Selected PET Radioligands for Ion Channel Linked Neuroreceptor Imaging: Focus on GABA, NMDA and nACh Receptors

Abstract

Positron emission tomography (PET) neuroimaging of ion channel linked receptors is a developing area of preclinical and clinical research. The present review focuses on recent advances with radiochemistry, preclinical and clinical PET imaging studies of three receptors that are actively pursued in neuropsychiatric drug discovery: namely the γ-aminobutyric acid-benzodiazapine (GABA) receptor, nicotinic acetylcholine receptor (nAChR), and N-methyl-d-aspartate (NMDA) receptor. Recent efforts to develop new PET radioligands for these targets with improved brain uptake, selectivity, stability and pharmacokinetics are highlighted.

Graphical abstract

Introduction

Positron emission tomography (PET) is a non-invasive functional imaging technique used to probe biological processes in vivo, via administration of radiotracers. Positron-emitting radionuclides such as carbon-11 (¹¹C; t_1/2 = 20.3 min) and fluorine-18 (¹⁸F; t_1/2 = 109.7 min) decay with the emission of a positron, which subsequently annihilates upon contact with an electron to produce two 511 keV annihilation photons emitted at approximately 180° to each other.^1,2,3,4 These photons can be observed by detectors positioned in an array around the visualized object, and when both are detected simultaneously the emission event can be traced back to its location in vivo by analysis of the coincidence lines. In order to image a particular biological target using PET, the positron-emitting radionuclide must be “embedded” in a chemical scaffold, constituting the radiotracer (imaging probe), with the desired biological properties to both transport the radionuclide over the existing biological obstacles such as the blood brain barrier and reach the desired tissue, and interact with the molecular target of interest. PET imaging has been applied to a variety of biological processes, and can be used to diagnose and monitor the progression of numerous disease states, including cancers, cardiac disease and neurological disorders.⁵ PET imaging probes can also be used to guide medicinal chemistry and drug development efforts at both preclinical and clinical stages by providing in vivo insights into drug binding and correlating receptor occupancy with pharmacological response. The quantitative data provided by PET is particularly useful for facilitating drug development to follow disease progression, treatment monitoring and longitudinal studies.⁶

Ion channels are membrane proteins which control the flow of ions passing through the cell membrane in almost all living species. Ion channel linked receptors are bound in cell membranes and mediated via the conformational interaction between ion channels and chemical ligands. Despite a large number of putative ion channels and related receptors proposed and identified in human genome, only few have been thoroughly studied and characterized.⁷ Although PET ligand development and imaging studies in ion channel related receptors have been reviewed in the past,^8,9,10 the present review is focused on recent advances (2010 – present) with three of these receptor protein targets that we and others are interested for neuropsychiatric PET radiopharmaceutical development: the γ-aminobutyric acid-benzodiazapine (GABA) receptor, the nicotinic acetylcholine receptor (nAChR), and the N-methyl-d-aspartate receptor (NMDA) receptor from publications. This review highlights selected radiochemical scaffolds with emphasis on promising preclinical and clinical PET neuroimaging studies with lead tracers.

γ-Aminobutyric Acid-Benzodiazapine Receptor

Recent Preclinical and Clinical Research

The most commonly used PET radiotracer for imaging the γ-Aminobutyric Acid-Benzodiazapine Receptor (GABA-BZD) is radiolabeled flumazenil, an imidazo-benzodiazapine derivative which binds allosterically to the receptor.¹¹ This tracer can be radiolabeled with either ¹⁸F (Figure 1, compound a; [¹⁸F]flumazenil) or ¹¹C (Figure 1, compound b; [¹¹C]flumazenil) and an azide derivative of flumazenil, Ro15-4513 (Figure 1, compound c; [¹¹C]Ro15-4513) is also widely used. Table 1 summarizes recent preclinical PET imaging studies with [¹¹C]flumazenil, [¹⁸F]flumazenil, or [¹¹C]Ro15-4513 carried out in rodents or nonhuman primates. Rodent studies were used to investigate binding and saturation of [¹¹C]flumazenil to the GABA receptor, and demonstrated that both receptor density and binding affinity of the tracer could be obtained in a single PET scan using a novel full saturation method.¹² [¹¹C]Flumazenil brain uptake was also monitored in anesthetized and awake (minimally restrained) nonhuman primates, and differences between the groups were shown to be minimal, though cortisol levels were significantly higher in awake animals.¹³ Interestingly, [¹¹C]flumazenil brain uptake was found to be influenced by the P-glycoprotein, a blood-brain barrier brain efflux transporter, in rodents.¹⁴

