Victor W. Pike

Characterisation of the Appearance of Radioactive Metabolites in Monkey and Human Plasma from the 5-HT1A Receptor Radioligand, [carbonyl-11C]WAY-100635—Explanation of High Signal Contrast in PET and an Aid to Biomathematical Modelling

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl)++ +cyclohexanecarboxamide (WAY-100635), labelled in its amido carbonyl group with 11C (t1/2 = 20.4 min), is a promising radioligand for the study of brain 5-HT1A receptors with positron emission tomography (PET). Thus, in PET experiments in six cynomolgus monkeys and seven healthy male volunteers, [carbonyl-11C]WAY-100635 was taken up avidly by brain. Radioactivity was retained in regions rich in 5-HT1A receptors, such as occipital cortex, temporal cortex and raphe nuclei, but cleared rapidly from cerebellum, a region almost devoid of 5-HT1A receptors. [Carbonyl-11C]WAY-100635 provides about 3- and 10-fold higher signal contrast (receptor-specific to nonspecific binding) than [O-methyl-11C]WAY-100635 in receptor-rich areas of monkey and human brain, respectively. To elucidate the effect of label position on radioligand behaviour and to aid in the future biomathematical interpretation of the kinetics of regional cerebral radioactivity uptake in terms of receptor-binding parameters, HPLC was used to measure [carbonyl-11C]WAY-100635 and its radioactive metabolites in plasma at various times after intravenous injection. Radioactivity cleared rapidly from monkey and human plasma. Parent radioligand represented 19% of the radioactivity in monkey plasma at 47 min and 8% of the radioactivity in human plasma at 40 min. [Carbonyl-11C]desmethyl-WAY-100635 was below detectable limits in monkey plasma and at most a very minor radioactive metabolite in human plasma. [11C]Cyclohexanecarboxylic acid was identified as a significant radioactive metabolite. In human plasma this maximally represented 21% of the radioactivity at 10 min after radioligand injection. All other major radioactive metabolites in monkey and human plasma were even more polar. No-carrier-added [carbonyl-11C]cyclohexanecarboxylic acid was prepared in the laboratory and after intravenous administration into cynomolgus monkey was shown with PET to give only a low uptake of radioactivity into brain tissue. The acid rapidly gave rise to several radioactive metabolites of higher polarity in plasma. The observed lack of any significant metabolism of [carbonyl-11C]WAY-100635 to highly lipophilic or pharmacologically potent radioactive compounds is consistent with its high signal contrast in primate brain.

Imaging the GABA-Benzodiazepine Receptor Subtype Containing the α5-Subunit In Vivo with [11C]Ro15 4513 Positron Emission Tomography:

There is evidence of marked variation in the brain distribution of specific subtypes of the GABA-benzodiazepine receptor and that particular subtypes mediate different functions. The α5-containing subtype is highly expressed in the hippocampus, and selective α5 inverse agonists (which decrease tonic GABA inhibition) are being developed as potential memory-enhancing agents. Evidence for such receptor localization and specialization in humans in vivo is lacking because the widely used probes for imaging the GABA-benzodiazepine receptors, [11C]flumazenil and [123I]iomazenil, appear to reflect binding to the α1 subtype, based on its distribution and affinity of flumazenil for this subtype. The authors characterized for positron emission tomography (PET) a radioligand from Ro15 4513, the binding of which has a marked limbic distribution in the rat and human brain in vivo. Competition studies in vivo in the rat revealed that radiolabeled Ro15 4513 uptake was reduced to nonspecific levels only by drugs that have affinity for the α5 subtype (flunitrazepam, RY80, Ro15 4513, L655,708), but not by the α1 selective agonist, zolpidem. Quantification of [11C]Ro15 4513 PET was performed in humans using a metabolite-corrected plasma input function. [11C]Ro15 4513 uptake was relatively greater in limbic areas compared with [11C]flumazenil, but lower in the occipital cortex and cerebellum. The authors conclude that [11C]Ro15 4513 PET labels in vivo the GABA-benzodiazepine receptor containing the α5 subtype in limbic structures and can be used to further explore the functional role of this subtype in humans.

