Imaging the 5-HT 2C receptor with PET: Evaluation of 5-HT 2C and 5-HT 2A affinity of pimavanserin in the primate brain

Abstract

Two complimentary techniques were used to estimate occupancy of pimavanserin (a selective 5-HT_2A/2C inverse agonist) to 5-HT_2A and 5-HT_2C receptors in non-human primate brains. One employed the 5-HT_2A/2C selective radioligand [¹¹C]CIMBI-36 combined with quantification of binding potentials in brain regions known to be enriched in 5-HT_2A (cortex) or 5-HT_2C (choroid plexus) receptors to estimate occupancy. Pimavanserin was 6–10 fold more potent displacing [¹¹C]CIMBI-36 from cortex (ED₅₀ = 0.007 mg/kg; EC₅₀ = 0.6 ng/ml) than from choroid plexus (ED₅₀ =0.046 mg/kg; EC₅₀ = 6.0 ng/ml). The assignment of [¹¹C]CIMBI-36 binding to 5-HT_2A and 5-HT_2C receptors by anatomical brain structure was confirmed using the 5-HT_2A selective inverse agonist MDL 100,907 and the 5-HT_2C selective antagonist SB 242584 to displace [¹¹C]CIMBI-36. The second technique employed a novel, 5-HT_2C selective tracer called [¹¹C]AC1332. [¹¹C]AC1332 bound robustly to choroid plexus, moderately to hippocampus, and minimally to cortex. Pimavanserin displaced [¹¹C]AC1332 with similar potency (ED₅₀ = 0.062 mg/kg; EC₅₀ = 2.5 ng/ml) as its potency displacing [¹¹C]CIMBI-36 binding from choroid plexus. These results demonstrate the feasibility of simultaneously estimating drug occupancy of 5-HT_2A and 5-HT_2C receptors in vivo, and the utility of a novel 5-HT_2C receptor selective tracer ligand.

Keywords

pimavanserin positron emission tomography and serotonin

Introduction

The 5-HT_2C receptor (5-HT_2CR) is an important target for neuropsychiatric drug action.¹ Preclinical studies have demonstrated that 5-HT_2CRs regulate dopamine release in the brain, and modulation of 5-HT_2C activity is linked to appetite control,² regulation of mood,³ anxiety and stress response,⁴ and addictive behaviors.⁵ A large number of antipsychotics, antidepressants and anxiolytics have substantial 5-HT_2CR affinity.⁶ Lorcaserin (Lorqess; Belviq), a selective 5-HT_2C receptor agonist, was approved by the FDA in 2012 for the treatment of obesity.⁷

The role of 5-HT_2CRs in drug action and clinical effect remains poorly defined. One reason for this is the lack of selective PET ligands to quantify drug binding to 5-HT_2CRs in vivo. Thus, the major goal of this study was to demonstrate feasible methods to quantify drug binding to 5-HT_2CRs in vivo that would be translatable to human use. Two complementary approaches were employed. The first involved using a tracer called [¹¹C]CIMBI-36 which is approved for use in humans, and has similar affinity for the 5-HT_2A and 5-HT_2CR in vitro,⁹ and the second employed the use of an experimental, highly selective 5-HT_2C tracer called [¹¹C]AC1332 (listed as compound #18).⁸ By studying both ligands we hoped to validate a tracer already approved for human use for quantifying drug occupancy of 5-HT_2CRs and simultaneously demonstrate the potential utility of a novel 5-HT_2CR selective tracer.

To study [¹¹C]CIMBI-36, we relied on the principle that PET signal (reported as binding potential) is a function of both B_max (receptor density) and K_D (radioligand equilibrium dissociation constant),⁹ and therefore when a ligand has appreciable specific binding to two targets, the fractional contribution of PET signal will be proportional to the B_max/K_D ratio of each target. For a radioligand with limited receptor selectivity, accurate quantification of a target in a specific region is possible if the relative density of the desired target within that region is significantly higher than that of competing targets. By using receptor subtype-selective unlabeled ligands, coupled with the known differential brain distribution of 5-HT_2A and 5-HT_2CRs, it was previously shown in non-human primates that [¹¹C]CIMBI-36 binding could be assigned to each receptor subtype within discreet brain regions, specifically 5-HT_2AR binding in the cortex and 5-HT_2CR binding in sub-cortical regions, and particularly the choroid plexus.¹⁰

