New Trends and current status of PET and SPECT radioligands for

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New Trends and current status of PET and SPECT radioligands for Neuronal 5-HT Receptors (5-HTRs) and serotonin transporter (SERT) Puja Panwar Hazari, Ankita Pandey, Shubhra Chaturvedi, and Anil Kumar Mishra Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00243 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Bioconjugate Chemistry

New Trends and current status of PET and SPECT radioligands for Neuronal 5HT Receptors and serotonin transporter (SERT) Puja Panwar Hazari, Ankita Pandey, Shubhra Chaturvedi, Anil K Mishra* Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig S.K. Mazumdar Road, Delhi, India

*Corresponding Author Anil Kumar Mishra, Scientist G, Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig S.K. Mazumdar Road, Delhi-11054, India Email: [email protected], [email protected] Tel: 91 11 2391 4374; Fax: 91 11 2391 9509

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ACS Paragon Plus Environment


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Abstract The critical role of serotonin (5-hydroxytryptamine; 5-HT) and its receptors (5-HTRs) in the pathophysiology of diverse neuropsychiatric and neurodegenerative disorders render them attractive diagnostic/therapeutic targets for brain disorders. Therefore, in vivo assessment of binding of 5-HT receptor ligands under a multitude of physiologic and pathologic scenario may support more accurate identification of disease and its progression, patient’s response to therapy, as well as screening of novel therapeutic strategies. The present review aims to focus at the current status of radioligands used for positron emission tomography and single photon emission computerized tomography (SPECT) imaging of human brain serotonin (5-HT) receptors. We further elaborate and emphasize upon the attributes that qualify a radioligand for theranostics on the basis of its frequency of use in clinics, its benefit to risk assessment in humans, and its continuous evolution, along with the major limitations.

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Introduction Significant growth in the field of nuclear medicine and molecular imaging is witnessed with the availability of a number of novel radiopharmaceuticals with high specificity and radionuclides with excellent radio-physical properties. The functional brain molecular imaging of neurotransmitters and neuro-receptors using nuclear medicine imaging methods [Positron emission tomography (PET) and Single Photon computed Tomography (SPECT)] and with newer radiopharmaceuticals has made possible to study cognitive functions of the human brain across a wide range of behaviors. The researchers and clinicians have extensively used PET and SPECT as the neuroreceptor mapping technique for last few decades to study living brain and various neurotransmitter systems in health and disease. PET and SPECT are the most frequently used in vivo brain imaging techniques for pharmacological purpose and quantification of brain receptor concentrations.1-4 In the brain, PET and SPECT have also been widely used to study glucose metabolism,5 blood–brain barrier transport,6,

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and neurotransmitter release.8,

9

The

pharmacokinetic and

pharmacodynamics properties of new chemical entities or drugs as well as their receptor occupancy pattern in the brain can be easily tracked by PET and SPECT imaging. Novel candidate compounds can be evaluated and optimized for their therapeutic and diagnostic doses by utilizing these techniques. Of the various neuroreceptors studied, serotonin receptors remain one of the most exploited target. Serotonin is involved in critical physiological phenomena in the brain, involving appetite regulation, mood, perception, pain, and anxiety.10 Serotonin is highly distributed outside the central nervous system11, thereby affecting a variety of physiologic processes in diverse organs like cardiovascular function, gastrointestinal functions, platelet functions, smooth muscle contraction, and growth of smooth muscle of uterus.12-14 Although only 2% of serotonin is present in the CNS,15 nevertheless it has played a predominant role in the etiology of numerous neurodegenerative ailments. Serotonin has a family of receptors that is divided into seven main categories depending on their structural,12 biochemical (signaling mechanism involved) and pharmacological differences, and constitutes fourteen different receptors.12 Serotonin activates several 5-Hydroxytryptamine receptors via their respective signaling mechanisms, while its reuptake from the synaptic cleft is regulated by the 5-HT transporter. Any perturbation in the structure, function, or distribution of serotonin receptor 3



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and/or serotonin transporter may be associated with mental dysfunction. Advancement in imaging techniques like PET and SPECT have allowed non-invasive detection and monitoring of alterations in serotonin receptor/ SERT thus, offering an opportunity to decipher the role of serotonin in mental disorders. Because of Serotonin’s involvement in a wide variety of brain functions, and its central role in mood and anxiety disorders makes serotonin studies of great importance. Serotonin receptors are ubiquitously present in the CNS, particularly in cortical and subcortical regions.16 The presence of serotonin receptors in these regions forms the structural explanation for the regulation of many psychiatric disorders. 5-HT neurons innervate the neuronal structures in the cortex, hypothalamus, thalamus, basal ganglia, substantia nigra, caudate putamen, amygdala, hippocampus, septum, tegmentum, mammillary bodies and spinal dorsal horns.17 The highest levels of serotonin fibers are seen in limbic and sensory areas. Serotonin signaling arises from the interaction of the serotonin neurotransmitter with serotonin receptors. The first step, which also happens to be rate-limiting, is the serotonin (5HT) synthesis through the hydroxylation of L-tryptophan to L-5-hydroxytryptophan (L-5HTP). This reaction is catalyzed by tryptophan hydroxylase (TPH).18 L-5-HTP is converted to 5-HT via the enzyme aromatic L-amino acid decarboxylase. The monoamine transporter collects 5-HT molecules and stores them in the synaptic vesicles, from where it is released into the synaptic cleft using [Ca2+]-dependent pathway. The released serotonin interacts with the receptor mediating signaling pathways. The reuptake of released 5-HT from synaptic cleft is carried by the membrane-bound 5-HT transporter (SERT) of 5-HT neurons. As true with biological systems, serotonin receptor also displays diversity (a) through subtle sequence variations resulting in varied ligand selectivity and (b) through the existence of multiple states as seen in case of GPCRs.

19

The various subtypes of serotonin receptors and

PET/SPECT radioligands in pre-clinical and clinical applications have been discussed exhaustively in the past.20

Key Summarization From the literature survey it may be concluded that SPECT based tracers present a bleaker picture than PET based tracers and 99mTc based ligands are mostly antagonist based. Based on

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the above observations the research focus towards novel 5-HT ligands narrows down to the following major areas: 1. Radiochemistry plays a significant role in the new tracer development. Radiosynthesis should be fast and simple with high specific activity 2. The radioligand should be designed in such a way that it must bind selectively to the target of interest. To obtain meaningful data the binding potential measured by PET or SPECT using the radioligand must be obtained from binding to the desired target only. Number of receptors available for binding (Bavail) and the affinity (1/KD) must be considered when determining the selectivity of a radioligands towards a target receptor. 3. Improvement of the metabolic stability to enable tracer-kinetic modeling. Fast rate of metabolism results in low levels of parent compound in plasma at late times. This can initially lead to small errors in the measurement of the fraction of labeled metabolites and which amplifies to relatively large errors in the determination. 4. Choice of radionuclide can be challenging. The half-life of the radionuclide is an important prerequisite for studying receptor function. The relatively short half-life of [11C] (t1/2= 20.4 min) is suitable for following quite rapid pharmacokinetics, and may permit more than one study session in the same subject in a single day. [11C]-labelling is usually feasible because of the presence of carbon atoms in all organic compounds. However, the necessity to produce [11C]-labeled radioligands on-site is a logistical disadvantage. Longer lived [18F] (t1/2= 109.8 min) for PET and [123I] (t1/2= 13.22 h) for SPECT do not require on-site production and are suitable for following slower pharmacokinetics. The introduction of the small electronegative [18F] atom in place of hydrogen or hydroxyl can be unpredictably beneficial or detrimental to the pharmacological properties of the ligand in terms of its affinity and selectivity. Introduction of a bulky [123I] atom in place of hydrogen generally affects pharmacology and will increase ligand lipophilicity. Alternative labels are being investigated, such as the SPECT label [99mTc] (t1/2= 6 h) because of its relative safety and availability. Metallic radionuclides will however, result in even more unpredictable pharmacological properties of the ligand. 5. Search for agonist based ligands for functional imaging of the receptors.

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The reported studies of 5-HTRs radioligand development clearly show the continuous efforts to either identify novel ligands or improve the existing ones in order to quantify 5-HT receptors (and subtypes) in physiological as well as pathological conditions. The present review emphasized upon the considerations in the design and development of radioprobes for in vivo brain imaging. A majority of radiotracers fail to even pass the developmental phase despite their potential for in vivo brain imaging in pre-clinical studies. The primary reasons for their failure are inability to cross blood–brain barrier, high non-specificity, unnecessary metabolite formation, too strong binding to be displaced by the target specific ligand or difficulty to quantitate due to poor pharmacokinetic attributes. Moreover the efficacy and potential of candidate compounds in preclinical studies in animal models often fail to translate as expected in clinical situations. Therefore, the design and synthesis of a candidate compound and its thorough assessment in pre-clinical as well as clinical set up to confirm its suitability as an imaging agent is extremely complicated.

5HT RECEPTOR FAMILY The 5-HT1 receptor sub family 5-HT receptors are classified into seven families (5-HT1 to 5-HT7) and at least 14 different subtypes. These are classified based on their structural (amino acid sequence), biochemical (functional status of receptor and mechanisms of signal transduction) and pharmacological differences. A family of five distinct gene products contributes towards the 5-HT1 receptor family, viz: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F receptors. The genes for all the 5-HT1 receptors are known to be present in all the mammalian species. The 5-HT1 receptors share significant sequence homology among them and each receptor is encoded by a single, intron-less reading frame. Among all the receptor subtypes, 5-HT1A is the most well studied and characterized and has been shown to regulate the mechanisms underlying the anxiety and anxiolytic action more frequently than the remaining members of the 5-HT1 family. The G-protein-coupled 5-HT1A receptor belongs to the family of adenylyl cyclase containing 5-HT1 receptors. 5-HT1A autoreceptors when activated inhibit release of serotonin from distal axon terminals. The coupling of 5-HT1A receptor to Gi/o, with Gi activation leads to an inhibition of adenylate cyclase, an intracellular enzyme which catalyzes the formation of cyclic AMP 21 from ATP. The cAMP molecule is a second messenger which 6


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can influence a variety of cellular processes including kinases. Activation of the receptor leads to closing of calcium channels via the Go subunit and opening of potassium channels via the βγ subunits in addition to inhibiting adenylate cyclase. 5HT1A receptors are primarily concentrated in the hippocampus, superficial layers of cortex and the raphe nuclei. 5HT1B receptors are located in the axon terminals in regions of basal ganglia. 5HT1C receptors are now known to be 5HT2C receptor because it shares 78% homology with 5-HT2 receptors family.22 The 5-HT1DR is a heteroreceptor and a serotonin nerve terminal autoreceptor, predominantly expressed in basal ganglia and substantia nigra. The 5-HT1ER is concentrated in caudate putamen hippocampus (dorsal striatum) and cortex with lower levels in amygdale and globus pallidus. Expression of 5-HT1FRs is very low and is found in hippocampus, cortex and dorsal raphe nucleus. The 5HT2A receptors are located mainly in the neocortex, caudate nucleus, nucleus accumbens, hippocampus, and vascular and non-vascular smooth muscle cells. Very high levels of 5HT2C receptors are present in the choroid plexus with much lower levels in substantia nigra, globus pallidus, hippocampus and ventromedial hypothalamus. 5HT3 receptors are concentrated to the lower brainstem nuclei, while its densities are significantly lower in forebrain regions including amygdala and hippocampus. 5 HT4 receptors are found in high densities in the regions of basal ganglia and striatal projections with lower levels in the hippocampal formations and the isocortex. 5HT5 receptors are expressed in various brain regions including cortex, hippocampus, cerebellum and striatum. 5-HT6 receptors are found exclusively in the CNS and are predominantly expressed in the striatum, nucleus accumbens as well as in hippocampus. 5HT7 receptors are mainly concentrated in the hippocampal formation, and lower densities in cortex and amygdala (Figure 1). 23

