John L. Neumeyer

Graphical, Kinetic, and Equilibrium Analyses of in vivo [123I]β-CIT Binding to Dopamine Transporters in Healthy Human Subjects

The in vivo kinetics of the dopamine (DA) transporter probe 123I-labeled 2β-carboxymethoxy-3β-(4-iodophenyl)tropane ([123I]β-CIT) in striatum was investigated with single-photon emission computerized tomography (SPECT) in five healthy human subjects. The aim of this study was to derive an adequate measure of the DA transporter density that would not be affected by regional cerebral blood flow or peripheral clearance of the tracer. SPECT data were acquired on the day of injection (day 1) from 0 to 7 h and on the following day (day 2) from 19 to 25 h. Arterial sampling on day 1 was used to measure the input function. Graphical, kinetic, and equilibrium analyses were evaluated. Graphical analysis of day 1 data, with the assumption of negligible dissociation of the tracer–receptor complex (k4 = 0), was found to be blood flow-dependent. A three-compartment kinetic analysis of day 1 data were performed using a three (k4 = 0)- and a four (k4 > 0)-parameter model. The three-parameter model estimated the konBmax product at 0.886 ± 0.087 min−1. The four-parameter model gave a binding potential (BP) of 476 ml g−1, a value consistent with in vitro measurements. The stability of the regional uptake on day 2 allowed direct measurement of the specific to nonspecific equilibrium partition coefficient (V3″ = k3/k4 = 6.66 ± 1.54). Results of day 1 kinetic analysis and day 2 equilibrium analysis were well correlated among subjects. Simulations indicated that the error associated with the day 2 equilibrium analysis was acceptable for plasma tracer terminal half-lives >10 h. We propose the equilibrium analysis on day 2 as the method of choice for clinical studies since it does not require multiple scans or the measurement of the arterial plasma tracer concentrations.

Recent Progress in Development of Dopamine Receptor Subtype-Selective Agents: Potential Therapeutics for Neurological and Psychiatric Disorders

Subtype-Selective Agents: Potential Therapeutics for Neurological and Psychiatric Disorders Na Ye,† John L. Neumeyer,‡ Ross J. Baldessarini, Xuechu Zhen, and Ao Zhang*,† †CAS Key Laboratory of Receptor Research, and Synthetic Organic & Medicinal Chemistry Laboratory (SOMCL), Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai, China 201203 ‡Medicinal Chemistry Laboratory, McLean Hospital, Harvard Medical School, Massachusetts 02478, United States Psychopharmacology Program, McLean Hospital, Massachusetts 02478, United States Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, China 215123

Differences in the nature of the stereotyped behaviour induced by aporphine derivatives in the rat and in their actions in extrapyramidal and mesolimbic brain areas

Apomorphine, (minus)-N-n-propylnorapomorphine [ (minus)-NPA ] and (plus or minus)-N-n-propylnorapomorphine [ (plus or minus)-NPA ] each caused stereotyped behaviour patterns in the rat which could be differentiated into two components, sniffing and repetitive head and limb movements (low intensity component) and gnawing, biting and licking (high intensity component). Low intensity components occurred at low doses of apomorphine and high intensity components at larger doses but the two components never occurred independently for (minus)-NPA or (plus or minus) NPA. Biting was the predominant effect of these agents which were shown to be at least twenty times more potent than apomorphine. The (minus)-isomer of NPA was the more potent. The two components of stereotypy were differentiated both pharmacologically (using amantadine, reserpine plus alpha-methyl-p-tyrosine and haloperidol) and by lesions placed in areas of the extrapyramidal (caudate--putamen, globus pallidus, substantia nigra) and mesolimbic (nucleus accumbens septi, tuberculum olfactorium, nucleus amygdaloideus centralis) systems. However, both sniffing and biting responses were reduced by lesions of the serotonergic raphe nuclei. The two stereotypic components were differentially induced by intracerebral injections of apomorphine and (minus)-NPA into the caudate--putamen, nucleus accumbens septi and tuberculum olfactorium. Injections into the central nucleus of the amygdala were ineffective. The degree of involvement of the different areas was shown to differ for apomorphine and (minus)-NPA, in particular the nucleus accumbens septi appeared more important for the action of (minus)-NPA and the tuberculum olfactorium for apomorphine. Intracaudate (minus)-NPA was less active than apomorphine but, generally, intracerebrally applied (minus)-NPA was twice as potent as apomorphine. Both (minus)-NPA and apomorphine caused circling behaviour in animals with asymmetric medial raphe nucleus lesions (contralateral) or unilateral lesions of the substantia nigra (ipsilateral). In these experiments (minus)-NPA was ten times more potent than apomorphine.