Figure 1. Radiolabeled compounds targeting the GABA receptor.

a) [¹⁸F]Flumazenil; b) [¹¹C]Flumazenil; c) [¹⁸F]Ro15-4513; d) Nitromazenil e) [¹⁸F]AH114726; f) [¹⁸F]GEH120348; g) quinoline derivative developed by Moran et al.¹⁵.

Table 1.

Preclinical Applications of Flumazenil and Ro15-4513

Tracer	PET Nuclide	Animal	Reference
Flumazenil	[18F]	Rhesus Monkey	16
Flumazenil	[18F]	Rat	17
Flumazenil	[11C]	Rhesus Monkey	13
Flumazenil	[11C]	Mouse and Rat	14
Flumazenil	[11C]	Rat	12
Ro15-4513	[3H]	Rat	18

[¹⁸F]flumazenil was recently employed in a rat model of temporal lobe epilepsy, which elucidated a decline in hippocampal receptor density in status epilepticus rats when compared with healthy controls.¹⁷ In Rhesus monkeys, socially dominant females were shown to have lower GABA receptor density in the prefrontal cortex than socially submissive animals by PET studies using [¹⁸F]flumazenil, but administration of the corticotropin-releasing hormone astressin B to submissive females eliminated this effect.¹⁶ [¹¹C]Ro15-4513 and [³H]Ro15-4513 were used in in vitro studies of rat brain tissue to investigate the effects of vigabatrin, tiagabine, and SNAP-5114 on receptor agonist distribution.¹⁸

¹¹C- and ¹⁸F-labeled flumazenil have also been used extensively in clinical research studies, as summarized in Table 2. For instance, a significant decrease in cerebellar binding of [¹¹C]flumazenil was reported in three patients with cerebellar ataxia compared with healthy controls.¹⁹ PET imaging with [¹¹C]flumazenil was also used to determine enhanced cognition effect of the specific GABA-α5 receptor agonist a5IA (LS-193,268) in patients without demonstrating the anxiogenic effects produced by nonspecific GABA agonists.²⁰ Low cerebellar binding of [¹¹C]flumazenil was also reported in infants with epileptic seizures.²¹ Tiagibine was demonstrated to increase [¹¹C]flumazenil binding in a dose-dependent manner.²² [¹¹C]Flumazenil PET imaging detected a decrease in GABA receptor expression and affinity in patients with primary dystonia.²³ The effectiveness of [¹⁸F]flumazenil as a PET radiotracer was recently assessed in patients with temporal lobe epilepsy.²⁴ [¹⁸F]Flumazenil imaging was used in stroke patients to monitor GABA neuroplasticity during the recovery phase, and increased GABA receptor density was correlated with the recovery of upper extremity motor function.²⁵ Men at ultra-high risk for psychosis showed significantly lower uptake of [¹⁸F]flumazenil in the right caudate region of the brain.²⁶ Schizophrenic men taking aripiprazole had decreased [¹⁸F]flumazenil uptake in several regions of the prefrontal cortex as compared with patients taking risperidone and healthy controls.²⁷ Differences in GABA receptor binding potential with [¹⁸F]flumazenil were observed in several regions of the brain when subject awareness was directed internally verses externally.²⁸ [¹⁸F]Flumazenil measurements of neuronal density were used to elucidate differences between MRI-based measurements of surface cortical thickness and actual cytoachitectonics in several brain structures.²⁹ [¹¹C]Ro15-4513 has also been used in clinical studies. This tracer was recently used to detect acute increases in synaptic GABA following the administration of tiagibine.³⁰ Individuals with a history of smoking showed higher distribution volume in limbic regions than nonsmokers even after a long period of abstinence from smoking.³¹ [¹¹C]Ro15-4513 was shown to have higher specificity for the GABA-α5 receptor subtype than flumazenil as demonstrated by dosage with the GABA-α1 selective agonist zolpidem.³²

Table 2.