Striatal and temporal cortical D2/D3 receptor occupancy by olanzapine and sertindole in vivo: a [123I]epidepride single photon emission tomography (SPET) study.

Abstract. Rationale: Previous work suggests clozapine preferentially targets limbic cortical dopamine systems, which could help account for its lack of extrapyramidal side effects (EPS) and superior therapeutic efficacy. Objectives: To test the hypothesis that olanzapine, a novel atypical antipsychotic drug, occupies temporal cortical D2/D3 receptors to a greater extent than striatal D2/D3 receptors in vivo. Methods: Nine schizophrenic patients taking either olanzapine [(n=5; mean (SD) age: 32.5 (6.5)xa0years; daily dose: 18.3 (2.6)xa0mg] or sertindole [(n=4; mean (SD) age: 30.3 (7.4)xa0years; daily dose: 16 (5.6)xa0mg] were studied with [123I]epidepride ((S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxy-benzamide) and single photon emission tomography (SPET). An estimate of [123I]epidepride specific binding to D2/D3 receptors was obtained in patients and age-matched healthy volunteers. A summary measure was generated representing striatal and temporal cortical relative %D2/D3 receptor occupancy by antipsychotic drugs. Occupancy data were compared with previously studied groups of patients receiving typical antipsychotic drugs (n=12) and clozapine (n=10). Results: Mean striatal and temporal cortical %D2/D3 receptor occupancy in olanzapine-treated patients was 41.3% (SD 17.9) and 82.8% (SD 4.2), respectively. Unexpectedly low levels of striatal relative %D2/D3 receptor occupancy were seen in two patients with typical antipsychotic-drug-induced movement disorder prior to switching to olanzapine. In the temporal cortex, mean D2/D3 dopamine receptor occupancy levels above 80% were seen for all antipsychotic drugs studied. Conclusions: The atypical antipsychotic drugs olanzapine and sertindole, in common with clozapine, demonstrate higher occupancy of temporal cortical than striatal D2/D3 dopamine receptors in vivo at clinically useful doses. This could help mediate their atypical clinical profile of therapeutic efficacy with few extrapyramidal side effects. Limbic selective blockade of D2/D3 dopamine receptors could be a common action of atypical antipsychotic drugs.

New synthesis of diaryliodonium sulfonates from arylboronic acids

Abstract Diaryliodonium salts, precursors to [ 18 F]fluoroaromatics, have been prepared, in a regioselective manner, from readily available arylboronic acids eliminating the need for acid sensitive and toxic organotin intermediates.

[carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is a potent and selective radioligand for central 5-HT1A receptors in vitro and in vivo.

