Dopamine (D2/3) receptor agonist positron emission tomography radiotracer [11C]-(+)-PHNO is a D3 receptor preferring agonist in vivo

[11C]PHNO is a recently introduced agonist to image DA D2-like receptors with Positron Emission Tomography (PET). In cats and humans, [11C]PHNO revealed an atypical distribution compared to radiolabeled D2-like antagonists (such as [11C]raclopride) or other D2-like agonists (such as [11C]NPA), as it displayed unusual high binding in the globus pallidus (GP). The goal of this study was to assess the pharmacological nature of the binding of [11C]PHNO in the GP in nonhuman primates. As previously reported in humans, [11C]PHNO equilibrium specific to nonspecific equilibrium partition coefficients (V3') in baboons was much higher in GP (3.88 +/- 1.15) than in the dorsal striatum (DST, 2.07 +/- 0.43), whereas the reverse was true for [11C]raclopride (1.48 +/- 0.41 in GP, 2.56 +/- 0.91 in DST) and [11C]NPA (0.87 +/- 0.19 in GP, 1.02 +/- 0.13 in DST). Administration of unlabeled raclopride resulted in similar reductions of [11C] PHNO V3' and [11C]raclopride V3' in both the GP and the DST. This observation demonstrated that the [11C]PHNO binding in the GP was specific to D2-like receptors. To evaluate the respective contribution of D3 and D2 receptors to the binding potential (BP) of [11C]PHNO and [11C]raclopride, experiments were carried out with the selective D3 partial agonist 1-(4(2-Napthoylamino)butyl)-4-(2-methoxyphenyl)-1A-piperazine HCL (BP897). BP897 reduced [11C]raclopride V3' by 29% +/- 9%, 19% +/- 8%, and 10% +/- 7% in GP, VST, and DST, respectively, a result consistent with expectation from postmortem studies (D3/D2 ratio in GP > VST > DST). BP897 reduced [11C]PHNO V3' by 57% +/- 11%, 30% +/- 11%, and 13% +/- 8% in GP, VST, and DST, respectively, indicating that the D3 receptor contribution to [11C]PHNO signal is higher than that of [11C]raclopride. From these experiments we conclude that [11C]PHNO is a D3 preferring agonist, and that this property explains the high GP signal not observed with [11C]raclopride or [11C]NPA. This property might contribute to its higher vulnerability to endogenous DA compared to [11C]raclopride and [11C]NPA.     

Measurement error analysis for the determination of dopamine D2 receptor occupancy using the agonist radioligand [11C]MNPA

The purpose of this study is to investigate errors in quantitative analysis for estimating dopamine D₂ receptor occupancy of antipsychotics with agonist radioligand [¹¹C]MNPA by numerical simulation, with particular attention to the validity of a quantitative approach based on the use of a reference region. Synthetic data were validated using clinical data combined with a bootstrap approach. Time–activity curves (TACs) of [¹¹C]MNPA were simulated, and the reliability of binding potential (BP_ND) and occupancy estimated by nonlinear least square (NLS) fitting and a simplified reference tissue model (SRTM) were investigated for various noise levels and scan durations. In the human positron emission tomography (PET) study with and without antipsychotic, risperidone, the uncertainty of BP_ND and occupancy estimated by SRTM was investigated using resampled TACs based on bootstrap approach with weighted residual errors of fitting. For both NLS and SRTM, it was possible to have <3% of bias in occupancy estimates of [¹¹C]MNPA by 60 mins. However, shortened scan duration degrades the quantification of very small binding potentials, especially in case of SRTM. Observations were replicated on the clinical data. Results showed that dopamine D₂ receptor occupancy by antipsychotics can be estimated precisely in region of interest analysis by SRTM with a longer than 60-min [¹¹C]MNPA PET scan duration.     

Estimating the effect of endogenous dopamine on baseline [11C]‐(+)‐PHNO binding in the human brain