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Figure 1. 5HT receptor distribution in Brain (redrawn from Brichta et al23) A majority of these receptors belong to the metabotropic receptor family (transmitting signals through G proteins), except for 5-HT3 receptors included into the ionotropic receptor family. Not all of these receptor subtypes have identified physiological roles in the brain, and selective agonists and antagonists have not been identified for all the receptor subtypes. Nevertheless, the existence of so many receptor subtypes for a single transmitter permits a great diversity of signaling so that the same neurotransmitter can produce very different effects on different neurons and on different parts of the same neuron. BRAIN IMAGING Molecular imaging merges molecular biology and in vivo imaging with the potential for integrating diagnosis and drug development, as well as opening up the possibility of personalized medicine. Other techniques like MRI or CT are not competent to provide information about the location and quantity of specific receptors or binding sites in the living human brain. PET is more suitable for quantification than SPECT owing to its higher resolution and higher sensitivity.

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However, low cost and use of radiotracers with longer half-lives have 8


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facilitated more availability of SPECT in hospitals. Therefore, SPECT radioligands are in great demand. 24 The major disadvantage of SPECT isotopes is that they require several hours to get rid of nonspecific binding and reach equilibrium owing to their longer physical halflives.

24

With the advancement of multimodalities like SPECT/CT and PET/CT, the three

dimensional anatomical CT images can be compared with PET or SPECT scans, thereby furnishing detailed information about biochemical and functional status of the receptors under both physiological and pathological situations. 25 Brain is organized into complex functional networks and complicated regional distribution of receptors, which is cumbersome to image by conventional morphometric analysis. The criteria to be a successful PET or SPECT radioligand for brain imaging involves stable radioisotope labeling, high receptor affinity, high selectivity along with low nonspecific binding. Rapid penetration through the blood– brain barrier allows the radiotracer to be readily accessible to receptors, clearance.

27

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and low rate of metabolite formation along with their rapid

The developments of selective PET and SPECT radioligands for serotonergic

system in human brain have provided critical insights. For example, it was observed that in patients with social anxiety and panic disorder, the level of 5-HT1A receptor was decreased 28, 29

whereas the depression patients exhibited an elevation in 5-HT2A receptor density in

response to treatment.

30

However, it is essential to design and develop specific radioligands

for all the 5-HT receptors in order to validate and finalize the 5-HT associated treatment strategy. 1) 5-HT1A receptor radioligands: 1.1) Development of 5HT1A radioligands The 5HT1A ligand comprises of 5HT1A agonists, partial agonists, antagonists, homodimeric and heterodimeric ligands, based on their reactivity and biochemical functions. Based on aryl piperazine pharmacophore, agents such as [11C]WAY-100635(N-[2-[4-(2-methoxyphenyl)-1piperazin‐yl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide) was developed. With regard to commonly employed long-chain arylpiperazine, they have the two structural features necessary for recognition of ligands by the 5 HT1A and dopaminergic sites, viz, an aromatic ring and a strongly basic nitrogen atom at a distance of 5.2-5.6 A°.

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It has been observed that the incorporation of an o-methoxy substituent in the aromatic ring leads to the development of arylpiperazine ligands possessing high affinity for 5- HT1A and binding sites. The influence of the side chain length on receptor affinities appears to be variable depending on prime skeleton of the compound. The HT1A ligands are expected to provide a better tool for estimation of occupancy of agonist drugs at the 5-HT1A receptor, estimation of the intrasynaptic levels of endogenous serotonin, as well as imaging rapid internalization of 5-HT1A autoreceptors after the administration of a selective serotonin reuptake inhibitor as a measure of its treatment efficacy. Following section describes the PET and SPECT radioligands for 5HT1AR and summarized in Table 1. a) [11C]CUMI-101: 11

C-CUMI-101 is believed to selectively bind the G-protein-coupled state of the serotonin-1A

(5-HT1A) receptor. It is a high affinity 5-HT1A agonist (Ki= 1.36 nM) as shown in studies using

11

C-labelled tracer, in rats, primates and humans.

31-33

In a recent study involving

healthy human subjects [11C]CUMI101 was used for distribution and quantification of 5HT1A in humans for the first time.33 (Figure 2) However subsequent studies reported that [11C]CUMI-101 exhibits only partial agonism at 5-HT1A receptors,

34-36

which may be a

pharmacological drawback pertaining to the interpretation of PET images, and hence warrants the search for alternative PET 5-HT1A agonists. Recent studies had shown that CUMI-101 behaved as a potent 5-HT1A receptor antagonist in rat brain. CUMI-101 also has moderate affinity (Ki = 6.75 nM) for α1 adrenoceptors measured in vitro. CUMI-101 is now treated as a 5-HT1A receptor antagonist after its distribution in primate brain, with α1 adrenoceptor cross-reactivity, limiting its potential use as a PET radioligand in humans.37 b) [18F]FECUMI-101 In comparison of

11

C based tracer, development of an F-18 labeled tracer can be a better

strategy as the half-life of F-18 tracer is 110 min and hence it can be transported to other locations and also allows the PET modeling studies for a longer duration of time with the highest resolution. FECUMI-101, (2-(4-(4-(2-(2-fluoro-ethoxy)phenyl)piperazin-1-yl)butyl)4-methyl-1,2,4-triazine-3,5(2H,4H)-dione), was synthesized as the fluoroethyl analogue of CUMI-101 and PET studies were performed in baboons for 5-HT1AR imaging.

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It

effectively crossed the blood brain barrier and its retention in brain was found to be in accordance with the spatial distribution of 5-HT1AR in brain, with highest uptake in 10


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hippocampus, anterior cingulate cortex, insular cortex and amygdala. However, the ligand was also found to bind thalamus and subsequent autoradiographic studies in human brain slices suggested that [18F]FECUMI-101 may not be an ideal 5-HT1AR ligand for further clinical applications.

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Radiotracer distribution in non-human primate is shown in Figure

2(B). c) [18F]F13714 In

rats,

F15599

(({1-[3-chloro-4-fluorobenzoyl]-4-fluoropiperidin-4-yl}methyl)

[(5-

methylpyrimidin-2-yl)methyl]amine) exhibited a particularly high selectivity for 5-HT1A receptors but insufficient affinity (Ki=2.24nM).

40, 41

Since the most preferred radioligand

affinity to achieve a high contrast image is usually kept lower than 1 nM, therefore F15599 was modified by replacing the pyrimidine ring by a 2-methylaminopyridine ring, leading to the

development

of

F13714

(6-{[({1-[3-chloro-4-fluoroben-zoyl]-4-fluoropiperidin-4-

yl}methyl)amino]methyl}-N,3-dimethylpyridin-2-amine). 42 (Figure 2C) F13714 exhibited a particularly high affinity for 5-HT1A receptors, with a Ki of 0.1 nM or less (0.06 nM for rat cortical 5-HT1A receptors, 0.10 nM for rat hippocampal 5-HT1A receptors, and 0.04 nM for recombinant human 5-HT1A receptors. In a recent study in marmosets,

43

it was observed

18

that [ F]F13714 as a biased agonist displayed remarkably distinct binding distribution when compared to [18F]-MPPF. [18F]-MPPF showed highest binding in hippocampus and amygdala, while [18F]F13714 showed increased activity at pre-synaptic 5-HT1A receptor including insular and cingulate cortex, thalamus, raphe, caudate nucleus, and putamen. Such study suggests that [18F]F13714 may have a higher sensitivity than known antagonist [18F]MPPF for identifying a subset of 5HT-1A receptors, thereby anticipating a better response in patients undergoing antidepressants or antipsychotic regimen. Such novel strategies may promote

new

avenues

for

the

treatment

of

neuropsychiatric

and

neurological

pathophysiologies.

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Figure 2. PET imaging of 5HT1AR [11C]CUMI101 in human44(A) , 18-FECUMI in non human primate

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(B). PET imaging of 5HT1AR [18F]F13714 in cat brain42 and marmoset

brain43 (C).

d) [11C]WAY-100635 Selective

HT1A receptor

agonists

and

partial

agonists

such

as

NAN-190

(l-(2

methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine), p-MPPI (4-(2’-methoxy phenyl)-1[2’-(N-2”-pyridyl)-p-iodobenzamidol-ethyl-piperazine) and WAY100635 (4-(2’-methoxypheny1)-l-[2’-(N-2”-pyridyl)-cyclohexanecarboxamidO]-ethylpiperazine)

have

made

possible the elucidation of the functional role of this receptor subtype in the CNS. WAY100635 is one of the most successful and clinically used 5-HT1A receptor imaging agents. It is highly selective towards 5-HT1A receptor with high binding affinity, (Kd = 0.20.4 nM). The promising potential of [11C] WAY 100635 as a 5-HT1A radiotracer in brain has been shown by innumerable studies in rodents, monkeys and humans (Figure 3). The spatial distribution of 5-HT1A receptors in human brain was first described in detail using its analogue, namely [O-methyl-11C]WAY-100635 Although it was found to be highly selective 12


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for 5HT1A receptor with a high signal to noise ratio (3.1), however it suffered from the major limitation of its hepatic biotransformation resulting in formation of numerous radio metabolites that bind non-specifically in brain and hence degrade image quality as well as disturbs PET modeling and quantification. Thus, the synthesis and development of novel 5HT1A radioligands is further warranted. DWAY is one of the putative metabolite and the desmethylated analog of [11C]WAY 100635. It is a highly selective radioligand that displayed similar radiochemistry and pharmacology to that of [11C]WAY-100635, with a high affinity for 5-HT1A receptors (IC50 = 1.4 nM) than[carbonyl-11 C]WAY-100635.

45, 46

45

and a higher signal to noise ratio in human

RWAY (Ki = 0.6 nM), has a reverse amide linkage

that reduces hydrolysis in vivo and improves metabolic stability. [11C]-RWAY has several radiosynthesis advantages over [11C]WAY100635 or [11C]DWAY apart from its promising in vivo binding in rodents and non-human primates.

