Julie A. McCarron

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [3H]WAY-100635 and [11C]WAY-100635

The distribution of 5-HT1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT1A receptor antagonist [3H]WAY-100635 ([O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT1A receptors with [3H]WAY-100635 was antagonised by the addition of 5-HT1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 microM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [3H]WAY-100635 has a Kd of approximately 2.5 nM. The selective labeling of 5-HT1A receptors with [3H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT1a receptor subtype in vitro [11C]WAY-100635 is used for the characterization of 5-HT1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t1/2 = 20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [11C]WAY-100635 gave images qualitatively similar to those of [3H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 microM), blocked all binding of [11C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [11C]Way-100635. These data provide a strong basis for expecting [11C]WAY-100635 to behave as a highly selective radioligand in vivo.

Exquisite delineation of 5-HT1A receptors in human brain with PET and [carbonyl-11C]WAY-100635

The 5-HT1A receptor antagonist, WAY-100635 [N-(2-(4-(2-methoxyphenyl)- 1-piperazinyl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide], was labelled in its carbonyl group with carbon-11 (t1/2 = 20.4 min), injected intravenously into healthy male volunteers and studied with positron emission tomography (PET). The acquired data provide exquisite delineation of 5-HT1A receptors in brain, with the ratio of radioactivity uptake in receptor-rich regions, such as medial temporal cortex, to that in receptor-devoid cerebellum reaching 25 by 60 min after radioligand injection. Application of biomathematical modelling to the data revealed high values (7.8) for binding potential, a measure of Bmax/Kp, in receptor-rich regions. Only very polar radioactive metabolites were present in plasma, a finding consistent with the low level of nonspecific binding seen in cerebellum. [carbonyl-11C]WAY-100635 is concluded to be far superior to the previously reported [0-methyl-11C]WAY-100635 as a radioligand for PET studies of 5-HT1A receptors in human brain.

First delineation of 5-HT1A receptors in human brain with PET and [11C]WAY-100635

The selective 5-HT1A receptor radioligand, [11C]WAY-100635 ([11C]N-2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-2- pyridyl)cyclohexanecarboxamide), has been injected intravenously into healthy male volunteers and studied by PET (positron emission tomography). The results provide the first delineation of 5-HT1A receptors in living human brain and demonstrate the potential to use [11C]WAY-100635 for the study of central 5-HT1A receptors in patients with psychiatric and neurological disorders and for the investigation of the pharmacology of drugs acting on the central nervous system.

Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methl-11C]WAY-100635, in monkey and human plasma by HPLC: Comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t1/2 = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-11C]WAY-100635 into humans, radioactivity was found to clear rapidly from blood and plasma. By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-11C]WAY-100635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-11C]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha 1-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-11C]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-11 in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake.

PET-characterization of [carbonyl-11C]WAY-100635 binding to 5-HT1A, receptors in the primate brain

Abstract [carbonyl-11C]WAY-100635 is a new radioligand which can be used with positron emission tomography (PET) to provide high contrast delineation of human brain regions that are rich in 5-HT1A receptors. In the present PET study, the binding of [carbonyl-11C]WAY-100635 was characterized in the cynomolgus monkey brain. Pretreatment with each of the two reference compounds, WAY-100635 and 8-OH-DPAT, as well as the drugs buspirone and pindolol, induced a marked inhibition of [carbonyl-11C]WAY-100635 binding in the neocortex and the raphe nuclei. A preliminary Scatchard analysis yielded 5-HT1A receptor density values of the same order as those that have been reported in vitro. The study shows that [carbonyl-11C]WAY-100635 binds specifically to 5-HT1A receptors in the primate brain and has potential for determination of 5-HT1A receptor occupancy and density in psychiatric patients.

Synthesis of a [6-Pyridinyl-18F]-labelled fluoro derivative of WAY-100635 as a candidate radioligand for brain 5-HT1A receptor imaging with PET

In recent years, considerable effort has been spent on the design, synthesis and pharmacological characterization of radiofluorinated derivatives of the 5-HT(1A) receptor antagonist, WAY-100635, for the in vivo study of these receptors in human brain with PET. (Pyridinyl-6)-fluoro- and (pyridinyl-5)-fluoro-analogues of WAY-100635 (6-fluoro and 5-fluoro-WAY-100635, 5a/6a) were synthesized as well as the corresponding chloro-, bromo- and nitro-derivatives as precursors for labelling (5b-d and 6b-d). Comparative radiolabelling of these precursors with fluorine-18 (positron-emitting isotope, 109.8 min half-life) clearly demonstrated that only ortho-fluorination in this pyridine series, and not meta-fluorination, is of interest for the preparation of a radioligand by nucleophilic heteroaromatic substitution. 6-[(18)F]Fluoro-WAY-100635 ([(18)F]5a) can be efficiently synthesized in one step, either from the corresponding 6-bromo precursor (using conventional heating at 145 degrees C for 10 min) or from the corresponding 6-nitro precursor (using microwave activation at 100 W for 1 min). Typically, 15-25 mCi (0.55-0.92 GBq) of 6-[(18)F]fluoro-WAY-100635 ([(18)F]5a, 1-2 Ci/micromol or 37-72 GBq/micromol) were obtained in 50-70 min starting from a 100 mCi (3.7 GBq) aliquot of a batch of cyclotron-produced [(18)F]fluoride. This (18)F-labelled radioligand is now being evaluated in PET studies.

