Skip to main content
PMC home page
Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2009 Dec 23;30(5):985–993. doi: 10.1038/jcbfm.2009.269

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

Ana M Catafau 1,*, Graham E Searle 2, Santiago Bullich 3, Roger N Gunn 2,4,5, Eugenii A Rabiner 2,5, Raul Herance 3, Joaquim Radua 3,6, Magi Farre 7, Marc Laruelle 2,5
PMCID: PMC2949183  PMID: 20029452

Abstract

[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-HT2A 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-HT2A R. Seven healthy volunteers underwent [11C]NNC112 PET scans at baseline and after a 5-HT2A R-blocking dose of ketanserin (0.15 mg/kg, i.v.). Percent BPND change between the post-ketanserin and baseline scans was calculated. Irrespective of the quantification method used, ketanserin pretreatment led to significant decrease of BPND in the cortical (∼30%) and limbic regions (∼20%) but not in the striatum, which contains a much lower amount of 5-HT2A R. Therefore, ketanserin allows D1 R signal to be detected by [11C]NNC112 PET without significant 5-HT2A R contamination. These data confirm the presence of a significant 5-HT2A 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.

Keywords: [11C]NNC112, D1 receptors, ketanserin, PET, selectivity

Introduction

[11C]NNC112 (8-chloro-7-hydroxy-3-methyl-5-(7-benzofuranyl)-2,3,4,5-tetrahydro-IH-3-benzazepine) has been used as a positron-emission tomography (PET) ligand for in vivo study of dopamine D1 receptors (R). In preclinical studies, a relationship between [11C]NNC112-binding potential (BPND) and prefrontal cortex dopamine concentrations has been shown both in rats (Guo et al, 2003) and monkeys (Tsukada et al, 2005). In clinical studies, increased D1 R availability by means of [11C]NNC112 PET has been reported both in schizophrenic patients with working memory deficits (Abi-Dargham et al, 2002) and in chronic ketamine abusers (Narendran et al, 2005), thus linking low prefrontal dopamine function to both cognitive impairment in schizophrenia and chronic glutamatergic hypofunction. All these studies were relying on the 100-fold in vitro selectivity of this ligand for D1 R (KD=0.18) over 5-HT2A R (KD=18) (Andersen et al, 1992). However, more recent in vivo studies using 5-HT2A R antagonist challenges both in baboons and in humans, with saturating doses of MDL100907 (0.1 to 1 mg/kg, intravenous (i.v.)) (Ekelund et al, 2007) and risperidone (2 mg, per os (p.o.)) (Slifstein et al, 2007), have shown a 25% to 30% reduction of [11C]NNC112 BPND in the cortical regions, suggesting that approximately one quarter of the cortical signal was due to binding to 5-HT2A R. In the absence of a selective ligand for in vivo D1 R imaging, pretreatment with a 5-HT2A R blocker could be a practical method to enable accurate quantification of D1 R in vivo. Unfortunately, none of the already used challenges are optimal for human studies. MDL100907 is not approved for human use, and risperidone is not a selective 5-HT2A antagonist. Moreover, the i.v. route of administration may be preferable for PET study challenges, as it should lead to more consistent plasma levels than oral administration. Ketanserin provides an acceptable alternative, as it is a selective 5-HT2A blocker (Ki=0.38 nmol/L) (Israilova et al, 2002) and can be administered intravenously to humans. I.v. ketanserin has been used as a challenge to study the 5-HT2A selectivity of PET and SPECT radioligands, both in baboons (Sadzot et al, 1995) and healthy volunteers (Travis et al, 2000; Pinborg et al, 2003; Catafau et al, 2006b), showing ∼90% blockade of 5-HT2A R with a 0.1-mg/kg dose. Although ketanserin is not completely devoid of D1 R affinity (Ki=190 to 464 nmol/L in cloned human D1 R using [3H]SCH23390; Sunahara et al, 1991; Toll et al, 1998), pretreatment with ketanserin at doses of 1 mg/kg (Kassiou et al, 1995a, 1995b, 2001, 2002) and 200 mg/kg (Duffy et al, 2000) has shown to be unable to displace other D1 R PET ligands in in vivo preclinical studies. In this study, we examined the utility of a ketanserin challenge in conjunction with [11C]NNC112 to quantify D1 R binding without contamination of 5-HT2A R binding in healthy volunteers. We hypothesized that ketanserin would induce a decrease of [11C]NNC112 binding in the cortical areas, supporting previous evidence of the 5-HT2A R component. Given the low affinity of ketanserin for D1 R, we also expected no significant change in [11C]NNC112 binding in the striatum, where the density of 5-HT2A R is much lower than that of D1 R (Pazos et al, 1985).

Materials and methods

Subjects

The study was approved by the local Ethics Committees and the Spanish Ministry of Health. All subjects provided written informed consent before inclusion in the study.

Seven male human volunteers aged 25±2 years (range 22 to 28 years) were enrolled in this study. None had past or present neurological or psychiatric disorders as assessed by the Structured Clinical Interview for DSM IV (SCID II) questionnaire (First et al, 1997). None were taking psychotropic drugs or other relevant medication. All had normal findings on physical examination, 12-lead electrocardiography, clinical chemistry, and hematology. Absence of illegal drug abuse was assessed by interviews with independent physicians in two different occasions, and supported by urine screening using a commercial on-site rapid test (MultiClin Cassette for Urine; SureScreen Diagnostic Ltd, Derby, UK) at screening and before PET scanning sessions. The screening procedure included determination of amphetamines, barbiturates, benzodiazepines, cocaine, tetrahydrocannabinol, methylenedioxymethamphetamine, opiates, oxycodone, phencyclidine, propoxyphene, and tricyclic antidepressants. Subjects had to withdraw from caffeine and alcohol 24 h before the study day and were not allowed to take any medications during the 14 days before the study day.

