In Vivo DA D1 Receptor Selectivity of NNC 112 and SCH 23390

  • Jesper Ekelund
  • Mark Slifstein
  • Raj Narendran
  • Olivier Guillin
  • Hemant Belani
  • Ning-Ning Guo
  • Yuying Hwang
  • Dah-Ren Hwang
  • Anissa Abi-Dargham
  • Marc Laruelle
Research Article

Abstract

Purpose

[11C]NNC 112 and [11C]SCH 23390 are selective positron emission tomography (PET) tracers for visualizing dopamine D1 receptors. It is known that both have some affinity for serotonin 2A receptors, but previous studies have suggested this is negligible compared to D1 affinity. We sought to verify this property in vivo.

Procedures

Two baboons were scanned to measure the selectivity of both tracers with a displacement paradigm. Four baboons were scanned to directly assess [11C] NNC 112 affinity for both receptors.

Results

In vivo, D1 to 5-HT2A selectivity is six to fourteenfold, not 100-fold as previously reported by other investigators.

Conclusion

We conclude that about 1/4 of the cortical signal of both [11C]NNC 112 and [11C]SCH 23390 is due to binding to 5-HT2A receptors. If confirmed in humans, this suggests caution should be exercised when drawing conclusions from studies using either tracer. These results also indicate the need for more selective tracers for the D1 receptor.

Key words

NNC 112 SCH 23390 Dopamine D1 receptor Serotonin 2A receptor Position emission tomography In vivo selectivity 

Introduction

D1 receptors are the most abundant dopaminergic receptor subtype in neocortical areas including the prefrontal cortex (PFC), as well as in the hippocampus (for review, see [1]). Studies in nonhuman primates have shown that activation of D1 receptors in the PFC is involved in working memory [2, 3, 4], and that D1 receptors in the hippocampus are implicated in short- and long-term memory [5, 6, 7, 8]. In conditions such as schizophrenia, a deficit in prefrontal D1 receptor function might contribute to the cognitive problems presented by these patients [9, 10, 11]. Therefore, an accurate and reliable method for measuring these receptors in extrastriatal regions of the living human brain with positron emission tomography (PET) is desirable to study their role in mediating cognition in health and disease states.

The radio-labeled benzazepine [11C]SCH 23390 (KD = 0.14 nM in vitro [12]) was the first radiotracer developed to image D1 receptors with PET [13, 14, 15]. Studies in humans have demonstrated that [11C]SCH 23390 is an appropriate radiotracer for measurement of D1 receptors in the striatum, where these receptors are present in high density [16, 17]. [11C]SCH 23390 displays relatively low specific to nonspecific binding ratios, which might compromise the sensitivity and reliability of the D1 receptor measurement in extrastriatal areas such as the PFC [18], where the density of these receptors is approximately fivefold lower than in the striatum [19, 20, 21]. Despite this, a recent test/retest study demonstrated appropriate reproducibility of the measurement of [11C]SCH23390 binding potential (BP) in the human PFC [22].

More recently, a new benzazepine, [11C]NNC 112 (KD = 0.18 nM, in vitro [23]), has been developed as a PET radiotracer to image D1 receptors [23, 24, 25, 26, 27]. [11C]NNC 112 provides higher specific to nonspecific binding ratios compared to [11C]SCH 23390. PET studies in humans have confirmed the potential of this radiotracer [26, 28].

Two studies have been performed with [11C]SCH 23390 to assess D1 receptor availability in patients with schizophrenia. The first reported decreased [11C]SCH 23390 BP in the PFC [29], and the other reported no change [18]. One study was performed with [11C]NNC 112, and reported increased [11C]NNC 112 BP in dorsolateral prefrontal cortex (DLPFC) in patients with schizophrenia [30]. The results of the three studies may diverge because of differences in patient populations, cameras, regions of interest boundaries, or method of analysis (for review, see [31]). However, these discrepant results might also stem from differences in the in vivo behavior of these radiotracers. For example, chronic dopamine depletion has different effects on the in vivo binding of both tracers. Guo et al. [32] showed that [11C]NNC 112 in vivo binding was upregulated after dopamine depletion, supporting the biological plausibility of the hypothesis that, in schizophrenia, upregulated [11C]NNC 112 BP is secondary to sustained dopamine depletion. However, no change of [3H]SCH 23390 binding was observed, suggesting that the in vivo binding of both radiotracers is differentially regulated by chronic dopamine depletion, and potentially accounting for the discrepant results observed in schizophrenia.

A possible difference between the two tracers is their in vivo selectivity for D1 receptors versus serotonin (5-HT) 5-HT2A receptors. A relative modest selectivity for 5-HT2A receptors is not likely to be problematic when imaging D1 receptors in the striatum with these ligands, as the density of D1 receptors is high and that of 5-HT2A receptors is negligible in that region. However, in the primate cortex, the density of 5-HT2A receptors is about twice that of D1 receptors [33]. Thus, the in vivo binding of [11C]NNC 112 and/or [11C]SCH 23390 to 5-HT2A receptors potentially presents a greater confound in cortex than in striatum.