Abstract.u2007[carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is possibly a low-level metabolite appearing in plasma after intravenous administration of [carbonyl-11C]WAY-100635 to human subjects for positron emission tomographic (PET) imaging of brain 5-HT1A receptors. In this study we set out to assess the ability of DWAY to enter brain in vivo and to elucidate its possible interaction with 5-HT1A receptors. Desmethyl-WAY-100635 was labelled efficiently with carbon-11 (t1/2xa0=xa020.4xa0min) in high specific radioactivity by reaction of its descyclohexanecarbonyl analogue with [carbonyl-11C]cyclohexanecarbonyl chloride. The product was separated in high radiochemical purity by high-performance liquid chromatography (HPLC) and formulated for intravenous injection. Rats were injected intravenously with DWAY, sacrificed at known times and dissected to establish radioactivity content in brain tissues. At 60xa0min after injection, the ratios of radioactivity concentration in each brain region to that in cerebellum correlated with previous in vitro and in vivo measures of 5-HT1A receptor density. The highest ratio was about 22 in hippocampus. Radioactivity cleared rapidly from plasma; HPLC analysis revealed that DWAY represented 55% of the radioactivity in plasma at 5xa0min and 33% at 30xa0min. Only polar radioactive metabolites were detected. Subsequently, a cynomolgus monkey was injected intravenously with DWAY and examined by PET. Maximal whole brain uptake of radioactivity was 5.7% of the administered dose at 5xa0min after injection. The image acquired between 9 and 90xa0min showed high radioactivity uptake in brain regions rich in 5-HT1A receptors (e.g. frontal cortex and neocortex), moderate uptake in raphe nuclei and low uptake in cerebellum. A transient equilibrium was achieved in cortical regions at about 60xa0min, when the ratio of radioactivity concentration in frontal cortex to that in cerebellum reached 6. The corresponding ratio for raphe nuclei was about 3. Radioactive metabolites appeared rapidly in plasma, but these were all more polar than DWAY, which represented 52% of the radioactivity in plasma at 4xa0min and 20% at 55xa0min. In a second PET experiment, in which a cynomolgus monkey was pretreated with the selective 5-HT1A receptor antagonist, WAY-100635, at 25xa0min before DWAY injection, radioactivity in all brain regions was reduced to that in cerebellum. Autoradiography of post mortem human brain cryosections after incubation with DWAY successfully delineated 5-HT1A receptor distribution. Receptor-specific binding was eliminated in the presence of the selective 5-HT1A receptor agonist, 8-OH-DPAT [(±)-8-hydroxy-2-dipropylaminotetralin]. These findings show that: (a) intravenously administered DWAY is well able to penetrate brain in rat and monkey, (b) DWAY is a highly effective radioligand for brain 5-HT1A receptors in rat and monkey in vivo and for human brain in vitro, and (c) the metabolism and kinetics of DWAY appear favourable to successful biomathematical modelling of acquired PET data. Thus, DWAY warrants further evaluation as a radioligand for PET studies of 5-HT1A receptors in human brain.

Radioligands for the study of brain 5-HT1A receptors in vivo–development of some new analogues of way

[Carbonyl-(11)C]WAY-100635 (WAY) has proved to be a very useful radioligand for the imaging of brain 5-HT(1A) receptors in human brain in vivo with positron emission tomography (PET). WAY is now being applied widely for clinical research and drug development. However, WAY is rapidly cleared from plasma and is also rapidly metabolised. A comparable radioligand, with a higher and more sustained delivery to brain, is desirable since these properties might lead to better biomathematical modelling of acquired PET data. There are also needs for other types of 5-HT(1A) receptor radioligands, for example, ligands sensitive to elevated serotonin levels, ligands labelled with longer-lived fluorine-18 for distribution to satellite PET centres, and ligands labelled with iodine-123 for single photon emission computerised tomography (SPECT) imaging. Here we describe our progress toward these aims through the exploration of WAY analogues, including the development of [carbonyl-(11)C]desmethyl-WAY (DWAY) as a promising, more brain-penetrant radioligand for PET imaging of human 5-HT(1A) receptors, and (pyridinyl-6-halo)-analogues as promising leads for the development of radiohalogenated ligands.

Evaluation of [11C]GB67, a novel radioligand for imaging myocardial α1-adrenoceptors with positron emission tomography