Endogenous dopamine (DA) levels at dopamine D_2/3 receptors (D_2/3R) have been quantified in the living human brain using the agonist radiotracer [¹¹C]‐(+)‐PHNO. As an agonist radiotracer, [¹¹C]‐(+)‐PHNO is more sensitive to endogenous DA levels than antagonist radiotracers. We sought to determine the proportion of the variance in baseline [¹¹C]‐(+)‐PHNO binding to D_2/3Rs which can be accounted for by variation in endogenous DA levels. This was done by computing the Pearson's coefficient for the correlation between baseline binding potential (BP_ND) and the change in BP_ND after acute DA depletion, using previously published data. All correlations were inverse, and the proportion of the variance in baseline [¹¹C]‐(+)‐PHNO BP_ND that can be accounted for by variation in endogenous DA levels across the striatal subregions ranged from 42‐59%. These results indicate that lower baseline values of [¹¹C]‐(+)‐PHNO BP_ND reflect greater stimulation by endogenous DA. To further validate this interpretation, we sought to examine whether these data could be used to estimate the dissociation constant (Kd) of DA at D_2/3R. In line with previous in vitro work, we estimated the in vivo Kd of DA to be around 20 nM. In summary, the agonist radiotracer [¹¹C]‐(+)‐PHNO can detect the impact of endogenous DA levels at D_2/3R in the living human brain from a single baseline scan, and may be more sensitive to this impact than other commonly employed radiotracers.     

In vivo vulnerability to competition by endogenous dopamine: comparison of the D2 receptor agonist radiotracer (-)-N-[11C]propyl-norapomorphine ([11C]NPA) with the D2 receptor antagonist radiotracer [11C]-raclopride

(-)-N-Propyl-norapomorphine (NPA) is a full dopamine (DA) D2 receptor agonist and [11C]NPA is a suitable radiotracer to image D2 receptors configured in a state of high affinity for agonists with positron emission tomography (PET). In this study the vulnerability of the in vivo binding of [11C]NPA to acute fluctuation in synaptic DA was assessed with PET in baboons and compared to that of the reference D2 receptor antagonist radiotracer [11C]raclopride. Three male baboons were studied with [11C]raclopride and [11C]NPA under baseline conditions and following administration of the potent DA releaser amphetamine (0.3, 0.5, and 1.0 mg kg(-1) i.v.). Kinetic modeling with an arterial input function was used to derive the striatal specific-to-nonspecific equilibrium partition coefficient (V3"). [11C]Raclopride V3" was reduced by 24 +/- 10%, 32 +/- 6%, and 44 +/- 9% following amphetamine doses of 0.3, 0.5, and 1.0 mg kg(-1), respectively. [11C]NPA V3" was reduced by 32 +/- 2%, 45 +/- 3%, and 53 +/- 9% following amphetamine doses of 0.3, 0.5, and 1.0 mg kg(-1), respectively. Thus, endogenous DA was more effective at competing with [11C]NPA binding compared to [11C]raclopride binding, a finding consistent with the pharmacology of these tracers (agonist vs. antagonist). These results also suggest that 71% of D2 receptors are configured in a state of high affinity for agonists in vivo. In conclusion, [11C]NPA might provide a superior radiotracer to probe presynaptic DA function with PET in health and disease.     

Positron emission tomography imaging of amphetamine‐induced dopamine release in the human cortex: A comparative evaluation of the high affinity dopamine D2/3 radiotracers [11C]FLB 457 and [11C]fallypride

The use of PET and SPECT endogenous competition binding techniques has contributed to the understanding of the role of dopamine in several neuropsychiatric disorders. An important limitation of these imaging studies is the fact that measurements of acute changes in synaptic dopamine have been restricted to the striatum. The ligands previously used, such as [¹¹C]raclopride and [¹²³I]IBZM, do not provide sufficient signal to noise ratio to quantify D₂ receptors in extrastriatal areas, such as cortex, where the concentration of D₂ receptors is much lower than in the striatum. Given the importance of cortical DA function in cognition, a method to measure cortical dopamine function in humans would be highly desirable. The goal of this study was to compare the ability of two high affinity DA D₂ radioligands [¹¹C]FLB 457 and [¹¹C]fallypride to measure amphetamine‐induced changes in DA transmission in the human cortex. D₂ receptor availability was measured in the cortical regions of interest with PET in 12 healthy volunteers under control and postamphetamine conditions (0.5 mg kg^?1, oral), using both [¹¹C]FLB 457 and [¹¹C]fallypride (four scans per subjects). Kinetic modeling with an arterial input function was used to derive the binding potential (BP_ND) in eight cortical regions. Under controlled conditions, [¹¹C]FLB 457 BP_ND was 30–70% higher compared with [¹¹C]fallypride BP_ND in cortical regions. Amphetamine induced DA release led to a significant decrease in [¹¹C]FLB 457 BP_ND in five out the eight cortical regions evaluated. In contrast, no significant decrease in [¹¹C]fallypride BP_ND was detected in cortex following amphetamine. The difference between [¹¹C]FLB 457 and [¹¹C]fallypride ability to detect changes in the cortical D₂ receptor availability following amphetamine is related to the higher signal to noise ratio provided by [¹¹C]FLB 457. These findings suggest that [¹¹C]FLB 457 is superior to [¹¹C]fallypride for measurement of changes in cortical synaptic dopamine. Synapse 63:447–461, 2009. © 2009 Wiley‐Liss, Inc.     