Available data suggest that the contribution of 5-HT2A receptors to [11C]NNC 112 and [11C]SCH 23390 cortical binding should be negligible (Table 1). The only published study reporting affinities of both agents for both receptors suggests a least 100-fold selectivity of both tracers for D1 relative to 5HT2A receptors. Anderson et al. [23] measured NNC112 in vitroKD for D1 and 5HT2A receptors of 0.18 and 18 nM, respectively, resulting in a selectivity ratio of 100. These investigators also measured SCH23390 in vitroKD for D1 and 5HT2A receptors of 0.14 and 37 nM, respectively, resulting in a selectivity ratio of 250 [23]. Assuming a Bmax ratio of 2, such a selectivity would result in a negligible contribution of 5HT2A receptors to the cortical binding of both radiotracers (2% for [11C]NNC 112, 1% for [11C]SCH 23390). Reports of in vivo data suggest negligible 5-HT2A receptor contributions to the cortical uptake of these tracers. Halldin et al. [26] observed no change in the cortical time–activity curves of [11C]NNC 112 after administration of the 5-HT2A receptor antagonist ketanserin (2 mg/kg i.v.). Suhara et al. [34] reported the in vivo selectivity of SCH23390 by showing that pretreatment with up to 1 mg/kg ketanserin did not alter the binding of SCH23390 in mice.
Table 1

Review of in vitroKD values reported in the literature for SCH 23390 and NNC 112 for the 5HT2A and DA D1 receptors

KD

Hot ligand

Species

Tissue

Reference

KD of SCH 23390 for the 5-HT2A receptor

37

[3H]Ketanserin

Rat

Brain

[27]

13.1

[3H]Ketanserin

Rat

Frontal cortex

[47]

37

[3H]Ketanserin

Rat

Striatal membranes

[23]

14

[3H]Ketanserin

Rat

Cortical membranes

[48]

8.51

[3H]Ketanserin

Rat

Cortex

[49]

19.9

[3H]Ketanserin

Rat

Brain

[50]

10.8

[3H]Ketanserin

Rat

Brain w/o cerebellum

[51]

KD of SCH 23390 for the D1 receptor

0.35

[3H]SCH23390

Human

Cloned

[52]

0.14

[3H]SCH23390

Mouse

Brain

[53]

0.37

[3H]SCH23390

Rat

Cloned

[54]

0.14

[3H]SCH23390

Rat

Brain

[27]

0.8

[3H]SCH23390

Rat

Brain

[55]

0.12

[3H]SCH23390

Rat

Brain

[51]

0.2

[3H]SCH23390

Rat

Striatum

[56]

0.34

[3H]SCH23390

Rat

Striatum

[57]

0.4

[3H]SCH23390

Rat

Striatum

[47]

0.14

[3H]SCH23390

Rat

Frontal cortex

[12]

1.4

[3H]SCH23390

Rat

Retina

[58]

0.37

[3H]SCH23390

Rat

Striatal membranes

[48]

0.14

[3H]SCH23390

Rat

Striatal membranes

[23]

10,000

[3H]SCH23390

Bovine

Caudate

[59]

0.57

[3H]SCH23390

Bovine

Pineal

[60]

1.3

[3H]SCH23390

Monkey

Brain

[55]

KD of NNC 112 for the 5-HT2A receptor

18

[11H]Ketanserin

Rat

Striatal membranes

[23]

KD of NNC 112 for the D1receptor

0.18

[11H]SCH23390

Rat

Striatal membranes

[23]

Nonetheless, the available data are limited. In vitro affinities and selectivities are not always predictive of the in vivo situation, and neither of the in vivo studies was performed using rigorous quantitative analysis. Also, the Suhara et al. [34] study was performed in mice and might not be an appropriate source of inference regarding cortical uptake in humans. Because of these limitations and because of the importance of this question for the interpretation of the scans performed with these ligands (especially in view of the results reported in schizophrenia described above), a more rigorous evaluation of this question was warranted.

In this study, we report results of PET studies conducted in baboons aimed at determining the extent of the 5-HT2A receptor contribution to striatal and cortical uptake of [11C]NNC 112 and [11C]SCH 23390. Initially, scans were obtained at baseline and after pretreatment with receptor saturating doses of the selective 5-HT2A receptor antagonist MDL 100907 (specific aim 1, see “Methods” section). Because the results of these studies were inconsistent with predictions based on in vitroKD values, we then undertook to determine the in vivo affinity of [11C]NNC 112 for both D1 (specific aim 2) and 5-HT2A receptors (specific aim 3). In vivo determination of D1 and 5-HT2A receptor affinities were performed only for [11C]NNC 112 because the rapid metabolization of [11C]SCH 23390 in plasma precludes accurate measurement of in vivoKD.

