William B. Mathews

Quantitative PET studies of the serotonin transporter in MDMA users and controls using [11C]McN5652 and [11C]DASB

(±)3,4-Methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) is a widely used illicit drug that produces toxic effects on brain serotonin axons and axon terminals in animals. The results of clinical studies addressing MDMAs serotonin neurotoxic potential in humans have been inconclusive. In the present study, 23 abstinent MDMA users and 19 non-MDMA controls underwent quantitative positron emission tomography (PET) studies using [11C]McN5652 and [11C]DASB, first- and second-generation serotonin transporter (SERT) ligands previously validated in baboons for detecting MDMA-induced brain serotonin neurotoxicity. Global and regional distribution volumes (DVs) and two additional SERT-binding parameters (DVspec and DVR) were compared in the two subject populations using parametric statistical analyses. Data from PET studies revealed excellent correlations between the various binding parameters of [11C] McN5652 and [11C]DASB, both in individual brain regions and individual subjects. Global SERT reductions were found in MDMA users with both PET ligands, using all three of the above-mentioned SERT-binding parameters. Preplanned comparisons in 15 regions of interest demonstrated reductions in selected cortical and subcortical structures. Exploratory correlational analyses suggested that SERT measures recover with time, and that loss of the SERT is directly associated with MDMA use intensity. These quantitative PET data, obtained using validated first- and second-generation SERT PET ligands, provide strong evidence of reduced SERT density in some recreational MDMA users.

In Vivo Detection of Short- and Long-Term MDMA Neurotoxicity—A Positron Emission Tomography Study in the Living Baboon Brain

The present study evaluated short‐ and long‐term effects of MDMA (3,4‐methylenedioxymethamphetamine) in the baboon brain using PET and [11C](+)McN 5652, a potent 5‐HT transporter ligand, as well as [11C]RTI‐55, a cocaine derivative which labels both 5‐HT and dopamine transporters. Following baseline PET scans with [11C](+)McN5652, [11C](−)McN5652 (the inactive enantiomer of the active enantiomer [11C](+)McN5652) and [11C]RTI‐55, a baboon was treated with MDMA (5 mg/kg, s.c., twice daily for four consecutive days). PET studies at 13, 19, and 40 days post‐MDMA revealed decreases in mean radioactivity levels in all brain regions when using [11C](+)McN 5652, but not with [11C](−)McN5652 or [11C]RTI‐55. Reductions in specific [11C](+)McN5652 binding (calculated as the difference in radioactivity concentrations between (+) and (−)[11C]McN5652) ranged from 44% in the pons to 89% in the occipital cortex. PET studies at 9 and 13 months showed regional differences in the apparent recovery of 5‐HT transporters, with increases in some brain regions (e.g., hypothalamus) and persistent decreases in others (e.g., neocortex). Data obtained from PET studies correlated well with regional 5‐HT axonal marker concentrations in the CNS measured after sacrifice of the animal. The results of these studies indicate that PET imaging of the living nonhuman primate brain with [11C](+)McN 5652 can detect changes in regional 5‐HT transporter density secondary to MDMA‐induced neurotoxicity. Using PET, it should also be feasible to use [11C](+)McN5652 to determine whether human MDMA users are also susceptible to MDMAs neurotoxic effects. Synapse 29:183–192, 1998.

Positron Emission Tomographic Studies of Brain Dopamine and Serotonin Transporters in Abstinent (±) 3,4-Methylenedioxymethamphetamine (“Ecstasy”) Users: Relationship to Cognitive Performance

