DOI 10.1515/revneuro-2014-0003 Rev. Neurosci. 2014; 25(3): 357–365
Luc Zimmer* and Thierry Billard
Molecular imaging of the serotonin 5-HT7 receptors: from autoradiography to positron emission tomography Abstract: Serotonin and its various receptors are involved in numerous brain functions and neuropsychiatric disorders. Of the 14 known serotoninergic receptors, the 5-HT7 receptor is the most recently identified and characterized. It is closely involved in the pathogenesis of depression, anxiety, epilepsy and pain and is therefore an important target for drug therapy. It is a crucial target in neuroscience, and there is a clear need for radioligands for in vitro and in vivo visualization and quantification, first in animal models and ultimately in humans. This review focuses on the main radioligands suggested for in vitro and in vivo imaging of the 5-HT7 receptor. Keywords: autoradiography; 5-HT7; neurotransmission; positron emission tomography; radiopharmaceutical. *Corresponding author: Luc Zimmer, University of Lyon 1, CNRS, INSERM, Lyon Neuroscience Research Center (CRNL), Lyon, France, e-mail: [email protected]; and Hospices Civils de Lyon, 59 Boulevard Pinel, 69003 Lyon, France Thierry Billard: University of Lyon 1, CNRS, Institute of Chemistry and Biochemistry (ICBMS), Lyon, France
Introduction The serotoninergic (5-HT) system plays a key role in many central nervous system functions. The 5-HT receptors are phylogenetically one of the oldest systems and also one of the most diverse groups of brain receptors in the human genome. Currently, 14 5-HT receptors have been described structurally and pharmacologically (IUPAHR Database, 2013). These 14 subtypes are divided into seven classes (5-HT1 to 5-HT7) according to structure and functional characteristics. All are members of the transmembrane GPCR (G protein-coupled receptor) family except for the 5-HT3 receptor, which is a ligand-gated ion channel. Even though the functions of these receptors remain to be elucidated, it is known that each of these targets has its own role in the central nervous system. Physiologically, serotoninergic functions are involved in the control of sensory
transmission, feeding behavior, sleep and mood. Pathophysiologically, serotoninergic dysfunction is implicated in psychiatric disorders including depression, anxiety and schizophrenia and neurological disorders including Alzheimer’s disease and Parkinson’s disease (Meneses, 1999; Jones and Blackburn, 2002). 5-HT7 is the most recently described 5-HT receptor subtype. 5-HT7 receptors are increasingly reported to be involved in pathophysiological processes and possible therapeutics, and there is a critical need for tools for the molecular imaging of these receptors, as will be described in this article.
General overview of the 5-HT7 receptors 5-HT7 receptors have been cloned from several animal species including humans (Bard et al., 1993; Lovenberg et al., 1993; Plassat et al., 1993). They are located in the periphery, mainly in blood vessels, the gastrointestinal tract (Schoeffter et al., 1996; Irving et al., 2007) and the brain, with highest densities in the thalamus, hypothalamus, amygdala, cortex, hippocampus and dorsal raphe (To et al., 1995; Gustafson et al., 1996; Duncan and Franklin, 2007). It is noteworthy that, whereas three splice variants of human 5-HT7 receptor have been found (Krobert et al., 2001), they do not seem to differ markedly in pharmacological profile. Based on this brain location pattern, the availability of 5-HT7 knockout (KO) mice (Guscott et al., 2005; Hedlund et al., 2005) and the introduction of selective 5-HT7 antagonists and, lately, agonists (Pittalà et al., 2007), involvement in physiological and behavioral functions has been confirmed. Thus, experimental data indicate that 5-HT7 receptors can influence sleep physiology. Administered to rodents at onset of sleep, the 5-HT7 antagonists SB-269970 and SB-656104-A increased the latency to rapid eye movement (REM) sleep and reduced REM sleep duration (Hagan et al., 2000). Accordingly, 5-HT7 KO mice showed modified
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358 L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors sleep architecture with shorter REM periods (Hedlund et al., 2005). Another physiological function involving 5-HT7 receptors is thermoregulation. The first evidence came from experiments in rodents, in which hypothermia induced by the nonselective 5-HT receptor agonist 5-carboxamido tryptamine (5-CT) was antagonized by selective and nonselective 5-HT7 receptor antagonists (Hagan et al., 2000). The antagonist SB-269970 was also able to block hypothermia induced in rodents by the 5-HT1A/7 receptor agonist 8-OHDPAT (Hedlund and Sutcliffe, 2004; Faure et al., 2006). In a more integrated manner, numerous studies have demonstrated the role of 5-HT7 receptors in various memory processes, short and long term, in rats (Meneses, 2004; Pérez-García et al., 2006), in mice (Freret et al., 2014) and in 5-HT7 receptor KO mice (Roberts et al., 2004). The therapeutic potential of 5-HT7 receptors in mood disorder was also explored. For example, 5-HT7 KO mice were reported to display antidepressant-like behavior (Guscott et al., 2005; Hedlund et al., 2005). A similar result was obtained after pharmacological blockade with SB-269970, a 5-HT7 antagonist, in conventional rodents (Hedlund et al., 2005; Wesołowska et al., 2006; Bonaventure et al., 2007; Hedlund, 2009). Because several classes of antidepressant have been shown to promote hippocampal neurogenesis (Drew and Hen, 2006), this effect was studied under 5-HT7 modulation. Cell proliferation in rat hippocampus is enhanced after 2–3 weeks of treatment with classical antidepressants such as fluoxetine (Malberg et al., 2000), but with just a 1-week treatment using SB-269970 (Mnie-Filali et al., 2011). This may be another crucial indication of a faster antidepressant-like profile for 5-HT7 receptor antagonists (Mnie-Filali et al., 2007). In terms of anxiety, only a few papers are available on 5-HT7 manipulation in animal models. For example, systemic administration of the 5-HT7 antagonist SB-269970 produced an anxiolytic-like effect in behavioral tests in rats (Wesołowska et al., 2006). 5-HT7 receptors have also been implicated in schizophrenia, as a large number of antipsychotic drugs are shown to have high affinity for the 5-HT7 receptor, e.g., risperidone (Roth et al., 1994). Preclinical results in rodents are preliminary and sometimes discrepant (Matthys et al., 2011), but additional evidence comes from a postmortem study showing decreased levels of 5-HT7 receptor mRNA in cortical areas of schizophrenics (Dean et al., 2006). Finally, a role for the 5-HT7 receptor has been suggested in animal models of hyperactivity and impaired attention (Ruocco et al., in press), seizure (Bourson et al., 1997; Graf et al., 2004) and pain (Rocha-González et al., 2005; Brenchat et al., 2009).
