The automated radiosynthesis and purification of the opioid receptor antagonist, [6‐O‐methyl‐11C]diprenorphine on the GE TRACERlab FXFE radiochemistry module

[6‐O‐Methyl‐¹¹C]diprenorphine ([¹¹C]diprenorphine) is a positron emission tomography ligand used to probe the endogenous opioid system in vivo. Diprenorphine acts as an antagonist at all of the opioid receptor subtypes, that is, μ (mu), κ (kappa) and δ (delta). The radiosynthesis of [¹¹C]diprenorphine using [¹¹C]methyl iodide produced via the ‘wet’ method on a home‐built automated radiosynthesis set‐up has been described previously. Here, we describe a modified synthetic method to [¹¹C]diprenorphine performed using [¹¹C]methyl iodide produced via the gas phase method on a GE TRACERlab FX_FE radiochemistry module. Also described is the use of [¹¹C]methyl triflate as the carbon‐11 methylating agent for the [¹¹C]diprenorphine syntheses. [¹¹C]Diprenorphine was produced to good manufacturing practice standards for use in a clinical setting. In comparison to previously reported [¹¹C]diprenorphine radiosyntheisis, the method described herein gives a higher specific activity product which is advantageous for receptor occupancy studies. The radiochemical purity of [¹¹C]diprenorphine is similar to what has been reported previously, although the radiochemical yield produced in the method described herein is reduced, an issue that is inherent in the gas phase radiosynthesis of [¹¹C]methyl iodide. The yields of [¹¹C]diprenorphine are nonetheless sufficient for clinical research applications. Other advantages of the method described herein are an improvement to both reproducibility and reliability of the production as well as simplification of the purification and formulation steps. We suggest that our automated radiochemistry route to [¹¹C]diprenorphine should be the method of choice for routine [¹¹C]diprenorphine productions for positron emission tomography studies, and the production process could easily be transferred to other radiochemistry modules such as the TRACERlab FX C pro. © 2014 The Authors. Journal of Labelled Compounds and Radiopharmaceuticals Published by John Wiley & Sons Ltd.     

Exploring regulation and function of dopamine D3 receptors in alcohol use disorder. A PET [11C]-(+)-PHNO study

Preclinical studies support an important role of dopamine D3 receptors (DRD3s) in alcohol use disorder (AUD). In animals, voluntary alcohol consumption increases DRD3 expression, and pharmacological blockade of DRD3s attenuates alcohol self-administration and reinstatement of alcohol seeking. However, these findings have yet to be translated in humans. This study used positron emission tomography (PET) and [¹¹C]-(+)-PHNO to compare receptor levels in several dopamine D2 receptor (DRD2) and DRD3 regions of interest between AUD subjects in early abstinence (n = 17; 6.59 ± 4.14 days of abstinence) and healthy controls (n = 18). We recruited non-treatment seeking subjects meeting DSM-5 criteria for AUD. We examined the relationship between DRD2/3 levels and both alcohol craving and alcohol motivation/wanting, using a cue reactivity procedure and an intravenous alcohol self-administration (IVASA) paradigm, respectively. [¹¹C]-(+)-PHNO binding levels in AUD subjects were significantly lower than binding in HCs when looking at all DRD2/3 ROIs jointly (Wilk’s Λ = .58, F(6,28) =3.33, p = 0.013, η²_p = 0.42), however there were no region-specific differences. Binding values demonstrate −12.3% and −16.1% lower [¹¹C]-(+)-PHNO binding in the SMST and SN respectively, though these differences did not withstand Bonferroni corrections. There was a positive association between [¹¹C]-(+)-PHNO binding in the SN (almost exclusively reflective of DRD3) and alpha (lower values reflect higher alcohol demand) in the APT after Bonferroni corrections (r = 0.66, p = 0.0080). This demonstrates that AUD subjects with lower DRD3 levels in the SN exhibit increased demand for alcohol. These results replicate previous findings demonstrating reduced DRD2/3 levels while also supporting a lack of DRD3 upregulation and potential downregulation in early abstinent AUD. Furthermore, the finding that binding in the SN is associated with alcohol demand warrants further examination.Subject terms: Neurotransmitters, Addiction, Translational research     