Study design

All subjects underwent two [11C]NNC112 PET scans, before (baseline scan) and 15 mins (min) after ketanserin challenge (0.15 mg/kg, i.v.), as well as a structural magnetic resonance imaging (MRI) to allow co-registration and region of interest (ROI) drawing. Five subjects underwent both PET scans on the same day, 4 to 6 h apart. For the remaining two subjects, post-ketanserin scan was performed 5 and 11 days after the baseline scan. For safety reasons, the subjects rested lying on the PET bed with continuous monitoring of electrocardiography, blood pressure, and heart rate from 15 mins before ketanserin injection until the end of the post-ketanserin scan. A follow-up phone call was performed 3 to 5 days after the ketanserin PET scan.

Radiochemistry

[11C]NNC112 was synthesized according to the following procedure: [11C]CO2 was generated in an IBA Cyclone 18/9 cyclotron by bombardment of an N2/O2 (95%/5%) gas mixture with high-energy (18 MeV) protons. [11C]CH3I was formed by reduction of [11C]CO2 in the presence of lithium aluminium hydride (300 μL, 0.1 M solution in dry tetrahydrofuran (THF); ABX, Radeberg, Germany) and distillation after solvent evaporation and addition of hydriodic acid (300 μL, 57% aqueous solution; ABX). [11C]CH3I was trapped in a 2-mL stainless steel high-performance liquid chromatography (HPLC) loop pre-charged with a solution of desmethyl-NNC112 (free base, 1 mg; ABX) in dimethylsulfoxide (80 μL). After complete trapping of [11C]CH3I, the reaction was allowed to occur for 4 mins at room temperature. The reaction mixture was purified by means of HPLC using an RP-C18 column (Mediterranea Sea18, 9.6 × 250 mm, 5 μm particle size) and using sodium phosphate buffer (pH 8)/acetonitrile (35/65) mixture as the mobile phase at 5 mL/minute, using UV (λ=254 nm) and isotopic detectors connected in series. The purified fraction (retention time=8 to 10 mins) was collected in a 40-mL vial pre-charged with 20 mL of physiological saline solution. The resulting mixture was passed through a C-18 cartridge (Sep-Pak plus; Waters, Milford, MA, USA) to retain [11C]NNC112. The radiotracer was eluted with ethanol (2 mL) and collected in a sterile 10-mL vial. The solution was evaporated at 70°C under continuous nitrogen flow until a final volume of approximately 100 μL was obtained. The residue was reconstituted with 7 mL of physiological saline solution. After double filtration through 0.22-μm sterile filters, a fraction of the final solution was subjected to quality control. Residual solvents were determined by means of gas chromatography; radiochemical purity was measured by means of HPLC and product identification was performed by HPLC and co-elution with standard reference solution. In both cases, chromatography was performed using an RP-C18 column (Mediterranea Sea18, 4.6 × 150 mm, 5 μm particle size) and using sodium phosphate buffer (pH 8)/acetonitrile (35%/65%) mixture as the mobile phase at 1 mL/minute, using UV (λ=254 nm) and isotopic detectors connected in series. Specific activity average was 41.8 (±19.9) GBq/μmol, with radiochemical purity higher than 95% in all cases.

PET and MRI Protocol

PET was performed using a Siemens ECAT-EXACT HR+ camera equipped with transmission sources of 68Ge. Prior to [11C]NNC112 injection, a 10-minute transmission scan was obtained for attenuation correction. After injection of a [11C]NNC112 dose (baseline scan: 520.7±88.3 MBq, post-ketanserin scan: 487.3±54.1 MBq), 100 mins of emission data were acquired as 27 dynamic frames (8 × 15 secs, 3 × 60 secs, 5 × 120 secs, 5 × 300 secs, 6 × 600 secs). The PET data were reconstructed using filtered back projection with a Hanning filter (kernel FWHM of 4.9 mm). Corrections were applied for attenuation (through transmission data), scatter, randoms, and dead time. The volumes produced consisted of 128 × 128 × 63 voxels; each voxel being 2.57 × 2.57 × 2.43 mm.

A structural T1-weighted MRI scan was performed for all subjects and co-registered with PET images for definition of striatal ROIs. An axial three-dimensional spoiled gradient-echo slab was positioned to include the entire head and the images were acquired with the following parameters: repetition time, 25 ms; echo time, 6 ms; flip angle, 28 degrees; field of view, 25 × 25 cm; matrix size, 256 × 256; section thickness, 2 mm, with no interslice gap; and number of excitations, 1.

Input Function Measurement

Arterial blood activity was measured every second for the first 15 mins after injection using a continuous arterial blood sampling system (ABSS Allogg, Mariefred, Sweden). Thereafter, 14 manual samples were collected at the following time points: 5, 10, 14, 17, 22.5, 27.5, 32.5, 37.5, 45, 55, 65, 75, 85, 95 mins. The plasma was centrifuged over 10 mins at 1800g and collected in 200-mL aliquots. Each aliquot's activity was counted with a gamma counter (Perkin Elmer Wallac, Waltham, MA, USA; Wizard 1470 Automatic Gamma Counter). Three samples collected at 5, 10, and 14 mins after injection, which overlapped the continuous blood sampling period, were used to calibrate continuous blood samples to match discrete samples. The discrete and continuous whole-blood data were thus combined into a single whole-blood activity curve. The sampled plasma data were divided by the corresponding whole-blood measurements and the average of these values was taken as the plasma-over-blood ratio. The whole blood curve was multiplied by this value to obtain a total plasma activity curve spanning the duration of the scan.