Clinical Applications of Flumazenil and Ro15-4513

Tracer	PET Nuclide	Experimental Condition	Reference
Flumazenil	[18F]	Unilateral Ischemic Stroke	25
Flumazenil	[18F]	Temporal Lobe Epilepsy	24
Flumazenil	[18F]	Children with Cerebral Palsy	33
Flumazenil	[18F]	Ultra-High Risk for Psychosis	26
Flumazenil	[18F]	Schizophrenics taking Aripiprazole	27
Flumazenil	[18F]	Aversive-Aversive Stimulus	34
Flumazenil	[18F]	Internal vs. External Awareness	28
Flumazenil	[18F]	Healthy – Cortical Thickness	29
Flumazenil	[11C]	Acute Triazolophthalazine α51A Dose	20
Flumazenil	[11C]	Cerebellar Ataxia	19
Flumazenil	[11C]	Epileptic Newborns	21
Flumazenil	[11C]	Tiagibine GAT-1 Blockade	22
Flumazenil	[11C]	Primary Distonia	23
Ro15-4513	[11C]	Induced Acute GABA increase	30
Ro15-4513	[11C]	History of Cigarette Smoking	31
Ro15-4513	[11C]	Acute Zolpidem Dose	32
Ro15-4513	[11C]	Alcohol-Dependent Men	35
Ro15-4513	[11C]	Autism Spectrum Disorder	36

Novel PET Tracers and Radiochemistry

Though the vast majority of PET imaging studies of the GABA receptor are performed with the well characterized radiotracers flumazenil and Ro15-4513, new PET ligands and radiochemistry have been reported and much of the new research focuses on the development of derivatives of the imidazo-benzodiazapine core present in both of these known tracers, with the aim to show improvement of binding affinity and radiochemical method to improve yield in a variety of ways. The synthesis of [¹⁸F]flumazenil has been previously reported,³⁷ but low yields are reported and variability is high. In our experience, the synthesis of [¹⁸F]flumazenil is complicated by the relatively large amounts of nitro-precursor (nitromazenil; Figure 1, compound d) required (5-10 mg) as decomposition rates compete with the radiolabeling step. An improved radiosynthesis for [¹⁸F]flumazenil is therefore desirable.

Recent research by Jackson et al. focused on improving upon existing methods for the radiofluorination of flumazenil while simultaneously investigating related structures with more accessible routes of fluorination. The flumazenil derivatives were synthesized in 13-24% radiochemical yield and specific activity around 2 GBq/μmol. Eleven of these derivatives were deemed to be suitable for initial in vivo PET imaging studies. Two of the original compounds ([¹⁸F]AH114726 and [¹⁸F]GEH120348; Figure 1, compounds e and f) showed radiofluorination results at levels comparable with [¹⁸F]flumazenil, and all showed improved capacity for radiofluorination.³⁸ A blocking study performed in rodents showed that these compounds showed tracer kinetics with similar or improved quality compared with [¹⁸F]flumazenil. A blocking study was also carried out for the tracers in Rhesus monkey, and one compound ([¹⁸F]AH114726) displayed pharmacodynamics similar to those of [¹⁸F]flumazenil.³⁸ Though most novel radiochemistry focuses on imidazo-benzodiazapine derivatives, some have attempted to produce radioligands from other core structures. Our laboratory has focused on a novel class of tracers based on a quinoline core (Figure 1, compound g).¹⁵ Two members of this class were synthesized and shown to be more selective to specific receptor subtype, i.e., GABA_A, than flumazenil and related benzodiazepine derivatives in in vivo PET imaging studies in rodents. These quinolines represent a new class of PET radiotracers for imaging the benzodiazepine site of GABA_A. In particular, [¹¹C]compound g readily penetrated the rat brain (>1 standard uptake value in cortical regions), had an appropriate regional brain distribution and reversible binding for GABA_A receptors.

