Judit Sóvágó

Specific in vivo binding to the norepinephrine transporter demonstrated with the PET radioligand, (S,S)-[11C]MeNER.

(S,S)-2-(alpha-(2-Methoxyphenoxy)benzyl)morpholine (MeNER), an O-methyl analog of the selective and potent norepinephrine transporter (NET) inhibitor, (S,S)-reboxetine, and its less active enantiomer, (R,R)-MeNER, have each been radiolabeled by O-methylation of their corresponding phenolic precursors in good yields from [(11)C]methyl iodide or [(11)C]methyl triflate. Radiochemical purities were >99% and specific radioactivity at time of injection was about 74 GBq/micromol. Autoradiographic examination of (S,S)-[(11)C]MeNER binding to human brain slices post mortem indicated specific binding in a brain region including the locus coeruleus. PET examination of both [(11)C]MeNER enantiomers in a cynomolgus monkey demonstrated a higher specific binding of the (S,S)-enantiomer with ratios of 1.4-1.6 in the lower brainstem, mesencephalon and thalamus to striatum. Pretreatment with the NET ligand, desipramine, decreased the specific binding of (S,S)-[(11)C]MeNER. Labeled metabolites of [(11)C]MeNER were all more polar. (S,S)-[(11)C]MeNER is a good lead compound in the search for a selective radioligand for quantitation of NET in the human brain in vivo.

Drug distribution in man: a positron emission tomography study after oral administration of the labelled neuroprotective drug vinpocetine

Abstract. Direct information on the distribution of a drug requires measurements in various tissues. Such data have until now been obtained in animals, or have indirectly been calculated from plasma measurements in humans using mathematical models. Here we suggest the use of positron emission tomography (PET) as a method to obtain direct measurements of drug distribution in the human body. The distribution in body and brain of vinpocetine, a neuroprotective drug widely used in the prevention and treatment of cerebrovascular diseases, was followed after oral administration. Vinpocetine was labelled with carbon-11 and radioactivity was measured by PET in stomach, liver, brain and kidney in six healthy volunteers. The radioactivity in blood and urine as well as the fractions of [11C]vinpocetine and labelled metabolites in plasma were also determined. After oral administration, [11C]vinpocetine appeared immediately in the stomach and within minutes in the liver and the blood. In the blood the level of radioactivity continuously increased until the end of the measurement period, whereas the fraction of the unchanged mother compound decreased. Radioactivity uptake and distribution in the brain were demonstrable from the tenth minute after the administration of the labelled drug. Brain distribution was heterogeneous, similar to the distribution previously reported after intravenous administration. These findings indicate that vinpocetine, administered orally in humans, readily enters the bloodstream from the stomach and gastrointestinal tract and, consequently, passes the blood-brain barrier and enters the brain. Radioactivity from [11C]vinpocetine was also demonstrated in the kidneys and in urine, indicating that at least a part of the radioactive drug and labelled metabolites is eliminated from the body through the kidneys. This study is the first to demonstrate that PET might be a useful, direct and non-invasive tool to study the distribution and pharmacokinetics of orally administered labelled CNS drugs in the living human body.

Positron emission tomographic evaluation of the putative dopamine-D3 receptor ligand, [11C]RGH-1756 in the monkey brain.

The dopamine-D3 receptor is of special interest due to its postulated role in the pathophysiology and treatment of schizophrenia and Parkinsons Disease. Increasing evidences support the assumption that the D3 receptors are occupied to a high degree by dopamine at physiological conditions. Research on the functional role of the D3 receptors in brain has however been hampered by the lack of D3 selective ligands. In the present Positron Emission Tomography (PET) study the binding of the novel, putative dopamine-D3 receptor ligand, [11C]RGH-1756 was characterized in the cynomolgus monkey brain. [11C]RGH-1756 was rather homogenously distributed in brain and the regional binding potential (BP) values ranged between 0.17 and 0.48. Pretreatment with unlabelled RGH-1756 decreased radioligand binding to the level of the cerebellum in most brain areas. The regional BP values were lower after intravenous injection of a higher mass of RGH-1756, indicating saturable binding of [11C]RGH-1756. The D2/D3 antagonist raclopride partly inhibited the binding of [11C]RGH-1756 in several brain areas, including the striatum, mesencephalon and neocortex, whereas the 5HT(1A) antagonist WAY-100635 had no evident effect on [11C]RGH-1756 binding. Despite the promising binding characteristics of RGH-1756 in vitro the present PET-study indicates that [11C]RGH-1756 provides a low signal for specific binding to the D3 receptor in vivo. One explanation is that the favorable binding characteristics of RGH-1756 in vitro are not manifested in vivo. Alternatively, the results may support the hypothesis that the dopamine-D3 receptors are indeed occupied to a high extent by dopamine in vivo and thus not available for radioligand binding.