Elevation of Dopamine Induced by Cigarette Smoking: Novel Insights from a [11C]-(+)-PHNO PET Study in Humans

Positron emission tomography (PET) has convincingly provided in vivo evidence that psychoactive drugs increase dopamine (DA) levels in human brain, a feature thought critical to their reinforcing properties. Some controversy still exists concerning the role of DA in reinforcing smoking behavior and no study has explored whether smoking increases DA concentrations at the D3 receptor, speculated to have a role in nicotine''s addictive potential. Here, we used PET and [¹¹C]-(+)-PHNO ([¹¹C]-(+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol) to test the hypothesis that smoking increases DA release (decreases [¹¹C]-(+)-PHNO binding) in D2-rich striatum and D3-rich extra-striatal regions and is related to craving, withdrawal and smoking behavior. Ten participants underwent [¹¹C]-(+)-PHNO scans after overnight abstinence and after smoking a cigarette. Motivation to smoke (smoking topography), mood, and craving were recorded. Smoking significantly decreased self-reported craving, withdrawal, and [¹¹C]-(+)-PHNO binding in D2 and D3-rich areas (−12.0 and −15.3%, respectively). We found that motivation to smoke (puff rate) predicted magnitude of DA release in limbic striatum, and the latter was correlated with decreased craving and withdrawal symptoms. This is the first report suggesting that, in humans, DA release is increased in D3-rich areas in response to smoking. Results also support the preferential involvement of the limbic striatum in motivation to smoke, anticipation of pleasure from cigarettes and relief of withdrawal symptoms. We propose that due to the robust effect of smoking on [¹¹C]-(+)-PHNO binding, this radiotracer represents an ideal translational tool to investigate novel therapeutic strategies targeting DA transmission.     

GABA-benzodiazepine receptor function in alcohol dependence: a combined <Superscript>11</Superscript>C-flumazenil PET and pharmacodynamic study

Rationale Gamma-aminobutyric acid (GABA)-benzodiazepine receptor function is hypothesised to be reduced in alcohol dependence. Objectives We used positron emission tomography (PET) with [¹¹C]flumazenil, a non-selective tracer for brain GABA-benzodiazepine (GABA-BDZ) receptor binding, to determine in vivo the relationship between BDZ receptor occupancy by an agonist, midazolam, and its functional effects. Methods Abstinent male alcohol dependent subjects underwent [¹¹C]flumazenil PET to measure occupancy of BDZ receptors by midazolam whilst recording its pharmacodynamic effects on behavioural and physiological measures. Rate constants describing the exchange of [¹¹C]flumazenil between the plasma and brain compartments were derived from time activity curves. Results A 50% reduction in electroencephalography (EEG)-measured sleep time was seen in the alcohol dependent group despite the same degree of occupancy by midazolam as seen in the control group. The effects of midazolam on other measures of benzodiazepine receptor function, increasing EEG beta1 power and slowing of saccadic eye movements, were similar in the two groups. No differences in midazolam or flumazenil metabolism were found between the groups. Conclusions In summary, our study suggests that alcohol dependence in man is associated with a reduced EEG sleep response to the benzodiazepine agonist, midazolam, which is not explained by reduced BDZ receptor occupancy, and is consistent with reduced sensitivity in this measure of GABA-BDZ receptor function in alcohol dependence. The lack of change in other functional measures may reflect a differential involvement of particular subtypes of the GABA-BDZ receptor.     