Nicotinic Acetylcholine Receptor

Central neuronal nicotinic acetylcholine receptors are involved in various neurological process and neurodegenerative diseases, including epilepsy, depression, schizophrenia and dementia. Among all the 17 identified subtypes, α4β2-nAChR and α7-nAChR are two most prominent targets in human brain. The development of PET ligand targeting subtype α4β2 has been reviewed thoroughly^9a and we aim to provide a brief and recent summary of preclinical and clinical use of these PET probes and recent ligand development therein.

Recent Preclinical and Clinical Research

Several radiotracers targeting the nicotinic acetylcholine receptor (nAChR) have recently been characterized in a variety of preclinical and clinical studies (Tables 3 and 4). There are four PET radiotracers, namely, [¹¹C]nicotine, [¹⁸F]2-FA, [¹⁸F]6-FA and [¹⁸F]AZAN that have been used in the imaging of α4β2 subtype in human brain.^9a While an ideal PET ligand for this receptor has not yet been definitively established, we summarize here recent preclinical and clinical advances of PET ligands targeting nAChR, including [¹⁸F]2-FA (Figure 2, compound a), [¹⁸F]Nifene (compound b), [¹⁸F]flubatine (compound c) and [¹¹C]CHIBA-1001(compound d). Specifically, [¹⁸F]2-FA is a nicotinic acetylcholine receptor PET ligand with high affinity for the β2 subunit. The clinical applications of this tracer have recently included studies of the effects of smoking and psychiatric and degenerative disorders. PET imaging with this ligand indicated lower nAChR density in the peripheral vasculature of individuals with Parkinson’s disease or multiple system atrophy.³⁹ A low dose of varenicline in smokers was shown by PET to fully saturate brain nAChR but had no effect on the reduction of nicotine withdrawal symptoms.⁴⁰ Tracer binding potential in the thalamus was significantly lower in paranoid schizophrenic smokers than in healthy controls.⁴¹ Patients with Alzheimer’s disease and mild cognitive impairment were demonstrated to have lower tracer binding potential than controls in regions of the brain affected by the disease.⁴² [¹⁸F]Nifene is a derivative of 2-FA with a dihydropyrrole ring (five-membered cycloamine) rather than an azetidine ring (four-membered cycloamine). This tracer has not yet been approved for clinical use but has demonstrated improved binding kinetics in preclinical studies over 2-FA, which often requires several hours to obtain a high resolution PET scan. Imaging with [¹⁸F]nifene was used to map nAChR in the brain of rats, and a potential role for this receptor in sensory-cognitive function was evaluated.⁴³ Brain distribution of tracer uptake was investigated in rhesus monkeys,⁴⁴ and pharmacokinetics of the tracer was also evaluated,⁴⁵ and a blocking study was conducted.⁴⁶ [¹⁸F]Flubatine is a nAChR PET radioligand with specificity for the α4β2 subtype.⁴⁷ This tracer was demonstrated to detect differences in synaptic acetylcholine concentration induced by receptor inhibitors in Rhesus monkeys.⁴⁸ The first fully automated radiosynthesis of this compound validated for human use⁴⁹ was reported in 2013 and first-in-human results of administration of the radiotracer showed no adverse effects in humans.⁵⁰ Tracer binding to plasma proteins in human blood was demonstrated in vitro and ex vivo to show no differences between patients with Alzheimer’s disease and healthy controls.⁵¹ [¹¹C]CHIBA-1001 is a PET radiotracer for the α-7 subtype of nAChR.⁵² The tracer demonstrated has low specific binding for the α-7 subtype.⁵³ However, tracer biodistribution and kinetics have been shown to be very different in humans from the analogous rodent properties, and clinical results showed homogeneous brain uptake with low specificity.⁵⁴ Dosage with tropesitron, an α-7 receptor agonist, decreased overall brain uptake of CHIBA-1001 in humans.⁵⁵ Other radiotracers for this target include [¹⁸F]NS-10743 (Figure 2, compound e), [¹⁸F]AZAN (compound f), [¹¹C]NS-14492 (compound g), [¹⁸F]nifzetidine and [¹⁸F]ZW-104, and have been overviewed in recent publications.⁵⁶

Table 3.