The fraction of plasma activity representing unmetabolized (parent) [11C]NNC112 was measured by HPLC for seven plasma samples (5, 10, 17.5, 27.5, 45, 65.5, and 95 mins) using the method described by Abi-Dargham et al (2000). These parent fraction data were fitted to a single exponential decay plus a constant term. The fitted parent fraction data were multiplied by the total plasma activity data to produce a sampled parent plasma activity curve. This curve was smoothed post-peak by fitting to a tri-exponential function, and thus an arterial parent plasma input function was obtained for use in kinetic modeling. The plasma clearance of unmetabolized [11C]NNC112 was calculated as the ratio of injected activity to the area under the fitted parent plasma input function. The plasma free fraction was also measured, using the method described by Abi-Dargham et al (2000).

Image Analysis

Dynamic PET images were registered to the subject's MRI and corrected for motion by realigning each time frame to the averaged frame using a mutual information-based algorithm as implemented in the SPM5b software (Wellcome Trust Centre for Neuroimaging; http://www.fil.ion.ucl.ac.uk/spm). ROIs were defined on each subject's MRI using a combination of manual and automated methods. The cortical (orbitofrontal cortex; medial and dorsolateral prefrontal cortices; anterior cingulate gyrus; and parietal, temporal, and occipital cortices), amygdala, and cerebellar regions were defined through nonlinear registration (using SPM5b) of a template MRI and the corresponding atlas (developed in-house on the basis of the Harvard–Oxford atlas included with FSL 4.1; http://www.fmrib.ox.ac.uk/fsl). The striatal regions (precommissural dorsal caudate and putamen, ventral striatum, and postcommissural caudate and putamen) were defined manually as described by Martinez et al (2003). A gray matter segmentation was applied to each of the cortical ROIs. Each ROI was then applied to the dynamic PET data to derive regional time–activity curves.

Receptor Parameter Estimation

A two-tissue-compartment (2TC) model was fitted to each ROI and the total volume of distribution (VT) was estimated. BPND was derived using the indirect method as BPND=(VT,ROI−VT,CER)/VT,CER, where VT,ROI and VT,CER are the total volumes of distribution in a region with specific uptake and in the reference region (cerebellum), respectively. The binding potential was also estimated by the simplified reference tissue method (SRTM) using the cerebellum as reference region (Lammertsma and Hume, 1996). The percentage of ketanserin-induced displacement (%Disp) was computed as %Disp=100%·(BPND,baseline−BPND,post-ketanserine)/BPND,baseline.

Parametric Maps

BPND maps obtained from the basis function implementation of SRTM (Gunn et al, 1997) were first co-registered with each individual' structural MRI using the SPM5b software. The BPND maps were then warped into stereotaxic space using deformation parameters derived from non-linear registration of the individual MRI to an MRI template in stereotaxic space and then smoothed with an 8-mm FWHM kernel. Paired t-tests with no proportional scaling were performed to compare baseline to post-ketanserin BPND maps. T maps were thresholded at P<0.001, as this level has been considered a significant threshold for statistical difference maps when there are a priori hypotheses regarding the location of findings (Friston et al, 1991).

Statistics

Differences in non-receptor-related scan parameters (e.g. injected dose) in baseline versus post-ketanserin scans were tested by two-tailed paired t-tests. Whether the percentage BPND change in each separate region was significantly different from 0, was also tested by two-tailed paired t-tests, correcting for multiple comparisons by means of the false discovery rate. The statistical significance of different kinetic analyses (2TC versus SRTM) and different regions in BPND percentage change from baseline to post-ketanserin were tested by means of repeated-measures analysis of variance in SPSS (Chicago, IL, USA). Three post hoc contrasts comparing whole cortex with amygdala, whole cortex with whole striatum, and amygdala with whole striatum, with Dunnet T3 correction were then performed to investigate the differences in the regional selectivity of [11C]NNC112.

Results

Safety data recorded at the beginning and end of the scanning session, including heart rate, blood pressure, and QTc intervals, showed that these parameters remained within reference ranges during the entire scanning period. At baseline, the ranges were 101 to 131 mm Hg (systolic)/49 to 81 mm Hg (diastolic) for blood pressure; 388 to 426 ms for QTc; and 58 to 77 b.p.m. for heart rate. At the end of the experiment, the respective ranges were 98 to 141 mm Hg (systolic)/41 to 82 mm Hg (diastolic); 390 to 418 ms; and 50 to 84 b.p.m.

Out of the seven subjects who completed the study, one showed excessive motion during scanning, which precluded reliable motion correction and subsequent image analysis. Therefore, the PET results reported below correspond to the sample of six subjects with evaluable PET scans. No differences were found for injected dose, specific activity, injected mass, plasma free fraction, VT,CER, or clearance between conditions (Table 1).

Table 1. Scan parameters.

  ID (MBq) SA (MBq/mmol) IM (μg) fP (unit-less) VT,CER (ml/cm3) Clearance (L/hour)
Baseline 513±94 4.54E+7±2.67E+7 4.7±2.2 2.65±2.22% 2.11±0.56 68±31
Post-ketanserin 504±36 4.17E+7±1.94E+7 4.5±2.0 2.80±1.84% 2.01±0.56 62±17
P 0.77 0.56 0.66 0.85 0.42 0.63
Open in a new tab

Abbreviations: fP, free fraction in plasma; ID, injected dose; IM, injected mass; SA, specific activity; VT,CER, volume of distribution of the non-displaceable tissue uptake (cerebellum).