This short summary of therapeutic applications of 5-HT7 receptors reveals a lack of clinical studies: preliminary indications depend mainly on preclinical data. This explains why there is a clear need for radioligands for 5-HT7 receptor imaging, for future translational studies linking animal studies to clinical applications.
The need for radiotracers for 5-HT7 receptor imaging In vitro imaging (e.g., autoradiography) allows postmortem visualization and accurate delineation and quantification of receptors in animal models and in humans, including healthy subjects and patients in neurology or psychiatry (Heckl et al., 2004). In complement, in vivo imaging [e.g., positron emission tomography (PET)], is often used to quantify receptor concentration, allowing in vivo pharmacology in anesthetized animals or awake human subjects (Lancelot and Zimmer, 2010). PET is also useful for tracking the pharmacokinetic and pharmacodynamic properties of drugs and can determine the occupancy of therapeutic drugs, which ultimately can be used to estimate optimal doses in Phase II studies. This technology is therefore considered crucial to accelerating the development of brain drug candidates (Pien et al., 2005). It is noteworthy that results from in animal models have been confirmed in the 5-HT system in the human brain following the development of selective PET radioligands. The hypothesis previously developed, that the 5-HT7 receptor may be a novel therapeutic target, later generated work on the development of 5-HT7 receptor antagonists and agonists (Pittalà et al., 2007). Despite the existence of pharmacological ligands, it has to be kept in mind that there is still a bottleneck between available molecules and radiotracers that are usable for imaging in animal models or humans (Zimmer and Luxen, 2012). For example, in 5-HT neurotransmission, only 5 of the 14 5-HT receptors have a dedicated PET radiotracer currently usable in humans (Zimmer and Le Bars, 2013). The explanation is that transfer from in vitro to in vivo radioligands (known as radiopharmaceuticals if used in humans) is demanding. In order to generate interpretable images of brain receptors, radioligands must possess certain properties (Halldin et al., 2001). From a radiochemical point of view, radiosynthesis should be as simple and fast as possible, especially with short-lived carbon-11, leading to high specific radioactivity to avoid any pharmacological blockade of the receptor with unlabeled compound, according to the tracer dose principle. From a radiopharmacological point of view, the
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L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors 359
ligand must possess high affinity toward the receptor, normally in the nanomolar range for 5-HT receptors, according to their density value (Bmax). In the case of 5-HT7 receptor, the brain density of which is particularly low in comparison to other 5-HT receptors (e.g., less than one-tenth of that of 5-HT1A receptors), affinity should ideally be subnanomolar. The association rate (kon) should be high enough to maximize the target-to-background ratio, and the dissociation rate (koff) should match the scanning frame. The radiotracer must bind selectively to the 5-HT7 receptor, with an affinity that is markedly higher than its affinities for a wide spectrum of other receptors and binding sites. The radioligand should also be moderately lipophilic for adequate brain penetration and should ideally not be a substrate for efflux transporters at the blood-brain barrier (i.e., P-glycoprotein). Finally, the radioactive metabolites produced after injection should be excluded from the brain so as not to interfere with the signal measured by the PET camera. Various 5-HT7 ligand candidate situations are found in the literature. The first is that the 5-HT7 ligand does not have a sufficiently higher affinity compared to other 5-HT families or other targets. The second situation is that the 5-HT7 radioligand candidate was tested favorably in vitro but failed in vivo because of the characteristics of its pharmacokinetics (rapid metabolism and radioactive metabolites in the brain, etc.), its pharmacodynamics (brain exclusion via efflux transporter, etc.) or unknown reasons (low target-to-background ratio, etc.). Finally, it has to be borne in mind that transfer to humans of a radiotracer used in animals involves ever increasing radiopharmaceutical prerequisites. All these heterogeneous situations will be illustrated in this review, explaining the numerous failures or insufficient advances in the development of radiotracers for 5-HT7 brain imaging.
In vitro imaging of 5-HT7 receptors Nonselective radioligands As previously mentioned, knowledge of the biological role of central 5-HT7 receptors is in part based on results from studies of their anatomical distribution. Discrete regional distribution has been described for cell populations expressing mRNA coding for 5-HT7 receptors in rat and guinea pig brain (To et al., 1995; Gustafson et al., 1996; Mengod et al., 1996; Kinsey et al., 2001; Neumaier et al., 2001). These studies reported an encouraging abundance of 5-HT7 receptor mRNA and 5-HT7 immunoreactivity, contrasting sharply with the low density of 5-HT7 receptors.
Assessment of the anatomical distribution of 5-HT7 receptor binding sites in brain tissue has long been hampered by the lack of a specific radioligand. Several attempts have been made using available radioligands in rat brain homogenates (Stowe and Barnes, 1998; Hemedah et al., 1999) or brain sections (To et al., 1995; Waeber and Moskowitz, 1995; Gustafson et al., 1996; Mengod et al., 1996). These autoradiography studies used [3H]5-CT, a nonselective 5-HT receptor agonist ligand, in the presence of specific antagonists to mask other binding sites, theoretically enabling full visualization of the receptor. An attempt to visualize 5-HT7 receptors in hamster brain used [3H]8-OH-DPAT, a 5-HT1A/7 ligand, in the presence of pindolol for specific 5-HT1A receptor blockade (Duncan et al., 1999). However, it turned out that the added masking compounds induced incomplete blockade of non-5-HT7 sites, introducing a bias in the interpretation and quantification of the results. An additional drawback was the poor selectivity of the blocking compounds used, with partial blockade of 5-HT7 receptors, resulting in an underestimation of the 5-HT7 binding component. Another strategy uses 5-HT1A and 5-HT1A/1B KO mice to visualize the anatomical location of 5-HT7 binding sites in mouse brain, using [3H]5-CT and [3H]8-OH-DPAT (Bonaventure et al., 2002). The rationale of this approach was that in mice lacking 5-HT1A receptors, the radioligands would bind more specifically 5-HT7 receptor binding sites. However, results revealed that despite its high affinity for 5-HT7 receptors, [3H]5-CT is not a good tracer for autoradiographic assessment of 5-HT7 receptors. Another team chose the nonselective antagonist [3H] mesulergine as 5-HT7 radioligand, as it has a high affinity for 5-HT7 receptors (Martín-Cora and Pazos, 2004). Autoradiographic studies were performed in the presence of adapted concentrations of raclopride and DOI to mask [3H] mesulergine binding to D2, 5-HT2A and 5-HT2C receptors. In addition, [3H]mesulergine is an antagonist (unlike previous agonists such as [3H]5-CT and [3H]8-OH-DPAT), which should be more favorable to stable binding. Therefore, in the case of an agonist, if multiple agonist affinity states of the receptor are present, only a fraction of the total 5-HT7 receptor population may be labeled. Thus, [3H]mesulergine enabled the anatomical distribution of 5-HT7 receptors to be mapped in human brain and compared with that observed in guinea pig and rat brain.