Methods

Design of Experiments

A total of 38 PET experiments in four different adult male baboons (referred to as baboons A through D) are presented in this study. The scans were acquired in 19 experimental sessions, and each session included two PET scans: one baseline scan and one scan obtained after a pharmacological challenge. The study included three specific aims:
  1. Specific aim 1:

    Blocking studies of [11C]NNC 112 and [11SCH 23390 by MDL 100907. In these experiments, [11C]NNC112 and [11C]SCH23390 in vivo binding were measured at baseline and after administration of receptor saturating doses of the selective 5HT2A antagonist MDL 100907. The goal of these experiments was to measure the proportion of cortical and striatal binding of both tracers accounted for by binding to 5-HT2A receptors. Two adult male baboons were studied. Both animals were studied twice using two different doses of MDL100907 (0.1 and 1 mg/kg). In previous studies with [11C]MDL 100907 in baboons, we established that the lower of these doses (0.1 mg/kg) resulted in complete blockade of 5-HT2A receptors (data not shown). We used this dose as well as a dose 10 times higher to verify that we had reached a plateau and no further radiotracer blockade could be observed at the higher dose. Both baseline and postchallenge scans were acquired after single bolus injections (SBI) of the radiotracers.

     
  2. Specific aim 2:

    In vivo affinity of NNC 112 for D1receptors. In these experiments, [11C]NNC112 in vivo striatal binding was measured at baseline and after various doses of unlabelled NNC112. The goal of these experiments was to measure the in vivo affinity of NNC112 for D1 receptors. Four adult male baboons were studied, in a total of seven experimental sessions. Each session included two PET scans with [11C]NNC 112, a baseline scan and a scan during administration of unlabelled NNC 112. The baseline (tracer dose) scan was obtained after SBI of [11C]NNC 112. The challenge scan was obtained during a bolus plus constant infusion (BCI) using [11C]NNC112 with a high mass of nonradioactive NNC112 added (carrier added experiment). Three pairs of experiments were obtained in baboon A, two in baboon B, and one each in baboons C and D.

    The decrease of [11C]NNC 112 binding in the striatum between the two experiments was taken to reflect the occupancy of D1 receptors by NNC 112, based on the assumption that 5HT2A receptors contribute a negligible fraction to [11C]NNC 112 binding in the striatum. The results of specific aim 1 were used to test the validity of this assumption.

    The reason for performing the carrier added experiment as BCI was to achieve a verifiable stable level of NNC112 in the plasma and the brain, allowing determination of the relationship between free level and occupancy under equilibrium conditions. BCI experiments were acquired with a Kbol of 160 minutes (the bolus dose corresponded to 160 minutes of constant infusion). [11C]NNC 112 reached equilibrium after 80 minutes of scanning, and outcome measures were derived from the data points in the 90- to 120-minute interval. The tracer dose experiments were performed as a single bolus, allowing derivation of receptor parameters in a shorter scanning session compared to a BCI experiment at tracer dose.

     
  3. Specific aim 3:

    In vivo affinity of NNC 112 for 5-HT2Areceptors. In these experiments, [11C]MDL 100907 in vivo cortical binding was measured at baseline and after the same doses, in the same animals, of unlabelled NNC112 as used in specific aim 2. The goal of these experiments was to measure the in vivo affinity of NNC112 for 5-HT2A receptors. Three adult male baboons were studied, in a total of four experimental sessions. Each session consisted of two SBI [11C]MDL100907 PET scans at tracer dose: one baseline scan, and a second scan during the steady state phase of an infusion of unlabeled NNC112, administered with a BCI schedule designed to attain the same steady state level as in the carrier added experiments in specific aim 2. As the same animals were engaged in specific aims 2 and 3, it was assumed that for a given animal, the free level of NNC112 during the [11C]MDL 100907 experiments of specific aim 3 was the same as the free level of NNC112 measured in the carrier added experiments of specific aim 2.

     

Radiochemistry

[11C]NNC112 and [11C]SCH23390 were synthesized as previously described [15, 26].

Scan Protocols

Experiments were performed according to protocols approved by the Columbia-Presbyterian Medical Center Institutional Animal Care and Use Committee. Fasted animals were immobilized with ketamine (10 mg/kg i.m.) and anesthetized with 1.5–2.5% isoflurane via an endotracheal tube, as required for adequate level of anesthesia. The animals’ vital signs were monitored every 10 minutes and temperature was kept constant at 37°C with heated water blankets. An i.v. infusion line was used for hydration and injection of radiotracers and nonradioactive drugs. In experiments involving [11C]NNC 112 and [11C]MDL 100907, a catheter was inserted in a femoral artery for arterial blood sampling. The head was positioned at the center of the field-of-view as defined by embedded laser lines.

PET imaging was performed with an ECAT EXACT HR+ scanner (Siemens/CTI, Knoxville, TN). In 3-D mode, this camera provides in-plane resolution of 4.3, 4.5, 5.4, and 8.0 mm full width at half maximum at distances of 0, 1, 10, and 20 cm from the center of the field-of-view [35]. A 10-minute transmission scan was obtained before radiotracer injection for attenuation correction. The injected radioactivity had an upper limit of 6 mCi for bolus experiments and 10 mCi for constant infusion. Emission data were collected in 3-D mode for 60 minutes ([11C]SCH23390 SBI experiments), 90 minutes ([11C]NNC 112 and [11C]MDL 100907 SBI experiments), and 120 minutes ([11C]NNC 112 BCI experiments) as successive frames of increasing duration.