Parameters from quantifiable subjects (n=6). The P-values are from two-tailed, paired t-tests.

Individual pre and post-ketanserin BPND values for cortical regions, amygdala, and striatum, as well as the percentage of change between conditions using both 2TC and SRTM methods both in this study and that in the study of Slifstein et al (2007), are presented in Tables 2 and 3. BPND parametric images at baseline and after ketanserin challenge are shown in Figure 1. Irrespective of the quantification method used, ketanserin pretreatment led to greater BPND decrease in the cortical regions (25% to 35%, all statistically significant) than that in the amygdala (19% to 20%, statistically significant in SRTM analysis) and striatum (5% to 13%, not statistically significant with the exception of the postcommissural putamen in SRTM analysis). Repeated-measures analysis of variance showed a significant effect of brain region (F=10.228, P<0.001) but not method (2TC versus SRTM analysis, F=0.004 P=0.950), with significantly larger BPND percent decreases in the cortical regions (taken together: ∼30%) than in the striatal regions (taken together, ∼10% Dunnet T3 P<0.001). The limbic region (amygdala) did not show statistically significant differences with either cortical regions or striatum. Accordingly, T maps of BPND change between baseline and post-ketanserin conditions showed significant decreases in the cortical regions, with no significant changes in the striatal regions (Figure 2).

Table 2. BPND results from 2TC modeling.

  [11C]NNC112+ketanserin, i.v. (this study) [11C]NNC112+risperidone, p.o. (Slifstein et al, 2007)
  Baseline Post-ketanserin % Change P % Change P
Cortical
 ANT CING 0.55±0.06 0.41±0.07 −25±13 0.012 −21±11 0.003
 DLPFC 0.41±0.05 0.27±0.06 −35±12 0.007 −30±20 0.011
 MPFC 0.46±0.05 0.31±0.07 −32±13 0.005 −28±13 0.002
 ORB FC 0.47±0.08 0.32±0.08 −29±19 0.022 −26±13 0.004
 TEM CTX 0.47±0.05 0.31±0.04 −33±13 0.006 −27±08 0.000
 PAR CTX 0.42±0.02 0.28±0.04 −33±12 0.012 −27±13 0.002
 OCC CTX 0.39±0.03 0.27±0.03 −30±12 0.006 −23±12 0.004
             
Limbic
 AMYG 0.46±0.08 0.37±0.12 −20±24 0.158 −10±11 0.231
             
Striatal
 Pre-DCA 2.48±0.34 2.33±0.27 −5±13 0.395 −9±21 0.302
 Pre-DPU 2.69±0.29 2.50±0.33 −7±13 0.323 0±13 0.992
 Post-CA 2.42±0.65 2.03±0.35 −13±19 0.227 −3±26 0.791
 Post-PU 2.64±0.32 2.45±0.25 −7±11 0.254 4±11 0.355
 VST 2.34±0.32 2.01±0.22 −12±23 0.297 5±14 0.635
Open in a new tab

Abbreviations: AMYG, amygdala; ANT CING, anterior cingulate gyrus; BP, binding potential; DLPFC, dorsolateral prefrontal cortex; i.v., intravenous; MPFC, medial prefrontal cortex; OCC CTX, occipital cortex; ORB FC, orbitofrontal cortex; PAR CTX, parietal cortex; Pre-DCA, precommissural dorsal caudate; Pre-DPU, precommissural dorsal putamen; Post-CA, postcommissural caudate; Post-PU, postcommissural putamen; 2TC, two-tissue-compartment; TEM CTX, temporal cortex; VST, ventral striatum.

Results from 2TC modeling analysis with arterial input function for six subjects. The P-values are from two-tailed paired t-tests, corrected by multiple comparisons by FDR.

Table 3. BPND results from SRTM analysis.

  [11C]NNC112+ketanserin, i.v. (this study) [11C]NNC112+risperidone, p.o. (Slifstein et al, 2007)
  Baseline Post-ketanserin % Change P % Change P
Cortical
 ANT CIN 0.54±0.04 0.40±0.06 −26±09 0.001 −22±18 0.012
 DLPFC 0.42±0.04 0.27±0.05 −35±07 0.000 −35±18 0.002
 MPFC 0.46±0.04 0.32±0.06 −31±08 0.000 −31±15 0.001
 ORB FC 0.47±0.05 0.32±0.06 −31±08 0.001 −33±15 0.002
 TEM CTX 0.45±0.04 0.31±0.03 −32±05 0.000 −29±10 0.000
 PAR CTX 0.42±0.03 0.28±0.03 −33±05 0.000 −32±17 0.002
 OCC CTX 0.40±0.02 0.28±0.02 −30±02 0.000 −27±11 0.001
             
Limbic
 AMYG 0.41±0.06 0.34±0.06 −19±11 0.012 −17±21 0.145
             
Striatal            
 Pre-DCA 1.98±0.25 1.85±0.18 −7±07 0.078 −4±24 0.632
 Pre-DPU 2.21±0.25 2.05±0.23 −7±08 0.076 2±18 0.792
 Post-CA 1.75±0.19 1.60±0.22 −9±08 0.068 4±38 0.765
 Post-PU 2.17±0.27 2.02±0.22 −6±05 0.043 5±01 0.336
 VST 1.89±0.20 1.65±0.14 −12±14 0.088 9±19 0.388
Open in a new tab

Abbreviations: AMYG, amygdala; ANT CING, anterior cingulate gyrus; BP, binding potential; DLPFC, dorsolateral prefrontal cortex; i.v., intravenous; MPFC, medial prefrontal cortex; OCC CTX, occipital cortex; ORB FC, orbitofrontal cortex; PAR CTX, parietal cortex; Pre-DCA, precommissural dorsal caudate; Pre-DPU, precommissural dorsal putamen; Post-CA, postcommissural caudate; Post-PU, postcommissural putamen; SRTM, simplified reference tissue method; TEM CTX, temporal cortex; VST, ventral striatum.