Blunted Endogenous Opioid Release Following an Oral Amphetamine Challenge in Pathological Gamblers

Pathological gambling is a psychiatric disorder and the first recognized behavioral addiction, with similarities to substance use disorders but without the confounding effects of drug-related brain changes. Pathophysiology within the opioid receptor system is increasingly recognized in substance dependence, with higher mu-opioid receptor (MOR) availability reported in alcohol, cocaine and opiate addiction. Impulsivity, a risk factor across the addictions, has also been found to be associated with higher MOR availability. The aim of this study was to characterize baseline MOR availability and endogenous opioid release in pathological gamblers (PG) using [¹¹C]carfentanil PET with an oral amphetamine challenge. Fourteen PG and 15 healthy volunteers (HV) underwent two [¹¹C]carfentanil PET scans, before and after an oral administration of 0.5 mg/kg of d-amphetamine. The change in [¹¹C]carfentanil binding between baseline and post-amphetamine scans (ΔBP_ND) was assessed in 10 regions of interest (ROI). MOR availability did not differ between PG and HV groups. As seen previously, oral amphetamine challenge led to significant reductions in [¹¹C]carfentanil BP_ND in 8/10 ROI in HV. PG demonstrated significant blunting of opioid release compared with HV. PG also showed blunted amphetamine-induced euphoria and alertness compared with HV. Exploratory analysis revealed that impulsivity positively correlated with caudate baseline BP_ND in PG only. This study provides the first evidence of blunted endogenous opioid release in PG. Our findings are consistent with growing evidence that dysregulation of endogenous opioids may have an important role in the pathophysiology of addictions.     

[11C]Ro 15-4513, a ligand for visualization of benzodiazepine receptor binding

Ro 15-4513, a partial inverse agonist at the benzodiazepine (BZ) receptor site was labelled with¹¹C and used for in vitro autoradiography on human post mortem brain sections and for positron emission tomography (PET) on Cynomolgus monkeys. The total radiochemical yield of [¹¹C]Ro 15-4513 was 30–40% with an overall synthesis time of 40 min. The specific radioactivity was about 1000 Ci/mmol at end of synthesis. In vitro autoradiography showed that [¹¹C]Ro 15-4513 bound specifically predominately in the neocortex of the human brain. Specific binding was also demonstrated in the basal ganglia and the cerebellar cortex. Flumazenil (Ro 15-1788) and clonazepam inhibited the binding in cerebral regions, but a significant proportion in the cerebellum was not inhibited by these agents. This proportion may represent ₆-containing BZ receptors. PET examination of [¹¹C]Ro 15-4513 binding in Cynomolgus monkeys demonstrated high uptake of radioactivity in neocortex. The uptake of radioactivity was markedly displaced by high doses of Ro 15-4513 or clonazepam. [¹¹C]Ro 15-4513 should be a useful ligand to examine BZ receptor characteristics in the living human brain by PET.     

Measuring Cigarette Smoking-Induced Cortical Dopamine Release: A [11C]FLB-457 PET Study

Striatal dopamine (DA) is thought to have a fundamental role in the reinforcing effects of tobacco smoking and nicotine. Microdialysis studies indicate that nicotine also increases DA in extrastriatal brain areas, but much less is known about its role in addiction. High-affinity D_2/3 receptor radiotracers permit the measurement of cortical DA in humans using positron emission tomography (PET). [¹¹C]FLB-457 PET scans were conducted in 10 nicotine-dependent daily smokers after overnight abstinence and reinstatement of smoking. Voxel-wise [¹¹C]-FLB-457-binding potential (BP_ND) in the frontal lobe, insula, and limbic regions was estimated in the two conditions. Paired t-tests showed BP_ND values were reduced following smoking (an indirect index of DA release). The overall peak t was located in the cingulate gyrus, which was part of a larger medial cluster (BP_ND change −12.1±9.4%) and this survived false discovery rate correction for multiple comparisons. Clusters were also identified in the left anterior cingulate cortex/medial frontal gyrus, bilateral prefrontal cortex (PFC), bilateral amygdala, and the left insula. This is the first demonstration of tobacco smoking-induced cortical DA release in humans; it may be the result of both pharmacological (nicotine) and non-pharmacological factors (tobacco cues). Abstinence increased craving but had minimal cognitive effects, thus limiting correlation analyses. However, given that the cingulate cortex, PFC, insula, and amygdala are thought to have important roles in tobacco craving, cognition, and relapse, these associations warrant investigation in a larger sample. [¹¹C]FLB-457 PET imaging may represent a useful tool to investigate individual differences in tobacco addiction severity and treatment response.     