Selective radioligand: [3H]SB-269970 Nonselective radioligands have proved useful in the initial characterization of cloned and native 5-HT7 receptors, as
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360 L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors well as providing information on their native distribution, but none selectively label 5-HT7 receptors. There is therefore still a need for a selective 5-HT7 antagonist radioligand. SB-269970 (3-{[(2R)-2-[2-(4-methylpiperidin-1-yl)ethyl] pyrrolidin-1-yl]sulfonyl}phenol), an analog of SB-258719 (Forbes et al., 1998), has been identified and described as a potent 5-HT7 receptor antagonist displaying at least 50- to 100-fold greater selectivity than 50 different receptors, enzymes and ion channels (Lovell et al., 2000). This molecule was radiolabeled with tritium and its binding was first characterized with respect to both cloned human 5-HT7 receptors and 5-HT7 receptors in guinea pig cerebral cortex (Thomas et al., 2000). The same team went on to investigate [3H]SB-269970 binding to brain tissue homogenates from a number of other species: mouse, rat, pig, marmoset and human (Thomas et al., 2002). [3H] SB-269970 bound saturably to mouse forebrain and rat, pig and marmoset cortex, labeling a homogenous population of sites. KD values were between 1 and 2 nm, according to species. The Bmax values for [3H]SB-269970 binding to mouse forebrain and rat, pig and marmoset cortex tissues were 20, 30, 31 and 14 fmol per milligram protein, respectively. However, preliminary characterization of [3H] SB-269970 binding to human cerebral cortex membranes revealed a too low level of 5-HT7 receptor expression to be accurately quantifiable. In the following study (Varnäs et al., 2004), 5-HT7 receptor distribution was analyzed using [3H]SB-269970 and human brain whole-hemisphere autoradiography. The results indicated that 5-HT7 receptors are most abundantly located in the anterior thalamus and dentate gyrus. Intermediate concentrations were found in the hypothalamus, anterior cingulate gyrus, hippocampus, amygdala and certain brainstem nuclei. This study confirmed the very low density of 5-HT7 receptors in the human brain, with specific binding ranging from 16 pmol/g (anterior thalamus) to 2 pmol/g (isocortex). Another study showed significant lower [3H]SB-269970 binding in Brodmann’s area 9 in subjects with schizophrenia than controls, consistent with the hypothesis that decreased levels of 5-HT7 receptors may be involved in the pathological processes of schizophrenia (Dean et al., 2006). However, this study confirmed the low signal-tonoise ratio obtained with the only tritiated radioligand currently available for 5-HT7 imaging. More recently, this radiotracer was used to quantify the binding of lurasidone, a new antipsychotic, to 5-HT7 receptors in rat brain by autoradiography (Horisawa et al., 2013). These recent findings confirmed that binding to 5-HT7 receptors might play some role in beneficial
pharmacological action in schizophrenia, confirming the interest in developing 5-HT7 radioligands for in vivo imaging.
Toward in vivo imaging of 5-HT7 receptors [11C]DR4446 A Japanese consortium developed the first 5-HT7 radio tracer for PET imaging in 2002 (Zhang et al., 2002). DR4446 is a tetrahydrobenzindole derivative that was selected from a series of molecules for its high selectivity for 5-HT7 over the other 5-HT receptors (Kikuchi et al., 2002). [11C]DR4446 was therefore synthesized by N-methylation of its desmethyl precursor with [11C]methyl iodide ([11C]CH3I). The radiotracer obtained was only tested in anesthetized monkeys, but showed good cerebral uptake, consistent with its lipophilicity (log p = 3.7). Radioactivity was slightly higher in the thalamus, an area rich in 5-HT7 receptors, than in other regions, and a blocking study with unlabeled DR4446 compound showed the reversibility of [11C]DR4446 binding. However, to our knowledge, no further reports have been published using this 5-HT7 PET radiotracer. This can probably be explained by the moderate affinity of [11C]DR4446 for 5-HT7 receptors, Ki 9.7 nm, which, given the low density of 5-HT7 receptors in the brain, leads to a low signal-to-noise signal.
[18F]SB-269970 derivatives The second attempt to develop a 5-HT7 PET radiotracer was made in 2010 in the University of Lyon, France (Andriès et al., 2010). Two molecules were envisaged, inspired by the literature results. The first was a piperazine derivative adapted from Yoon et al.’s (2008) findings. The second was based on SB-269970, previously described as a successful tritiated ligand (Thomas et al., 2000), but modified in this case with an ester moiety in the piperidine core. Both candidates presented intermediate affinity toward 5-HT7 receptors (pKB, 7.26 and 7.10, respectively) and relatively high lipophilicity (log D at pH 7.4, 3.27 and 4.13, respectively). Both were radiolabeled by nitro group nucleophilic aromatic substitution. Fluorinated piperazine was abandoned because radiolabeling did not give satisfactory radiochemical yields. The second candidate, [18F]SB-269970 derivative, was evaluated by in
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L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors 361
vitro autoradiography in rat brain. Binding proved homogeneous throughout the brain, with no specific binding in regions rich in 5-HT7 receptors; development was therefore stopped.
[18F]2FP3 and derivatives More recently, the same team from the University of Lyon developed four other SB269970 analogs. To avoid the undesirable in vivo metabolization previously observed (Andriès et al., 2010), 18F labeling was introduced onto a less reactive part of SB269970, the phenylsulfonyl moiety. Close analogs were synthesized with a fluorophenylsulfonyl group on the pyrrolidine and with a piperazine group instead of the piperidine heterocycle (Andriès et al., 2011). The nitro precursors of these analogs were radiolabeled by 18 F nucleophilic substitution. All presented suitable affinity for 5-HT7 receptors (apparent dissociation constant KD between 1.6 and 14 nm), although two were abandoned because of their agonist activity for 5-HT1A receptors. In vitro autoradiography in rats revealed that only two radioligand candidates were displaced by the 5-HT7 antagonist SB 269970. Therefore, subsequent in vivo study focused only on these two. According to PET scans in cats and competition studies, [18F]2FP3 appeared to be the best PET radiotracer for in vivo imaging of 5-HT7 receptors in animal models (Lemoine et al., 2011). Optimally, better affinity for 5-HT7 receptors would be preferable, because specific binding is a function of a radiotracer’s affinity for the receptor relative to the density of the binding site (which is low for 5-HT7). Consequently, the development of [18F]2FP3 analogs was pursued by this team, with chemical structures modified to optimize PET tracer properties, particularly in terms of affinity, which should be subnanomolar. Unfortunately, although one of these new radioligand candidates showed promising characteristics in vitro (5-HT7 Ki 50 nm), it interacted unpredictably with P-glycoprotein, with consequently poor brain penetration in vivo (Colomb et al., in press).