Input Function Measurements

The rapid metabolization of [11C]SCH 23390 prevents reliable measurement of the arterial input function for this tracer. Therefore, arterial plasma input function measurements were obtained only for the [11C]NNC 112 and [11C]MDL 100907 scans. Arterial samples were collected every 10 seconds for the first two minutes and every 20 seconds from two to four minutes using an automated blood sampling system, and drawn manually thereafter at various intervals. For [11C]NNC 112 and [11C]MDL 100907, the total number of samples collected were 28 and 21, respectively. After centrifugation (1,100 g for 10 minutes), plasma was collected and radioactivity measured in 0.2 mL aliquots using an automatic gamma counter (Wallac 1480 Wizard 3M, Perkin–Elmer, Boston, MA).

Selected plasma samples were further processed by extraction with methanol followed by HPLC analysis to determine the fraction of plasma activity representing the unmetabolized parent tracer. The percent of parent was determined as follows: 0.4 mL of the plasma sample was mixed with methanol (1 mL) in a microcentrifuge tube. After vortexing, the tube was centrifuged at 15,000 g for four minutes. The liquid phase was separated from the precipitates. The amount of radioactivity in 0.1 mL of the liquid phase was determined using the gamma counter and the rest was injected onto HPLC. For the analysis of [11C]NNC 112, an ODS-Prep analytical column (10 micron, 4.6 × 250 mm, Phenomenex, Torrance, CA, USA) was eluted with a solvent mixture of 35% ACN and 65% AMF at a flow rate of 2 mL/minute. For the analysis of [11C]MDL 100907, an Econocil C-18 analytical column (10 micron, 4.6 × 250 mm, Alltech, Deerfield, IL, USA) was eluted with 40% ACN and 60% AMF (0.1 M with 0.5% acetic acid) and a flow rate of 2 mL/minute.

Before plasma sample analysis, the retention time of the parent tracer was established by the injection of a small amount of the tracer. The HPLC eluate was fraction-collected and counted in the Wallac gamma counter. The percent parent fraction was calculated as the ratio of the radioactivity in the fractions containing the parent tracer to the total radioactivity of all collected fractions.

A biexponential function was fitted to the six measured parent fractions, and used to interpolate values between the measurements. The smallest exponential of the parent fraction curve, λpar, was constrained to the difference between λcer, the terminal washout rate of the cerebellar activity, and λtot, the smallest elimination rate constant of the total plasma. The input function was then calculated as the product of total counts and interpolated parent fraction at each time point. The measured input function values, starting from the time of the measured peak plasma concentration, were fitted to a sum of three exponentials. The fitted values were used as input for the kinetic analyses.

For the determination of the plasma free fraction (f1), triplicate aliquots of plasma (0.2 mL), separated from the blood sample (collected before tracer injection) spiked with the radiotracer, were pipetted into ultrafiltration units (Amicon Centrifree, Millipore, Bedford, MA, USA) and centrifuged (1,100 g) at room temperature for 20 minutes. The amount of radioactivity in the filter unit and the filtrate were counted. The fraction f1 was calculated as the ratio of the concentration of the filtrate to that of the total (radioactivity/mL). Triplicate aliquots of the radiotracer in pH 7.4 Tris buffer were also processed to determine the filter retention of the free tracer.

Image Analysis

A magnetic resonance imaging (MRI) of each baboon’s brain was obtained for the purpose of identifying the regions of interest (ROIs) (T1-weighted axial MRI sequence, acquired parallel to the anterior–posterior commissure, TR 34 milliseconds, TE 5 milliseconds, flip angle of 45°, slice thickness 1.5 mm, zero gap, matrix 1.5 × 0.86 × 0.86 mm3 voxels). The ROIs were delineated on the MRI based on the criteria defined in [36]. PET emission data were attenuation-corrected using the transmission scan and decay corrected frames were reconstructed with filtered backprojection, using a Shepp filter (cutoff 0.5 cycles/projection ray). Reconstructed image files were then processed using the image analysis software MEDx (Sensor Systems, Sterling, VA, USA). An image was created by summing all the frames and this summed image was used to define the registration parameters for use with the MRI, using the between-modality automated image registration (AIR) algorithm [37]. Registration parameters were then applied to the individual frames for registration to the MR dataset. ROI boundaries were transferred to the individual registered PET frames and time–activity curves were formed as the mean activity in each ROI from each frame. Included ROIs were striatum, frontal, temporal, parietal and occipital cortices, cingulate, hippocampus, and cerebellum. Right and left ROIs were averaged. For a given animal the same regional boundaries were used for all experiments. Brain activity was corrected for the contribution of plasma activity assuming a 5% blood volume [38].