Results from SRTM analysis of six subjects. The P-values are from two-tailed paired t-test, corrected by multiple comparisons by FDR.

Figure 1.

Figure 1

Open in a new tab

Co-registered MRI: [11C]NNC112 PET BPND parametric images (mean from all subjects, n=6), at baseline (top) and after ketanserin 0.15 mg/kg, i.v. (bottom). The decrease of [11C]NNC112 signal in the cortical regions, without modification in the striatum, is appreciated.

Figure 2.

Figure 2

Open in a new tab

T maps of BPND change between baseline and post-ketanserin conditions. Threshold: P<0.001. A significant BPND decrease was shown in the cortical areas, demonstrating [11C]NNC112 displacement from 5-HT2A R, whereas no significant changes are seen in the striatum (area devoid of 5-HT2A R).

Discussion

This study contributes to the previous evidence of limited in vivo selectivity of [11C]NNC112 for D1 R compared to 5-HT2A R (Ekelund et al, 2007; Slifstein et al, 2007). This is not a problem in the striatum, given the poor concentration of 5-HT2A R as shown by PET and SPECT studies with selective ligands for 5-HT2A R (Travis et al, 2000; Pinborg et al, 2003; Catafau et al, 2006a), and the fourfold higher density of D1 R over 5-HT2A R in this region (List and Seeman, 1981; McBride et al, 1997). However, the reverse is true in the cortex, where the density of 5-HT2A R is four- to five-fold that in the striatum (Pazos et al, 1985), and therefore the [11C]NNC112 signal is significantly contaminated by 5-HT2A R. This study also provides a method for imaging cortical D1 R without significant contamination from 5-HT2A R.

The ketanserin dose was chosen on the basis of previous experience of ketanserin displacement of selective 5-HT2A R PET and SPECT ligands. Using a 0.1-mg/kg i.v. ketanserin dose, more than 90% displacement has been reported in monkey using [18F]altanserin (Sadzot et al, 1995). In humans, the ketanserin-induced displacement of the SPECT ligand [123I]R91150 was reported to be dose-dependent following an Emax model relationship where doses beyond 0.1 mg/kg, i.v., would not induce significantly higher ligand displacements (Catafau et al, 2006b). This is supported by the lack of significant differences in [11C]NNC112 displacement found using a 0.1-mg/kg dose and that using a 10-fold higher dose (1 mg/kg) of MDL100907 in baboons (Ekelund et al, 2007). These data were used as the rationale for the 0.15-mg/kg ketanserin dose as a blocking dose in this study, since it lies in the plateau of the described Emax model.

It must be noted that ketanserin is already used for patients with hypertension to lower their blood pressure, likely because of its α1-adrenergic antagonism. The compound has been shown to be well-tolerated and severe side effects after treatment have not been reported (Distler, 1990). The clinical oral dose for hypertension is 20 to 80 mg/day, and an intravenous dose of 10 mg has been reported effective in the treatment of post-anesthetic shivering in humans (Joris et al, 1993). In our study, the maximum ketanserin dose administered was 11.96 mg, and no safety issues were found with any of the doses tested. Blood pressure, heart rate, and electrocardiogram, including the QTc interval, were maintained within the reference ranges for the entire scanning period, thus suggesting that ketanserin 0.15 mg/kg is a safe dose that could be administered to healthy volunteers for diagnosis or research procedures. To generalize the use of i.v. ketanserin as a PET challenge for D1 R measurement, it has to be taken into account that it is only marketed in some countries. However, it may be imported for its use in other countries, with appropriate regulatory permissions. The latter was the procedure followed in this study. Another limitation of the use of ketanserin as a PET challenge is that it should not be administered to subjects who are at risk of suffering side effects of ketanserin (i.e., cardiovascular diseases, diabetes, renal insufficiency, hypotension, vascular headache, cerebrovascular disease, carcinoid syndrome, and hepatic diseases) or those for whom ketanserin is contraindicated, as indicated in the ketanserin data sheet.

The ketanserin-induced [11C]NNC112 displacement in the cortical regions found in this study supports previous evidence of a 5-HT2A R component in the in vivo signal of [11C]NNC112. The magnitude of ligand displacement in the cortical areas and amygdala are in close agreement with that in the study by Slifstein et al (2007), who used a 5-HT2A R-blocking dose of risperidone 2 mg p.o. (see Tables 1 and 2). Also in agreement with that study, no significant displacement was found in the striatum. However, the effect of ketanserin seemed to be larger than that of risperidone in this region (Tables 2 and 3). Ketanserine affinity for D1 R could be a potential explanation. Assuming that 0.15 mg/kg i.v. ketanserin induces 90% of 5-HT2A R occupancy, the corresponding D1 R occupancy would be 1.77% to 0.85% for the reported in vitro, Ki=190 to 464nmol/L (Sunahara et al, 1991; Toll et al, 1998), which would have a negligible contribution to the D1 R measurement. However, limitations of the in vitro-based calculations have to be taken into account before extrapolating to the in vivo effects of ketanserin on D1 striatal binding. An alternative explanation could be that i.v. ketanserin was able to displace [11C]NNC112 even from the low amount of 5-HT2A R present in the striatum.