Abstract. Dysfunction of the sympathetic nervous system underlies a number of myocardial disorders. Positron emission tomography (PET) offers a way of assessing receptor function non-invasively in humans, but there are no PET radioligands for assessing myocardial α-adrenoceptors. GB67, a structural and pharmacological analogue of the α1-adrenoceptor antagonist prazosin, was labelled with positron-emitting carbon-11 (t1/2=20.4 min) by 11C-methylation of N-desmethylamido-GB67 (GB99). [11C]GB67 was injected intravenously into conscious rats. Serial arterial blood samples were taken. Rats were killed and tissues removed to determine radioactivity. The percentages of unchanged [11C]GB67 and its radioactive metabolites in plasma and tissues were assessed by HPLC. Plasma clearance of radioactivity was rapid. Myocardial uptake was maximal at 1–2 min and decreased slowly during 60 min. Predosing with adrenoceptor antagonists demonstrated selectivity for myocardial α1-adrenoceptors. GB67 and prazosin blocked uptake of radioactivity; the non-selective antagonist, phentolamine, partially blocked uptake; the α2-adrenoceptor antagonist, RX 821002, only blocked uptake at high dose and the β-adrenoceptor antagonist, CGP 12177, had no effect. Additionally, injection of prazosin at 20 min after radioligand displaced radioactivity. In vivo competition curves obtained by injecting [11C]GB67 with varying amounts of either unlabelled GB67 or its precursor GB99 were fitted to a competitive binding model to provide estimates of the maximum number of binding sites (Bmax) and half saturation doses (K) for myocardium. Assuming a tissue protein content of 10%, the values of Bmax [∼13 pmol·(g tissue)–1] were similar to those [50–170 fmol·(mg protein)–1] reported for myocardial α1-adrenoceptors assessed in vitro. Both GB67 and its precursor GB99 had high affinity for α1-adrenoceptors [KGB67=1.5 nmol·(kg body weight)–1, KGB99=4.8 nmol· (kg body weight)–1]. HPLC demonstrated four radioactive metabolites in plasma. [11C]GB67 was 80% of the radioactivity at 5 min and 50% at 45 min. No radioactive metabolites were detected in myocardium up to 60 min after injection. [11C]GB67 was assessed in two male human volunteers. PET demonstrated high myocardial uptake. The profile of radioactive metabolites in plasma was comparable to that in the rat, although metabolism was slower in humans. Thus, [11C]GB67 is a promising radioligand for assessing α1-adrenoceptors in human myocardium with PET.

Radioligands for the study of brain 5-HT 1A receptors in vivo

Publisher Summary This chapter reviews that serotonin (5-hydroxytryptamine, 5-HT, 1) is a major brain neurotransmitter that elicits a multitude of physiological functions by interactions with various receptors. The 5-HT1A receptor is structurally, pharmacologically, and functionally the best characterized of all known serotonin receptors. There is substantial evidence, mainly from post-mortem studies, to associate changes in this receptor population with several neuropsychiatric disorders, including depression and suicide, schizophrenia, alcoholism, and Parkinsons disease. The brain 5-HT1A receptor is also a functional target in both established and new drug therapies. The chapter is concerned with important recent advances for the quantitative imaging of brain 5-HT1A receptors in living animal and human brain, based on the use of novel radioligands, primarily with the non-invasive technique of positron emission tomography (PET), but also with single photon emission tomography. It addresses the development of the required radioligands and also indicates why such imaging can be valuable in clinical research and drug development. It is valuable to consider the identity, structure, location and function of brain 5-HT1A receptors, and understanding of their molecular interactions with various ligands.

The synthesis of [18F]fluoroarenes from the reaction of cyclotron-produced [18F]fluoride ion with diaryliodonium salts

Diaryliodonium salts have been shown to react with fluoride ion at 80xa0°C in acetonitrile to generate aryl fluorides. The regioselectivity is controlled electronically and by the bulk of the ortho-substituents on the rings, with the latter the dominant factor such that electron-rich rings can be fluorinated. ortho-Substituted aryl fluorides can be selectively produced from unsymmetrical diaryliodonium salts. The process has been used to synthesise [18F] labelled aromatics by the use of cyclotron generated [18F]fluoride ion.

Improved syntheses of the PET radioligands, [11C]FLB 457, [11C]MDL 100907 and [11C]β-CIT-FE, by the use of [11C]methyl triflate

The highly reactive labelling agent, [11C]methyl triflate, was used to synthesise three recently developed PET radioligands. The labelling reactions were 11C-methylations of phenols for the preparations of the D2 receptor radioligand, [11C]FLB 457, and the 5-HT2A receptor radioligand, [11C]MDL 100907, and 11C-methylation of a carboxylic acid for the preparation of the dopamine transporter radioligand, [11C]β-CIT-FE. The synthesis of the 5-HT1A receptor radioligand, [O-methyl-11C]WAY-100635, was used to establish general reaction conditions for the methylation of phenols with [11C]methyl triflate. Compared to the previous use of [11C]methyl iodide in these radiosyntheses, [11C]methyl triflate demanded less precursor, allowed faster reactions and improved radiochemical yields. Normal-phase HPLC was used to speed up product purifications (except for [11C]β-CIT-FE). Hence, preparation times were shorter, resulting in radioligands with higher specific radioactivity.