Derivation of Binding Parameters

Outcome Measures

The regional total tissue distribution volume (VT, mL/g) was defined as the ratio of the ligand concentration in a region (CT, mCi/g) to the concentration of unmetabolized ligand in arterial plasma (CA, mCi/mL) at equilibrium:
$$ V_{{\text{T}}} = \frac{{C_{{\text{T}}} }} {{C_{{\text{A}}} }} $$
(1)
The total distribution volume in regions of interest (VT ROI) included the nondisplaceable distribution volume (free and nonspecific binding, V2) and the specific binding distribution volume, or binding potential (BP). The concentration of D1 receptors and 5-HT2A receptors are negligible in the cerebellum [20]. Therefore, only free and nonspecifically bound radiotracer were considered to contribute to VT in the cerebellum (VT CER), and VT CER was assumed to be equal to the nondisplaceable distribution volume (V2) in other regions. Receptor availability was estimated with the specific to nonspecific equilibrium partition coefficient, \( V^{\prime{\prime}}_{ 3} \). \( V^{\prime^{\prime}}_{3} \) is the ratio of BP to V2, and was computed indirectly as VT ROI/VT CER −1 when the arterial plasma function was available, and with the simplified reference tissue method (SRTM) [39] otherwise. \( V^{\prime^{\prime}}_{3} \) is related to receptor parameters by:
$$ V^{\prime^{\prime}}_{3} = f_{2} \frac{{B_{{{\text{max}}}} }} {{K_{{\text{D}}} }} $$
(2)
where Bmax is the concentration of receptors (nanomoles per gram of tissue), KD is the in vivo equilibrium dissociation constant of the radiotracer (nanomoles per milliliter of brain water) and f2 is the free fraction of free plus nonspecifically bound ligand in brain. \( V^{\prime^{\prime}}_{3} \) is the only parameter of receptor availability that can be derived in the absence of arterial input function. The change in receptor availability was measured as
$$ \Delta V^{\prime^{\prime}}_{3} = 100\% \times {\left( {1 - \frac{{V^{\prime^{\prime}}_{3} {\left( {{\text{challenge}}} \right)}}} {{V^{\prime^{\prime}}_{3} {\left( {{\text{baseline}}} \right)}}}} \right)} $$
(3)

Assuming a purely competitive model with reporting ligand given at tracer dose, \( \Delta V^{\prime^{\prime}}_{3} \) is equivilant to the occupancy of receptors by the competitor. For all specific aims, \( \Delta V^{\prime^{\prime}}_{3} \) in cortical regions (all ROIs other than striatum and cerebellum) are reported as a single average.

For specific aim 1, derivation of [11C]NNC 112 and [11C]SCH 23390 \( V^{\prime^{\prime}}_{3} \) were performed with kinetic analysis using SRTM with cerebellum as reference region. \( \Delta V^{\prime^{\prime}}_{3} \) was used to calculate the reduction in [11C]NNC 112 and [11C]SCH 23390 \( V^{\prime^{\prime}}_{3} \) due to occupancy of 5-HT2A receptors by MDL 100907.

Because arterial plasma data were available for the [11C] NNC 112 data, kinetic analysis was performed by the indirect method with these data as well, using a two-tissue compartment model [24].

For specific aim 2, SBI experiments were analyzed with kinetic modeling using the arterial input function and a two-tissue compartment model, and BCI experiments were analyzed with equilibrium analysis [40]. Striatal \( \Delta V^{\prime^{\prime}}_{3} \) was used to calculate the reduction in D1 receptor availability due to occupancy of D1 receptors by NNC 112.

For each of the seven pairs of experiments, an estimate of the in vivo affinity of NNC112 for D1 receptors was derived according to
$$ K_{{\text{D}}} = \frac{{F{\left( {1 - {\text{occupancy}}} \right)}}} {{{\text{occupancy}}}} $$
(4)
where F is the concentration of free radioligand (at low specific activity with carrier added) at equilibrium. Assuming that NNC 112 crosses the blood brain barrier by passive diffusion, the free level of NNC 112 in plasma and in brain equilibrate under steady state conditions. Therefore, F was estimated as f1CSS, where f1 was the plasma free fraction and CSS was the unmetabolized [11C]NNC 112 plasma concentration at steady state.

For specific aim 3, [11C]MDL 100907 SBI experiments were analyzed with kinetic modeling using the arterial input function and a two-tissue compartment model [41] Cortical \( \Delta V^{\prime^{\prime}}_{3} \) was used to calculate the reduction in 5-HT2A receptor availability due to occupancy of 5-HT2A receptors by NNC 112 (Eq. 3). For each of the four pairs of experiments, an estimate of the in vivo affinity of NNC112 for 5-HT2A receptor was derived according to Eq. 4. As experiments were performed in the same animals as in specific aim 2, with BCI infusion schedule designed to reach the same steady state values as in specific aim 2, the values of F measured in specific aim 2 were used in Eq. 4 to derive the in vivo affinity of NNC112 for 5-HT2A receptors.