Preclinical Nicotinic Acetylcholine Receptor PET Studies

Tracer	PET Nuclide	Animal	Reference
Nifene	[18F]	Mice	57
Nifene	[18F]	Rat	58
Nifene	[18F]	Rat	59
Nifene	[18F]	Rat	43
Nifene	[18F]	Rhesus Monkey	45
Nifene	[18F]	Rhesus Monkey	44
Nifene	[18F]	Rhesus Monkey	46
Flubatine	[18F]	Rhesus Monkey	48
Flubatine	[18F]	Rhesus Monkey	49
NS10743	[18F]	Pig	60
AZAN	[18F]	Baboon	61
CHIBA-1001	[11C]	Rat	53
CHIBA-1001	[131I]	Monkey	52
ASEM	[18F]	Mouse	62
ASEM	[18F]	Baboon	63
XTRA	[18F]	Baboon	64

Table 4.

Clinical Nicotinic Acetylcholine Receptor PET Studies

Tracer	PET Nuclide	Experimental Condition	Reference
2FA	[18F]	Neurodegenerative Disorders	39
2FA	[18F]	Low Varinicline Dose, Smokers	40
2FA	[18F]	Schizophrenic Smokers	41
2FA	[18F]	Mild Cognitive Impairment, Alzheimer’s Disease	42
Flubatine	[18F]	Healthy	50
Flubatine	[18F]	Alzheimer’s Disease	51
CHIBA-1001	[11C]	Healthy	54
CHIBA-1001	[11C]	Tropisetron Dose	55
ASEM	[18F]	Healthy	65

Figure 2. Radiolabeled compounds targeting the nACh receptor.

a) [¹⁸F]2FA; b) [¹⁸F]Nifene; c) [¹⁸F]Flubatine; d) [¹¹C]CHIBA-1001; e) [¹⁸F]NS10743; f) [¹⁸F]AZAN; g) [¹¹C]NS14492; h) [¹⁸F]AZ11637326; i) [¹⁸F]Niofene; j) quinuclidine derivative developed by Pin et al.⁶⁶ ; k) [¹⁸F]ASEM; l) [¹¹C]Rac-1; m) [¹¹C]A-833834; n) [¹¹C]A-752274; o) [¹⁸F]NS14490; p) [¹⁸F]XTRA.