Oxindole derivatives A Danish team (University of Copenhagen), in collaboration with a Hungarian pharmaceutical company, chose to use phenylpiperazinyl-butyloxindoles as a lead structure for 5-HT7 radiotracers, because this structure displays an inhibition constant (Ki) that is 2000-fold lower for 5-HT7 receptors than for 5-HT1A receptors (Volk et al.,
2008, 2011). In particular, they investigated the influence of various substitution patterns on the affinity toward the 5-HT7 receptor and concentrated on structure-activity relationships at the arylpiperazine moiety (Herth et al., 2012a). Two of the 16 candidate compounds synthesized were tested toward 37 receptors. Although both displayed a promising in vitro profile for PET imaging of the 5-HT7 receptor, their adrenergic α1 receptor affinity may contaminate the signal detected from the 5-HT7 receptor. At the time of writing, none of these oxydole derivatives is yet used in vivo as a 5-HT7 PET ligand.
[11C]Cimbi-806 In 2011–2012, the same Danish team (University of Copenhagen) developed an alternative strategy to obtain a 5-HT7 radioligand. They focused on another lead structure, biphenethylamines. Cimbi-806 is a biphenethylamine with a profile initially characterized at the University of Caen, France (Paillet-Loilier et al., 2007). Its affinity for 5-HT7 is significantly higher than for 5-HT1A (Ki = 8.8 vs. 4826 nm). This large difference in Ki is especially useful because of the low density (Bmax) of 5-HT7 receptors compared to 5-HT1A receptors and because of the brain colocalization of 5-HT7 and 5-HT1A receptors. [11C]Cimbi-806 was therefore radiolabeled and its in vivo binding characteristics were subsequently assessed in pigs (Herth et al., 2012b). After intravenous injection of [11C]Cimbi-806, there was rapid high brain uptake, with a maximum in the thalamus and striatum and a minimum in the cerebellum. However, no significant change in [11C]Cimbi-806 after SB-269970 pretreatment was found in any region, so that in vivo binding could not be claimed to be selective for the 5-HT7 receptor. The authors concluded that [11C]Cimbi-806 was not suitable for imaging the 5-HT7 receptor.
Arylpiperazinic ligands A group of the University of Bari (Italy) designed piperazine derivatives and characterized one of them as a 5-HT7 receptor agonist (Leopoldo et al., 2008). This same group claimed in a recent patent the invention of 1-arylpiperazinic ligands of 5-HT7 receptors that present high affinity and selectivity for the receptor (Leopoldo et al., 2012). However, although binding assays are presented in the patent (with 5-HT7 pKi ranging from 7.70 to 8.79), no other biological testing is detailed. Particularly, no radiolabeling or in vivo imaging data have as yet been reported.
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362 L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors
[3H]5-CT
[3H]8-OH-DPAT
[3H]SB-269970
[11C]DR4446
[18F]SB-269970 derivative
Piperazine derivative
[18F]2FP3
[3H]mesulergine
[11C]Cimbi-806
[11C]1-[2-(4methoxyphenyl) phenyl]piperazine
Figure 1 Chemical structures of the radioligands developed for the 5-HT7 receptor imaging.
[11C]methoxyphenyl)phenyl]piperazine
Conclusions
The Japanese team that 10 years ago developed [11C] DR4446 as the first putative 5-HT7 radiotracer recently reported a new 5-HT7 candidate (Shimoda et al., 2013). 1-[2-(4-Methoxyphenyl)phenyl]piperazine was developed as a PET candidate because it showed high binding affinity to human recombinant 5-HT7 receptors expressing in mammalian cells (Ki = 2.6 nm), whereas its binding affinity to 5-HT1A receptor was 476 nm. It also displayed a weak affinity for adrenergic 1 receptor (Ki = 156 nm), in contrast to previous PET candidates. Furthermore, it has a methoxyl group in its molecule and can be easily labeled by 11 C without changing its chemical structure or pharmacological functions. In vitro autoradiographic studies have been encouraging, but a small-animal PET study showed homogenous whole-brain distribution of radioactivity, inconsistent with the brain distribution of 5-HT7 receptors; in particular, uptake in the 5-HT7-rich thalamus was not higher than in the other brain regions. Moreover, a blocking experiment using a 5-HT7-selective compound did not reduce but increased brain uptake, precluding the pursuit of the use of this radiotracer.
The 5-HT7 receptor represents a crucial target in neuroscience, justifying a need for radioligands for visualization and quantification, first in animal models and ultimately in humans. Although no precise data regarding the Bmax of 5-HT7 receptors have been reported, 5-HT7 receptor density was assessed at less than one-tenth that of the other 5-HT families. Therefore, brain visualization is particularly challenging, requiring radiotracer candidates with subnanomolar 5-HT7 affinity and particularly high specificity. Despite numerous characterizations of radiotracer candidates (see Figure 1) and encouraging results, there are currently no PET radioligands for the exploration of 5-HT7 receptor in humans. Development of 5-HT7 brain radiopharmaceuticals is therefore still an exciting field, involving interdisciplinary research bringing together chemistry, neuropharmacology and imaging science.
Received November 25, 2013; accepted January 9, 2014; previously published online February 5, 2014
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Luc Zimmer received a pharmacist education at the University of Strasbourg (1986–1992); he completed training during his internship and residency in radiopharmacy and radiopharmacology (1993–1999) at the Hospital University of Tours (Nuclear Medicine Department) and at the National Institute for Nuclear Sciences and Technology, Saclay. He received his PharmD in radiopharmacology (1998) and his PhD in neuroscience (1999) from the University of Tours. He is currently professor of pharmacology at the University of Lyon (Université Claude Bernard Lyon 1) and is radiopharmacist at the University Hospital of Lyon (Hospices Civils de Lyon). He is head of the preclinical department of the CERMEP-imaging platform and is in charge of the laboratory ‘Radiopharmaceutical and neurochemical biomarkers’ at the Lyon Neuroscience Research Center (CRNL). His research interests include radiopharmacology and in vivo imaging of neurotransmissions with PET.
Thierry Billard, after education in chemistry, completed by an engineer diploma from ‘Institut de Chimie et Physique Industrielles de Lyon’ (now ‘CPE Lyon’) in 1993. He obtained his PhD in organic chemistry from the University of Lyon in 1996. After a postdoctoral internship with Prof. L. Ghosez (Université Catholique de Louvain, Belgium), he was appointed in 1999 as a CNRS permanent researcher. He was promoted Research Director by CNRS in 2008. He is currently in charge of a research team at the Institute of Chemistry and Biochemistry (ICBMS). His research activities focus on fluorine chemistry, medicinal chemistry, radiochemistry and their applications in medical imaging.