To complement and validate studies with [¹¹C]CIMBI-36, we repeated these studies using an experimental, highly selective 5-HT_2C tracer called [¹¹C]AC1332 which has high affinity for 5-HT_2C receptors (Ki = 2.2 nM) and 100-fold or greater selectivity over other receptors including 5-HT_2A/2B receptors (affinities for the different subtypes of 5-HT₂ receptor were determined in HEK 293 expressing cloned human receptors as described previously^8,11). Preliminary PET data with [¹¹C]AC1332 has shown high uptake in the choroid plexus, with moderate uptake in hippocampus and amygdala, which was blocked with the 5-HT_2CR selective antagonist SB 242084.¹² Despite the limited data on this tracer, its profile suggested it could be a valuable control for the studies with [¹¹C]CIMBI-36 and itself potentially a tool for measuring drug binding to 5-HT_2C receptors. The expectation was that [¹¹C]AC1332 would primarily label binding sites in non-human primate brain regions like choroid plexus that express high levels of 5-HT_2C receptors, with lower-level binding in regions like hippocampus that express mixtures of both 5-HT_2A and 5-HT_2C receptors, and little binding in brain regions like cortex that express mainly 5-HT_2A receptors.

To evaluate both approaches, we studied pimavanserin as a candidate drug of interest. Pimavanserin is FDA approved for treating hallucinations and delusions in Parkinson’s disease and is a highly selective 5-HT_2A inverse agonist with lesser, but appreciable affinity for 5-HT_2C receptors, and no appreciable affinity for any other receptor targets.^13,14 We chose pimavanserin because its receptor profile was ideal for testing our methodology for quantifying 5-HT_2A and 5-HT_2C receptor binding with one tracer, as well as demonstrating the potential utility of a novel, 5-HT_2C selective tracer. We also employed unlabelled ligands with high selectivity for 5-HT_2ARs (MDL 100,907) and 5-HT_2CRs (SB 242084) to further validate and confirm binding patterns of [¹¹C]CIMBI-36 and [¹¹C]AC1332 in the non-human primate brain (see Table 1 for relative affinity of the study blocking compounds and ligands). Using these complimentary approaches, the feasibility of estimating drug binding to 5-HT_2A and 5-HT_2C receptors in vivo using [¹¹C]CIMBI-36 as well as the potential utility of [¹¹C]AC1332 for quantifying drug occupancy of 5-HT_2C receptors in vivo is demonstrated below.

Table 1.

In vitro binding affinities of study ligands.

Ligand	5-HT_2A pKi	5-HT_2C pKi	2A/2C selectivity	Source
Pimavanserin	9.3; 10.0	8.8; 9.3	3X; 5X	Vanover et al., 2006;¹⁴ Acadia data on file
CIMBI-36	9.0	8.8	2X	Ettrup et al., 2011²³
AC1332	6.1	8.8	0.002X	Hamprecht et al., 2007⁸
MDL 100,907	10.5; 9.1	8.4; 7.1	200X; 100X	Vanover et al., 2006¹⁴; Kehne et al., 1996³²
SB 242584	6.8	9.0	0.006X	Kennett et al., 1997³³

Material and methods

Preparation of [¹¹C]MDL 100,907 and [¹¹C]CIMBI-36

Unlabelled CIMBI-36 and its precursor (tert-butyl 4-bromo-2,5-dimethoxyphenethyl (2-hydroxybenzyl)carbamate, CIMBI-37 were produced by Department of Drug Design and Pharmacology (Denmark).

N-(2[11C-OCH3]methoxybenzyl)-2,5-dimethoxy-4-bromophenethylamine ([¹¹C]CIMBI-36) was prepared essentially as described elsewhere¹⁰ with minor modifications. In brief, [¹¹C]CIMBI-36 was radiolabelled using [¹¹C]methyl triflate which was prepared from in-target produced [¹¹C]methane. [¹¹C]CH3OTf was trapped at room temperature in a reaction vessel containing the precursor CIMBI-37 (0.4–0.6 mg), acetonitrile (300 µl), acetone (300 µl) and aqueous sodium hydroxide (3 M) (2–3 µl). After reaction (ca. 1 min), 1 ml of trifluoroacetic acid/acetonitrile 1/2 v/v mixture was added to the reaction vessel and the mixture was heated to 80°C for 5 min. After cooling to room temperature, the reaction mixture was neutralized with aqueous sodium hydroxide (3 M) (1000 µl) and purified using HPLC system equipped with ACE 5 C18-HL column (250 × 10 mm) with acetonitrile/0.1M aq. ammonium formate 400:600 as mobile phase (flow 6 ml/min). The desired fraction was collected into a vial containing 60 ml of water. The resulting solution was pushed through tC18 Short SPE cartridge (Waters), the cartridge was rinsed with 8 ml of distilled water. The product was then eluted with 3.0 ml of 30% of EtOH in propylene glycol and collected in a sterile receiving vial prefilled with 12 ml sterile physiological phosphate buffer solution (PBS) (pH = 7.4). The solution was finally filtered through a Millex-GV filter into the product vial. The resulting product was analyzed for radiochemical purity and specific radioactivity. The purity of [¹¹C]CIMBI-36 was determined using an ACE 5 C18-HL HPLC column (250 × 4.6 mm, 5 µm) with acetonitrile/0.1M aq. ammonium formate 50:50 v/v as eluent, with UV absorbance monitoring at 220 nm. Radiochemical purity was in the range of 90 to 99%, MA was in the range of 280 to 1600 GBq/µmol at the time-of-injection, corresponding to injected mass range from 0.19 to 0.03 µg.