47

However apart from 5-HT1AR, it

possesses significant affinity to 5-HT2BR (Ki = 7.2 nM), α-1AR (Ki =10.35 nM), D2R (Ki = 34.5 nM), D3R (Ki =5.1 nM), and D4R (Ki =15.6 nM). Moreover a number of studies in rodents and primates have shown the presence of a lipophilic radioactive metabolite as well as its slow washout from the body, thereby restricting its use in clinics.

48, 49

[11C]6FPWAY

and [11C]6BPWAY are the halogenated analogs of WAY, labeled in their respective carbonyl position: namely, [11C]6FPWAY and [11C]6BPWAY N-(6-fluoro(or bromo)pyridin-2-yl)-N{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}cyclohexane-carboxamide.50 11

Studies

in

11

primates have shown that binding of both [ C]6FPWAY and [ C]6BPWAY corresponds very well to the distribution of 5-HT1A receptors.50 [11C]6FPWAY is the more promising molecule among the two owing to high uptake in brain as well as high target to non-target ratio. However, the major hindrance in developing both [11C]6FPWAY and [11C]6BPWAY as effective PET radiotracers is attributed to their active metabolism into lipophilic radiolabeled metabolites that penetrates the blood-brain barrier. 50 e) [11C]CPC-222 [O-methyl-11C]-CPC-222 is another analogue of WAY-100635. In vitro, CPC-222 has a high affinity for 5-HT1A receptors (IC50 = 4.2 nM). Moreover, in preliminary studies, it was found that in healthy human volunteers, the uptake of [11C]CPC-222 in brain was high with the maximum value of target to nontarget ratios to be 4 at 45 min.

51

[11C]CPC-222 may be a

promising 5-HT-1A radioimaging agent however due to its low target to background ratio, it was not further developed for clinical studies. 13



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f) [11C]NAD-299 NAD-299([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5 carboxamide) has high binding affinity (Kd 50.17nM), and high selectivity as shown in rat brain, and in postmortem human brain. characteristics in the nonhuman primate

53

52

[11C]NAD-299 exhibited promising PET

as well as healthy human volunteers,

54

which

include rapid accumulation in brain and high uptake in 5HT1A-rich brain areas like frontal cortex and raphe nuclei.

53, 54 11

[ C]NAD-299 was also shown to undergo hepatic metabolism

resulting in more polar radiometabolites than the parent radioligand, with slower accumulation as compared to that of WAY-100635. However, [11C]NAD-299 has not been used in clinics till date. g) [18F] MeFWAY: In an another attempt to improve the in vivo stability of 5-HT1A agents, N-{2-[4-(2methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-18F-fluoromethylcyclohexane) carboxamide (18F-MeFWAY) was synthesized, which contains a 18F on a primary carbon to make the compound more stable to defluorination.55 [18F]MeFWAY is a fluoromethyl analogue of WAY100635, and can be labelled in either cis or trans position at the 4cyclohexyl site of the compound. In monkeys, it was shown that -[18F]MeFWAY radiolabeled at trans position showed higher binding to 5-HT1AR which is comparable to WAY100635 in vivo.56, 57 In a more recent evaluation of [18F](trans)-MeFWAY in human subjects it was shown that [18F]MeFWAY can be a promising PET radiotracer for assay of 5HT1A receptors owing to its simple radiochemical production, high specific radioligand uptake, and lack of PET signal in bone. 58, 59 (Figure 3) h) [18F]FCWAY This analog of WAY 100635 has a trans-[18F] 4-fluoro group in the cyclohexane ring and exhibits high affinity for 5-HT1A receptor and high hippocampus-to-cerebellum ratio. 60 [18F] FCWAY is difficult to synthesise because the yield of 4-fluorocyclo-hexanecarboxylic acid is quite poor. It also undergoes in vivo defluorination, leading to bone radioactivity uptake.

61

Therefore despite of its high selectivity and high affinity towards 5HT1A receptor, [18F] FCWAY has limited use. 62 Moreover, it is a weak substrate for efflux transport nevertheless Pgp rapidly clears it. FCWAY, being a poor substrate of efflux transporter, could be used to 14


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assess its function for which the available radioligands for transporters such as [11]Cloperamide and [11]C-verapamil are not very useful.62 i) [18F]MPPF [18F]MPPF (Ki = 3.3 nM,) is a fluorophenyl analogue of WAY100635.

63

[18F]MPPF was

successfully assessed in human volunteers to label 5-HT1A receptors in the brain

64, 65

(Figure 3) with a target-to-background ratio of 3-4 and its radiopharmacology was explored in rodent and cat brain.

66

A [18F] MPPF PET database, including various age groups of

healthy males and females, was constructed as a prerequisite for subsequent clinical PET studies in epilepsy, Alzheimer's disease, migraine and Parkinson’s disease. 67 In patients with refractory temporal lobe epilepsy,

68

it was observed that the binding of [18F]MPPF to 5-

HT1A receptors was decreased in the epileptogenic temporal lobe.

69

Moreover in patients

with Alzheimer’s disease the 5-HT1A receptor density in the medial temporal lobe in was shown to be attenuated with concomitant increase of ß-amyloid deposits using [18F]MPPF. 70 Although [18F]MPPF is a useful radioligand for the quantification of 5-HT1A receptors in human, it is also a substrate for p-glycoprotein (p-gp) which means the amount of [18F]MPPF reaching the brain is relatively low (0.05% injected dose/gram (ID/g) at 30 min in rats) compared to [11C]WAY100635 (0.46% ID/g at 30 min in rats) (Figure 3).

71

However,

[18F]MPPF has the advantage of a longer half-life of [18F] (109.8 min) compared to [11C] (20 min), and hence can be effectively utilized by the hospitals without requiring any cyclotron. A recent study performed on a marmoset brain depicted significant differences in the receptor binding distribution pattern between the antagonist probe, [18F]MPPF and a new agonist PET radioligand [18F]F13714. It is worthwhile to note that [18F]F13714 uptake in the brain was strongly influenced by anesthesia.43 The functional state of 5-HT1A receptors on susceptibility to isoflurane, should be grossly analyzed for accurate interpretation of results on serotonin receptor PET imaging in anesthetized animals. Further studies on biased agonist activity at 5-HT1A receptors should be extended to humans for the treatment of neuropsychiatric and neurological disorders involving serotonergic systems.

15



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Page 16 of 70

Figure 3. PET imaging of 5HT1AR [11C]WAY100635. 18F]MPPF and [18F]MEFWAY in non human primates57 and PET Imaing of [18F]MefWay in rodents, non human primate and human59 j) [18F]DMPPF: In order to improve the brain uptake of [18F]MPPF, a desmethylated analogue, [18F]DMPPF(4-fluoro-N-{2-[4-(2-hydroxyphenyl) yl)benzamide)[, was synthesized.

72

piperazin-1-yl]ethyl}-N-(pyridin-2-

In vivo studies in rats showed that an overall brain uptake

of 0.31% ID/g was measured at 15 min after tracer injection with a better brain penetration, better contrast, and a slower clearance than [18F]MPPF.

72

Further studies are warranted to

demonstrate the in vivo ability of [18F]DMPPF to be a PET tracer for 5-HT1A receptors. Preclinical studies in rodent revealed better brain penetration, better contrast, slower clearance and slower metabolism than with [18F]MPPF. The binding experiments in rat brain slices showed that p-DMPPF is highly selective for 5-HT(1A) receptors, with a Ki value of 7 nM on these receptors.

73

[18F]DMPPF probably achieves higher brain uptake than its

methoxy-analog [18F]MPPF because DMPPF is a poor substrate of P-glycoprotein. However, despite these promising properties, there has been no further development of [18F] DMPPF. 1.2 Bivalent approach for high affinity radioligands The homo/heterodimeric bivalent ligands are widely used containing two sets of pharmacophoric entities linked through a spacer. It is envisaged that duplication of the pharmacophoric groups according to the bivalent ligand approach leads to a supra-additive 16


Page 17 of 70

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increase in its efficacy and binding affinity. The bivalent ligand approach in the design of homo/heterodimeric ligands targeting GPCRs has proven to be promising to improve selectivity as well as pharmacokinetic profile of compounds.74 Bivalent ligands binding neighboring binding sites of two physically interacting GPCRs may serve as pharmacological tools to study the quaternary structure of receptor dimers and to gain insights into the role of GPCR oligomerization on biosynthesis, receptor function and internalization. On the other hand, bivalent ligands with a dual steric binding mode facilitate the generation of subtypeselective agonists or antagonists because allosteric regions are frequently less conserved than the orthosteric binding pocket, which is usually very similar for all subtypes of a receptor family. Herein we describe such agents used for PET and SPECT imaging based on bivalent approach 2-methoxyphenylpiprazine with enhanced affinity and selectivity towards 5HT1A in preclinical studies. 1.2.1 [18F]BMPPSiF A bivalent homodimeric SiFA-derivatized radioligand, [18F] BMPPSiF, with high affinity was developed as a neuroimaging PET tracer with high uptake in 5-HT1A receptor rich regions.

75

The design of the homodimeric system has been made on the basis of structure–

activity relationship studies reported in the literature for enhanced affinities toward GPCR receptors. Homology modeling and molecular docking studies of BMPPSiF and known antagonists (WAY-100635, MPPF, and MefWAY) with dimeric and multimeric 5-HT1A receptor models were documented. The radiotracer accumulation was in corroborating with the known 5-HT1A receptor distribution, with high uptake of radioactivity in the hippocampus (HIP), cingulated cortex (CG), and caudate putamen (CPu) and moderate accumulation in the nucleus raphe dorsalis (Figure 4). 75 This could be the promising strategy to develop homo/heterodimeric radioimaging agents for GPCRs for the quantification of receptor.

17



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Page 18 of 70

Figure 4. (A) PET ligand 5HT1AR. Bivalent homodimeric ligand. 0–60 min summation PET images of the [18F]-BiMPPSiF in Rat brain75 (B)[68Ga]-DO3A-butyl-MPP) showed the significantly high uptake in hippocampus region in the PET imaging studies in rat brain.76

1.3 Metal Based radioligands

1.3.1 [99m] Tc based radioligands In the past, metallic radiotracers viz., [99mTc]DTPA (pentetate) or [99mTc]-pertechnetate have been used for brain imaging. Efforts are on to develop metal based radiotracers for mapping neuro-receptors. [99mTc] based radioligands for serotonin were reported as early as 1999. Detailed description is presented in the recent review.77 Our group has also contributed to the development of metal- based radioligands for imaging of 5HT1A. Following section describes the metal based radiopharmaceuticals in detail. These examples demonstrate the different approaches that have been used in designing the ligands, namely the bifunctional chelating approach, bivalent ligands, agonist derived ligand and theranostics.