The results of this study were similar, irrespective of the quantification method used (2TC and SRTM), to those reported by Slifstein et al (2007). The similarity of results replicated by two different groups may support the use of SRTM as a simpler method for within-subject comparison studies, avoiding the need for arterial blood sampling and thus increasing the availability of this methodology. The cerebellum was used as a reference region for both methods, given the known lower density of D1 R in this region and its previous use as a reference region for D1 R PET measurements (Ekelund et al, 2007; Slifstein et al, 2007). Although some in vitro studies have reported a small presence of 5-HT2A R in the cerebellum (Pazos et al, 1987; Eastwood et al, 2001), data from 5-HT2A R PET and SPECT studies support the use of the cerebellum as a reference region for 5-HT2A R measurement (Pinborg et al, 2003; Catafau et al, 2006b). In this study, there was no significant change in VT,CER between the pre and post-ketanserin scans, further supporting this region as a suitable reference region for [11C]NNC112 PET scanning with ketanserin challenge.

These data contribute to the growing evidence of discrepancies between in vitro and in vivo studies (Slifstein et al, 2007) and support the importance of the in vivo studies to assess PET ligand's selectivity. Moreover, the finding of [11C]NNC112's lack of D1 R selectivity calls for caution in the interpretation of previously published results on D1 R availability assessed by [11C]NNC112 PET without any drug challenge to block 5-HT2A R (Abi-Dargham et al, 2002; Guo et al, 2003; Narendran et al, 2005; Tsukada et al, 2005). Research on D1 R availability in patients with schizophrenia using PET has reported inconsistent results, with some authors finding increased D1 R availability (Abi-Dargham et al, 2002), whereas other finding it decreased (Okubo et al, 1997), and still others finding it to be unchanged in comparison with a control group (Karlsson et al, 2002). Since different PET ligands for D1 R ([11C]NNC112 or [11C]SCH23390) were used in those studies, a ligand selectivity issue was discussed as a potential explanation for these discrepancies. However, this seems unlikely given that although a 200-fold selectivity for D1 R (KD=0.14 nmol/L) over 5-HT2A R (KD=37 nmol/L) has been reported in vitro for [11C]SCH23390 (Andersen, 1988), the in vivo selectivity of this ligand has been found to be similar to that of [11C]NNC112 (Ekelund et al, 2007).

The lack of ketanserin displacement in striatal [11C]NNC112 binding supports the D1 R selectivity of the ligand in this region. However, a higher-affinity and more selective D1 R ligand is still warranted, given the low BPND values obtained with this ligand in the cortex both at baseline (∼0.5) and after ketanserin (∼0.3). In the absence of a better ligand, a suitable 5-HT2A R blocker such as ketanserin (0.15 mg, i.v.) would be required to image pure D1 R signal in the cortical regions.

In conclusion, data from this study support previous evidence of significant contribution from 5-HT2A R in the [11C]NNC112 signal in the cortex, calling for caution in the interpretation of previously published [11C]NNC112 PET findings on D1 R from humans in this cerebral region. Ketanserin pretreatment may be used to pre-block 5-HT2A R contribution to the [11C]NNC112 PET signal, without affecting the D1 R signal. Therefore, in the absence of a purely selective ligand for D1 R, i.v. ketanserin challenge provides a suitable method for D1 R imaging using [11C]NNC112 PET.

Acknowledgments

This study was funded by GlaxoSmithKline. We thank Stefano Zancan and Elisabet Tahull for study coordination; the IAT, CRC-CIM and IMIM staff for execution of the PET scans; and all the volunteers who participated in this study.

Ana M Catafau, Graham E Searle, Roger N Gunn, Eugenii A Rabiner, and Marc Laruelle are GlaxoSmithKline employees.

