 900–1200 mL of buffer) for 60 min (37 °C or room temperature). Adjacent sections were incubated in the same buffer with 1 μM WAY100635, 1 μM prazosin, or 1 μM SB269970 to determine nonspecific binding. Sections were washed in same buffer at 4 °C (2× 1 min) and briefly dipped in ice-cold water to remove salts. Slides were quickly dried under a stream of cold air and exposed to ST-phosphor-imaging screen (Packard, wrapped in Mylar film) for 60 min. Screens were scanned with a Packard Cyclone phosphor-imaging system and analyzed with OptiQuant Acquisition and Analysis software (Packard).

Acknowledgments

This work was partially funded by PHS grants MH40210, MH90964, and MH62185.

Author Contributions

∥ These authors contributed equally to this work.

The authors declare no competing financial interest.

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