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Luc Zimmer* and Thierry Billard
Molecular imaging of the serotonin 5-HT7 receptors: from autoradiography to positron emission tomography Abstract: Serotonin and its various receptors are involved in numerous brain functions and neuropsychiatric disorders. Of the 14 known serotoninergic receptors, the 5-HT7 receptor is the most recently identified and characterized. It is closely involved in the pathogenesis of depression, anxiety, epilepsy and pain and is therefore an important target for drug therapy. It is a crucial target in neuroscience, and there is a clear need for radioligands for in vitro and in vivo visualization and quantification, first in animal models and ultimately in humans. This review focuses on the main radioligands suggested for in vitro and in vivo imaging of the 5-HT7 receptor. Keywords: autoradiography; 5-HT7; neurotransmission; positron emission tomography; radiopharmaceutical. *Corresponding author: Luc Zimmer, University of Lyon 1, CNRS, INSERM, Lyon Neuroscience Research Center (CRNL), Lyon, France, e-mail: [email protected]; and Hospices Civils de Lyon, 59 Boulevard Pinel, 69003 Lyon, France Thierry Billard: University of Lyon 1, CNRS, Institute of Chemistry and Biochemistry (ICBMS), Lyon, France
Introduction The serotoninergic (5-HT) system plays a key role in many central nervous system functions. The 5-HT receptors are phylogenetically one of the oldest systems and also one of the most diverse groups of brain receptors in the human genome. Currently, 14 5-HT receptors have been described structurally and pharmacologically (IUPAHR Database, 2013). These 14 subtypes are divided into seven classes (5-HT1 to 5-HT7) according to structure and functional characteristics. All are members of the transmembrane GPCR (G protein-coupled receptor) family except for the 5-HT3 receptor, which is a ligand-gated ion channel. Even though the functions of these receptors remain to be elucidated, it is known that each of these targets has its own role in the central nervous system. Physiologically, serotoninergic functions are involved in the control of sensory
transmission, feeding behavior, sleep and mood. Pathophysiologically, serotoninergic dysfunction is implicated in psychiatric disorders including depression, anxiety and schizophrenia and neurological disorders including Alzheimer’s disease and Parkinson’s disease (Meneses, 1999; Jones and Blackburn, 2002). 5-HT7 is the most recently described 5-HT receptor subtype. 5-HT7 receptors are increasingly reported to be involved in pathophysiological processes and possible therapeutics, and there is a critical need for tools for the molecular imaging of these receptors, as will be described in this article.
General overview of the 5-HT7 receptors 5-HT7 receptors have been cloned from several animal species including humans (Bard et al., 1993; Lovenberg et al., 1993; Plassat et al., 1993). They are located in the periphery, mainly in blood vessels, the gastrointestinal tract (Schoeffter et al., 1996; Irving et al., 2007) and the brain, with highest densities in the thalamus, hypothalamus, amygdala, cortex, hippocampus and dorsal raphe (To et al., 1995; Gustafson et al., 1996; Duncan and Franklin, 2007). It is noteworthy that, whereas three splice variants of human 5-HT7 receptor have been found (Krobert et al., 2001), they do not seem to differ markedly in pharmacological profile. Based on this brain location pattern, the availability of 5-HT7 knockout (KO) mice (Guscott et al., 2005; Hedlund et al., 2005) and the introduction of selective 5-HT7 antagonists and, lately, agonists (Pittalà et al., 2007), involvement in physiological and behavioral functions has been confirmed. Thus, experimental data indicate that 5-HT7 receptors can influence sleep physiology. Administered to rodents at onset of sleep, the 5-HT7 antagonists SB-269970 and SB-656104-A increased the latency to rapid eye movement (REM) sleep and reduced REM sleep duration (Hagan et al., 2000). Accordingly, 5-HT7 KO mice showed modified
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358 L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors sleep architecture with shorter REM periods (Hedlund et al., 2005). Another physiological function involving 5-HT7 receptors is thermoregulation. The first evidence came from experiments in rodents, in which hypothermia induced by the nonselective 5-HT receptor agonist 5-carboxamido tryptamine (5-CT) was antagonized by selective and nonselective 5-HT7 receptor antagonists (Hagan et al., 2000). The antagonist SB-269970 was also able to block hypothermia induced in rodents by the 5-HT1A/7 receptor agonist 8-OHDPAT (Hedlund and Sutcliffe, 2004; Faure et al., 2006). In a more integrated manner, numerous studies have demonstrated the role of 5-HT7 receptors in various memory processes, short and long term, in rats (Meneses, 2004; Pérez-García et al., 2006), in mice (Freret et al., 2014) and in 5-HT7 receptor KO mice (Roberts et al., 2004). The therapeutic potential of 5-HT7 receptors in mood disorder was also explored. For example, 5-HT7 KO mice were reported to display antidepressant-like behavior (Guscott et al., 2005; Hedlund et al., 2005). A similar result was obtained after pharmacological blockade with SB-269970, a 5-HT7 antagonist, in conventional rodents (Hedlund et al., 2005; Wesołowska et al., 2006; Bonaventure et al., 2007; Hedlund, 2009). Because several classes of antidepressant have been shown to promote hippocampal neurogenesis (Drew and Hen, 2006), this effect was studied under 5-HT7 modulation. Cell proliferation in rat hippocampus is enhanced after 2–3 weeks of treatment with classical antidepressants such as fluoxetine (Malberg et al., 2000), but with just a 1-week treatment using SB-269970 (Mnie-Filali et al., 2011). This may be another crucial indication of a faster antidepressant-like profile for 5-HT7 receptor antagonists (Mnie-Filali et al., 2007). In terms of anxiety, only a few papers are available on 5-HT7 manipulation in animal models. For example, systemic administration of the 5-HT7 antagonist SB-269970 produced an anxiolytic-like effect in behavioral tests in rats (Wesołowska et al., 2006). 5-HT7 receptors have also been implicated in schizophrenia, as a large number of antipsychotic drugs are shown to have high affinity for the 5-HT7 receptor, e.g., risperidone (Roth et al., 1994). Preclinical results in rodents are preliminary and sometimes discrepant (Matthys et al., 2011), but additional evidence comes from a postmortem study showing decreased levels of 5-HT7 receptor mRNA in cortical areas of schizophrenics (Dean et al., 2006). Finally, a role for the 5-HT7 receptor has been suggested in animal models of hyperactivity and impaired attention (Ruocco et al., in press), seizure (Bourson et al., 1997; Graf et al., 2004) and pain (Rocha-González et al., 2005; Brenchat et al., 2009).
This short summary of therapeutic applications of 5-HT7 receptors reveals a lack of clinical studies: preliminary indications depend mainly on preclinical data. This explains why there is a clear need for radioligands for 5-HT7 receptor imaging, for future translational studies linking animal studies to clinical applications.