[11C]β-CIT, a cocaine analogue. Preparation, autoradiography and preliminary PET investigations

beta-CIT (2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane) is a cocaine analogue with a high affinity for the dopamine transporter. [11C] beta-CIT was prepared by N-methylation of nor-beta-CIT with [11C]methyl iodide. The total radiochemical yield of [11C] beta-CIT was 40-50% with an overall synthesis time of 35-40 min. The radiochemical purity was > 99% and the specific radioactivity at the time of injection was about 1000 Ci/mmol (37 GBq/mumol). Autoradiographic examination of [11C] beta-CIT binding in human brains post-mortem demonstrated a high level of specific binding in the striatum. PET examination of [11C] beta-CIT in a Cynomolgus monkey showed a marked accumulation of radioactivity in the striatum. The ratio of radioactivity in the striatum-to-cerebellum approached 5 after 87 min. In a displacement experiment, radioactivity in the striatum but not in the cerebellum, was markedly reduced after injection of unlabelled cocaine. [11C] beta-CIT has a potential as ligand for PET examination of cocaine effects in man.

Radiosynthesis of [18F] N-3-fluoropropyl-2-β-carbomethoxy-3-β-(4-iodophenyl) nortropane and the first human study with positron emission tomography☆

A procedure for the routine preparation of [18F]FP-CIT has been developed. Purification of the final product was achieved by preparative HPLC using phenethyl column without decomposition or epimerization. [18F] labeled-N-fluoropropyl-2 beta-carbomethoxy-3 beta-(4-iodophenyl) nortropane was prepared and PET imaging was performed on human subjects. A high uptake into striatal regions was observed. HPLC plasma analysis using [18F]FP-CIT indicated the presence of only one metabolite. By directly comparing the behavior of these three radiotracers ([18F]DOPA, [123I]FP-CIT, and [18F]FP-CIT) in the same subjects, we can enhance our understanding of the dopaminergic system as well as the relative potential of these techniques in a clinical research setting.

Evaluation of the monoamine uptake site ligand [131I]methyl 3β-(4-Iodophenyl)-tropane-2β-carboxylate ([123I]β-CIT) in non-human primates: Pharmacokinetics, biodistribution and SPECT brain imaging coregistered with MRI