Imaging cortical dopamine D1 receptors using [11C]NNC112 and ketanserin blockade of the 5-HT2A receptors

[¹¹C]NNC112 (8-chloro-7-hydroxy-3-methyl-5-(7-benzofuranyl)-2,3,4,5-tetrahydro-IH-3-benzazepine), a selective positron-emission tomography (PET) ligand for the D₁ receptor (R) over the 5-HT_2A R in vitro, has shown lower selectivity in vivo, hampering measurement of D₁ R in the cortex. [¹¹C]NNC112 PET and intravenous (i.v) ketanserin challenge were used to (1) confirm the previous findings of [¹¹C]NNC112 in vivo D₁ R selectivity, and (2) develop a feasible methodology for imaging cortical D₁ R without contamination by 5-HT_2A R. Seven healthy volunteers underwent [¹¹C]NNC112 PET scans at baseline and after a 5-HT_2A R-blocking dose of ketanserin (0.15 mg/kg, i.v.). Percent BP_ND change between the post-ketanserin and baseline scans was calculated. Irrespective of the quantification method used, ketanserin pretreatment led to significant decrease of BP_ND in the cortical (∼30%) and limbic regions (∼20%) but not in the striatum, which contains a much lower amount of 5-HT_2A R. Therefore, ketanserin allows D₁ R signal to be detected by [¹¹C]NNC112 PET without significant 5-HT_2A R contamination. These data confirm the presence of a significant 5-HT_2A R contribution to cortical [¹¹C]NNC112 signal, and call for caution in the interpretation of published [¹¹C]NNC112 PET findings on cortical D₁ R in humans. In the absence of more selective ligands, [¹¹C]NNC112 PET with ketanserin can be used for cortical D₁ R imaging in vivo.     

Differential effects of stress on [11C]raclopride and [11C]MNPA binding to striatal D2/D3 dopamine receptors: A PET study in conscious monkeys

It has been reported that stress and facilitation of dopamine neuronal system are closely related. In the present study, the effects of stress on the binding of antagonist‐based [¹¹C]raclopride and agonist‐based (R)‐2‐CH3O‐N‐n‐ propylnorapomorphine ([¹¹C]MNPA) to D₂/D₃ receptors were evaluated in the striatum of conscious monkey brain. The stress state assessed from plasma cortisol level was negatively correlated with [¹¹C]raclopride binding as expected. It was noteworthy that [¹¹C]MNPA binding exhibited a positive correlation with stress state; thus, the animals with higher cortisol levels showed higher binding to D₂/D₃ receptors. Synapse 65:84–89, 2011. © 2010 Wiley‐Liss, Inc.     

Measurement of density and affinity for dopamine D2 receptors by a single positron emission tomography scan with multiple injections of [11C]raclopride

Positron emission tomography (PET) with [¹¹C]raclopride has been used to investigate the density (B_max) and affinity (K_d) of dopamine D₂ receptors related to several neurological and psychiatric disorders. However, in assessing the B_max and K_d, multiple PET scans are necessary under variable specific activities of administered [¹¹C]raclopride, resulting in a long study period and unexpected physiological variations. In this paper, we have developed a method of multiple-injection graphical analysis (MI-GA) that provides the B_max and K_d values from a single PET scan with three sequential injections of [¹¹C]raclopride, and we validated the proposed method by performing numerous simulations and PET studies on monkeys. In the simulations, the three-injection protocol was designed according to prior knowledge of the receptor kinetics, and the errors of B_max and K_d estimated by MI-GA were analyzed. Simulations showed that our method could support the calculation of B_max and K_d, despite a slight overestimation compared with the true magnitudes. In monkey studies, we could calculate the B_max and K_d of diseased or normal striatum in a 150 mins scan with the three-injection protocol of [¹¹C]raclopride. Estimated B_max and K_d values of D₂ receptors in normal or partially dopamine-depleted striatum were comparable to the previously reported values.     

Ketamine alters the availability of striatal dopamine transporter as measured by [(11)C]beta-CFT and [(11)C]beta-CIT-FE in the monkey brain.