18


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1.3.1 [99mTc] based radioligands a) [99mTc]DTPA–bis(MPBA) As the name suggests, the ligand was synthesized by appending two units of identical moieties of 1-(2-methoxyphenyl)piperazine moiety of WAY-100635 (N-(2-(4- (2methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-cyclohexane carboxamide) to an acyclic polyaminopolycarboxylate chelator, DTPA. The resulting homodimeric 5HT1A ligand, DTPA–bis(MPBA) showed high selectivity, and binding affinity for the 5-HT1A receptor due to the multivalent interactions.78 The radiolabeling yield (>95%) and purity (>98%) of [99m]TcDTPA–bis(MPBA) was very high even at a very low concentration of the ligand. In vitro studies carried out on hippocampal cultures and PC3 cell lines expressing 5-HT1A receptors revealed that the selectivity of the complex for 5-HT1A receptors was 1000 times more than it is for 5-HT2A receptors, with KD in the picomolar range. Moreover, in vitro studies in rats displayed that the complex bears high affinity for 5-HT1A receptors. The uptake of radiolabeled DTPA–bis(MPBA) was found to be initiated within 2 min of injection as evidenced from dynamic imaging experiments. Thus such an imaging agent developed, based on the strategy of bivalent ligands may serve a promising role in clinics for imaging 5HT1A receptors using SPECT.78

b) Serotonin-DTC Agonist based imaging can hold more potential for assessing the functional status of the receptors. This is because agonist display discriminate binding to the active form of the receptor as opposed to the indiscriminate binding of the antagonist. Taking leads from the fact that the natural ligand of the 5HT receptor, serotonin is also an agonist, our group modified serotonin as a radiotracer. Thus serotonin, an agonist as well as a natural ligand for 5HT1A was modified as dithiocarbamate and evaluated for imaging of 5HT1A.79 Theoretical studies suggested the retention of agonistic binding even after modification. The ligand was synthesized in a single step reaction. Excellent radiolabeling above 95% was reported. The ligand was reported to have preferential uptake in the hippocampus of the brain, thereby confirming its selectivity. The peak brain uptake was approximately 1.10%ID/g 5 min p.i. in mice.79 1.3.2 [68Ga]- based radioligand – 19



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Page 20 of 70

a) [68Ga]DO3A-butyl-MPP Gallium based ligands can serve as excellent PET tracers. PET owing to high resolution and better sensitivity can prove to be a more effective imaging modality for tracing the neuroreceptor. Thus, 1-(2-methoxyphenyl)piperazine was conjugated with DO3A for targeting 5HT1ARs.76 The advantage was that the same radioligand could be used for loading with Gd(III) and Eu(III) thereby serving as MRI and Optical Imaging respectively.

68

In rat

brain, [68Ga]-DO3A-butyl-MPP) displayed promising uptake and also showed the significantly high affinity towards hippocampus region in the PET imaging studies,76 thereby substantiating its affinity towards 5- HT1A receptors (Figure 4B). Moreover, DO3A-butylMPP also exhibited therapeutic efficacy as evidenced by the behavioral studies conducted in CMS rat model in which it displayed significant antidepressant potential and was able to reverse the level of depression completely. It is believed that the mechanism of action of the DO3A-butyl-MPP on 5-HT1A is via SSRI effect, which could be of significant advantage in treating neurological disorders like depression. The preclinical assessment of DO3A-butylMPP as a CNS imaging system, however, warrants further elaborate investigations.

Table 1. 5HT1A receptor radioligands for PET and SPECT 5HT1A Receptor Radioligand

Modality

Strength High affinity

[11C]CUMI-101

[18F]FECUMI101

18

[ F]F13714

Limitations

PET

Earlier presumed as agonist, but later identified as a 5HT1A antagonist; showed α1 adrenoceptor cross-reactivity

PET

Showed highest uptake in Non-specific as it also binds regions concomitant to 5- thalamus. HT1AR distribution in brain, namely hippocampus, cortex and amygdala

PET

Showed higher specificity Requires further validation in towards pre-synaptic 5-HT1A human receptors; may exhibit better response in patients with 20


Page 21 of 70

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depression and psychosis. [11C]WAY-100635

PET

Clinically most successful 5- Undergo biotransformation into HT1A radioligand till date various metabolites Showed a similar Further validation and clinical radiopharmacy to [11C]WAY- studies are awaited 100635; DWAY gives about

[11C]DWAY

PET

twice the level of radioactivity in all brain region thus conferring better radiation safety than [11C]WAY-100635

PET

Better radiosynthesis than Showed affinity towards other WAY and DWAY and 5-HT, Dopamine and adrenergic promising in vivo binding receptors; lipophilic radioactive metabolites, slow washout from the body

PET

High uptake and target to non- Actively metabolized into target ration lipophilic radiolabeled metabolites penetrating the blood brain barrier

[11C]CPC-222

PET

High brain uptake as evidenced Low target to background ratio in human study

[11C]NAD-299

PET

Rapid brain uptake in 5-HT1A Forms polar radiometabolites rich regions with lower uptake than WAY.

[18F] MeFWAY

PET

Easy radiosynthesis, high More clinical data, especially in uptake and minimum patients is warranted. background in bone, in human studies

[18F]FCWAY

PET

[11C]-RWAY

[11C]6FPWAY and [11C]6BPWAY

High selectivity affinity

and

high Difficult radiochemistry, poor yield, in vivo defluorination with bone uptake.

[18F]MPPF

PET

Longer half life, high affinity Substrate for p-glycoprotein, and selectivity; innumerable resulting in lower brain uptake studies on healthy and patients available.

[18F]DMPPF

PET

Better brain penetration and More pre-clinical and clinical slower clearance than studies are warranted 18 [ F]MPPF, a poor substrate for 21



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Page 22 of 70

p-gp [68Ga]DO3Abutyl-MPP

PET

High brain penetration and More pre-clinical and clinical slower clearance studies are warranted

PET

Sub-nanomolar affinity for 5- More in HT1AR, high uptake in brain warranted

vivo

studies

are

vivo

studies

are

SPECT

High radiolabellin yield and More in purity, significantly high warranted affinity and brain uptake in very less time

SPECT

Agonist based probe, thereby More studies are warranted useful to evaluate the functional status of receptors, good radiochemistry, preferential uptake in hippocampus

Bivalent Ligands [18F]BMPPSiF

[99mTc]DTPA– bis(MPBA)

SerDTC

2) 5-HT 1B receptor radioligands a) [11C]AZ10419369 [11C]5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic

acid

(4-

morpholin-4-yl-phenyl)-amide ([11C]AZ10419369) is a PET ligand for 5-HT1BRs (Ki = 0.8 nM,).

80

PET studies in macaques and human subjects show high tracer uptake (3–4%) in

brain (Figure 5). The highest uptake was found in occipital cortex and basal ganglia followed by temporal and frontal cortical regions, less in thalamus and the lowest in cerebellum.

80

[11C]AZ10419369 shows dose-dependent binding of AZD3783, a 5-HT1BR antagonist with potential antidepressant properties in non-human primates and human subjects indicating its potential for receptor occupancy measurement for drug development and dose-finding in clinical studies. 81 b) [11C] P943 R-1-[4-(2-methoxy-isopropyl)-phenyl]-3-[2-(4-methyl-piperazin-1-yl)benzyl]-pyrrolidin-2one, P943 is a highly selective antagonist

with very high affinity to the human 5-

HT1B receptor (Ki = 0.77 nM), while affinity for other 5-HT receptors is very low.

82-84

([11C]P943) (Ki = 1.2 nM,) is a selective PET tracer for 5-HT1BR in non human primates and 22


Page 23 of 70

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human subjects (Figure 5).

82-84

[11C]P943 when evaluated in patients with depression,

posttraumatic stress disorder (PTSD)

86

or subjects addicted to cocaine

87

85

showed lower

11

binding when compared to healthy subjects. On the contrary, [ C] P943 exhibited enhanced binding in subjects with alcohol dependence and pathological gambling. 88, 89

Figure 5. 5HT1BR PET Ligands. PET Scan of [11C]AZ1041936981 (A) and [11C]P94384 (B) in human 3) 5-HT1C/1D/E/F receptor radioligands 5-HT1C receptors are now included in 5-HT2R family because these receptors possess a 78% sequence homology with 5-HT2R.22 The 5-HT1DR agonists are used for migraine treatment.90 The 5-HT1ER shares ~60% homology with 5HT1BR.Serotonin 5-HT1FRs, shows 70% sequence homology with 5-HT1E. The basic difference between 5-HT1ER and 5HT1BR or 5HT1FR is that the former is not expressed in rodents.

91

The 5-HT1FR is

primarily reported to be involved in the nociceptive function regulation, due to which a number of selective 5-HT1FR agonist have recently been developed to alleviate migraine pain. At present there is no SPECT or PET radioligand known for 5-HT1D, E, F receptors, thereby leaving a huge gap for basic research.

23



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Page 24 of 70

4) 5-HT 2A receptor radioligands. 5HT2A receptor radioligands for PET and SPECT are summarized in Table 2. 4.1 PET ligands: a) [11C]MDL 100,907: MDL 100,907 is a reversible, highly selective 5-HT2A ligand with subnanomolar affinity (Ki=0.38 nM).

92

Despite differences in their in vitro selectivity profiles, the binding of

[18F]altanserin and [3H]MDL 100,907 to the 5-HT2A receptor is quite comparable.

93

[11C]MDL 100,907 showed promise in nonhuman primates where it accumulated in 5-HT2A receptor-rich regions, with a neocortex-to-cerebellum ratio of 4.5 that was observed to be abolished after injection of ketanserin.

94

Subsequent in vitro radioligand binding and

3

autoradiography studies using [ H]MDL 100,907 and [11C]MDL 100,907, confirmed its selectivity and high specific binding in rat

95

and nonhuman primate,

96

thus projecting it to

be the first truly selective 5-HT2A receptor ligand. Simultaneously human studies highlighted its preferable PET characteristics (Figure 6 A). 92, 97 However, the applications of [11C]MDL 100,907 has remained limited in the clinical setting like in patients recovering from depression 30 and in patients with obsessive–compulsive disorder.

98

Furthermore it has been

recently used to characterize 5ht2A receptor density in preclinical models.99

Figure 6. 5HT2AR PET and SPECT Ligands. PET Scans of (A) [11C]MDL100907, 18

[ F]Setoperone

100

18

(C) [ F]Altanserin.

101

97

(B)

18

PET and SPECT scans of (D) [ F]MDL100907, 102

and (E) [123I] R91150 103 in humans respectively. 24


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b) [11C]CIMBI Compounds: The first radiolabelled high-affinity 5-HT2A receptor agonist was 2-(4-iodo-2,5dimethoxyphenyl)-N-(2-[11C-Omethyl]

methoxybenzyl)ethanamine

([11C]Cimbi-5).

5-

HT2A antagonists bind to the total pool of receptors, whereas 5-HT2A agonists bind only to the high-affinity functional state of the receptor and hence may be more important in disease states because the high affinity sites are the ones that transmit the intracellular signals. Furthermore, [11C]CIMBI-5, a potent and selective 5-HT2A agonist, has been shown to have similar cortex-to-cerebellum binding ratio as compared with [18F]altanserin in the pigs, and it was displaceable by ketanserin in both rats and pigs. 104 This series of compounds seem to be the first promising radioligands for the investigation of 5-HT2A agonist binding in the living human

brain.