Results

Displacement Studies

Striatal and cortical \( V^{\prime^{\prime}}_{3} \) values of [11C]NNC 112 and [11C]SCH 23390 at baseline and following blockade of 5-HT2A receptors are presented in Table 2. The average \( V^{\prime^{\prime}}_{3} \) of NNC112 (4.04 ± 0.20 in striatum and 0.8 ± 0.07 in cortex) and SCH23390 (2.98 ± 0.25 in striatum and 0.60 ± 0.09 in cortex) are consistent with previously reported values from PET studies in primates [22, 26]. There was a significant effect of region on \( \Delta V^{\prime^{\prime}}_{3} \), so that the decrease in cortex was significantly larger than in the striatum (p = 0.007). This effect remained significant when covarying for animal and tracer (p = 0.04). Neither animal nor tracer in themselves showed a significant effect on change in \( V^{\prime^{\prime}}_{3} \) (p = 0.29 and 0.90, respectively). Also, there was no detectable region by animal or region by tracer interaction (p = 0.58 and 0.13, respectively).
Table 2

Decrease in \( V^{\prime^{\prime}}_{3} \) of [11C]NNC 112 and [11C]SCH 23390 after blocking dose of MDL100907

Animal

Dose of MDL100907 (mg/kg)

Striatum baseline

Striatum challenge

Striatum change (%)

Cortex baseline

Cortex challenge

Cortex change (%)

[11C]SCH23390 (SRTM)

A

0.1

2.94

2.97

1.0

0.48

0.43

−9.8

A

1

3.25

3.01

−7.4

0.61

0.50

−18.9

B

0.1

3.08

2.68

−12.8

0.69

0.60

−12.1

B

1

2.65

1.92

−27.4

0.62

0.36

−41.8

Average ± SD

   

−11.7 ± 11.9

  

−20.7 ± 14.6

p value

   

0.125

  

0069

[11C]NNC112 (SRTM)

A

0.1

4.16

4.01

−3.4

0.74

0.49

−34.0

A

1

4.24

4.03

−4.9

0.75

0.54

−28.8

B

0.1

3.81

3.59

−5.8

0.82

0.61

−26.2

B

1

3.95

3.65

−7.4

0.88

0.65

−25.8

Average ± SD

   

−5.4 ± 1.7

  

−28.7 ± 6.4

p value

   

0.006

  

<0.001

[11C]NNC112 (2TCM)

A

0.1

6.33

5.90

−6.8

0.97

0.79

−19.4

A

1

6.81

5.94

−12.8

0.94

0.65

−30.7

B

0.1

6.27

7.21

10.7

0.86

0.70

−18.8

B

1

5.94

5.71

−3.8

0.97

0.71

−27.0

Average ± SD

   

−3.2 ± 10.0

  

24.0 ± 5.8

p value

   

0.55

  

0.005

The p values are from paired t tests comparing baseline to the blocking condition.

While the repeated measures design showed no significant effect of tracer on \( \Delta V^{\prime^{\prime}}_{3} \), t tests on each ligand and region separately (with MDL100907 doses pooled) showed significant reduction of [11C] NNC 112 binding in cortex (p = 0.00016) and trend level reduction for [11C] SCH 23390 (p = 0.069). Thus MDL100907 reduced cortical [11C] NNC 112 and [11C] SCH 23390 to a similar extent in the mean, albeit with different effect sizes, while causing much less reduction in striatum with both tracers.

There were no significant differences in \( \Delta V^{\prime^{\prime}}_{3} \) between the 0.1 and 1.0mg/kg MDL100907 experiments in any region.

Two tissue modeling of the [11C] NNC 112 data was qualitatively similar to the SRTM analysis: binding reduction after MDL 100907 administration was 24% in cortex (p = 0.005) and 3% in striatum (p = 0.55) (Fig. 1). The actual \( V^{\prime^{\prime}}_{3} \) values were somewhat larger for the arterial plasma input based model than for SRTM; this is a property of these methods that has been observed with several radioligands [42, 43, 44]. There was no difference in nondisplaceable distribution volume (cerebellum) between the baseline and MDL 100907 conditions (baseline = 7.60 ± 1.98, MDL condition = 7.36 ± 1.46, p = 0.78, paired t test).
Fig. 1.

Decrease in \( V^{\prime^{\prime}}_{3} \) after blocking dose of MDL100907. The figure represents the mean ± SD for four pairs of experiments in two baboons.

Injected doses, specific activities and injected masses of [11C]NNC 112 in specific aim 1 experiments were 5.03 ± 0.22 mCi, 873.8 ± 184.5 Ci/mmol, and 6.0 ± 1.3 μg, respectively.

Injected doses, specific activities and injected masses of [11C]SCH 23390 in specific aim 1 experiments were 4.84 ± 0.58 mCi, 983.3 ± 706.2 Ci/mmol, and 9.6 ± 8.3 μg, respectively.

In vivo Affinity of NNC112 for D1 Receptors

Table 3 lists striatal \( V^{\prime^{\prime}}_{3} \) measured during pairs of tracer only and carrier added experiments as well as KD estimates. The average estimate of KD from all seven experiments was 0.018 ± 0.006 nM (Fig. 2).
Table 3

In vivo affinity of [11C]NNC 112 for D1 receptors

Animal

Striatal [11C]NNC 112 \( V^{\prime^{\prime}}_{3} \)

Occupancy D1 receptors (%)

Free NNC112 (nM)

KD (nM)

Baseline

Carrier added

A

6.13

2.86

53

0.016

0.014

A

6.36

1.88

70

0.029

0.012

A

6.90

1.16

83

0.054

0.011

B

6.70

2.35

65

0.040

0.021

B

5.43

1.46

73

0.067

0.025

C

6.38

1.21

81

0.092

0.022

D

10.28

1.80

83

0.104

0.022

Average

    

0.018 ± 0.006

Fig. 2.