References

  1. Abi-Dargham A, Martinez D, Mawlawi O, Simpson N, Hwang DR, Slifstein M, Anjilvel S, Pidcock J, Guo NN, Lombardo I, Mann JJ, Van Heertum R, Foged C, Halldin C, Laruelle M. Measurement of striatal and extrastriatal dopamine D1 receptor binding potential with [11C]NNC 112 in humans: validation and reproducibility. J Cereb Blood Flow Metab. 2000;20:225–243. doi: 10.1097/00004647-200002000-00003. [DOI] [PubMed] [Google Scholar]
  2. Abi-Dargham A, Mawlawi O, Lombardo I, Gil R, Martinez D, Huang Y, Hwang DR, Keilp J, Kochan L, Van Heertum R, Gorman JM, Laruelle M. Prefrontal dopamine D1 receptors and working memory in schizophrenia. J Neurosci. 2002;22:3708–3719. doi: 10.1523/JNEUROSCI.22-09-03708.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andersen PH. Comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding to dopamine receptors in vivo in mouse brain. Eur J Pharmacol. 1988;146:113–120. doi: 10.1016/0014-2999(88)90492-x. [DOI] [PubMed] [Google Scholar]
  4. Andersen PH, Gronvald FC, Hohlweg R, Hansen LB, Guddal E, Braestrup C, Nielsen EB. NNC-112, NNC-687 and NNC-756, new selective and highly potent dopamine D1 receptor antagonists. Eur J Pharmacol. 1992;219:45–52. doi: 10.1016/0014-2999(92)90578-r. [DOI] [PubMed] [Google Scholar]
  5. Catafau AM, Danus M, Bullich S, Llop J, Perich J, Cunningham VJ, Plaza P, Penengo MM, Eersels JL, Squassante L, Ros D, Barbanoj M. Characterization of the SPECT 5-HT2A receptor ligand 123I-R91150 in healthy volunteers: part 1—pseudoequilibrium interval and quantification methods. J Nucl Med. 2006a;47:919–928. [PubMed] [Google Scholar]
  6. Catafau AM, Danus M, Bullich S, Nucci G, Llop J, Abanades S, Cunningham VJ, Eersels JL, Pavia J, Farre M. Characterization of the SPECT 5-HT2A receptor ligand 123I-R91150 in healthy volunteers: part 2—ketanserin displacement. J Nucl Med. 2006b;47:929–937. [PubMed] [Google Scholar]
  7. Distler A. Clinical aspects during therapy with the serotonin antagonist ketanserin. Clin Physiol Biochem. 1990;8:64–80. [PubMed] [Google Scholar]
  8. Duffy RA, Hunt MA, Wamsley JK, McQuade RD. In vivo autoradiography of [3H]SCH 39166 in rat brain: selective displacement by D1/D5 antagonists. J Chem Neuroanat. 2000;19:41–46. doi: 10.1016/s0891-0618(00)00045-4. [DOI] [PubMed] [Google Scholar]
  9. Eastwood SL, Burnet PW, Gittins R, Baker K, Harrison PJ. Expression of serotonin 5-HT(2A) receptors in the human cerebellum and alterations in schizophrenia. Synapse. 2001;42:104–114. doi: 10.1002/syn.1106. [DOI] [PubMed] [Google Scholar]
  10. Ekelund J, Slifstein M, Narendran R, Guillin O, Belani H, Guo NN, Hwang Y, Hwang DR, Abi-Dargham A, Laruelle M. In vivo DA D1 receptor selectivity of NNC 112 and SCH 23390. Mol Imaging Biol. 2007;9:117–125. doi: 10.1007/s11307-007-0077-4. [DOI] [PubMed] [Google Scholar]
  11. First MB, Gibbon M, Spitzer RL, Williams JBW, Benjamin LS. Structured Clinical Interview for DSM-IV Axis II Personality Disorders, (SCID-II) Washington, DC: American Psychiatric Press Inc.; 1997. [Google Scholar]
  12. Friston KJ, Frith CD, Liddle PF, Frackowiak RS. Comparing functional (PET) images: the assessment of significant change. J Cereb Blood Flow Metab. 1991;11:690–699. doi: 10.1038/jcbfm.1991.122. [DOI] [PubMed] [Google Scholar]
  13. Gunn RN, Lammertsma AA, Hume SP, Cunningham VJ. Parametric imaging of ligand–receptor binding in PET using a simplified reference region model. Neuroimage. 1997;6:279–287. doi: 10.1006/nimg.1997.0303. [DOI] [PubMed] [Google Scholar]
  14. Guo N, Hwang DR, Lo ES, Huang YY, Laruelle M, Abi-Dargham A. Dopamine depletion and in vivo binding of PET D1 receptor radioligands: implications for imaging studies in schizophrenia. Neuropsychopharmacology. 2003;28:1703–1711. doi: 10.1038/sj.npp.1300224. [DOI] [PubMed] [Google Scholar]
  15. Israilova M, Suzuki F, Tanaka T, Nagatomo T, Taniguchi T, Muramatsu I. Binding and functional affinity of sarpogrelate, its metabolite m-1 and ketanserin for human recombinant alpha-1-adrenoceptor subtypes. Pharmacology. 2002;65:69–73. doi: 10.1159/000056189. [DOI] [PubMed] [Google Scholar]
  16. Joris J, Banache M, Bonnet F, Sessler DI, Lamy M. Clonidine and ketanserin both are effective treatment for postanesthetic shivering. Anesthesiology. 1993;79:532–539. doi: 10.1097/00000542-199309000-00017. [DOI] [PubMed] [Google Scholar]
  17. Karlsson P, Farde L, Halldin C, Sedvall G. PET study of D(1) dopamine receptor binding in neuroleptic-naive patients with schizophrenia. Am J Psychiatry. 2002;159:761–767. doi: 10.1176/appi.ajp.159.5.761. [DOI] [PubMed] [Google Scholar]
  18. Kassiou M, Loc'h C, Bottlaender M, Mardon K, Ottaviani M, Coulon C, Katsifis A, Maziere B. (+)-[76Br]A-69024: a non-benzazepine radioligand for studies of dopamine D1 receptors using PET. Nucl Med Biol. 2002;29:295–302. doi: 10.1016/s0969-8051(01)00306-7. [DOI] [PubMed] [Google Scholar]
  19. Kassiou M, Mardon K, Mattner F, Katsifis A, Dikic B. Pharmacological evaluation of (+)-2-[123I]A-69024: a radioligand for in vivo studies of dopamine D1 receptors. Life Sci. 2001;69:669–775. doi: 10.1016/s0024-3205(01)01156-0. [DOI] [PubMed] [Google Scholar]