2-(4-Bromo-2,5-dimethoxyphenyl)-N-(2-[11C]methoxybenzyl)ethanamine

([11C]CIMBI-36), a bromo analog of [11C]CIMBI-5, has also been developed as a tool for studying 5-HT2A agonist binding in the brain.

105

It showed the maximum target-to-

background binding ratio along with a high brain uptake compared to its other analogues. 105 The cortical binding of [11C]Cimbi-36 was decreased by pretreatment with ketanserin, supporting 5-HT(2A) receptor selectivity in vivo in pigs, while in primate brains, it was shown that [11C]Cimbi-36 is suitable for examination of 5-HT2A receptors in the cortical regions and of 5-HT2C receptors in the choroid plexus.

105, 106

c) [18F]FECIMBI-36: Owing to its limitations like slow pharmacokinetics and a low cortex to cerebellum ratio (90 min), especially when the equilibrium is attained between blood and tissue. Earlier time points may provide erroneous BPND values. It was therefore suggested that such error can be avoided by substituting the aryl fluoride on MDL100907 with fluorine-18 (t1/2 = 109.8 min)102. Therefore, Ren H et al reported a novel strategy to synthesize [18F] MDL 100907 via nickel mediated oxidative fluorination that gave high yield along with high specific activity.102 Comparative study was conducted in non-human primates to assess its reliability for quantification of 5HT2A receptors (Figure 6D). A higher BPND of [18F] MDL 100907 demonstrates superior 5HT2A distribution/density estimates to [11C] MDL100907, however the work is still under progress to validate its clinical applications. 4.2) SPECT LIGANDS a) [123I]DOI [123I]DOI is a potent 5-HT2A selective agonist as evidenced from in vitro studies of 5HT2A/2C receptors in rat brains using autoradiography.

136, 137 137

In vitro ligand binding

studies in rats demonstrated that the enantiomer R(–)[123I]DOI exhibited a better affinity (Kd=1.26 nM) and higher selectivity than the S–enantiomer.

138

Therefore, [123I]R-DOI was

synthesized and evaluated in rats and baboon using SPECT modality.

139

[123I]R-DOI

exhibited rapid brain uptake and regional distribution in accordance with the localization of 5-HT2A receptor in brain. However in both the species, it showed a non-specific character as displayed by its very low target to non-target ratio as well as its inability to displace the cold ligand.

139

Eventually [123I]DOI was rendered unsuitable as a SPECT radioligand for clinical

purpose. b) [123I]MSP

27



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Page 28 of 70

[123I]MSP (8-[3-(4-iodobenzoyl)propyl]-1-methyl-1,3,8-triazaspiro[4,5]decan-4-one) is the spiperone derived analogue that showed better selectivity towards 5HT2A over other receptors. In mice, the pharmacokinetic and binding studies indicated that the regional distribution of the radioligand was corresponding to that of 5HT2A receptor rich regions, and also exhibited significant brain uptake with good retention capability. 140 [123I]MSP displayed promising selectivity and specificity towards 5HT2A receptors as validated by the attenuation in its uptake following treatment of mice with IMSP and ritanserin.

140

Although the

preliminary studies have shown that [123I]MSP could be a promising candidate for SPECT imaging of 5HT2A receptors, however detailed investigations are still warranted. c) [123I]-R91150 Radioiodinated

R91150

(4-amino-N-1-[3-(4-fluorophenoxy)propyl]-4-methyl-4-

piperidinyl]5-iodo-2-methoxybenzamide) when evaluated in vitro, exhibited

promising

affinity (KD = 0.11 nM) and selectivity towards 5-HT2A receptors. It showed high brain uptake in rats with good retention capability in the frontal cortex. The target to-background ratio was observed to be 10. In baboons, the pilot SPECT studies with [123I]-R91150 showed very high brain uptake in the cortex, which was reversed by the 5-HT2A receptor antagonist ketanserin.

141

In preliminary studies with 5 human volunteers, the ratio of frontal cortex-to-

cerebellum was found to remain stable at 1.4. The multi-slice SPECT sequences displayed that binding of [123I]-R91150 followed the pattern similar to autoradiography studies regarding

the

distribution

cortex>striatum>cerebellum).

of 103, 142

5-HT2A

receptors

in

human

brain

(cerebral

When dynamic SPECT was performed on 6 healthy

men, it was observed that [123I]-R91150 is sensitive enough to measure the displacement of ketanserin in a dose-dependent manner in cerebral regions dense in 5-HT(2A) receptors, thereby highlighting the promising selectivity of [123I]R91150 towards 5-HT(2A) receptors (Figure 6E).103 The ease of access of SPECT facilities has popularised the clinical applications of [123I]-R91150 as evidenced by studies in disease cases like anxiety, depression,

144

parkinson’s disease,

145

Alzheimer’s disease,

suicidal behaviour, 148 and anorexia nervosa.

149

146

Asperger’s syndrome,

143 147

In a more recent study R91150 was labelled

with 18F and evaluated as a potential PET radioligand in mice. 150 However it was later found to be a pGP substrate as it exhibited a 3 times increase in its brain uptake upon tariquidar treatment as demonstrated by SPECT imaging.151

28


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d) [123I]-3-I-CO A number of novel derivatives from altanserin and MDL 100,907 were synthesized as a new series of 4′-substituted phenyl-4-piperidinylmethanol and benzoyl-4-piperidine derivatives. A number of compounds showed significantly high affinity towards 5-HT2A receptors, among which three were radioiodinated and further evaluated as potential SPECT ligands. [123I]-3I-CO was the most promising ligand with its high affinity (Ki = 0.51 nM) and selectivity toward 5-HT2A receptors.

152

In vivo, it readily entered the rat brain and its binding was

displaceable by ketanserin. In addition, in rats no radiometabolites entered the brain. However, target-to background ratio was low, and it is possible that [123I]-3-I-CO is a target to be effluxed by P-Glycoprotein. 152 Table 2. 5HT2A receptor radioligands for PET and SPECT Radioligand

Modality Strength

Limitations

[11C]MDL 100,907

PET

Delayed imaging not possible due to mismatch of High selectivity and affinity, pharmacokinetic profile of claimed to be the first truly radiotracer and half-life of selective 5-HT2A receptor Clinical isotope [11C], ligand applications remains limited to depression and OCD

[11C]CIMBI Compounds

PET

Agonist based radioligand, high brain uptake with high target to Studies in human are warranted non-target ratio

[18F]Setoperone

PET

Highly successful 5-HT2A PET Produces radiometabolites, probe used for various clinical require inhouse facility of conditions cyclotron

PET

Produces radiometabolites One of the most frequently used which cross BBB, thereby PET radioligand complicating quantification

I]DOI

SPECT

Clinically unsuitable owing to Rapid brain uptake and high its very low target to non-target affinity ratio.

[123I]MSP

SPECT

Significant brain uptake and Detailed retention warranted

[123I]-R91150

SPECT

18

[ F]Altanserin

[

123

investigations

High selectivity, high brain Further studies are warranted uptake and retention. Clinical 29



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Page 30 of 70

application in a number of neurological disorders

[

123

I]-3-I-CO

SPECT

High affinity and selectivity

Poor target to background ratio, probable substrate of pgp

5) 5-HT-2B receptor radioligands 5-HT2B receptors are less abundant in CNS however some studies indicate that 5-HT2BRs might be involved in anxiety, cognition, food intake and neuroendocrine regulation in rodents. There are not many selective ligands known for 5-HT2BR, and hence no PET ligands are currently available for this receptor subtype. 6) 5-HT-2C receptor radioligands The choroid plexus and spinal cord shows abundant expression of 5HT-2C receptor,

68, 153

with their primary function being regulation of ion exchange between the brain and the cerebrospinal fluid.

91, 154

That is why the 5-HT-2C receptors are considered to be poor PET

imaging target owing to their absence in other major brain regions. However recently a number of 5-HT2CR PET tracers were tested with limited success, which include a low affinity azetidine analogue of pyrimidoazepine (Ki = 75 nM) and antagonists WAY-163909 (Ki = 10 nM) and vabicaserin (Ki = 3 nM). 155, 156 7) 5-HT3 receptor radioligands 7.1) 5-HT3 PET Radioligands a) [11C]MDL 72222 MDL 72222 ((8-methyl-8-azabicyclo[3.2.1]octan-3-yl)3,5-dichlorobenzoate) is a selective 5HT3 receptor antagonist that was radiolabeled with 11C and was further evaluated in rats157and baboons 158 in preliminary studies. Although [11C]MDL 72222 rapidly crossed the blood brain barrier, it was found to diffuse heterogeneously in brain, with gradual retention preferably in cerebral cortex. The radioligand suffered from major limitations of high lipophilicity and low specificity as evidenced from non-displacement of unlabelled MDL 72222 from brain areas with 5-HT3 receptors in pre-treated rats. 30


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b) [11C]YM060, [11C]Y-25130, and [11C]KF17643 Y-25130 [(azasetron) hydrochloride, (+)-N-(l-azabicyclo[2.2.2]oct - 3-yl)-6-chloro-3-oxo3,4-dihydro- 2H-1,4-benzoxazine-8-carboxamide] and YM060 [(R)-5-[l-methyl- 3 indolyl)carbonyl] -4, 5, 6, 7 - tetrahydro- 1 H- benzimidazole] showed very high affinity towards 5-HT3 in vitro, however owing to their high lipophilicity their brain uptake was very low in vivo and hence PET imaging using these two radioligands has not been successful.11, 159

Moreover

KF17643

[endo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl

2-(npropyloxy)-4-

quinolinecarboxylate] when used for PET imaging in mice did not show any specific binding to the 5-HT3 receptor despite of its high uptake and metabolic stability. 11 c) [11C]S21007 [11C]S21007 (5-(4-benzylpiperazin-1-yl)4Hpyrrolo[1,2-a]thieno[3,2-e]pyrazine) is a partial agonist of 5-HT3 that showed very high affinity (Ki=1.4nM) and selectivity, and therefore was investigated for PET imaging in rat and baboon.

160

[11C]S21007 exhibited rapid and

high brain uptake with favourable metabolic features. However, both phosphorplate ex vivo autoradiography in rats as well as PET imaging in baboons suggested that the regional distribution of [11C]S21007 was not concomitant to the spatial pattern of 5-HT3 receptors in rodents or primates brain. Its non-specificity was further confirmed when [11C]S21007 failed to displace the unlabelled S21007 in the brain of baboons. 160 d) [11C]NMQ ([11C]NMQ, (2-[1-(4-methyl)-piperazinyl]quinoline)), showed good affinity for 5-HT3 receptors (IC50 = 4.7 nM), and was synthesized by the N-methylation of quipazine, using [11C]CH3I. In rats, the biodistribution of [11C]NMQ showed its uptake in both 5-HT3 rich and non 5-HT3 regions. In monkeys, although uptake was observed in structures known to contain 5-HT 3 receptors, both its distribution pattern and pharmacology indicate that the radiotracer also interacts with non-5-HT 3 sites and showed non-specificity. Therefore, more arylpiperazine derivatives with improved specificity may be further screened for putative 5HT3 imaging radioligand for the PET studies in man. 161 e) [18F]MR18445

31



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MR18445

Page 32 of 70

(4-[4-(4-fluor-obenzyl)piperazino]-7-methoxypyrrolo[1,2-α]quinoxaline)

was

synthesized by N-alkylation of precursor with 4-[18F]fluorobenzyl iodide. In both rats and baboons it was observed that [18F] MR18445 could not be a suitable PET radioligand due to its lack of specific binding in 5-HT3 rich brain regions. Although the ligand showed high brain uptake without any indication of radiolabeled metabolites, hence can be further studied and modified to synthesize better 5-HT3 ligands with improved radiochemistry and specificity. 162 f) [18F]Fesetron Fesetron

was synthesized

by coupling (S)-3-aminoquinuclidinylamine 10 with 2,3-

dimethoxy-5-(3′-hydroxypropyl) benzoic acid.