Boxplot of KD values obtained from seven and four pairs of experiments for the D1 and 5-HT2A receptors, respectively. Note that the y-axis is presented on a log10 scale.

An example of data from one animal that received multiple carrier added doses of NNC 112 (baboon A) is shown in Fig. 3. For display purposes, both the curves fit to all data points using the Michaelis–Menten equation and a Scatchard plot are shown.
Fig. 3.

Michaelis Menten equation fit in one animal (baboon A) that received multiple carrier added doses of NNC 112. The two baseline scans from specific aim 1 are included in the fit (five tracer dose scans and three carrier added). Estimated KD (from this animal’s data only) is 0.011 nM with nonlinear least squares estimation and 0.012 nM with Scatchard plot (inset). Estimated Bmax is 37.9 and 39.5 nM by nonlinear least squares and Scatchard plot, respectively.

In the carrier added experiments, the free level of NNC112 in the plasma showed a linear relationship with injected dose in all animals (R2 = 0.92).

The rate of change of [11C]NNC 112 activity in striatum and cortex was 4%/hour ± 4% between 90 and 120 minutes (not significantly different from zero).

Injected doses, specific activities, and injected masses of [11C]NNC 112 in SBI tracer only experiments were 4.60 ± 1.00 mCi, 863.5 ± 329.9 Ci/mmol, and 5.5 ± 2.0 μg, respectively. Injected doses, specific activities, and injected masses of [11C]NNC 112 in BCI carrier added experiments were 6.20 ± 1.68 mCi, 13.45 ± 13.71 Ci/mmol, and 696.6 ± 349.0 μg, respectively.

Plasma clearance was 63.0 ± 15.2 L/hour in tracer only experiments. Plasma free fraction was 2.0 ± 0.7 and 1.9 ± 0.3% in tracer only and carrier added experiments, respectively (p = 0.77). V2 was 6.88 ± 1.15 and in 9.41 ± 4.01 in tracer only and carrier added experiments, respectively (p = 0.19).

In Vivo Affinity of NNC112 for 5-HT2A Receptors

The average KD of NNC112 for the 5-HT2A receptor was 0.26 ± 0.07 nM (Fig. 2). We chose to use a mass of NNC112 that corresponds to a 70–85% occupancy of the DA D1 receptor based on the experiments from specific aim 1, which was 1 μmol for all three animals. Given the KBOL of 160 minutes, and the longer time of infusion (90 minutes for equilibration, 90 minutes for [11C]MDL100907 scan) we injected 1.21 μmol in total, 0.578 μmol as bolus and then 0.2121 μmol/hour as infusion during 180 minutes. Full results are provided in Table 4.
Table 4

In vivo binding data for NNC112 for the 5HT2A receptor and within subject selectivity ratio (D1 vs 5-HT2A, Baboons B, C, and D only)

Animal

Cortical [11C]MDL 100907 V3

Occupancy 5HT2A receptors (%)

Free NNC112 (nM)

KD (nM)

Selectivity (within subject)

Baseline

During NNC 112 infusion

B

0.94

0.86

10

0.040

0.36

12.2

B

1.09

0.83

25

0.067

0.20

 

C

1.56

1.12

28

0.092

0.24

10.9

D

1.04

0.73

30

0.104

0.24

10.9

Average

    

0.26 ± 0.07

11.3 ± 0.7

Injected doses, specific activities and injected masses of [11C]MDL 100907 experiments were 4.49 ± 0.49 mCi, 2585 ± 853 Ci/mmol, and 0.69 ± 0.16 μg, respectively. [11C]MDL 100907 plasma clearance was 59.6 ± 16.2 L/hour and in 64.2 ± 39.8 L/hour in baseline and challenge experiments, respectively (p = 0.81). [11C]MDL 100907 Plasma free fraction was 0.43 ± 0.05 and 0.41 ± 0.02 in baseline and challenge experiments, respectively (p = 0.35). This is consistent with the plasma free fraction of 0.34 ± 0.04 reported previously [41]. [11C]MDL 100907 V2 was 15.1 ± 1.2 and in 18.4 ± 2.4 in baseline and challenge added experiments, respectively (p = 0.11). This is somewhat higher than reported in a previous study in rhesus monkeys (9.8 ± 0.049) [41].

Discussion

This study was designed to quantify the in vivo binding to 5-HT2A receptors of the putatively D1 specific PET tracers [11C]NNC112 and [11C]SCH23390. It was previously known that the second highest affinity of both NNC112 and SCH23390 is to the 5-HT2A receptor. However, assuming a KD ratio of 100, even in the primate cortex the binding to the 5-HT2A receptor would constitute less than 2% of the total binding signal and therefore be completely negligible. These assumptions were based on in vitro binding studies and a small number of in vivo studies with technical limitations.