Background(±)3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) is a recreational drug and brain serotonin (5-HT) neurotoxin. Under certain conditions, MDMA can also damage brain dopamine (DA) neurons, at least in rodents. Human MDMA users have been found to have reduced brain 5-HT transporter (SERT) density and cognitive deficits, although it is not known whether these are related. This study sought to determine whether MDMA users who take closely spaced sequential doses, which engender high plasma MDMA concentrations, develop DA transporter (DAT) deficits, in addition to SERT deficits, and whether there is a relationship between transporter binding and cognitive performance.Materials and methodsSixteen abstinent MDMA users with a history of using sequential MDMA doses (two or more doses over a 3- to 12-h period) and 16 age-, gender-, and education-matched controls participated. Subjects underwent positron emission tomography with the DAT and SERT radioligands, [11C]WIN 35,428 and [11C]DASB, respectively. Subjects also underwent formal neuropsychiatric testing.ResultsMDMA users had reductions in SERT binding in multiple brain regions but no reductions in striatal DAT binding. Memory performance in the aggregate subject population was correlated with SERT binding in the dorsolateral prefrontal cortex, orbitofrontal cortex, and parietal cortex, brain regions implicated in memory function. Prior exposure to MDMA significantly diminished the strength of this relationship.ConclusionsUse of sequential MDMA doses is associated with lasting decreases in brain SERT, but not DAT. Memory performance is associated with SERT binding in brain regions involved in memory function. Prior MDMA exposure appears to disrupt this relationship. These data are the first to directly relate memory performance to brain SERT density.

Chemogenetics revealed: DREADD occupancy and activation via converted clozapine

DREADD not the designer compound Designer receptors exclusively activated by designer drugs (DREADDs) constitute a powerful chemogenetic strategy that can modulate nerve cell activity in freely moving animal preparations. Gomez et al. used radioligand receptor occupancy measurements and in vivo positron emission tomography to show that DREADDs expressed in the brain are not activated by the designer compound CNO (clozapine N-oxide). Instead, they are activated by the CNO metabolite clozapine, a drug with multiple endogenous targets. This may have important implications for the interpretation of results obtained with this popular technology. Science, this issue p. 503 Metabolically derived clozapine is the in vivo actuator of designer drug receptors expressed in the central nervous system. The chemogenetic technology DREADD (designer receptors exclusively activated by designer drugs) is widely used for remote manipulation of neuronal activity in freely moving animals. DREADD technology posits the use of “designer receptors,” which are exclusively activated by the “designer drug” clozapine N-oxide (CNO). Nevertheless, the in vivo mechanism of action of CNO at DREADDs has never been confirmed. CNO does not enter the brain after systemic drug injections and shows low affinity for DREADDs. Clozapine, to which CNO rapidly converts in vivo, shows high DREADD affinity and potency. Upon systemic CNO injections, converted clozapine readily enters the brain and occupies central nervous system–expressed DREADDs, whereas systemic subthreshold clozapine injections induce preferential DREADD-mediated behaviors.

2-[18F]fluoro-A-85380, an in vivo tracer for the nicotinic acetylcholine receptors

6-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]fluoro-A-85380 or 6-[18F]FA), a new tracer for positron emission tomography, was synthesized by no-carrier-added [18F] fluorination of 6-iodo-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine followed by acidic deprotection. 6-[18F]FA followed the regional densities of brain nicotinic acetylcholine receptors (nAChRs) reported in the literature. Evidence of binding to nAChRs and high specificity of the binding in vivo was demonstrated by inhibition with nAChR selective ligands as well as with unlabeled 6-FA. A preliminary toxicology study of the 6-FA showed a relatively low biological effect.