Immediate and Persistent Effects of Salvinorin A on the Kappa Opioid Receptor in Rodents,Monitored In Vivo with PET

Monitoring changes in opioid receptor binding with positron emission tomography (PET) could lead to a better understanding of tolerance and addiction because altered opioid receptor dynamics following agonist exposure has been linked to tolerance mechanisms. We have studied changes in kappa opioid receptor (KOR) binding availability in vivo with PET following kappa opioid agonist administration. Male Sprague–Dawley rats (n=31) were anesthetized and treated with the (KOR) agonist salvinorin A (0.01–1.8 mg/kg, i.v.) before administration of the KOR selective radiotracer [¹¹C]{"type":"entrez-nucleotide","attrs":{"text":"GR103545","term_id":"238230768","term_text":"GR103545"}}GR103545. When salvinorin A was administered 1 min prior to injection of the radiotracer, [¹¹C]{"type":"entrez-nucleotide","attrs":{"text":"GR103545","term_id":"238230768","term_text":"GR103545"}}GR103545 binding potential (BP_ND) was decreased in a dose-dependent manner, indicating receptor binding competition. In addition, the unique pharmacokinetics of salvinorin A (half-life ~8 min in non-human primates) allowed us to study the residual impact on KOR after the drug had eliminated from the brain. Salvinorin A was administered up to 5 h prior to [¹¹C]{"type":"entrez-nucleotide","attrs":{"text":"GR103545","term_id":"238230768","term_text":"GR103545"}}GR103545, and the changes in BP_ND were compared with baseline, 2.5 h, 1 h, and 1 min pretreatment times. At lower doses (0.18 mg/kg and 0.32 mg/kg) we observed no prolonged effect on KOR binding but at 0.60 mg/kg salvinorin A induced a sustained decrease in KOR binding (BP_ND decreased by 40–49%) which persisted up to 2.5 h post administration, long after salvinorin A had been eliminated from the brain. These data point towards an agonist-induced adaptive response by KOR, the dynamics of which have not been previously studied in vivo with PET.     

Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving

Rationale and objectives

Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial prefrontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence.

Methods

We trained rats to self-administer heroin or saline for 9?C10?days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone??s (0?C1.0?mg/kg) effect on extinction responding after 1 and 15?days.

Results

Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day?11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day?1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1?day.

Conclusions

Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving.     

A highly reproducible method for the measurement of [6‐O‐methyl‐11C]diprenorphine and its radio‐metabolites based on solid‐phase extraction and radio‐high‐pressure liquid chromatography

Described here is a method for the measurement of the radio‐metabolites of the positron emission tomography radiotracer [6‐O‐methyl‐¹¹C]diprenorphine ([¹¹C]diprenorphine) using in‐line solid‐phase extraction (SPE) combined with radio‐high‐pressure liquid chromatography analysis. We believe that this method offers a reliable and reproducible approach to [¹¹C]diprenorphine metabolite analysis. In addition, different SPE stationary phases are assessed for their efficiency for loading, retention and elution of the parent molecule and its metabolites. Having assessed C4, phenyl and C18 stationary phase, we concluded that a C18 SPE was optimal for our method. Finally, in silico predictions of diprenorphine metabolism were compared with in vivo metabolism of [¹¹C]diprenorphine induced by hepatic microsomal digestion and analysed by matrix‐assisted laser desorption/ionisation mass spectrometry. It was found that there was a high degree of agreement between the two methods and in particular the formation of the diprenorphine‐3‐glucuronide metabolite.     