The need for radiotracers for 5-HT7 receptor imaging In vitro imaging (e.g., autoradiography) allows postmortem visualization and accurate delineation and quantification of receptors in animal models and in humans, including healthy subjects and patients in neurology or psychiatry (Heckl et al., 2004). In complement, in vivo imaging [e.g., positron emission tomography (PET)], is often used to quantify receptor concentration, allowing in vivo pharmacology in anesthetized animals or awake human subjects (Lancelot and Zimmer, 2010). PET is also useful for tracking the pharmacokinetic and pharmacodynamic properties of drugs and can determine the occupancy of therapeutic drugs, which ultimately can be used to estimate optimal doses in Phase II studies. This technology is therefore considered crucial to accelerating the development of brain drug candidates (Pien et al., 2005). It is noteworthy that results from in animal models have been confirmed in the 5-HT system in the human brain following the development of selective PET radioligands. The hypothesis previously developed, that the 5-HT7 receptor may be a novel therapeutic target, later generated work on the development of 5-HT7 receptor antagonists and agonists (Pittalà et al., 2007). Despite the existence of pharmacological ligands, it has to be kept in mind that there is still a bottleneck between available molecules and radiotracers that are usable for imaging in animal models or humans (Zimmer and Luxen, 2012). For example, in 5-HT neurotransmission, only 5 of the 14 5-HT receptors have a dedicated PET radiotracer currently usable in humans (Zimmer and Le Bars, 2013). The explanation is that transfer from in vitro to in vivo radioligands (known as radiopharmaceuticals if used in humans) is demanding. In order to generate interpretable images of brain receptors, radioligands must possess certain properties (Halldin et al., 2001). From a radiochemical point of view, radiosynthesis should be as simple and fast as possible, especially with short-lived carbon-11, leading to high specific radioactivity to avoid any pharmacological blockade of the receptor with unlabeled compound, according to the tracer dose principle. From a radiopharmacological point of view, the
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L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors 359
ligand must possess high affinity toward the receptor, normally in the nanomolar range for 5-HT receptors, according to their density value (Bmax). In the case of 5-HT7 receptor, the brain density of which is particularly low in comparison to other 5-HT receptors (e.g., less than one-tenth of that of 5-HT1A receptors), affinity should ideally be subnanomolar. The association rate (kon) should be high enough to maximize the target-to-background ratio, and the dissociation rate (koff) should match the scanning frame. The radiotracer must bind selectively to the 5-HT7 receptor, with an affinity that is markedly higher than its affinities for a wide spectrum of other receptors and binding sites. The radioligand should also be moderately lipophilic for adequate brain penetration and should ideally not be a substrate for efflux transporters at the blood-brain barrier (i.e., P-glycoprotein). Finally, the radioactive metabolites produced after injection should be excluded from the brain so as not to interfere with the signal measured by the PET camera. Various 5-HT7 ligand candidate situations are found in the literature. The first is that the 5-HT7 ligand does not have a sufficiently higher affinity compared to other 5-HT families or other targets. The second situation is that the 5-HT7 radioligand candidate was tested favorably in vitro but failed in vivo because of the characteristics of its pharmacokinetics (rapid metabolism and radioactive metabolites in the brain, etc.), its pharmacodynamics (brain exclusion via efflux transporter, etc.) or unknown reasons (low target-to-background ratio, etc.). Finally, it has to be borne in mind that transfer to humans of a radiotracer used in animals involves ever increasing radiopharmaceutical prerequisites. All these heterogeneous situations will be illustrated in this review, explaining the numerous failures or insufficient advances in the development of radiotracers for 5-HT7 brain imaging.
In vitro imaging of 5-HT7 receptors Nonselective radioligands As previously mentioned, knowledge of the biological role of central 5-HT7 receptors is in part based on results from studies of their anatomical distribution. Discrete regional distribution has been described for cell populations expressing mRNA coding for 5-HT7 receptors in rat and guinea pig brain (To et al., 1995; Gustafson et al., 1996; Mengod et al., 1996; Kinsey et al., 2001; Neumaier et al., 2001). These studies reported an encouraging abundance of 5-HT7 receptor mRNA and 5-HT7 immunoreactivity, contrasting sharply with the low density of 5-HT7 receptors.
Assessment of the anatomical distribution of 5-HT7 receptor binding sites in brain tissue has long been hampered by the lack of a specific radioligand. Several attempts have been made using available radioligands in rat brain homogenates (Stowe and Barnes, 1998; Hemedah et al., 1999) or brain sections (To et al., 1995; Waeber and Moskowitz, 1995; Gustafson et al., 1996; Mengod et al., 1996). These autoradiography studies used [3H]5-CT, a nonselective 5-HT receptor agonist ligand, in the presence of specific antagonists to mask other binding sites, theoretically enabling full visualization of the receptor. An attempt to visualize 5-HT7 receptors in hamster brain used [3H]8-OH-DPAT, a 5-HT1A/7 ligand, in the presence of pindolol for specific 5-HT1A receptor blockade (Duncan et al., 1999). However, it turned out that the added masking compounds induced incomplete blockade of non-5-HT7 sites, introducing a bias in the interpretation and quantification of the results. An additional drawback was the poor selectivity of the blocking compounds used, with partial blockade of 5-HT7 receptors, resulting in an underestimation of the 5-HT7 binding component. Another strategy uses 5-HT1A and 5-HT1A/1B KO mice to visualize the anatomical location of 5-HT7 binding sites in mouse brain, using [3H]5-CT and [3H]8-OH-DPAT (Bonaventure et al., 2002). The rationale of this approach was that in mice lacking 5-HT1A receptors, the radioligands would bind more specifically 5-HT7 receptor binding sites. However, results revealed that despite its high affinity for 5-HT7 receptors, [3H]5-CT is not a good tracer for autoradiographic assessment of 5-HT7 receptors. Another team chose the nonselective antagonist [3H] mesulergine as 5-HT7 radioligand, as it has a high affinity for 5-HT7 receptors (Martín-Cora and Pazos, 2004). Autoradiographic studies were performed in the presence of adapted concentrations of raclopride and DOI to mask [3H] mesulergine binding to D2, 5-HT2A and 5-HT2C receptors. In addition, [3H]mesulergine is an antagonist (unlike previous agonists such as [3H]5-CT and [3H]8-OH-DPAT), which should be more favorable to stable binding. Therefore, in the case of an agonist, if multiple agonist affinity states of the receptor are present, only a fraction of the total 5-HT7 receptor population may be labeled. Thus, [3H]mesulergine enabled the anatomical distribution of 5-HT7 receptors to be mapped in human brain and compared with that observed in guinea pig and rat brain.