The data presented in this study strongly suggest that for in vivo measurements in primates, the selectivity ratio is much less than 100-fold. First, we showed that for both NNC112 and SCH23390, 5-HT2A binding does in fact contribute significantly to the cortical binding signal, about 1/4 of the total \( V^{\prime^{\prime}}_{3} \) for both tracers. There was no significant difference between tracers in the effect of blockade of the 5-HT2A receptors. Using the previously known densities of these two receptors in the primate brain, the blocking studies imply selectivity more in the range of 10-fold. Given this level of selectivity, binding to the 5-HT2A receptor significantly contributes to the measured \( V^{\prime^{\prime}}_{3} \) using either of the tracers. The fact that there were no significant differences in binding reduction in any region between the 1 and 0.1 mg doses of MDL 100907 strongly suggests that complete blocking of 5-HT2A receptors was achieved in all regions at both doses, and the smaller reduction in striatum compared to cortex is consistent with the known relative distributions of D1 and 5-HT2A receptors.

The density of 5-HT2A receptors is about twice that of D1 receptors in the primate cortex [33]. Because
$$V^{\prime^{\prime}}_{{3\_{\text{TOTAL}}}} = f_{2} {\left( {\frac{{B_{{\max D1}} }}{{Kd_{{D1}} }} + \frac{{B_{{\max 2A}} }}{{Kd_{{2A}} }}} \right)}$$
(5)
and 3/4 and 1/4 of the total binding was to the DA D1 and 5-HT2A receptors respectively, we can conclude that:
$$\frac{{B_{{\max 2A}} }}{{Kd_{{2A}} }} = \frac{1}{3} * \frac{{B_{{\max D1}} }}{{Kd_{{D1}} }}$$
(6)
and therefore:
$$\frac{{Kd_{{2A}} }}{{Kd_{{D1}} }} = 3 * \frac{{B_{{\max 2A}} }}{{B_{{\max D1}} }}$$
(7)
which, based on the known Bmax ratio is around 6 for both tracers. In this article, “cortex” refers to an average of the frontal, temporal, parietal, and occipital cortex as well as the cingulate and hippocampus.

In specific aims 2 and 3 of this study, direct measurement of the in vivoKD of NNC112 for D1 and 5-HT2A receptors indicated that the KD ratio for the two receptors is 14.4 when the mean estimates of the two KDs are compared (or 11.3 when measured within subject from the three animals that participated in specific aims 2 and 3), verifying the findings from the displacement study. The estimate of both KD and the ratio showed little variation between animals. It should be pointed out that measurement of the free fraction in plasma (f1) is technically difficult when this parameter is in the range of 2% or less, as was the case in this study. Given the model in Eq. 4, any error in the measurement of f1 would directly translate into an error in the estimate of KD. Therefore, the individual values of KD for NNC112 from the in vivo study should be interpreted with caution. However, this has no effect on the KD ratio and does not affect the main result of this study.

Recently, Chou et al. [45] have published a PET study in monkeys in which, after a challenge with the antipsychotic drug clozapine, they observed larger blockade of [11C] NNC 112 in cortex than in striatum. These authors interpreted their data as evidence that clozapine binds to D1-like receptors with higher affinity in cortex than striatum. However, clozapine exhibits high affinity for 5-HT2A receptors and significantly blocks 5-HT2A receptors when administered in the dose range used in that study [46]. In light of the results of the present study, the larger blockade of [11C] NNC 112 binding by clozapine in cortex compared to striatum is more likely due to blockade of 5-HT2A receptors in cortex, rather than a difference in affinity of clozapine between cortical and striatal D1 receptors.

Based on our study, we can conclude that a nonnegligible component of the binding potential of NNC112 in cortex derives from binding to the 5-HT2A receptor. SCH 23390 was tested less extensively than NNC 112 in this study, but the data we did collect are consistent with similar D1:5-HT2A selectivity for SCH 23390 to that observed for NNC 112, and this ligand should be tested more thoroughly for its in vivo selectivity as well.

Our results suggest that studies comparing clinical populations using either of these tracers need to be interpreted with caution because it is not known whether an observed difference between groups could reflect changes in D1 levels, 5-HT2A levels or some combination of both. It will be necessary to replicate this study in humans to assess how significant our observation is for D1 PET studies in humans. These data also suggest that development of a more selective D1 PET tracer is warranted.

Notes

Acknowledgments

The authors thank Elizabeth Hackett, Elizabeth Mitchell, Robyn Gonzales, Lyudmila Savenkova, and Kurt Sudeall for their excellent technical assistance.

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Copyright information

© Academy of Molecular Imaging 2007

Authors and Affiliations

  • Jesper Ekelund
    • 1
    • 3
  • Mark Slifstein
    • 1
  • Raj Narendran
    • 1
  • Olivier Guillin
    • 3
  • Hemant Belani
    • 3
  • Ning-Ning Guo
    • 1
    • 3
  • Yuying Hwang
    • 1
    • 3
  • Dah-Ren Hwang
    • 1
    • 3
  • Anissa Abi-Dargham
    • 1
    • 3
  • Marc Laruelle
    • 1
    • 2
    • 3
  1. 1.Department of PsychiatryColumbia UniversityNew YorkUSA
  2. 2.Department of RadiologyColumbia UniversityNew YorkUSA
  3. 3.Division of Functional Brain MappingNew York State Psychiatric InstituteNew YorkUSA

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