Lack of effect of reserpine-induced dopamine depletion on the binding of the dopamine-D3 selective radioligand, [11C]RGH-1756

The effect of reserpine induced dopamine depletion on the binding of the putative dopamine-D3 receptor ligand, [(11)C]RGH-1756 was examined in the monkey brain with positron emission tomography (PET). In a previous series of experiments, we have made an attempt to selectively label D3 receptors in the monkey brain using [(11)C]RGH-1756. Despite high selectivity and affinity of RGH-1756 in vitro, [(11)C]RGH-1756 displayed only low specific binding to D3 receptors in vivo. The aim of the present study was to examine whether low specific binding of [(11)C]RGH-1756 is caused by insufficient in vivo affinity of the ligand, or by high physiological occupancy of D3 receptors by endogenous dopamine (DA). PET experiments were performed in three monkeys under baseline conditions and after administration of reserpine (0.5 mg/kg). The results of the baseline measurements corresponded well to our earlier observations with [(11)C]RGH-1756. Reserpine caused no evident change in the regional distribution of [(11)C]RGH-1756 in the monkey brain, and no conspicuous regional accumulation of activity could be observed. After reserpine treatment there was no evident increase of specific binding and binding potential (BP) of [(11)C]RGH-1756. The lack of increased [(11)C]RGH-1756 binding after reserpine treatment indicates that competition with endogenous DA is not the predominant reason for the failure of the radioligand to label D3 receptors. Therefore, the low binding of [(11)C]RGH-1756 could largely be explained by the need for very high affinity of radioligand for D3 receptors in vivo, to obtain a suitable signal for the minute densities of D3 receptors expressed in the primate brain.

Cerebral uptake of [ethyl-11C]vinpocetine and 1-[11C]ethanol in cynomolgous monkeys: a comparative preclinical PET study

PET provides the potential to quantify the distribution of radiolabelled drugs in the human body. In cases when radiolabelled compounds undergo metabolic transformation after administration in vivo, it is necessary to examine the kinetics and distribution of both the labeled mother compound and labeled metabolites. The objective of this study was to assess the extent by which 11C-labeled ethanol, the product arising from the de-esterification of the neuroprotective drug vinpocetine (ethyl-apovincaminate), might contribute to the regional cerebral radioactivity measured by PET after the administration of [ethyl-11C]vinpocetine. In three cynomolgous monkeys PET measurements were made after intravenous bolus injection of both [11C]vinpocetine and 1-[11C]ethanol. There was a marked difference between the regional time-activity curves of [11C]ethanol and [11C]vinpocetine. The distribution pattern obtained with [11C]ethanol was similar to that observed with blood flow tracers such as [15O]water and [15O]butanol. The study shows that although [11C]ethanol may moderately contribute to the brain radioactivity distribution pattern of [11C]vinpocetine, the rapid degradation of [11C]ethanol makes it unlikely that the contribution of this metabolite is of importance. The distinct distribution patterns and kinetics of [11C]vinpocetine and [11C]ethanol also support the view, obtained from our previous observations, that vinpocetine may bind to specific sites in the monkey and human brain, especially in the thalamus.

Autoradiographic mapping of dopamine-D2/D3 receptor stimulated [35S]GTPγS binding in the human brain

Agonist stimulated [35S]guanosine 5′‐γ‐thiotriphosphate ([35S]GTPγS) binding autoradiography was established for the examination of dopamine‐D2/D3 receptors in human brain sections. The distribution of G proteins activated by dopamine‐D2/D3 receptors was studied in whole hemisphere cryosections. Dopamine stimulated [35S]GTPγS binding in brain regions with high densities of dopamine D2‐like receptors, i.e. putamen (23 ± 2%, mean ± SEM,% stimulation over basal binding), caudate (20 ± 0%) and substantia nigra (22 ± 2%), but also in regions with lower receptor densities such as amygdala (17 ± 8%), hippocampus (16 ± 6%), anterior cingulate (13 ± 3%), and thalamus (12 ± 2%). Dopamine stimulated [35S]GTPγS binding to significantly higher levels in the dorsal than in the ventral part of the striatum. Dopamine caused low or very low stimulation in all cortical areas. Raclopride, a selective D2/D3 receptor antagonist, potently inhibited dopamine stimulated [35S]GTPγS binding, whereas R(+)‐7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine hydrochloride (SCH23390), a selective D1 antagonist, did not block the [35S]GTPγS binding response stimulated by dopamine. Hence, the stimulatory effect of dopamine was primarily mediated by D2/D3 receptors. Quinpirole stimulated [35S]GTPγS binding in the same regions as dopamine. The maximal level of stimulation induced by dopamine and quinpirole was not significantly different. The present study demonstrates that agonist stimulated [35S]GTPγS binding autoradiography could be a suitable technique for the examination of dopamine‐D2/D3 receptors in the human brain. This functional assay could provide useful new information about dopamine receptor/G protein coupling in the postmortem human brain, and reveal possible disease related alterations of the interaction between D2/D3 receptors and G proteins.