The effects of ketamine anesthesia on the binding of [(11)C]-labeled cocaine analogs, [(11)C]beta-CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane) and [(11)C]beta-CIT-FE (N-(2-fluoroethyl)-2beta-carbomethoxy-3beta-(4-iodophenyl)tropane), to the striatal dopamine transporter (DAT) were evaluated in the monkey brain using positron emission tomography (PET). We sequentially measured the kinetics of these labeled compounds in the brains of five young-adult male rhesus monkeys (Macaca mulatta) in the conscious state, followed by those under ketamine anesthesia with continuous infusion (3 and 10 mg/kg/h). After intravenous injection, [(11)C]beta-CFT and [(11)C]beta-CIT-FE were predominantly accumulated in the striatum in both conscious and ketamine-anesthetized states. In the conscious state, the striatal uptake of [(11)C]beta-CFT and [(11)C]beta-CIT-FE continuously increased with time up to 91 min after injection. Continuous infusion of ketamine-induced higher levels of uptake of [(11)C]beta-CFT and [(11)C]beta-CIT-FE into the brain in a dose-dependent manner as compared with conscious state, and kinetic analysis with metabolite-corrected arterial input function indicated that the binding potentials (BP = k(3)/k(4)) of both compounds were elevated by ketamine. Not only [(11)C]beta-CIT-FE but also [(11)C]beta-CFT reached the equilibrium state of specific binding in the striatum within 40-50 min after injection. The present results demonstrated that ketamine significantly alters the DAT availability as measured with [(11)C]beta-CFT and [(11)C]beta-CIT-FE in the brain.     

Investigating the effects of norepinephrine α1 receptor blockade on dopamine levels: A pilot PET study with [11C]‐(+)‐PHNO in controls

Interest in a role for norepinephrine (NE) in substance use disorders has increased over recent years. In particular, its interaction with dopamine (DA) is of importance. In this study, positron emission tomography (PET) was used to explore the impact of prazosin (an alpha 1 NE antagonist) on DA levels. Healthy volunteers were administered prazosin for approximately 4 weeks at the daily dose of 15 mg to reach steady state. Participants were scanned with PET imaging and the [¹¹C]‐(+)‐PHNO tracer at baseline (before prazosin), at steady state, and after a wash out period. Prazosin administration was associated with an increase of [¹¹C]‐(+)‐PHNO binding potential in the dorsal caudate relative to baseline, which corresponds to a decrease in DA levels. This study is the first to demonstrate interactions between DA and NE in healthy humans.     

Characterization of [3H]nemonapride binding to mouse brain dopamine D2 receptors assessedin vivo andex vivo for metabolic modeling in PET studies

Summary We characterized [³H]nemonapride ([³H]NEM, [³H]YM-09151-2) binding to dopamine D₂ receptors. In mice given [³H]NEM with and without sulpiride, thein vivo specific binding of the [³H]NEM to the D₂ receptors in the striatum was assessed: SB_{in vivo-1}, striatal uptake minus cerebellar uptake; and SB_{in vivo-2}, uptake in the control mice minus uptake in the sulpiride-treated mice. Tissue homogenates were divided into cytosol and the membrane binding fraction (MB). When the MB was incubatedin vitro with sulpiride, the dissociated and nondissociated fractions were defined as theex vivospecific binding (SB_{ex vivo}) and theex vivo nonspecific binding (NB_{ex vovo}), respectively. HPLC revealed that most of the radioactivity in the MB was [³H]NEM, whereas metabolites were found in the cytosol. In the striatum, the SB_{ex vivo} increased with time (50% of the total tissue uptake at 60 min), and was equivalent to the SB_{in vivo-2}. The SB_{in vivo-1} was comparable to the MB. In the cerebral cortex and cerebellum, the SB_{ex vivo} decreased with time and the SB_{ex vivo}/free [³H]NEM ratios were smaller than those in the striatum.     

Binding of (+)PHNO and other D2-dopamine agonists to D1-dopamine receptors labelled by [3H]SCH 23390

Summary The affinity of (+)PHNO for the D₁-dopamine receptor subtype in striatal tissue was determined in a radioligand binding assay using the D₁-selective compound, [³H]SCH 23390. The IC₅₀ for (+)PHNO was 22M, a value considerably higher than obtained for reference agents (lisuride, apomorphine, pergolide, [–]EOE and bromocriptine). The low potency of (+)PHNO at the D₁-receptor subtype contrasts with the compound's high potency at the D₂-receptor subtype, as reported previously. (+)PHNO can, therefore, be classified as a very selective D₂-receptor agonist.     

Increased dopamine D2/D3 receptor binding after recovery from anorexia nervosa measured by positron emission tomography and [11c]raclopride.