Novel PET Tracers and Radiochemistry

Improvement in selectivity for the α7 subtype of the nicotinic acetylcholine receptor, which has been linked to several neurological disorders, is the focus of the majority of new radiochemistry and ligand development for this receptor. Ettrup et al. recently published a blocking study evaluating the in vivo characteristics of the nAChR α7 receptor subtype PET radioligand [¹¹C]NS-14492.⁶⁷ This radiotracer demonstrated high binding affinity for the target in vitro and high stability and selectivity in vivo in pigs, and was the first tracer to show dose-dependent blockade of this receptor subtype. [¹⁸F]AZ11637326 (Figure 2, compound h), an α7-nAChR radioligand, was developed by Ravert et al.⁶⁸ This radiotracer was prepared via nucleophilic [¹⁸F]fluorination with a subsequent decarboxylation step, resulting in about 3% overall radiochemical yield and specific activity of 140 GBq/μmol. This tracer was evaluated in vivo in rodents and nonhuman primates. While some level of brain uptake was observed in rodents, no specific binding was shown in nonhuman primates. Kuruvilla et al. developed an alternative radiotracer 2-fluoro-5-iodo-3-[2-(S)-3-dehydropyrrolinylmethoxy]pyridine, (Figure 2, compound i; [¹⁸F]niofene) in an attempt to improve the binding kinetics of the known tracer [¹⁸F]nifene and develop a compound suited to both PET and SPECT imaging.⁶⁹ Niofene exhibited two-fold improved binding affinity over nifene in vitro. In rodent PET studies, niofene showed rapid brain uptake and indicated some selectivity for the nAChR. A series of novel quinuclidine derivatives were investigated by Pin et al. as α7-nAChR radiotracers.⁶⁶ Amide derivatives within the series demonstrated promising in vitro binding results, and some of these compounds were selected for radiolabeling. One compound from the series (Figure 2, compound j) was evaluated in a rodent PET study and showed good penetration of the blood-brain-barrier, but also appeared to have fast clearance. [¹⁸F]ASEM (Figure 2, compound k) is a radioligand developed by Gao et. al targeting the α7 nAChr. The original synthesis of the tracer involved substitution from a nitro derivative, resulting in 16% radiochemical yield with greater than 99% radiochemical purity and specific activity ranging from 330-1260 GBq/μmol.⁶² More recently, an improved method using microwave synthesis has been reported⁷⁰ which results in 20% radiochemical yield with 856 GBq/μmol specific activity. In an initial blocking study in mice, [¹⁸F]ASEM was demonstrated to have higher in vivo binding potential to the target than previously developed radioligands.⁶² Specific binding to the receptor was later demonstrated to be in the range of 80-90% in baboons.⁶³ In a recent blocking study in healthy humans, the average binding potential of [¹⁸F]ASEM was 10.8%.⁶⁵ Another α7 nAChR radioligand, 5-(5-(6-[¹¹C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole (Figure 2, compound l; [¹¹C]rac-(1)) was also recently developed by the same group.⁷¹ The compound showed promising initial selectivity for the α7 receptor subtype in an ex vivo biodistribution study in rodents. Horti et al. also investigated [¹¹C]A-833834 (Figure 2, compound m) and [¹¹C]A-752274 (Figure 2, compound n), two radioligands for the α7-nAChR.⁷² In PET studies in rodents, both ligands showed somewhat low brain uptake but high specificity indicated by ex vivo localization in the thalamus. In a nonhuman primate PET study, there was very little brain uptake of [¹¹C]A-752274. A new synthesis method for the nAChR radiotracer [¹⁸F]NS14490 (Figure 2, compound o) proposed by Rotering et al. allows for the direct nucleophilic substitution of the precursor, resulting in 70% radiochemical yield.⁷³ This tracer demonstrated high target affinity as well as selectivity in vitro for the α7 receptor subtype. In rodent PET studies, low brain uptake for the tracer was observed, but the compound had high stability in brain tissue and in plasma. [¹⁸F]XTRA (Figure 2, compound p) is another radioligand developed by Gao et. al which targets the α4β2 nAChr (this compound was developed alongside [¹⁸F]AZAN).⁷⁴ The original synthesis of [¹⁸F]XTRA from a bromo precursor resulted in a radiochemical yield of 16-47%, radiochemical purity greater than 98%, and specific activity ranging from 185 GBq – 1.8TBq/μmol.⁶⁴ An improved synthetic strategy for the bromo precursor was later reported.⁷⁵ In a baboon blocking study, both [¹⁸F]XTRA and [¹⁸F]AZAN were demonstrated to have rapid, reversible brain kinetics.⁶⁴

N-methyl-d-aspartate Receptor

Recent Clinical Research

N-Methyl-d-aspartate receptor agonists have been shown to treat symptoms of Alzheimer’s disease, though no reduction in disease progression has been demonstrated. The response to NDMA receptor agonists has been monitored by PET using [¹⁸F]FDG.⁷⁶ No PET radioligands developed for this receptor have been suitable for clinical use,⁷⁷ though it has been investigated with SPECT.⁷⁸