Selective radioligand: [3H]SB-269970 Nonselective radioligands have proved useful in the initial characterization of cloned and native 5-HT7 receptors, as
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360 L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors well as providing information on their native distribution, but none selectively label 5-HT7 receptors. There is therefore still a need for a selective 5-HT7 antagonist radioligand. SB-269970 (3-{[(2R)-2-[2-(4-methylpiperidin-1-yl)ethyl] pyrrolidin-1-yl]sulfonyl}phenol), an analog of SB-258719 (Forbes et al., 1998), has been identified and described as a potent 5-HT7 receptor antagonist displaying at least 50- to 100-fold greater selectivity than 50 different receptors, enzymes and ion channels (Lovell et al., 2000). This molecule was radiolabeled with tritium and its binding was first characterized with respect to both cloned human 5-HT7 receptors and 5-HT7 receptors in guinea pig cerebral cortex (Thomas et al., 2000). The same team went on to investigate [3H]SB-269970 binding to brain tissue homogenates from a number of other species: mouse, rat, pig, marmoset and human (Thomas et al., 2002). [3H] SB-269970 bound saturably to mouse forebrain and rat, pig and marmoset cortex, labeling a homogenous population of sites. KD values were between 1 and 2 nm, according to species. The Bmax values for [3H]SB-269970 binding to mouse forebrain and rat, pig and marmoset cortex tissues were 20, 30, 31 and 14 fmol per milligram protein, respectively. However, preliminary characterization of [3H] SB-269970 binding to human cerebral cortex membranes revealed a too low level of 5-HT7 receptor expression to be accurately quantifiable. In the following study (Varnäs et al., 2004), 5-HT7 receptor distribution was analyzed using [3H]SB-269970 and human brain whole-hemisphere autoradiography. The results indicated that 5-HT7 receptors are most abundantly located in the anterior thalamus and dentate gyrus. Intermediate concentrations were found in the hypothalamus, anterior cingulate gyrus, hippocampus, amygdala and certain brainstem nuclei. This study confirmed the very low density of 5-HT7 receptors in the human brain, with specific binding ranging from 16 pmol/g (anterior thalamus) to 2 pmol/g (isocortex). Another study showed significant lower [3H]SB-269970 binding in Brodmann’s area 9 in subjects with schizophrenia than controls, consistent with the hypothesis that decreased levels of 5-HT7 receptors may be involved in the pathological processes of schizophrenia (Dean et al., 2006). However, this study confirmed the low signal-tonoise ratio obtained with the only tritiated radioligand currently available for 5-HT7 imaging. More recently, this radiotracer was used to quantify the binding of lurasidone, a new antipsychotic, to 5-HT7 receptors in rat brain by autoradiography (Horisawa et al., 2013). These recent findings confirmed that binding to 5-HT7 receptors might play some role in beneficial
pharmacological action in schizophrenia, confirming the interest in developing 5-HT7 radioligands for in vivo imaging.
Toward in vivo imaging of 5-HT7 receptors [11C]DR4446 A Japanese consortium developed the first 5-HT7 radio tracer for PET imaging in 2002 (Zhang et al., 2002). DR4446 is a tetrahydrobenzindole derivative that was selected from a series of molecules for its high selectivity for 5-HT7 over the other 5-HT receptors (Kikuchi et al., 2002). [11C]DR4446 was therefore synthesized by N-methylation of its desmethyl precursor with [11C]methyl iodide ([11C]CH3I). The radiotracer obtained was only tested in anesthetized monkeys, but showed good cerebral uptake, consistent with its lipophilicity (log p = 3.7). Radioactivity was slightly higher in the thalamus, an area rich in 5-HT7 receptors, than in other regions, and a blocking study with unlabeled DR4446 compound showed the reversibility of [11C]DR4446 binding. However, to our knowledge, no further reports have been published using this 5-HT7 PET radiotracer. This can probably be explained by the moderate affinity of [11C]DR4446 for 5-HT7 receptors, Ki 9.7 nm, which, given the low density of 5-HT7 receptors in the brain, leads to a low signal-to-noise signal.
[18F]SB-269970 derivatives The second attempt to develop a 5-HT7 PET radiotracer was made in 2010 in the University of Lyon, France (Andriès et al., 2010). Two molecules were envisaged, inspired by the literature results. The first was a piperazine derivative adapted from Yoon et al.’s (2008) findings. The second was based on SB-269970, previously described as a successful tritiated ligand (Thomas et al., 2000), but modified in this case with an ester moiety in the piperidine core. Both candidates presented intermediate affinity toward 5-HT7 receptors (pKB, 7.26 and 7.10, respectively) and relatively high lipophilicity (log D at pH 7.4, 3.27 and 4.13, respectively). Both were radiolabeled by nitro group nucleophilic aromatic substitution. Fluorinated piperazine was abandoned because radiolabeling did not give satisfactory radiochemical yields. The second candidate, [18F]SB-269970 derivative, was evaluated by in
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vitro autoradiography in rat brain. Binding proved homogeneous throughout the brain, with no specific binding in regions rich in 5-HT7 receptors; development was therefore stopped.
[18F]2FP3 and derivatives More recently, the same team from the University of Lyon developed four other SB269970 analogs. To avoid the undesirable in vivo metabolization previously observed (Andriès et al., 2010), 18F labeling was introduced onto a less reactive part of SB269970, the phenylsulfonyl moiety. Close analogs were synthesized with a fluorophenylsulfonyl group on the pyrrolidine and with a piperazine group instead of the piperidine heterocycle (Andriès et al., 2011). The nitro precursors of these analogs were radiolabeled by 18 F nucleophilic substitution. All presented suitable affinity for 5-HT7 receptors (apparent dissociation constant KD between 1.6 and 14 nm), although two were abandoned because of their agonist activity for 5-HT1A receptors. In vitro autoradiography in rats revealed that only two radioligand candidates were displaced by the 5-HT7 antagonist SB 269970. Therefore, subsequent in vivo study focused only on these two. According to PET scans in cats and competition studies, [18F]2FP3 appeared to be the best PET radiotracer for in vivo imaging of 5-HT7 receptors in animal models (Lemoine et al., 2011). Optimally, better affinity for 5-HT7 receptors would be preferable, because specific binding is a function of a radiotracer’s affinity for the receptor relative to the density of the binding site (which is low for 5-HT7). Consequently, the development of [18F]2FP3 analogs was pursued by this team, with chemical structures modified to optimize PET tracer properties, particularly in terms of affinity, which should be subnanomolar. Unfortunately, although one of these new radioligand candidates showed promising characteristics in vitro (5-HT7 Ki 50 nm), it interacted unpredictably with P-glycoprotein, with consequently poor brain penetration in vivo (Colomb et al., in press).