Two C-Methyl Derivatives of [11C]WAY-100635 – Effects of an Amido α-Methyl Group on Metabolism and Brain 5-HT1A Receptor Radioligand Behavior in Monkey*

Purpose[carbonyl-11C]N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl)ethyl)-N-pyridinyl)cyclohexanecarboxamide ([carbonyl-11C]WAY-100635) is an effective radioligand for imaging brain 5-HT1A receptors with positron emission tomography (PET). However, this radioligand has some drawbacks for deriving relative regional receptor densities, including rapid metabolism, which acts against accurate definition of an arterial input function for compartmental modeling, and very low nonspecific binding in brain, which detracts from the accuracy of modeling by a simplified reference tissue (cerebellum) approach. Here, in a search for a radioligand that overcomes these limitations, we investigated the effects of introducing a single methyl group at either of the carbon atoms alpha to the amide bond in [11C]WAY-100635.ProceduresLigands with a methyl group on the alpha carbon of the cyclohexyl group (SWAY) or the alpha carbon of the C2H4 linker ((R,S)-JWAY) were synthesized and tested for binding affinity and intrinsic activity at 5-HT1A receptors. SWAY was labeled with carbon-11 (t1/2 = 20.4 minutes; β+ = 99.8%) in its O-methyl group and (R,S)-JWAY in its carbonyl group. Each radioligand was evaluated by PET experiments in cynomolgus monkey.ResultsSWAY and (R,S)-JWAY were found to be high-affinity antagonists at 5-HT1A receptors. After injection of [11C]SWAY into monkey, radioactivity uptake in brain reached a maximum of 3% at 4.5 minutes and decreased to 0.7% at 72 minutes. However, over the time span of the experiment, radioactivity concentrations in 5-HT1A receptor-rich brain regions were only fractionally higher than in cerebellum. Radioactivity represented by parent radioligand in plasma was 39% at 45 minutes. After injection of [11C](R,S)-JWAY alone, radioactivity uptake in brain reached a maximum of 4.8% at 2.5 minutes and decreased to 1.2% at 90 minutes. At this time, radioactivity concentration in 5-HT1A receptor-rich brain regions was markedly greater than in cerebellum. In another PET experiment, the monkey was predosed with WAY-100635 before [11C](R,S)-JWAY injection. At 90 minutes after injection, the ratio of radioactivity in 5-HT1A receptor-rich regions to that in cerebellum was reduced to near unity. Radioactivity represented by parent radioligand in plasma was 12% at 45 minutes.Conclusions[11C](R,S)-JWAY, but not [11C]SWAY, gives a sizeable 5-HT1A receptor-selective PET signal in monkey. The presence of a C-methyl group adjacent to the amide bond in SWAY or (R,S)-JWAY fails to counter metabolism.

A novel principle of radiotracer administration in human brain mapping: pet studies on brain uptake and distribution of orally and intravenously administered [11C]vinpocetine

Introduction: Vinpocetine is a nootropic drug used world-wide in the prevention and treatment of ischaemic stroke and other cerebrovascular diseases. Former PET studies have indicated that [llC]vinpocetine, administered intravenously in monkeys, passes readily the blood-brain-barrier and about 5% of the injected radioactivity appears in the brain wherein the distribution of radioactivity is heterogeneous, indicating binding to specific sites (1). Objectives: The main objective of the present investigation was to test a novel principle in human brain mapping with PET: whether radiotracers can be administered orally in quantitative PET measurements. In addition, the experiments aimed at a comparison of global uptake and regional distribution values of [ 1 lC]vinpocetine in the human brain following oral and intravenous administration.

Synthesis of 11C-labeled (S,S)-CFMME and (R)-OHDMI and their evaluation as candidate radioligands for imaging norepinephrine transporters with PET

Introduction: (R)-1-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-3-methylamino-propan-2-ol ((R)-OHDMI) and (S,S)-1-cyclopentyl-2-(5-fluoro2-methoxy-phenyl)-1-morpholin-2-yl-ethanol ((S,S)-CFMME) were synthesized and found to be potent inhibitors of norepinephrine reuptake (inhibitory constants (K is) were 8.29 and 10.8 nM for (R)-OHDMI and (S,S)-CFMME, respectively, as compared with (S,S)-2-(a-(2methoxyphenoxy)benzyl)morpholine (S,S)-MeNER) which has a Ki of 7.93 nM). Here, we have explored the labeling of these ligands with carbon-11 for evaluation as candidate radioligands for imaging central norepinephrine transporters (NET).