Longitudinal imaging of metabotropic glutamate 5 receptors during early and extended alcohol abstinence

Chronic alcohol use has important effects on the glutamate system. The metabotropic glutamate 5 (mGlu5) receptor has shown promise in preclinical models as a target to reduce drinking-related behaviors and cue-induced reinstatement, motivating human studies of mGlu5 receptor negative allosteric modulators. The goal of this work was to measure levels of mGlu5 receptor availability with positron emission tomography (PET) imaging using the mGlu5 receptor-specific radiotracer [¹⁸F]FPEB during early and extended alcohol abstinence. Subjects who met DSM-5 criteria for alcohol use disorder (AUD; n = 17) were admitted inpatient for the study duration. [¹⁸F]FPEB PET scans were acquired first during early abstinence (6 ± 4 days after last drink) and a second time during extended abstinence (n = 13; 27 ± 6 days after last drink). A single scan was acquired in healthy controls matched for sex and smoking status (n = 20). [¹⁸F]FPEB total volumes of distribution (V_T) corrected for partial volume effects were measured using equilibrium analysis throughout the brain. A linear mixed model controlling for smoking status and sex identified significantly higher [¹⁸F]FPEB V_T in AUD subjects at early abstinence compared to controls (F_(1,32) = 7.23, p = 0.011). Post-hoc analyses revealed this effect to occur in cortical brain regions. No evidence for significant changes in [¹⁸F]FPEB V_T over time were established. These findings provide human evidence consistent with a robust preclinical literature supporting mGlu5 receptor drugs as pharmacotherapies for AUD.Subject terms: Addiction, Predictive markers     

[N-methyl-11C]Mirtazapine for positron emission tomography neuroimaging of antidepressant actions in humans

Rationale Many actions of antidepressant drugs cannot yet be studied using positron emission tomography (PET) neuroimaging due to lack of suitable radioligands. We believe that mirtazapine, radiolabeled with C-11, might be suitable for PET neuroimaging of ₂-adrenoceptors in selected regions of the living human brain.Objective To determine the regional central biodistribution and pharmacokinetics of [N-methyl-¹¹C]mirtazapine in humans.Methods Five healthy volunteers received an intravenous injection of [N-methyl-¹¹C]mirtazapine for evaluating its metabolism, biodistribution and pharmacokinetics.Results [N-methyl-¹¹C]Mirtazapine entered the brain readily, with initial clearance from blood to tissue (K₁) ranging from 0.31 ml/ml/min in amygdala to 0.54 ml/ml/min in thalamus. The rate of metabolism of [N-methyl-¹¹C]mirtazapine in the bloodstream was relatively slow, with 20–40% of [¹¹C]-derived radioactivity still present as parent compound at 60 min post-injection. The clearance of [N-methyl-¹¹C]mirtazapine from the tissue compartment (k₂) ranged from a low of 0.03 min^–1 in amygdala to a high of 0.06–0.07 min^–1 in thalamus and cerebellum. The volume of distribution (V_e) of [N-methyl-¹¹C]mirtazapine was markedly greater in hippocampus and amygdala (11.3–12.0) than in cerebellum (6.7), with intermediate levels in the thalamus (9.4).Conclusions [N-methyl-¹¹C]Mirtazapine has suitable properties for PET neuroimaging. We envision [N-methyl-¹¹C]mirtazapine as a molecular probe for PET imaging of antidepressant actions at sites such as ₂-adrenoceptors in the living human brain.     

Dose-dependent binding of AZD3783 to brain 5-HT1B receptors in non-human primates and human subjects: a positron emission tomography study with [11C]AZ10419369

Rationale

The serotonin 5-HT_1B receptor is a potential target for the pharmacologic treatment of depression. Positron emission tomography (PET) determination of 5-HT_1B receptor occupancy with drug candidates targeting this receptor in non-human primate and human subjects may facilitate translation of research from animal models and guide dose selection for clinical studies. AZD3783 is a recently developed, orally bioavailable 5-HT_1B receptor antagonist with potential antidepressant properties.

Objectives

To determine the relationship between plasma concentration of AZD3783 and occupancy at primate brain 5-HT_1B receptors using PET and the radioligand [¹¹C]AZ10419369.