Oxindole derivatives A Danish team (University of Copenhagen), in collaboration with a Hungarian pharmaceutical company, chose to use phenylpiperazinyl-butyloxindoles as a lead structure for 5-HT7 radiotracers, because this structure displays an inhibition constant (Ki) that is 2000-fold lower for 5-HT7 receptors than for 5-HT1A receptors (Volk et al.,
2008, 2011). In particular, they investigated the influence of various substitution patterns on the affinity toward the 5-HT7 receptor and concentrated on structure-activity relationships at the arylpiperazine moiety (Herth et al., 2012a). Two of the 16 candidate compounds synthesized were tested toward 37 receptors. Although both displayed a promising in vitro profile for PET imaging of the 5-HT7 receptor, their adrenergic α1 receptor affinity may contaminate the signal detected from the 5-HT7 receptor. At the time of writing, none of these oxydole derivatives is yet used in vivo as a 5-HT7 PET ligand.
[11C]Cimbi-806 In 2011–2012, the same Danish team (University of Copenhagen) developed an alternative strategy to obtain a 5-HT7 radioligand. They focused on another lead structure, biphenethylamines. Cimbi-806 is a biphenethylamine with a profile initially characterized at the University of Caen, France (Paillet-Loilier et al., 2007). Its affinity for 5-HT7 is significantly higher than for 5-HT1A (Ki = 8.8 vs. 4826 nm). This large difference in Ki is especially useful because of the low density (Bmax) of 5-HT7 receptors compared to 5-HT1A receptors and because of the brain colocalization of 5-HT7 and 5-HT1A receptors. [11C]Cimbi-806 was therefore radiolabeled and its in vivo binding characteristics were subsequently assessed in pigs (Herth et al., 2012b). After intravenous injection of [11C]Cimbi-806, there was rapid high brain uptake, with a maximum in the thalamus and striatum and a minimum in the cerebellum. However, no significant change in [11C]Cimbi-806 after SB-269970 pretreatment was found in any region, so that in vivo binding could not be claimed to be selective for the 5-HT7 receptor. The authors concluded that [11C]Cimbi-806 was not suitable for imaging the 5-HT7 receptor.
Arylpiperazinic ligands A group of the University of Bari (Italy) designed piperazine derivatives and characterized one of them as a 5-HT7 receptor agonist (Leopoldo et al., 2008). This same group claimed in a recent patent the invention of 1-arylpiperazinic ligands of 5-HT7 receptors that present high affinity and selectivity for the receptor (Leopoldo et al., 2012). However, although binding assays are presented in the patent (with 5-HT7 pKi ranging from 7.70 to 8.79), no other biological testing is detailed. Particularly, no radiolabeling or in vivo imaging data have as yet been reported.
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362 L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors
[3H]5-CT
[3H]8-OH-DPAT
[3H]SB-269970
[11C]DR4446
[18F]SB-269970 derivative
Piperazine derivative
[18F]2FP3
[3H]mesulergine
[11C]Cimbi-806
[11C]1-[2-(4methoxyphenyl) phenyl]piperazine
Figure 1 Chemical structures of the radioligands developed for the 5-HT7 receptor imaging.
[11C]methoxyphenyl)phenyl]piperazine
Conclusions
The Japanese team that 10 years ago developed [11C] DR4446 as the first putative 5-HT7 radiotracer recently reported a new 5-HT7 candidate (Shimoda et al., 2013). 1-[2-(4-Methoxyphenyl)phenyl]piperazine was developed as a PET candidate because it showed high binding affinity to human recombinant 5-HT7 receptors expressing in mammalian cells (Ki = 2.6 nm), whereas its binding affinity to 5-HT1A receptor was 476 nm. It also displayed a weak affinity for adrenergic 1 receptor (Ki = 156 nm), in contrast to previous PET candidates. Furthermore, it has a methoxyl group in its molecule and can be easily labeled by 11 C without changing its chemical structure or pharmacological functions. In vitro autoradiographic studies have been encouraging, but a small-animal PET study showed homogenous whole-brain distribution of radioactivity, inconsistent with the brain distribution of 5-HT7 receptors; in particular, uptake in the 5-HT7-rich thalamus was not higher than in the other brain regions. Moreover, a blocking experiment using a 5-HT7-selective compound did not reduce but increased brain uptake, precluding the pursuit of the use of this radiotracer.
The 5-HT7 receptor represents a crucial target in neuroscience, justifying a need for radioligands for visualization and quantification, first in animal models and ultimately in humans. Although no precise data regarding the Bmax of 5-HT7 receptors have been reported, 5-HT7 receptor density was assessed at less than one-tenth that of the other 5-HT families. Therefore, brain visualization is particularly challenging, requiring radiotracer candidates with subnanomolar 5-HT7 affinity and particularly high specificity. Despite numerous characterizations of radiotracer candidates (see Figure 1) and encouraging results, there are currently no PET radioligands for the exploration of 5-HT7 receptor in humans. Development of 5-HT7 brain radiopharmaceuticals is therefore still an exciting field, involving interdisciplinary research bringing together chemistry, neuropharmacology and imaging science.
Received November 25, 2013; accepted January 9, 2014; previously published online February 5, 2014
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L. Zimmer and T. Billard: Imaging of serotonin 5-HT7 receptors 363
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Luc Zimmer received a pharmacist education at the University of Strasbourg (1986–1992); he completed training during his internship and residency in radiopharmacy and radiopharmacology (1993–1999) at the Hospital University of Tours (Nuclear Medicine Department) and at the National Institute for Nuclear Sciences and Technology, Saclay. He received his PharmD in radiopharmacology (1998) and his PhD in neuroscience (1999) from the University of Tours. He is currently professor of pharmacology at the University of Lyon (Université Claude Bernard Lyon 1) and is radiopharmacist at the University Hospital of Lyon (Hospices Civils de Lyon). He is head of the preclinical department of the CERMEP-imaging platform and is in charge of the laboratory ‘Radiopharmaceutical and neurochemical biomarkers’ at the Lyon Neuroscience Research Center (CRNL). His research interests include radiopharmacology and in vivo imaging of neurotransmissions with PET.
Thierry Billard, after education in chemistry, completed by an engineer diploma from ‘Institut de Chimie et Physique Industrielles de Lyon’ (now ‘CPE Lyon’) in 1993. He obtained his PhD in organic chemistry from the University of Lyon in 1996. After a postdoctoral internship with Prof. L. Ghosez (Université Catholique de Louvain, Belgium), he was appointed in 1999 as a CNRS permanent researcher. He was promoted Research Director by CNRS in 2008. He is currently in charge of a research team at the Institute of Chemistry and Biochemistry (ICBMS). His research activities focus on fluorine chemistry, medicinal chemistry, radiochemistry and their applications in medical imaging.
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