163

The single step radiosynthesis gave a

moderate yield of [18F]fesetron that exhibited high specific activity. The in vivo microPET imaging in rats demonstrated low brain uptake of [18F] Fesetron, because a major percentage of radioactivity was unable to penetrate the blood brain barrier.

163

Although the very low

amount of activity was detected in brain but it was shown to be retained preferably in the 5HT3 dense regions. However, it’s very low uptake limits the usefulness of [18F]Fesetron as a PET radiotracer in rats.

163

Hence further experiments are warranted to improve the brain

uptake of [18F]Fesetron in order to develop it as a PET radiotracer for 5-HT3 receptors for clinical applications.

7.2) SPECT Radioligands a) Zacopride Derivatives Despite of high selectivity and affinity towards 5HT3 receptors over other 5-HT receptors, Zacopride analogues could not be developed and successfully deployed for SPECT imaging. [125I]DAIZAC

((S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2.2]oct-3-yl)benzamide)

was observed to be one of the promising candidate with more than 100 times high selectivity for 5HT-3 than 5-HT4. However the detailed investigation regarding its pre-clinical and clinical utility is still warranted.

8) 5-HT 4 receptor radioligand: 32


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5-HT4Rs are located predominantly in striatum (Bmax= 223 fmol/mg/protein), neocortex, thalamus, hippocampus and brainstem.

68, 153

These receptors are primarily involved in the

cognitive, memory, depression, ADHD, anorexia and obesity.

68, 153

Several agonist and

antagonist ligands are known for this receptor. Regarding PET imaging, [11C]SB207145 is the only antagonist radiotracer tested so far in human for 5-HT4R.

164-166

[11C]SB207145

belongs to the benzodioxine class of compounds with high affinity for 5-HT4R (Ki = 0.3 nM,) and show heterogeneous binding that corresponds to the known distribution of 5-HT4R binding in rodents, primates, pigs and human. The radioligand did not show sensitivity to endogenous competition studies with citalopram in comparison to control subjects.

164

No

11

significant difference of [ C]SB207145 was found in a small group of AD patients in comparison to controls.

167

However, [11C]SB207145 binding was positively correlated to

amyloid β burden and negatively correlated to MMSE score of AD patients. Several radioligands were emerged based on [11C]SB207145 and among these, [18F]MNI-698 and [18F]MNI-699, the fluoroalkyl analogues of SB207145, were tested in primates.

168, 169

[18F]MNI-698 exhibits excellent in vivo characteristics and it is used for the measurement of 5-HT4R in monkey.

170

Several C-11 labeled analogues of SB207145 derivatives were

synthesized for 5-HT4R, however, no in vivo data are reported for these tracers.

171

More

11

recently [ C]prucalopride, a potent 5-HT4R agonist has been developed and in vivo studies in rats show low uptake of the radioligand which is likely due to inadequate lipophilicity and possibility of being a P-gp substrate. 172 9) 5-HT5R: No therapeutically active and selective compounds acting at 5-HT5R are known till date. Though two selective antagonists are known to alter the circadian rhythm and function in vivo, however specific PET or SPECT agents to study 5-HT5R are not available.173, 174 10) 5-HT6 Radioligands 10.1) PET radioligands a) [11C]GSK215083 The 3-benzenesulfonyl-8-piperazine-1-yl-quinoline based compounds exhibited promising brain uptake and were further studied for their radioimaging capabilities. In stable HeLa cell lines expressing recombinant human 5-HT6 receptor, GSK215083 displayed very high 33



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Page 34 of 70

affinity (Ki=0.16 nM).175 Although it also showed high affinity towards 5HT2A receptor, however both the receptors are largely concentrated in different brain regions (5-HT6 in striatum and 5HT2A in cortex) and hence can be differentiated from each other effectively. The studies in pigs and nonhuman primates (NHPs), and its initial assessment in humans as well as in human kinetic analysis, suggested that [11C]-GSK215083 may be developed as a PET radioligand for 5-HT6 receptor for clinical applications.176, 177 b) [18F]12ST05 A number of compounds were derived from the 5-HT6 antagonist SB271046 and their selectivity towards the 5-HT6 receptor was evaluated. [18F]12ST05 ([18F]-N-[2-(1-[(4fluorophenyl)sulfonyl]-1H-indol-4-yloxy)ethyl]-N,N-dimethylamine) was identified to be one of the first potential 5-HT6 PET radioligand.178 In rat and cat studies, it showed a very promising brain uptake and kinetics. This was further substantiated by in vitro and ex vivo autoradiographic studies in rat, which displayed its high selectivity and affinity in 5-HT6 receptors dense regions. Unfortunately the in vivo studies in cat failed to show specific binding of [18F]12ST05 to 5-HT6 receptors in cerebral regions, and hence more detailed investigations were not undertaken to develop [18F]12ST05 as a PET radioligand.178 c) [18F]2FNQ1P: It is the derivative of GSK215083 with high affinity (Ki =0.8 nM) and antagonistic characterstics (IC50 =1.8 nM) towards 5HT6 receptors. In rat brains, [18F]2FNQ1P showed binding in regions which corresponded to 5-HT6 receptors dense regions, as assessed by semi-quantitative autoradiography in rat brains. In vitro PET autoradiographies in rat brain sections confirmed the 5-HT6 distribution pattern of [18F] 2FNQ1P. In vitro autoradiography in the rat model confirmed that [18F]2FNQ1P specifically binds 5-HT6 receptors, since addition of SB2258585 (the specific 5-HT6 antagonist) displaced the binding of [18F]2FNQ1P in a concentration dependent manner. However, the in vivo assessment in rats using microPET exhibited reduced brain uptake of [18F]2FNQ1P. In vivo PET studies in cat showed its high brain uptake particularly in 5-HT6 dense striatal regions and it was displaced successfully by unlabelled 2FNQ1P, highlighting its promising selectivity. Further PET scanning in macaque monkeys substantiated the high specificity of [18F]2FNQ1P towards 5HT6 receptor as evidenced by high SUV ratio between striatum and cerebellum as well as blocking studies , in which the binding of [18F]2FNQ1P in striatum was attenuated in the 34


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presence of the 5-HT6 receptor antagonist SB258585. Such pharmacological inconsistencies in different species may restrict development of a novel PET radiotracer if further evaluated in rodents. Such preclinical report in diverse species (rodents, cat, non-human primates) are also instrumental to further undertake [18F]2FNQ1P to next level of radioimaging in disease models and to develop further for clinical purpose.179 (Figure 7A)

11) 5-HT7 receptor radioligand: 5-HT7R and 5-HT1AR share a 49% sequence homology,

180, 181

which therefore presents

with the potential challenge to design and develop ligands that are extremely selective for either of these two receptors. The thalamus has higher density of 5-HT7R compared to 5HT1AR

182-184

however the temporal cortex is highly dense in 5-HT1AR but not in 5-

HT7R.182-184 A number of ligands have been reported, which allows further characterization of these receptors in native tissues.

Figure 7. PET Ligands of 5HT6R and 5HT7R. Representative PET Scan of (A) [18F]2FNQ1P179(5HT6R) and (B) [18F]2FP3(5HT7)in cat brain185

1) [18F]2FP3: 35



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SB 269970 has been shown to inhibit the serotonin induced hypothermia in guinea pigs

186

and has also reduced the time spent in paradoxical sleep in rats186, thereby suggesting the participation of 5-HT7 receptors in regulation of sleep disorders and depression. Therefore, a number of

18

F-ligands based on SB-269970 have been synthesized. In vitro studies in rats

showed that [18F]2FP3 (Ki = 8.4 nM) and [18F]4FP3 (Ki = 14 nM) possess specific binding as evidenced in brain sections

185

Furthermore 5-HT7 shares ~95% interspecies homology187

and studies in cats using [18F]2FP3 confirmed its significant uptake in brain, regional distribution and specificity

188

, thereby implicating it to be a promising PET radiotracer for

clinical applications.(Figure 7B) 2) [11C]DR4446: Preliminary

studies

on

[11C]DR4446

(1-methyl-2a-[4-(4,5,6,7-tetrahydrothieno[3,2-

c]pyridin-5-yl)butyl]-2a,3,4,5-tetrahydro-1H-benz[cd]indole-2-one) has indicated its decent affinity (Ki = 9.7 nM) and high selectivity for 5-HT7 receptors compared to other 5-HT receptors

189

. When tested in monkey brain, [11C] DR4446 exhibited

189

good penetration in

brain, with high metabolic stability, but low specificity. However, clinical studies using [11C] DR4446 are still awaited. 3) [11C]CIMBI compounds: Two selective phenylpiperazinyl butyloxindole derivatives, [11C]CIMBI-712 (Ki = 1.1 nM) and [11C]CIMBI-717 (Ki = 2.6 nM), were evaluated for 5-HT7R specificity 190. [11C]CIMBI717 showed higher uptake and specific binding than [11C]CIMBI-712. All these pre-clinical studies suggests that [18F] 2FP3 and [11C]CIMBI-717 are the two 5-HT7R PET ligands that may be further tested for their utility in clinics. 4) Biaryl derivatives: [11

C] PM20 (1-[2-(4-methoxyphenyl) phenyl]piperazine) was developed and evaluated as a

putative 5-HT7 specific PET imaging agent.191 Although the compound exhibited promising 5-HT7 affinity and high specifity in vitro autoradiography assays, as evidenced from significant affinity to human recombinant 5-HT7 receptors expressed in mammalian cells (Ki = 2.6 nM).191 However the in vivo PET evaluation was disappointing as the compound displayed limited specificity of [11C]PM20 for 5-HT7 as well as inconsistent distribution pattern of radioactivity, not corresponding to that of 5-HT7 in the brain. However PM20, in 36


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later studies, was used as a lead compound to derive other promising PET agent. It was shown that chemical compounds having biaryl system (5-(4-(methoxyphenyl)-1-methyl-4nitro-1H imidazole) has the potential as a promising 5-HT7 radioligand.192 The compound was observed to be highly selective for 5-HT7 (Ki=16.8±0.9nm) over 5-HT1A receptors. PET studies in mice and rats showed its high brain uptake. However, owing to the low receptor density of 5-HT7 in brain, the in vivo imaging was not up to the expectation, and hence require further optimization and detailed investigations to develop a more robust 5HT7 receptor imaging agent.