Methods

PET studies with [¹¹C]AZ10419369 were performed in three non-human primates at baseline and after intravenous injection of AZD3783. Subsequently, PET measurements were undertaken in six human subjects at baseline and after administration of different single oral doses of AZD3783 (1?C40?mg).

Results

After administration in non-human primates and human subjects, AZD3783 reduced regional [¹¹C]AZ10419369 binding in a dose-dependent and saturable manner. The AZD3783 plasma concentration required for 50% receptor occupancy (K _i,plasma) for monkeys was 25 and 27?nmol/L in occipital cortex and striatum, respectively. Corresponding estimates for human occipital cortex and ventral striatum were 24 and 18?nmol/L, respectively.

Conclusions

The potential antidepressant AZD3783 binds in a saturable manner to brain 5-HT_1B receptors with a similar in vivo affinity for human and monkey receptors. [¹¹C]AZ10419369 can be successfully used to determine occupancy at brain 5-HT_1B receptors in vivo and constitutes a useful tool for dose selection in clinical studies with 5-HT_1B receptor compounds.     

PET analysis of alcohol interaction with the brain disposition of [11C]flumazenil

Acute alcohol administration to rats has in preliminary studies been reported to drastically increase the binding of the benzodiazepine (BZ) receptor antagonist [³H]flumazenil (Ro 15-1788) to central BZ receptors. In the present study the effect of acute alcohol ingestion on the disposition of [¹¹C]flumazenil in the human brain and plasma was examined by positron emission tomography (PET) in four healthy volunteers. Neocortex, cerebellum and pons (reference region) were delineated using X-ray computerized tomography (CT). Alcohol did not increase either total radioactivity uptake or specific [¹¹C]flumazenil binding in neocortex or cerebellum. However, alcohol had a small but significant effect on [¹¹C]flumazenil in arterial blood. After alcohol the plasma radioactivity peak was higher, more narrow and occurred earlier than in the control experiments. The present experiments contradict the view that alcohol directly affects central BZ receptor binding in man. Thus the dramatic increase of flumazenil binding in rat brain reported previously could not be observed in the human brain.     

Effect of early and late compliance on the effectiveness of acamprosate in the treatment of alcohol dependence

Background

The aim of this study is to assess the influence of early and late compliance of acamprosate on attendance and abstinence duration in the treatment of alcohol dependence.

Methods

Individual patient data of 2,305 patients from 11 randomized controlled trials comparing acamprosate (n = 1,128) with placebo (n = 1,177) were used to predict early and late compliance and to study the effect of early and late compliance on attendance and abstinence duration using regression analysis and structural equation modeling.

Results

Early compliance was predicted by baseline motivation to become fully abstinent and baseline abstinence (R² = .26); late compliance was predicted by early compliance (R² = .13); treatment discontinuation was predicted by young age, marital status, compliance, and treatment condition (R² = .26); and abstinence duration was predicted by motivation to become fully abstinent early compliance and the interaction of early compliance and treatment condition (R² = .27). Structural equation modeling showed that abstinence duration was significantly associated with motivation at baseline, late compliance, and treatment condition (Goodness of Fit Index [GFI] χ²/df = 1.56; Parsimonious Goodness of Fit Index [PGFI] = 0.69).

Conclusions

Motivation to become fully abstinent and abstinence at the start of treatment are important for early compliance. Early compliance in turn predicts late compliance. Late compliance, in combination with motivation to become fully abstinent, and treatment condition (acamprosate vs. placebo) predict duration of abstinence. This suggests that motivational interventions directed toward full abstinence motivation and abstinence at the start of treatment are crucial for both compliance with acamprosate and successful treatment outcome.     