Abstract The in vivo properties of a new radioiodinated probe of the dopamine and serotonin transporter, [123I]methyl 3β-(4-iodophenyl)tropane-2β-carboxylate ([123I]β-CIT) were evaluated in baboons and vervet monkeys. The labeled product was prepared in 65.2 ± 2.8% yield (mean ± SEM; n = 18) by reaction of the tributylstannyl precursor with [123I]NaI in the presence of peracetic acid followed by high pressure liquid chromatography (HPLC) purification to give a product with radiochemical purity of 97.5 ± 0.5% and specific activity of 500–1200 Ci/mmol. After intravenous administration, whole brain activity peaked at 6–10% injected dose within 1 h post injection (p.i.) and washed out in a biphasic manner with clearance half-lives of 1–2 and 7–35 h for the rapid and slow components, respectively. Excretion occurred primarily through the hepatobiliary route, with about 30% of the injected dose appearing in the GI tract after 5 h. Estimates of radiation absorbed dose gave 0.01, 0.1, 0.2 and 0.03 mGy/MBq to the brain, gall bladder wall, lower large intestine wall and urinary bladder wall, respectively. High resolution SPECT imaging in a baboon demonstrated high uptake of tracer in the region of the striatum (striatum: cerebellum ratio 4.0), in the hypothalamus (ratio 2.6) and in a midbrain region comprising raphe, substantia nigra and superior colliculus (ratio 2.0), with regional brain uptakes measured at 210 min p.i. of [123I]β-CIT. The anatomical locations of the regions on the SPECT image were confirmed by coregistration with MRI. Plasma metabolites and pharmacokinetics were analyzed in baboons and vervets by ethyl acetate extraction and HPLC. The major metabolite was a polar, non-extractable fraction, which increased to > 50% of the plasma activity by 30–45 min p.i. A minor lipophilic (extractable) metabolite was also observed, increasing to about 4% at 2–3 h p.i. The plasma protein bound fraction, determined by ultrafiltration, was 74.8 ± 1.4% (n = 6). The arterial input function was characterized by the sum of three exponential terms with half-lives of 0.3–1.7, 9.7–24.9 and 77–166 min, respectively, for the concentration of free parent compound. [123I]β-CIT promises to be a useful marker for SPECT study of the monoamine uptake system in primate brain.

[O-methyl-11C]β-CIT-FP, a potential radioligand for quantitation of the dopamine transporter: Preparation, autoradiography, metabolite studies, and positron emission tomography examinations

beta-CIT-FP [N-(3-fluoropropyl)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)nortropane] is a cocaine analogue with a high affinity for the dopamine transporter. [O-methyl-11C]beta-CIT-FP ([11C]beta-CIT-FP) was prepared by O-alkylation of the free acid with [11C]methyl iodide. The total radiochemical yield of [11C]beta-CIT-FP was 50 to 60% with an overall synthesis time of 30 min. The radiochemical purity was > 99%, and the specific radioactivity at time of injection was about 37 GBq/mumol (1000 Ci/mmol). Autoradiographic examination of [11C]beta-CIT-FP binding in human brain postmortem demonstrated specific binding in the caudate nucleus and putamen. Positron emission tomography (PET) examination of [11C]beta-CIT-FP in a Cynomolgus monkey demonstrated accumulation in the striatum with a striatum-to-cerebellum ratio of about 8 after 60 min. Equilibrium in the striatum was attained within 70 to 90 min. The radioactivity ratios of thalamus/cerebellum and neocortex/cerebellum were about 2 and 1.5, respectively. In a displacement experiment, radioactivity in the striatum but not in the cerebellum was reduced after injection of beta-CIT, indicating that striatal radioactivity following injection of [11C]beta-CIT-FP is associated with dopamine transporter sites and that the binding is reversible. The fraction of the total radioactivity in plasma representing [11C]beta-CIT-FP determined by high-performance liquid chromatography (HPLC) was 84% at 15 min and 50% at 95 min. [11C]beta-CIT-FP should be a useful PET radioligand for the quantitation of dopamine transporters in the human brain in vivo.