BACKGROUND: Several lines of evidence support the possibility that disturbances of dopamine (DA) function could contribute to alterations of weight, feeding, motor activity, and reward in anorexia nervosa (AN). METHODS: To assess possibly trait-related disturbances but avoid confounding effects of malnutrition, 10 women who were recovered from AN (REC AN) were compared with 12 healthy control women (CW). Positron emission tomography with [(11)C]raclopride was used to assess DA D2/D3 receptor binding. RESULTS: The women who were recovered from AN had significantly higher [(11)C]raclopride binding potential in the antero-ventral striatum than CW. For REC AN, [(11)C]raclopride binding potential was positively related to harm avoidance in the dorsal caudate and dorsal putamen. CONCLUSIONS: These data lend support for the possibility that decreased intrasynaptic DA concentration or increased D2/D3 receptor density or affinity is associated with AN and might contribute to the characteristic harm avoidance or increased physical activity found in AN. Most intriguing is the possibility that individuals with AN might have a DA related disturbance of reward mechanisms contributing to altered hedonics of feeding behavior and their ascetic, anhedonic temperament.     

Characterization of in vivo pharmacokinetic properties of the dopamine D1 receptor agonist DAR-0100A in nonhuman primates using PET with [11C] NNC112 and [11C] raclopride

DAR-0100A, the active enantiomer of dihydrexidine, is a potent dopamine D1 agonist under investigation for treatment of cognitive impairment and negative symptoms of schizophrenia. We measured the dose–occupancy relationship for DAR-0100A at D1 receptors using positron emission tomography (PET) imaging in baboons with [¹¹C] NNC112 and its binding to D2 with [¹¹C] raclopride. Two baboons were scanned with [¹¹C] NNC112 at baseline and after three different doses of DAR-0100A. Two baboons were scanned with [¹¹C] raclopride at baseline and after one dose of DAR-0100A. Occupancy (ΔBP_ND) was computed in the striatum and cortex. A clear relationship was observed between plasma concentration of DAR-0100A and ΔBP_ND. ΔBP_ND was larger in the striatum than in the cortex, consistent with reports showing that 25% of [¹¹C] NNC112 BP_ND in the cortex is attributed to 5-HT_2A. Plasma EC₅₀ estimates ranged from 150 to 550 ng/mL according to the constraints on the model. There was no detectable effect of DAR-0100A on [¹¹C] raclopride BP_ND. These data suggest that at doses likely to be administered to patients, occupancy will not be detectable with [¹¹C] NNC112 PET and binding of DAR-0100A to D2 will be negligible. This is the first demonstration with PET of a significant occupancy by a full D1 agonist in vivo.     

Brain region binding of the D2/3 agonist [11C]-(+)-PHNO and the D2/3 antagonist [11C]raclopride in healthy humans

The D(2) receptors exist in either the high- or low-affinity state with respect to agonists, and while agonists bind preferentially to the high-affinity state, antagonists do not distinguish between the two states. [(11)C]-(+)-PHNO is a PET D(2) agonist radioligand and therefore provides a preferential measure of the D(2) (high) receptors. In contrast, [(11)C]raclopride is an antagonist radioligand and thus binds with equal affinity to the D(2) high- and low-affinity states. The aim was to compare the brain uptake, distribution and binding characteristics between [(11)C]-(+)-PHNO and [(11)C]raclopride in volunteers using a within-subject design. Both radioligands accumulated in brain areas rich in D(2)/D(3)-receptors. However, [(11)C]-(+)-PHNO showed preferential uptake in the ventral striatum and globus pallidus, while [(11)C]raclopride showed preferential uptake in the dorsal striatum. Mean binding potentials were higher in the putamen (4.3 vs. 2.8) and caudate (3.4 vs 2.1) for [(11)C]raclopride, equal in the ventral-striatum (3.4 vs. 3.3), and higher in the globus pallidus for [(11)C]-(+)-PHNO (1.8 vs. 3.3). Moreover [(11)C]-(+)-PHNO kinetics in the globus pallidus showed a slower washout than other regions. One explanation for the preferential binding of [(11)C]-(+)-PHNO in the globus pallidus and ventral-striatum could be the presence of a greater proportion of high- vs. low-affinity receptors in these areas. Alternatively, the observed distribution could also be explained by a preferential binding of D(3)-over-D(2) with [(11)C]-(+)-PHNO. This differential binding of agonist vs. antagonist radioligand, especially in the critically important region of the limbic striatum/pallidum, offers new avenues to investigate the role of the dopamine system in health and disease.