Novel PET Tracers and Radiochemistry

The N-methyl d-aspartate (NMDA) receptor is involved in neurodegenerative disease pathways, but currently has no PET-suitable radioligands available for monitoring its activity in vivo. Several ¹¹C- and ¹⁸F-labeled compounds have been assessed as potential ligands, but further improvement is necessary before a satisfactory PET radiotracer is developed. Since the development of PET imaging ligand for NMDA receptor has been recently reviewed,¹¹ we aim to provide a brief overview of these PET probes and recent ligand development particularly after 2010. Figure 3a showed several representative NMDA ligands targeting ion channel/PCP site (CNS1261,^{6, 56, 79} CNS 5161,^{7, 80} GE-179⁸¹ and GMOM⁸²), glycine binding site (L-703717⁸³) or NR2B subunit (MK-0657⁸⁴). In particular, Robins et al. fluoroalkylated two diarylguanidine derivatives (fluoroethyl derivative is called GE-179, Figure 3, compound c) using thiol precursors.⁸⁵ The [¹⁸F]fluoroalkyl compounds were prepared in 4-9% yield and up to 2.5 GBq/μmol specific activity, showing promising results regarding lipophilicity, binding affinity and selectivity for the PCP-binding site. The Sobrio research group has investigated fluoropiperidine derivatives as selective radioligands targeting NMDA receptors containing NR2B subunits. Greater brain penetration and target binding was exhibited by [¹⁸F]cis-4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]-3-fluoro-piperidine-1-carboxylate and its trans isomer (Figure 3, compound f; [¹⁸F]MK-0567) as indicated by ex vivo autoradiography experiments.⁸⁶ Radiochemical yields for synthetic trials ranged from 31-45%, and the trans compound was produced with higher specific activity than the cis compound (236 vs. 170 GBq/μmol). Although logD_7.4 values in in vitro assays indicated the potential for good brain penetration, low brain uptake of both ¹⁸F-isomers failed to provide satisfactory properties for imaging NMDA receptor in the living brain.^84b

Figure 3a. Radiolabeled compounds targeting the NMDA receptor.

a-d) ligands targeting ion channel/PCP binding site; e) ligand targeting glycine binding site and f) ligand targeting NR2B subunit.

A recent example of guanidine derivative targeting NMDA NR2B subunit, PK-209, showed more than 50 fold selectivity over other targets, including adrenergic, muscarinic and opioid receptors, as well as NMDA-PCP binding site, sigma-1 and -2, calcium and sodium ion channels. Although this tracer is metabolized rapidly in vivo, the distribution volume can be quantified in the primate brain (Figure 3b, compound a).⁸⁷ Two 4-(4-fluorobenzyl)piperidine compounds (Figure 3b, compounds b and c) were radiolabeled using nucleophilic aromatic substitution.⁸⁸ These compounds demonstrated very little brain uptake (0.035%-0.054% ID/g) in rat μPET experiments, as well as high uptake in bone tissue indicating radiodefluorination. Christiaans et al. recently developed a ¹¹C-labeled radiotracer with high uptake in the rodent brain.⁷⁷ N-((5-(4-Fluoro-2-[¹¹C]methoxyphenyl)pyridin-3-yl)methyl) cyclopentanamine (Figure 3b, compound d) was labeled in 49% yield and 78 GBq/μmol specific activity. Rodent PET studies indicated binding to the NR2B binding site in vivo, but selectivity was not ideal as some binding to the sigma-1 receptor was also observed. Ametamey et al. demonstrated a new class of NMDA ligandm namely NB1, targeting GluN2B/NR2B subunit.⁸⁹ The autoradiography studies showed specific binding in rat brain cryosections and blocking studies demonstrated an up to 32% reduction of tracer binding, which represents a promising radiotracer for imaging NR2B subunit (Figure 3b, compound e). It is worthy of mention that other approaches, including NMDA SPECT agents,⁹⁰ radiolabeled drug candidate ASP0777⁹¹, a NR2A selective radioligand [¹⁸F]FP-PEAQX⁹² and an array of candidate compounds based on CNS 1261,⁹³ are also disclosed in the literature in the pursue of NMDA imaging ligand.