Further characterization of [3H]ifenprodil binding in rat brain☆

The present study was undertaken to characterize [3H]ifenprodil binding in rat brain. [3H]Ifenprodil showed saturable, high-affinity binding at 4 degrees C. Specific binding, defined with 10 microM ifenprodil as a competitor, was inhibited biphasically by the s receptor ligands, GBR 12909, 1,3-di-o-tolylguanidine (DTG), and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP). At 4 degrees C, 3 microM GBR 12909, which inhibited about 50% of specific binding of [3H]ifenprodil was used to mask sigma receptors. Under these conditions, specific binding of [3H]ifenprodil was inhibited potently by ifenprodil, SL 82.0715, poly(L-arginine), poly(L-lysine), neomycin, ruthenium red, spermine, arcaine and spermidine. In the presence of 3 microM GBR 12909, Zn2+ and Mg2+ partially inhibited specific binding of [3H]ifenprodil at 4 degrees C. In contrast, in the absence of GBR 12909, at 37 degrees C specific binding of [3H]ifenprodil was partially inhibited by Zn2+, but not by Mg2+. The anatomical distribution of [3H]ifenprodil binding at 4 degrees C (GBR 12909 included) in rat brain closely paralleled that of [3H]MK-801 (dizocilpine) binding (r = 0.971, P < 0.005). Without GBR 12909, specific [3H]ifenprodil binding at 37 degrees C was inhibited potently by sigma ligands. In the presence of 3 microM GBR 12909, [3H]ifenprodil binding at 4 degrees C was highest in synaptosomal and myelin fractions; however, without GBR 12909, [3H]ifenprodil binding at 37 degrees C was highest in microsomal and myelin fractions, consistent with the subcellular distribution of sigma receptors. The results suggest that, in the presence of 3 microM GBR 12909, at 4 degrees C, [3H]ifenprodil binds to sites that are sensitive to polyamines and related compounds; and that without GBR 12909, at 37 degrees C, [3H]ifenprodil interacts with sigma receptors in rat brain.

Long-Term Effects of S(+)N-n-Propylnorapomorphine Compared with Typical and Atypical Antipsychotics: Differential Increases of Cerebrocortical D2-Like and Striatolimbic D4-Like Dopamine Receptors

Changes in D2-like dopamine (DA) receptor binding in rat brain regions were compared by quantitative in vitro receptor autoradiography after 21-d treatment with a typical (fluphenazine), atypical (clozapine), or candidate atypical antipsychotic (S[+]-N-n-propylnorapomorphine, [+]-NPA). Fluphenazine treatment significantly increased binding of the D2,3,4 radioligands [3H]nemonapride and [3H]spiperone in caudate-putamen (CPu: 22%, 32%), nucleus accumbens (ACC: 67%, 52%), olfactory tubercle (OT: 53%, 43%), and medial prefrontal cerebral cortex (MPC: 46%, 47%) but not dorsolateral frontal cortex (DFC). D2-like binding in MPC was also increased by (+)-NPA (49%, 39%) and clozapine (60%, 40%), but not in DFC, CPu, ACC, or OT. Binding of D2,3-selective [3H]raclopride increased less after fluphenazine in ACC (27%) and CPu (16%) than with the nonselective radioligands, and not after clozapine or (+)-NPA. D3-selective binding of [3H]R(+)-7-OH-DPAT was not changed with any treatment or region including islands of Calleja. Binding of [3H]nemonapride or [3H]spiperone under D4-selective conditions (with 300 nM S[−]-raclopride and other masking agents, at sites occluded by D4 ligand L-745,870), was increased by fluphenazine, (+)-NPA, clozapine in ACC (120%, 76%, 70%, respectively), and CPu (54%, 37%, 35%), but not in OT, DFC or MPC. These results support the hypothesis that cerebrocortical D2-like and striatolimbic D4-like receptors contribute to antipsychotic actions of both typical and atypical drugs and encourage further consideration of S(+)aporphines as potential atypical antipsychotics.

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under “Physiological” Conditions

Abstract: Methyl 3β‐(4‐[125I]iodophenyl)tropane‐2β‐carboxylate ([123I]β‐CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5‐HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [123I]β‐CIT in vivo binding to DA transporters had a much slower washout than binding to 5‐HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [125I]β‐CIT for DA and 5‐HT transporters. We now report [125I]β‐CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5‐HT transporters was compatible with one population of sites. At 22°C, [125I]β‐CIT showed similar affinity to high‐affinity DA (0.39 nM) and 5‐HT transporter sites (0.47 nM). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5‐HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [123 I]β‐CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.