Evaluation in rat of RS-79948-197 as a potential PET ligand for central α2-adrenoceptors

Tritium-labelled RS-79948-197 {(8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine} was evaluated in rat brain as an in vivo ligand for central α2-adrenoceptors, as a preliminary step in the development of a radioligand for positron-emission tomography (PET) studies. The maximal receptor-specific signal was achieved within 90–120 min after i.v. injection of [ethyl-3H]RS-79948-197 and was selective for the α2-compared with the α1-adrenoceptor, with no detectable binding to the imidazoline-I2 site. Estimates for binding potential (approximating to BmaxKd) ranged between 3.4 in entorhinal cortex and 0.5 in medulla oblongata. The results, which indicate a similarly localised but 2-fold increase in specific binding compared with that previously demonstrated using [3H]RX 821002 (2-methoxy-idazoxan), are sufficiently encouraging as to support further investment in the development of 11C-labelled RS-79948-197, or a close structural analogue, as a ligand for clinical PET.

Preclinical Development of a Radioligand for the Study of Central 5-HT1A Receptors with PET — [11C]Way-100635

N-(2-(1-(4-(2-Methoxyphenyl)-1 -piperazinyl)ethyl))-N-(2-pyridyl)cyclohexanecarboxamide (WAY-100635) has properties that favour its development as the first radioligand for studies of 5-HT1A receptors in vivo with positron emission tomography (PET). These include high potency, high selectivity, pure antagonist action and amenability to labelling with the positron-emitter, carbon-11 (t 1/2 = 20.4 min). Here the status of our preclinical development of [11 C] WAY-100635 as a PET radioligand is summarised. On the basis of studies in rats and human volunteers, it is concluded that [11C]WAY-100635 is a promising radioligand for the study of central 5-HT 1A receptors with potential for application to the study of neuropsychiatric disorders and to the human pharmacology of psychoactive drugs.

Chapter 7 - Radioactive Metabolites of the 5-HT 1A Receptor Radioligand, [ O-methyl - 11 C]WAY-100635, in Humans

[O-methyl- 11 C]WAY-100635 {[O-methyl- 11 C]N-2-(1-(4-(2-methoxypheny)-l-piperazinyl)-ethyl)-N-(2-pyridyl)-cyclohexanecarboxamide} is a promising radioligand for the study of 5-HT 1A receptors in human brain with positron emission tomography (PET). Mathematical modeling of the acquired PET data requires the knowledge of radioligand metabolism. After iv injection of [O-methyl- 11 C]WAY-100635 in five healthy male volunteers, radioactivity was found to clear rapidly from plasma. Radioactivity in serial plasma samples was analyzed by solid phase extraction and reverse phase high performance liquid chromatography (HPLC) of the retained components. In all samples, a polar fraction of plasma radioactivity was not retained by the solid phase extraction column. HPLC analyses of retained radioactivity revealed three major components, of which unchanged radioligandis the least polar. Of the radioactivity in plasma, on average 94.9% was unchanged radioligand at 2.5 min after iv injection and 4.5% at 60 min. One radioactive metabolite was found to coelute with descyclohexanecarbonyl-WAY-100635 (WA Y-100634). [ 11 c]WAY-100634 represented 35% of the radioactivity in plasma at 10 min and 26% at 60 min. At 60 min, polar radioactive metabolites represented 70.5% of the radioactivity in plasma. WAY-lO0634 is known to have high affinity for 5-HT 1A and α 1 -adrenoceptors and higher extraction into rat brain than WAY-lO0635. Thus, [O-methyl- 11 C]WAY-100634 may contribute to nonspecific and specific binding in human brain in PET studies with [O-methyl- 11 C]WAY-100635. It has been conclude that WAY-100635 labeled with carbon-11 in the amido carbonyl group might be the preferred for PET studies of 5-HT 1A receptors in humans because [ 11 C]WAY-100634 cannot be formed from this radioligand.

Development of PET Radioligands for the Quantitation of Serotonin Receptors in the Human Brain

Pharmacological studies have shown that there are multiple serotonin receptor subtypes, which are classified in at least seven classes. [1]. There has been a lack of subtype selective serotonin receptor radioligands suitable for PET imaging of the human brain. The 5-HT1a receptor is of particular interest as it may be involved in the pathophysiology of several neuropsychiatric disorders, like anxiety, depression and schizophrenia. Recently, a potent and selective 5-HT1a receptor antagonist, WAY-100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cyclohexanecarboxamide) was developed. Labelling of WAY-100635 in the methoxy position with 11C [2–3] provided the first radioligand for the delineation of 5-HT1a receptors in the human brain using positron emission tomography (PET) [4]. The descyclohexanecarbonyl analogue ([11C]WAY- 100634) was shown to be a labelled lipophilic metabolite in primates with high affinity to 5-HTlA receptors and high ability to enter the brain, so hampering the quantitation of the uptake of the radioligand in vivo [5]. WAY-100635 has recently been labelled with 11C also in the carbonyl position and examined in the human brain with PET [6].