New Radiochemistry for PET Imaging of Other Ion Channel linked Receptor Proteins

Several noteworthy biologically active radioligands for other receptor proteins⁹⁴ have been recently reported. Specifically, investigation of chemical probes for ion channels and receptors such as the transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor has yielded promising results, and a few of these results are discussed here. PET radioligands targeting TRPV1 have recently been investigated by van Veghel et al.⁹⁵ [¹¹C]DVV24 (Figure 4, compound a), a derivative of cinnamic acid, was obtained in up to 75% yield. This radiotracer, as well as the aminoquinazoline [¹⁸F]DVV54 (Figure 4, compound b) were evaluated for biological activity in mice. Though selectivity for TRPV1 was indicated by [¹¹C]DVV24 retention in the trigeminal nerve, the binding affinity of both tracers was just above 100 nM, not high enough to indicate success as PET radioligands. Derivatives of N-(3-methoxyphenyl)-4-chlorocinnamide (Figure 4, compound c; [¹¹C]SB366791) were subsequently synthesized by van Veghel et al. due to the high affinity of this molecule for TRPV1. Radiochemical yield and specific activity for [¹¹C]SB366791 were comparable to the previous tracers, but improved in vitro binding affinity to both mouse (280 nM) and human (780 nM) TRPV1 was observed for this tracer. Despite these improvements, binding affinity remained low for application of this tracer to PET. The voltage gated sodium ion channels (Na_vs) were recently targeted for PET imaging by Hoehne et al.^{96 18}F-radiolabeled saxitoxin (Figure 4, compound d) was shown to localize at the site of recent nerve injuries in rats by in vivo PET and ex vivo biodistribution studies. Tetrahydroisoquinolinium derivatives of the small conductance Ca²⁺-activated K⁺ (SK_Ca) channel blocker N-methyl-laudanosine (Figure 4, compound e) were investigated as PET radioligands of this ion channel by Gao et al.⁹⁷ These compounds were synthesized from substituted isoquinoline intermediates in 40-65% yields. This series has yet to be investigated for biological activity.

Figure 4b.

Recent examples in the development of NMDA imaging ligand

a-c) ligands targeting ion channel/PCP binding site; d-f) ligands targeting NR2B subunit.

Conclusions

The GABA receptor has well-characterized PET radiotracers available for clinical research studies. The current radiochemical synthesis of [¹⁸F]flumazenil could benefit from improvement and development of receptor subtype specific radiotracers for this target remain an ongoing area of development. Current nAChR radiotracer development focuses on improving specificity for the therapeutically relevant α7 subtype, while maintaining or improving brain uptake over known tracers. The NMDA receptor does not yet have a suitable PET radiotracer, and brain uptake remains a significant obstacle for this target. Further development still remains for satisfactory clinical PET radiotracers for ion channel linked receptors. New knowledge learned from PET imaging studies will be of importance to the design of radiopharmaceuticals for existing and new ion channel targets, and will guide neuropsychiatric and neurodegenerative drug development.

Figure 5. Radiolabeled compounds targeting other receptor proteins.

a) [¹¹C]DVV24, a TRPV1 radioligand; b) [¹⁸F]DVV54, a TRPV1 radioligand; c) [¹¹C]SB366791, a TRPV1 radioligand; d) a saxitoxin derivative developed by Hoehne et al. targeting the Na_vs⁹⁶; e) tetrahydroisoquinolinium derivative developed by Gao et al. targeting the SK_Ca.⁹⁷

LIST OF ABBREVIATIONS

FDG

2-Deoxy-2-[¹⁸F]fluoroglucose

GABA

γ-Aminobutyric Acid-Benzodiazapine

nAChR

Nicotinic Acetylcholine Receptor

Na_vs

Voltage-Gated Sodium Ion Channel

NMDA

N-Methyl D-Aspartate

PET

Positron Emission Tomography

SK_Ca

Small Conductance Ca²⁺-Activated K⁺ Channel

TRPV1

Transient Receptor Potential Vanilloid Subfamily Member 1