Positron emission tomographic measure of brain dopamine dependence to nicotine as a model of drugs of abuse

Rationale  Nicotine/tobacco are prototypic substances used throughout the world. Nicotine abstinence produces some depressive-like effects which are treated by the dopamine (DA) and norepinephrine reuptake inhibitor bupropion. A quantitative measure of the regional brain utilization of these catecholamines (CA) during nicotine dependence and withdrawal is important. Objective  The aim of this study was to prove that regional brain DA utilization by nicotine can be quantified by positron emission tomography (PET) using l-[β-¹¹C]DOPA. Materials and methods  Eight young Macaca mulatta monkeys were given 0.9% NaCl or nicotine in doses of 32 or 100 μg/kg i.m. bid for 9 days to produce minimal dependence. On the tenth day, PET measurements were repeated before and after i.v. nicotine administration. PET studies were done in habituated, trained, and fully conscious animals. Results  Compared to a 0.9% NaCl control, acute i.v. nicotine as a bolus plus infusion for 30 min in similar doses to maintain a steady-state level for 30 min did not affect the utilization rate constant (k ₃) in dorsal or ventral striatum as measured by l-[β-¹¹C]DOPA. When monkeys were given nicotine bid repeatedly after overnight nicotine abstinence, CA utilization was reduced. A subsequent nicotine dose normalized utilization to slightly above control levels. Changes in ventral striatum were similar to those in dorsal striatum. The reduced rate of utilization demonstrated with l-[β-¹¹C]DOPA after overnight nicotine abstinence and its reversal by nicotine the next day provides an important PET measure of brain nicotine dependence and withdrawal. This method can be applied to other substances of abuse that release DA. Research was done at the Central Research Laboratory, Hamamatsu Photonics K.K.     

Brain mu-opioid receptor binding: relationship to relapse to cocaine use after monitored abstinence

Rationale  Cocaine users have increased regional brain mu-opioid receptor (mOR) binding which correlates with cocaine craving. The relationship of mOR binding to relapse is unknown. Objective  To evaluate regional brain mOR binding as a predictor of relapse to cocaine use is the objective of the study. Materials and methods  Fifteen nontreatment-seeking, adult cocaine users were housed on a closed research ward for 12 weeks of monitored abstinence and then followed for up to 1 year after discharge. Regional brain mOR binding was measured after 1 and 12 weeks using positron emission tomography (PET) with [¹¹C]carfentanil (a selective mOR agonist). Time to first cocaine use (lapse) and to first two consecutive days of cocaine use (relapse) after discharge was based on self-report and urine toxicology. Results  A shorter interval before relapse was associated with increased mOR binding in frontal and temporal cortical regions at 1 and 12 weeks of abstinence (Ps < 0.001) and with a lesser decrease in binding between 1 and 12 weeks (Ps < 0.0008). There were significant positive correlations between mOR binding at 12 weeks and percent days of cocaine use during first month after relapse (Ps < 0.002). In multiple linear regression analysis, mOR binding contributed significantly to the prediction of time to relapse (R ²= 0.79, P < 0.001), even after accounting for clinical variables. Conclusions  Increased brain mOR binding in frontal and temporal cortical regions is a significant independent predictor of time to relapse to cocaine use, suggesting an important role for the brain endogenous opioid system in cocaine addiction.     

In vivo binding of the dopamine-1 receptor PET tracers [11C]NNC112 and [11C]SCH23390: a comparison study in individuals with schizophrenia

Rationale

A deficit in dopamine-1 (D₁) receptor function in the prefrontal cortex is suggested to play a role in the cognitive dysfunction observed in patients with schizophrenia. However, the results from positron emission tomography imaging studies of D₁ receptor levels in individuals with schizophrenia are mixed.

Objectives

The aim of this investigation was to determine whether the in vivo characteristics of the different D₁ receptor tracers used in previous reports, [¹¹C]SCH23390 and [¹¹C]NNC112, may have contributed to these discrepancies reported in the literature.

Methods

Eight patients with schizophrenia and 12 healthy control subjects were scanned with both [¹¹C]SCH23390 and [¹¹C]NNC112.

Results

[¹¹C]SCH23390 and [¹¹C]NNC112 binding potentials in both patients and control subjects were compared and no tracer by diagnosis interactions were observed.