12) Serotonin Transporters (SERT) The serotonin transporter (SERT) has the primary role in maintaining extracellular 5-HT concentrations through reuptake The SERT inhibitors have proved to be very promising for treating depression, obsessive compulsive disorder, schizophrenia, bipolar disorder, anxiety and eating disorders. Therefore, development of PET and SPECT ligands for the detailed investigation of SERT in human brain has remained an unattained objective. A number of SERT binding imaging agents for PET and SPECT have been designed. 193 The dynamics of serotonin transporter expression and functions in 5-HT reuptake are tightly regulated. 5-HT regulates its own reuptake as evidenced from attenuation in SERT internalization when exposed to increased concentrations of 5-HT while SERT is downregulated at low 5-HT concentrations. On the contrary, SSRIs augment the concentration of 5-HT in the extracellular space which consequently leads to reduced SERT binding in the cell membrane. With the advent of molecular imaging studies (PET, SPECT, CT), it has become possible to understand the critical interplay between SERT functions, pathomechanisms of depression and the pharmacological efficacy of antidepressants in clinical use. PET studies have enabled the accurate assessment of the expression and density of target molecules in a specified region of brain. For example, recent PET imaging studies along with cellular assays have shown that SERT genotype is directly associated with the risk for inherited depression by regulating its distribution and expression. With widespread clinical applications of SERT imaging may therefore enable the development of multifaceted diagnostics and may also help in screening and categorization of patients with distinct psychiatric disorders in the future. PET and SPECT radioligands for SERT are summarized in Table 3. 37



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Page 38 of 70

12.1) PET Radioligands for SERT a) [11C]McN5652: McN5652 (trans-1,2,3,5,6,10-β-hexahydro-6-[4-(methylthio)phenyl]pyrrolo-[2,1-a]isoquinoline) is an antidepressant developed by Maryanoff et al.194, whose (+) enantiomer has a strong binding potency to 5-HTT (Ki = 0.40 nM), in contrast to its (-) enantiomer which has shown ~150 times lower affinity.

195

[11C] McN5652 was the first selective PET radioligand for

imaging SERT in the human brain. This radioligand has been used for the assessment of SERT binding in depression,

196

ecstasy use

197

and mood disorders.

198

However it suffers

from the major limitations of low target-to-background ratio in vivo, as well as a slow brain uptake and irreversible kinetics that complicate quantification in high binding regions, and thus hampers its applicability as a PET imaging agent. Development of an S[18F]fluoromethyl analogue of (+)-McN5652, [18F]FMe-(+)-McN5652, showed promising PET characteristics for SERT imaging and in vivo quantification in humans. b) [11C]DASB: [11C]-DASB (3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile) has been used in PET for the in vivo quantification of serotonin transporters (SERTs).

199

When

evaluated in baboons and compared with [11C](+)McN5652, it was observed that [11C]DASB exhibited lower non-specific binding, less plasma protein binding and rapid clearance from blood with much improved brain uptake. All these characteristics of [11C]-DASB enabled efficient measurement of SERT in very less time,200 later confirmed in healthy volunteers201 showing a higher specific-to-nonspecific ratios thereby conferring [11C]-DASB as a superior PET ligand over [11C](+)McN5652.

202

PET studies of [11C]-DASB in humans

showed that the uptake distribution matched that expected for SERT and binding kinetics could be quantified with reference tissue models (Figure 8A). 202 In humans, [11C]DASB has good brain uptake; its ratio of specific binding relative to free and nonspecific binding is good and the latter has low between–subject variability. Multiple brain regions may be assessed with noninvasive methods, and the reliability of regional SERT binding potential measures is good. In summary, [11C]DASB PET imaging is the state of the art method in quantifying SERT in humans. [11C]-DASB PET has since been used to measure SERT occupancy at clinical doses of fluoxetine, citalopram, sertraline, duloxetine, and venlafaxine. These were followed by patient studies, investigating a multitude of conditions including 38


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MDMA use, depression, schizophrenia, obsessive–compulsive disorder, alcoholism, and bipolar disorder. [11C]-DASB binding has further been investigated in healthy individuals in relation to personality traits, seasonal changes, and familial risk for mood disorders. c) [11C]MADAM [11C]MADAM (N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine) was found to be a potential PET radioligand for quantitative studies of SERT binding in the nonhuman primate PET studies (Ki=1.65 nM).

203

In human subjects, the highest radioactivity

concentration was detected in the raphe nuclei, followed consecutively by the striatum, hippocampal complex, cingulate cortex, neocortex, and cerebellum, which was consistent with postmortem data acquired with [3H]-MADAM as well as with that of other reference ligands in vitro [paroxetine (Ki = 0.32 nM) and citalopram (Ki = 1.57 nM)].

204 205

In a test

retest reproducibility study done in 9 healthy male subjects [11C]MADAM was shown to have good to excellent reliability in measurements of SERT binding in brain in order to pursue research on psychiatric disorders (Figure 8B).206 In another PET imaging study using [11C]MADAM to focus on the role of gender on serotonin associated psychiatric conditions, such as depression, anxiety and suicide in men and women, it was shown that compared to men, women had significantly lower SERT binding potentials in the cortex and sub cortex region of brain. In women, there was a positive correlation between 5-HT(1A) receptor and SERT binding potentials for the region of hippocampus, thereby indicating biological distinctions in the serotonin system contributing to sex differences in the prevalence of psychiatric disorders such as depression and anxiety. 207 d) [18F]ADAM The 18F-labelled form of ADAM, 4-[18F]ADAM (N,N-dimethyl-2-(2-amino-4-[18F]fluorophenylthio)benzylamine) has shown very high selectivity and affinity for SERT when evaluated in cell lines based competitive binding studies and has shown 1,000-fold lower binding affinities than DAT and NET.

208

In rats the uptake of 4-[18F]ADAM in SERT-rich

regions was shown to be markedly inhibited by SERT inhibitors. 210

human demonstrated (Figure 8C)

209

Preliminary studies in

its clinical utility and is well supported by bio-

distribution, toxicity and dosimetry studies in monkeys.211 4-[18F]ADAM has been used to study SERT availability and binding in diseases such as depression, 214

212 213

suicidal behavior

and Parkinson’s disease model. 202 39



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Page 40 of 70

Figure 8 SERT PET Ligands. High activity concentration in dorsal raphe and amygdala on [11C]DASB scan215 Summation image from frames 6–20 showing radioactivity accumulation after intravenous injection of [11C]MADAM204 and [18F]ADAM.210 SPECT scan of [123I]ADAM in a control and depression subject.216 e) [18F] FPBM [18F]FPBM (2-(2′-((dimethylamino)methyl)-4′-(3-[18F]-fluoropropoxy)phenylthio) benzenamine), owing to its simple radiochemistry, can be easily prepared with good radiochemical yields and purity.217 The in vitro binding studies have registered the high affinity (Ki = 0.38 nM) and selectivity of [18F]FPBM,

218

which was further substantiated by

in vivo biodistribution, ex vivo autoradiography, and PET imaging studies in rats thereby exhibiting its high brain uptake and a promising target to non-target ratio (7.7 at 120 min post-injection). Subsequent studies in a rat model of Parkinson’s disease

219

induced by a 6-

hydroxydopamine (6-OHDA) and in p-chloroamphetamine (PCA) treated rat model further highlighted the high selectivity of [18F]FPBM and its utility for in vivo SERT imaging. 12.2) SPECT Radioligands for SERT a) ß-[123I]CIT and Nor-ß-[123I]CIT: ß-[123I]CIT (2-ß-carbomethoxy-3-β-(4-iodophenyl)tropane), is a non-selective radioligand which can be used simultaneously for 5-HT and dopamine (DA) transporter imaging in the 40


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living human brain with single photon emission computed tomography (SPECT).

220 123

[

I]b-

CIT has shown differential kinetics of binding to 5-HT and DA transporters. Moreover, the spatio-regional distribution of both transporters is quite distinct in human brain wherein hypothalamus contains much more 5-HT than DA transporters,

221

whereas the striatum

contains much more DA transporters than 5-HT transporters. Therefore, simultaneous imaging of both transporters is possible.

222

Nor-β-[123I]-CIT (2beta-Carbomethoxy-3beta-(4-

iodophenyl)nortropane) is a des-methyl analogue of beta-CIT, which in vitro has shown tenfold higher affinity (IC50=0.36 nM) to the serotonin transporter than beta-CIT (IC50=4.2 nM),223 however it remains a matter of discussion whether nor-β-[123I]CIT is a better radioligand for SERT than β-[123I]CIT. In healthy volunteers, single-photon emission tomography (SPECT) studies with [123I]nor-beta-CIT demonstrated very high accumulation in the striatum, the mid-brain and the thalamus, with an overall 33% higher specific binding in the mid-brain in comparison to [123I]beta-CIT, thus representing its accumulation in serotonin transporter rich regions in human brain.

224

A number of clinical studies using β-

[123I]CIT and nor-β-[123I]CIT have shown correlation between SERT densities and psychological pathologies like depression,225,226 autism, 227 Parkinson’s Disease, 228 obsessive compulsive disorder, 229 and mixed mania. 230 b) [123I]ADAM: [123I]ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine) is a highly selective SERT radioligand (Ki=0.013 nM), that has shown more than 1,000-fold selectivity for SERT over NET and DAT (Ki = 699 and 840 nM/L for norepinephrine transporter and DAT, respectively). Its binding affinity toward SERT is also very high, along with an excellent target-to-background ratio. [123I]ADAM is a clinically successful SPECT radioligand and has shown wide clinical applications for correlating SERT densities in depression wherein the lower uptake in midbrain was seen in MDD patients as compared to the healthy controls (Figure 8D).216 [123I]ADAM had also been evaluated in smoking,231 epilepsy,232 and night eating syndrome. 233, 234 In a parkinsonian primate model, it was shown that after a bolus injection, the uptakes of [123]I]ADAM in the striatum, thalamus, and frontal cortex were 31%, 31%, and 23% lower than those of normal monkeys at 210-240 min postinjection, respectively thus indicating the significant potential of [123I]ADAM for evaluating the serotonin transporter changes in human PD. [

123

235

These studies apparently suggest that

I]ADAM has significant advantage as a SPECT imaging agent for SERT in the brain, as 41



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Page 42 of 70

evidenced from its localization in the hypothalamus region of the brain where the concentration of SERT is the highest. [123I]ADAM has so far been the ligand of choice for SPECT imaging of serotonin transporter because of obvious advantages like lower costs and the easier-to-handle longer lived radioligands. The major disadvantage is its availability in clinics for routine imaging as [123I] labeled radioligand requires a cyclotron for production and an overnight shipment from the site of production.