Kinetic Analysis of [11C]McN5652: A Serotonin Transporter Radioligand

The impulse response function of a radioligand is the most fundamental way to describe its pharmacokinetics and to assess its tissue uptake and retention pattern. This study investigates the impulse response function of [11C](+)-McN5652, a radioligand used for positron emission tomography (PET) imaging of the serotonin transporter (SERT) in the brain. Dynamic PET studies were performed in eight healthy volunteers injected with [11C](+)McN5652 and subsequently with its pharmacologically inactive enantiomer [11C](–)-McN5652. The impulse response function was calculated by deconvolution analysis of regional time–activity curves, and its peak value (fmax), its retention value at 75 minutes (fT), and its normalized retention (frel = fT/fmax) were obtained. Alternatively, compartmental models were applied to calculate the apparent total distribution volume (DVT) and its specific binding component (DVs). Both the noncompartmental (fT, frel) and the compartmental parameters (DV) were investigated with and without correction for nonspecific binding by simple subtraction of the corresponding value obtained with [11C](–)-McN5652. The impulse response function obtained by deconvolution analysis demonstrated high tracer extraction followed by a slow decline in the form of a monoexponential function. Statistical analysis revealed that the best compartmental model in terms of analysis of variance F and condition number of the parameter variance–covariance matrix was the one that was based on a single tissue compartment with parameters k1 and k2 and that also included the parameter of regional cerebral blood volume (BV). The parameter frel demonstrated low between-subject variance (coefficient of variation [CV] = 19%), a midbrain to cerebellum ratio of 1.85, and high correlation with the known density of SERT (r = 0.787 where r is the coefficient of linear correlation between the parameter and the known density of SERT). After correction for nonspecific binding, frel demonstrated further improvement in correlation (r = 0.814) and midbrain to cerebellum ratio (3.09). The variance of the distribution volumes was acceptable when the logarithmic transform lnDV was used instead of DV (17% for the three-parameter model), but correlation of this compartmental parameter was slightly less (r = 0.652 for the three-parameter model) than the correlation of the noncompartmental frel with the known density of SERT, and the midbrain to cerebellum ratio was only 1.5 (uncorrected) and 1.8 (corrected). At the expense of increasing variance, the correlation was increased after correction for nonspecific binding using the inactive enantiomer (r = 0.694; CV = 22%). These results indicate that the kinetics of [11C](+)McN5652 can best be described by a one-tissue compartment model with three parameters (k1, k2, and BV), and that both the noncompartmental parameter frel and the compartmental distribution volumes have the potential for quantitative estimation of the density of SERT. Further validation of the radioligand in experimental and clinical situations is warranted.

Imaging of δ opioid receptors in human brain by N1′‐ ([11C]methyl)naltrindole and PET

Recently, we have developed the positron emitting radiotracer N1′‐([11C]methyl)naltrindole ([11C]MeNTI) and demonstrated its high selectivity for δ opioid receptors in the mouse brain [Lever et al. (1992) Eur. J. Pharmacol., 216:449‐450]. In the present study, we examined the selectivity of [11C]MeNTI for the δ opioid receptor in the human brain, using positron emission tomography (PET). The regional kinetics and distribution as well as the pharmacology confirmed the selectivity of [11C]MeNTI for δ opioid receptor in the human brain. First, the regional kinetics of [11C]MeNTI are in accordance with the density of the δ opioid receptor. Rapid washout in receptor‐poor areas and prolonged retention in receptor‐rich areas were observed. Second, the regional distribution of [11]MeNTI correlated well (r = 0.91) with the in vitro distribution of δ opioid sites but not with μ or κ site densities (r ≤ 0.008 or r ≤ 0.014, respectively). [11C]MeNTI binding was highest in regions of the neocortex (insular, parietal, frontal, cingulate, and occipital), caudate nucleus, and putamen. Binding was intermediate in the amygdala and lowest in the cerebellum and thalamus. Third, studies using the competitive antagonist naltrexone demonstrated the inhibition of [11C]MeNTI binding. Naltrexone inhibition of [11C]MeNTI binding was most effective in δ receptor‐rich regions, and its inhibitory potency correlated well (r = 0.88) with the regional distribution of δ opioid sites. [11C]MeNTI is the first radioligand which selectively labels δ opioid receptors in vivo in the human brain following systemic administration. The availability of [11C]MeNTI will enable a receptor specific analysis of the role of [11C]MeNTI receptors in normal and abnormal human brain.