Conclusions

The results of this study suggest that differences in the binding of [¹¹C]SCH23390 and [¹¹C]NNC112 observed in previous studies are not due to differences in the in vivo behavior of these tracers.     

Heightened D3 Dopamine Receptor Levels in Cocaine Dependence and Contributions to the Addiction Behavioral Phenotype: A Positron Emission Tomography Study with [11C]-(+)-PHNO

The dopamine system is a primary treatment target for cocaine dependence (CD), but research on dopaminergic abnormalities (eg, D₂ receptor system deficiencies) has so far failed to translate into effective treatment strategies. The D₃ receptor system has recently attracted considerable clinical interest, and D₃ antagonism is now under investigation as a novel avenue for addiction treatment. The objective here was to evaluate the status and behavioral relevance of the D₃ receptor system in CD, using the positron emission tomography (PET) radiotracer [¹¹C]-(+)-PHNO. Fifteen CD subjects (many actively using, but all abstinent 7–240 days on scan day) and fifteen matched healthy control (HC) subjects completed two PET scans: one with [¹¹C]-(+)-PHNO to assess D₃ receptor binding (BP_ND; calculated regionally using the simplified reference tissue model), and for comparison, a second scan with [¹¹C]raclopride to assess D_2/3 binding. CD subjects also completed a behavioral battery to characterize the addiction behavioral phenotype. CD subjects showed higher [¹¹C]-(+)-PHNO BP_ND than HC in the substantia nigra, which correlated with behavioral impulsiveness and risky decision making. In contrast, [¹¹C]raclopride BP_ND was lower across the striatum in CD, consistent with previous literature in ⩾2 week abstinence. The data suggest that in contrast to a D₂ deficiency, CD individuals may have heightened D₃ receptor levels, which could contribute to addiction-relevant traits. D₃ upregulation is emerging as a biomarker in preclinical models of addiction, and human PET studies of this receptor system can help guide novel pharmacological strategies for treatment.     

Opioid receptor agonists activate pertussis toxin-sensitive G proteins and inhibit adenylyl cyclase in canine cardiac sarcolemma

Although both opioid receptors and endogenous opioids are abundant in cardiac tissues, the signal transduction pathways of opioids in cardiac sarcolemmal membranes have yet to be identified. In highly purified canine cardiac sarcolemmal membranes, binding of the opioid receptor antagonist [³H]diprenorphine and effects of , and agonists on low K_m GTPase and adenylyl cyclase were measured. Equilibrium binding of [³H]diprenorphine revealed a maximal binding capacity of 7.2 pmol/mg protein and a K_d of 1.3 nmol/1. In the presence of GTP, (D-Pen^2,5, p-Cl-Phe⁴)enkephalin and (D-Arg⁶)dynorphin A 1-13 fragment both inhibited adenylyl cyclase by 20–25% (from 206 ± 30to164 ± 28 pmol·min^{– 1}·mgprotein^–1, EC₅₀6 mol/Landfrom254 ± 109to204 ± 90 pmol·min^{– 1}·mg protein^–, EC₅₀ 8 pmol/L, respectively; P<0.001). Both substances stimulated low Km GTPase by 20%and13%,respectively(from12.7 ± 3.0 to 15.2 ± 3.7 pmol·min^–1.mgprotein^–1,EC₅₀ 12 mol/L, P<0.01, and from 9.1 ± 2.8 to 10.4 ± 3.2 pmol-min^{– 1}·mg protein^–1, EC₅₀ 6 mol/L, P<0.05, respectively). These effects were blocked by the opioid receptor antagonist naltrexone and by pretreatment of sarcolemmal membranes with pertussis toxin. The opioid receptor agonists (D-AIa², Me Phe⁴, Gly-[ol]⁵)enkephalin and morphiceptin had no effect on either cardiac adenylyl cyclase or low Km GTPase activities. These data suggest that in cardiac sarcolemma, opioid receptors are coupled to pertussis toxin sensitive G proteins and mediate inhibition of adenylyl cyclase activity.