Table 3: SERT radioligands for PET and SPECT

Radioligands

Modality

Strength

Weakness

PET

Low target to background High affinity, has been used in ratio, slow brain uptake and depression, mood disorder and complicated PET ecstasy use quantification.

[ C]DASB

PET

High specificity and brain uptake More clinical studies are with rapid clearance from blood. warranted Good reliability, used in clinics

[11C]MADAM

PET

High specificity and brain uptake, More clinical studies are being used in clinics warranted

PET

High selectivity and affinity, clinically used in depression, More clinical studies are suicidal behavior, Parkinson’s warranted disease model

PET

Simple radiochemistry good clinical yield, high affinity and warranted selectivity, high brain uptake

[11C]McN5652

11

[18F]ADAM

[18F] FPBM

studies

are

Nor-ß-[123I]CIT

SPECT

Simultaneous imaging of SERT and DA transporters, clinically Non-selective used to correlate SERT density with psychological disorders

[123I]ADAM

SPECT

High selectivity and affinity, Requires an onsite access to highly successful as a SERT cyclotron imaging agent in wide clinical

ß-[123I]CIT and

42


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applications

43



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Future Perspective The studies based upon molecular imaging of 5-HTRs have been continuously evolving in an attempt to either identify novel ligands or improve the existing ones in order to quantify various 5-HT receptors subtypes in physiological as well as pathological conditions. The present review emphasizes upon the fact that the design and development of radioprobes for in vivo brain imaging in patients is a tedious process. A majority of radiotracers fail to even pass the developmental phase despite their potential for in vivo brain imaging in pre-clinical studies. The primary reasons for their failure are inability to cross blood–brain barrier, high non-specificity, unnecessary metabolite formation, too strong binding to be displaced by the target specific ligand or difficulty to quantitate due to poor pharmacokinetic attributes. Moreover the efficacy and potential of candidate compounds in preclinical studies in animal models often fail to translate as expected in clinical situations. Therefore the design and synthesis of a candidate compound and its thorough assessment in pre-clinical as well as clinical set up to confirm its suitability as an imaging agent is extremely complicated. Radiochemistry plays a significant role in the new tracer development. Radiosynthesis should be fast and simple with high specific activity for any significant pharmacological blockade of the receptor with the natural ligand for that receptor. The radioligand should be designed in such a way that it must bind selectively to the target of interest. To obtain meaningful data the binding potential measured by PET or SPECT using the radioligand must be obtained from binding to the desired target only. Number of receptors available for binding (Bavail) and the affinity (1/KD) must be considered when determining the selectivity of a radioligands towards a target receptor. [11C]-labelling is usually feasible because of the presence of carbon atoms in all organic compounds. The introduction of the small electronegative [18F] atom in place of hydrogen or hydroxyl can be unpredictably beneficial or detrimental to the pharmacological properties of the ligand in terms of its affinity and selectivity. Introduction of a bulky [123I] atom in place of hydrogen generally affects pharmacology and will increase ligand lipophilicity. The halflife of the radionuclide is an important prerequisite for studying receptor function. The relatively short half-life of [11C] (t1/2=520.4 min) is suitable for following quite rapid pharmacokinetics, and may permit more than one study session in the same subject in a single day. However, the necessity to produce [11C]-labelled radioligands on-site is a logistical 44


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disadvantage. Longer lived [18F] (t1/2=5109.8 min) for PET and [123I] (t1/2=513 hr) for SPECT do not require on-site production and are suitable for following slower pharmacokinetics. Alternative labels are being investigated, such as the SPECT label [99mTc] (t1/2=56 hr) because of its relative safety and availability. Presently the radiotracers for 5-HT1A, 5-HT1B, 5-HT2A and 5-HT6 receptors are widely accepted in clinics for imaging the serotonergic system in brain. [11C]WAY100635, [18F]MPPF, and [18F]FCWAY are currently used for PET studies of the 5-HT1A receptor but their major drawbacks like complex radiochemistry, fast metabolism, P-gp substrate and radiodefluorination hinders their clinical relevance. The most validated 5-HT2A receptor radiotracers include the non-selective PET tracer [18F]setoperone, and the selective tracers [18F]altanserin and [11C]MDL100,907. Some of the emerging radioligands that have shown promise in preliminary studies in humans include the 5-HT1AR antagonist [11C]trans-MeFWAY, the two 5-HT1B ligands [11C]AZ10419369 and [11C]P943, the 5-HT4 receptor radioligand [11C]SB207145. The partial agonist ligand [11C]CUMI-101 is promising in human and non-human primates (baboon) to measure the high affinity state of 5-HT1AR, however, no data is available so far to support its potential to image pathological conditions. However there are still gaps in development of successful PET or SPECT radioligands for the study of 5-HT3, 5-HT2B and 5-HT5 receptor in humans probably due to inadequate receptor density or location. Therefore, future development of suitable PET agonist/antagonist radiotracers is needed to study a variety of serotonin receptor subtypes as well as their role in the progression of various neurological disorders. A multitude of specific ligands have been identified for many of these therapeutic targets, thereby facilitating the development of novel radioprobes to study complex mechanisms of brain functions and disorders using in vivo imaging techniques. Few promising radioligands are presented in Table 4. Biased agonists present a novel strategy to manage psychiatric disorders that are associated with 5HT1A receptors for identifying a subset of 5HT-1A receptors than a known antagonist [18F]-MPPF thereby anticipating a better response in patients undergoing antidepressants or antipsychotic treatment. Such novel strategies may promote new avenues for the treatment of neuropsychiatric and neurological pathophysiologies. Imaging functional status of the receptor; first to know whether the compound is agonist, antagonist, biased agonist, partial agonist will play significant role in

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accurate interpretation of the existence of 2 distinct affinity states of 5-HT1Areceptors (high affinity, functional state and lower affinity, nonfunctional state) Over the past few decades the understanding of G-protein coupled receptor structure and function has revealed that GPCRs are able to form homo- and hetero-oligomeric complexes. One of the most intriguing possibilities raised by the discovery of GPCR dimerization is the amazing pharmacological diversity and potential for regulation that would be offered by the occurrence of homo- and heterodimerization. The dimerization–oligomerization of GPCRs poses a differentiated pharmacology from the monomers. The existence of homo- and heterodimers has been demonstrated for several classes of receptors. Investigating postmortem striatal sections from schizophrenia patients and striatal tissue of animal models of schizophrenia, the expression of serotonin dimers proved to be significantly enhanced, while expression of dopamine monomers was decreased. As evidenced from promising results obtained from the pre-clinical studies using BMPPSiF for PET and DTPA-BisMPPA and SerDTC for SPECT, it can be anticipated that this novel strategy of bivalent homodimeric and heterodimeric systems can be further applied so as to open new avenues in 5HT1A /x dimers (wherein x can be 5HT1A/5HT2/D2/D3/α1A under neurodegenerative disorders). New strategies to develop serotonin ligands are shown in Figure 9.

46


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Figure 9. New approaches to design serotonin receptor ligand CONCLUSION In conclusion, it is evident from this review that once a radioligand for imaging of a serotonergic target has been successfully evaluated, the clinical and commercial interest in using the radioligand for studies of a whole range of human conditions and for establishing its therapeutic doses becomes vast. Additionally, the potential use of agonist tracers for imaging “functional” receptors in vivo and improvement of the metabolic stability to enable tracer-kinetic modeling may provide us with additional information that cannot be obtained using antagonist tracers. The distribution and binding pattern of a radioligand may also be more susceptible to changes in endogenous neurotransmitter, a property that may provide 47



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new avenues regarding understanding of a complex pathophysiological conditions involving brain. With this in mind the continuing efforts to develop radioligands for imaging of serotonergic targets should be facilitated well.

48


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Table 4. MOST PROMISING PET AND SPECT LIGANDS AT PRE-CLINICAL AND CLINICAL STAGE

5-HT1A LIGANDS

N

N

N

N

N

N N

N 11 C

O

18F

O O

O

[11C]WAY-100635

[18F] MeFWAY

Ki 0.17 nM

Ki 0.9 nM

pGP inhibitor/ pGP KO on brain uptake 23 fold increase in rats treated with CsA236

pGP inhibitor/ pGP KO on brain uptake 160% higher uptake in tariquidar treated rat hippocampus237

t

Bu tBu F Si

OMe

18

N N

O O

O

N N N

O O

HN

N

N

N

Ga

O

O

N

O

N O

N O

MeO N N

N

N N MeO

[18F]BMPPSiF

[68Ga]DO3A-Butyl MPP

Ki 0.09 nM

Ki 0.64 nM

49



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5-HT1B LIGANDS

[11C]AZ10419369

[11C] P943

Ki 0.8 nM

Ki 1.2 nM

pGP inhibitor/ pGP KO on brain uptake Significant increase in CsA treated mice, rats and guinea pigs as well as in mdr1 KO mice238 5HT2A LIGANDS

[123I]-R91150 Ki 0.2 nM pGP inhibitor/ pGP KO on brain uptake Region dependent effect; 3 times higher in tariquidar treated rats151

[18F]Setoperone

[18F]Altanserin

Ki 0.3 nM

Ki 8.4 nM 50


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pGP inhibitor/ pGP KO on brain uptake Significant increase in CsA treated rats and monkeys. 5H7 LIGANDS N 18

F

N

N S O O

N

O11 CH 3 NH O

[18F]2FP3

[11C]CIMBI 717

Ki 1.1 nM,

Ki 0.013 nM

P-gp interaction of [18F]2FP3 analogs is known185 SERT LIGANDS CH3 N CH3

NH2

11

CH3 N CH3

NH2

S

S 123

I CN

[123I]ADAM

[11C]DASB

Ki 1.1 nM

Ki 1.65 nM

Exhibited adequate brain uptake in mdr1a KO mice237

[11C]MADAM

[18F]ADAM

Ki 4.8 nM

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AKNOWLEDGEMENTS This report was supported by YSC-CSASDA/INMAS/INM-319 and INMAS, Defense Research and Development Organization. The authors also extend their thanks to Dr A.K. Singh, Director, INMAS for constant encouragement and support.

ABBREVIATION 5-HT: 5-Hydroxytryptamine; 5-HTRs: 5-Hydroxytryptamine receptors; SERT: Serotonin transporter; PET: Positron Emission Tomography; SPECT: Single photon emission computerized tomography; CNS: Central nervous system; cAMP: cyclic adenosine monophosphate; ATP: Adenosine triphosphate; MRI: Magnetic Resonance Imaging; CT: Computed tomography; GPCRs: G-Protein coupled receptors; SiFA: Silyl fluorine acceptor; DTPA: diethylenetriaminepentaacetic acid; %ID/g: Percent injected dose per gram; ADHD: Attention deficit hyperactivity disorder; SSRIs: Selective serotonin reuptake inhibitor; REM: Rapid eye movement sleep; NET: Norepinephrine transporter; DAT: Dopamine transporter; PD: Parkinson’s disease; MDMA: Methylenedioxymethamphetamine ; 6-OHDA: 6-hydroxydopamine; PCA: p-chloroamphetamine; CsA: Cyclosporine A; KO: knock out

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