  20. Kassiou M, Scheffel U, Ravert HT, Mathews WB, Musachio JL, Lambrecht RM, Dannals RF. [11C]A-69024: a potent and selective non-benzazepine radiotracer for in vivo studies of dopamine D1 receptors. Nucl Med Biol. 1995a;22:221–226. doi: 10.1016/0969-8051(94)00086-y. [DOI] [PubMed] [Google Scholar]
  21. Kassiou M, Scheffel UA, Musachio JL, Stathis M, Dannals RF. [3H]A-69024: a non-benzazepine ligand for in vitro and in vivo studies of dopamine D1 receptors. Life Sci. 1995b;57:367–372. doi: 10.1016/0024-3205(95)02212-2. [DOI] [PubMed] [Google Scholar]
  22. Lammertsma AA, Hume SP. Simplified reference tissue model for PET receptor studies. Neuroimage. 1996;4:153–158. doi: 10.1006/nimg.1996.0066. [DOI] [PubMed] [Google Scholar]
  23. List SJ, Seeman P. Resolution of dopamine and serotonin receptor components of [3H]spiperone binding to rat brain regions. Proc Natl Acad Sci USA. 1981;78:2620–2624. doi: 10.1073/pnas.78.4.2620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martinez D, Slifstein M, Broft A, Mawlawi O, Hwang DR, Huang YY, Cooper T, Kegeles L, Zarahn E, Abi-Dargham A, Haber SN, Laruelle M. Imaging human mesolimbic dopamine transmission with positron emission tomography. Part II: amphetamine-induced dopamine release in the functional subdivisions of the striatum. J Cereb Blood Flow Metab. 2003;23:285–300. doi: 10.1097/01.WCB.0000048520.34839.1A. [DOI] [PubMed] [Google Scholar]
  25. Mcbride WJ, Chernet E, Russell RN, Chamberlain JK, Lumeng L, Li TK. Regional CNS densities of serotonin and dopamine receptors in high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats. Alcohol. 1997;14:603–609. doi: 10.1016/s0741-8329(97)00072-4. [DOI] [PubMed] [Google Scholar]
  26. Narendran R, Frankle WG, Keefe R, Gil R, Martinez D, Slifstein M, Kegeles LS, Talbot PS, Huang Y, Hwang DR, Khenissi L, Cooper TB, Laruelle M, Abi-Dargham A. Altered prefrontal dopaminergic function in chronic recreational ketamine users. Am J Psychiatry. 2005;162:2352–2359. doi: 10.1176/appi.ajp.162.12.2352. [DOI] [PubMed] [Google Scholar]
  27. Okubo Y, Suhara T, Suzuki K, Kobayashi K, Inoue O, Terasaki O, Someya Y, Sassa T, Sudo Y, Matsushima E, Iyo M, Tateno Y, Toru M. Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET. Nature. 1997;385:634–636. doi: 10.1038/385634a0. [DOI] [PubMed] [Google Scholar]
  28. Pazos A, Cortés R, Palacios JM. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res. 1985;346:231–249. doi: 10.1016/0006-8993(85)90857-1. [DOI] [PubMed] [Google Scholar]
  29. Pazos A, Probst A, Palacios JM. Serotonin receptors in the human brain—IV. Autoradiographic mapping of serotonin-2 receptors. Neuroscience. 1987;21:123–139. doi: 10.1016/0306-4522(87)90327-7. [DOI] [PubMed] [Google Scholar]
  30. Pinborg LH, Adams KH, Svarer C, Holm S, Hasselbalch SG, Haugbøl S, Madsen J, Knudsen GM. Quantification of 5-HT2A receptors in the human brain using [18F]altanserin-PET and the bolus/infusion approach. J Cereb Blood Flow Metab. 2003;23:985–996. doi: 10.1097/01.WCB.0000074092.59115.23. [DOI] [PubMed] [Google Scholar]
  31. Sadzot B, Lemaire C, Maquet P, Salmon E, Plenevaux A, Degueldre C, Hermanne JP, Guillaume M, Cantineau R, Comar D, et al. Serotonin 5HT2 receptor imaging in the human brain using positron emission tomography and a new radioligand, [18F]altanserin: results in young normal controls. J Cereb Blood Flow Metab. 1995;15:787–797. doi: 10.1038/jcbfm.1995.99. [DOI] [PubMed] [Google Scholar]
  32. Slifstein M, Kegeles LS, Gonzales R, Frankle WG, Xu X, Laruelle M, Abi-Dargham A. [11C] NNC 112 selectivity for dopamine D1 and serotonin 5HT2A receptors: a PET study in healthy human subjects. J Cereb Blood Flow Metab. 2007;27:1733–1741. doi: 10.1038/sj.jcbfm.9600468. [DOI] [PubMed] [Google Scholar]
  33. Sunahara RK, Guan HC, O'Dowd BF, Seeman P, Laurier LG, Ng G, George SR, Torchia J, Van Tol HH, Niznik HB. Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature. 1991;350:614–619. doi: 10.1038/350614a0. [DOI] [PubMed] [Google Scholar]
  34. Toll L, Berzetei-Gurske IP, Polgar WE, Brandt SR, Adapa ID, Rodriguez L, Schwartz RW, Haggart D, O'Brien A, White A, Kennedy JM, Craymer K, Farrington L, Auh JS. Standard binding and functional assays related to medications development division testing for potential cocaine and opiate narcotic treatment medications. NIDA Res Monogr. 1998;178:440–466. [PubMed] [Google Scholar]
  35. Travis MJ, Visvikis D, Erlandsson K, Mulligan RS, Matthiasson P, Waddington W, Costa DC, Pilowsky LS, Ell PJ, Kerwin RW. Displacement of the selective 5HT2A radioligand 123-5-I-R91150 by unlabelled ketanserin. Schizophr Res. 2000;41:253. [Google Scholar]
  36. Tsukada H, Nishiyama S, Fukumoto D, Sato K, Kakiuchi T, Domino EF. Chronic NMDA antagonism impairs working memory, decreases extracellular dopamine, and increases D1 receptor binding in prefrontal cortex of conscious monkeys. Neuropsychopharmacology. 2005;30:1861–1869. doi: 10.1038/sj.npp.1300732. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism are provided here courtesy of SAGE Publications

RESOURCES