Kinetic Analysis of l11CrMcN5652c A Serotonin Transporter Radioligand

The impulse response function of a radioligand is the most fundamental way to describe its pharmacokinetics and to assess its tissue uptake and retention pattern. This study investigates the impulse response function of [11C](+)-McN5652, a radioligand used for positron emission tomography (PET) imaging of the serotonin transporter (SERT) in the brain. Dynamic PET studies were performed in eight healthy volunteers injected with [11C](+)McN5652 and subsequently with its pharmacologically inactive enantiomer [11C](–)-McN5652. The impulse response function was calculated by deconvolution analysis of regional time–activity curves, and its peak value (fmax), its retention value at 75 minutes (fT), and its normalized retention (frel = fT/fmax) were obtained. Alternatively, compartmental models were applied to calculate the apparent total distribution volume (DVT) and its specific binding component (DVs). Both the noncompartmental (fT, frel) and the compartmental parameters (DV) were investigated with and without correction for nonspecific binding by simple subtraction of the corresponding value obtained with [11C](–)-McN5652. The impulse response function obtained by deconvolution analysis demonstrated high tracer extraction followed by a slow decline in the form of a monoexponential function. Statistical analysis revealed that the best compartmental model in terms of analysis of variance F and condition number of the parameter variance–covariance matrix was the one that was based on a single tissue compartment with parameters k1 and k2 and that also included the parameter of regional cerebral blood volume (BV). The parameter frel demonstrated low between-subject variance (coefficient of variation [CV] = 19%), a midbrain to cerebellum ratio of 1.85, and high correlation with the known density of SERT (r = 0.787 where r is the coefficient of linear correlation between the parameter and the known density of SERT). After correction for nonspecific binding, frel demonstrated further improvement in correlation (r = 0.814) and midbrain to cerebellum ratio (3.09). The variance of the distribution volumes was acceptable when the logarithmic transform lnDV was used instead of DV (17% for the three-parameter model), but correlation of this compartmental parameter was slightly less (r = 0.652 for the three-parameter model) than the correlation of the noncompartmental frel with the known density of SERT, and the midbrain to cerebellum ratio was only 1.5 (uncorrected) and 1.8 (corrected). At the expense of increasing variance, the correlation was increased after correction for nonspecific binding using the inactive enantiomer (r = 0.694; CV = 22%). These results indicate that the kinetics of [11C](+)McN5652 can best be described by a one-tissue compartment model with three parameters (k1, k2, and BV), and that both the noncompartmental parameter frel and the compartmental distribution volumes have the potential for quantitative estimation of the density of SERT. Further validation of the radioligand in experimental and clinical situations is warranted.

Positron emission tomography imaging of the serotonin transporter in subjects with a history of alcoholism

BACKGROUND Our purpose was to investigate the serotonin transporter (SERT) in various brain regions of alcoholics using positron emission tomography and C-11 McN5652. METHOD Thirty-two adult subjects were involved, 17 social drinkers as control subjects and 15 subjects who were abstinent or recovering alcoholics. Concomitant psychiatric diseases were ruled out based on DSM-IV criteria. The majority of subjects were men. Radioligand binding in 11 brain areas was expressed as the total distribution volume (DV), distribution volume of specific binding (DV(spec)), and distribution volume ratio (DVR). The cerebellum was used as reference tissue for calculation of DV(spec) and DVR. RESULTS In subjects with a history of alcoholism, DV was lower in all brain regions, with significant differences in the midbrain, thalamus, amygdala, pons, cingulate gyrus, frontal cortex, and cerebellum. Additionally, DV(spec) was lower in all brain regions, but differences were only significant in the midbrain; DVR was lower in nine regions but the differences did not reach statistical significance. CONCLUSIONS These studies demonstrate lower binding of [(11)C](+)McN5652 to the SERT in the brain of abstinent or recovering alcoholics compared with control subjects. Differences in the radioligand distribution volumes are more significant before than after correction for nonspecific binding of the radioligand.

Synthesis of a radiotracer for studying nicotinic acetylcholine receptors: 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]A-85380)

The radiochemical synthesis of 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]A-85380, [18F]1) was accomplished by Kryptofix® 222 assisted nucleophilic no-carrier-added [18F]fluorination of 2-iodo-3((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine, 2 followed by acidic deprotection. The average radiochemical yield was 10% and the average specific radioactivity was 1050 mCi/μmol, calculated at end-of-synthesis (EOS).