Unlabelled AC1332 and its desmethyl precursor for production of [¹¹C]AC1332 were synthesized by Red Glead Discovery. [¹¹C]AC1332 was prepared from the corresponding desmethyl precursor according to process summarized in supplementary Figure S1.

[¹¹C]7-chloro-2-[4-methoxy-3-(2-methylpiperidin-1-yl)ethoxy)pheny]isoindolin-1one ([¹¹C]AC1332) was prepared via 1-step carbon-11 labeling starting from desmethyl precursor AC1339 (0.3–0.5 mg), that was reacted with [¹¹C]MeOTf, obtained via reaction of [¹¹C]methane with elemental iodine, using DMF (500–600 µl) as the reaction solvent and Cs₂CO₃ (2.2–2.9 mg) as base, with reaction time of ca. 1 min at room temperature. Prior to reaction the mixture of precursor, carbonate and DMF was vortexed for 5–10 min until yellowish coloration became prominent. The resulting [¹¹C]AC1332 was purified on HPLC using Waters XBridge 250 × 10 mm column, with acetonitrile/0.05M aq. phosphoric acid 350:650 v/v as mobile phase (flow rate 6–7 ml/min), concentrated on SPE cartridge (Waters tC18 Vac 1 cc) and reconstituted in sterile mixture of saline with 0.5% Kolliphor HS15 (13.5 ml) and ethanol (99.5%, 1.3 ml – used to release labelled product from SPE), followed by sterile filtration though Millex GV 0.22 µm sterile filter. The resulting product was analyzed for radiochemical purity and specific radioactivity. The purity of [¹¹C]AC1332 was determined using a XBridge C18 HPLC column (150 × 4.6 mm, 5 µm) with acetonitrile/0.05M aq. phosphoric acid 320:680 v/v as eluent, with UV absorbance monitoring at 245 nm. Radiochemical purity was in the range of 95 to 98%, MA was in the range of 150 to 1100 GBq/µmol at the time-of-injection, corresponding to injected mass range from 0.33 to 0.05 µg.

Non-human primates

The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency (Dnr 10367-2019) and was performed according to “Guidelines for planning, conducting and documenting experimental research” (Dnr 4820/06-600) of Karolinska Institutet and is in accordance with ARRIVE guidelines 2.0 for how to REPORT animal experiments. Six cynomolgus non-human primates (NHPs), weighing 5.7–9.5 kg, were included in the study. The monkeys were housed in the Astrid Fagraeus Laboratory, Comparative Medicine, Karolinska Institutet, Solna, Sweden.

PET and MRI system

PET measurements were conducted using the High Resolution Research Tomograph system (Siemens Molecular Imaging, Knoxville, TN, USA). A six minute transmission scan using a single ¹³⁷Cs source was obtained immediately before the radioligand injection. List-mode data were acquired for 123 min. PET images were reconstructed with a series of frames of increasing duration (9 × 20 s, 3 × 60 s, 5 × 180 s, 17 × 360 s) using the ordinary Poisson 3- dimensional ordered-subset expectation maximization (OP-3D-OSEM) algorithm, with 10 iterations and 16 subsets, including modelling of the point spread function, after correction for attenuation, random and scatter. The in-plane resolution of the reconstructed images is approximately 1.5 mm.¹⁵ For each NHP an MRI scan was made available, either performed before the PET measurements, or an historical MRI.

PET experimental procedure

Anaesthesia of the monkeys was induced by intramuscular injection of ketamine hydrochloride (∼10 mg/kg, Ketaminol vet.; Intervet, Sollentuna, Sweden) and maintained by the administration of a mixture of sevoflurane (2%–8%, sevoflurane®; Abbot Scandinavia AB, Solna, Sweden), oxygen, and medical air after endotracheal intubation. The NHP’s heads were immobilized with a fixation device.¹⁶ Body temperature was maintained by a Bair Hugger device (model 505; Arizant Health Care, Eden Prairie, MN, USA) and monitored by an oral thermometer. Electrocardiogram, heart rate, respiratory rate, oxygen saturation, and arterial blood pressure were continuously monitored throughout the experiments. Fluid balance was maintained by continuous infusion of saline. The radioligand was intravenously administered by a bolus administration of radioligand (∼150 MBq in 4 mL). A cannula was inserted in the external iliac artery or in the femoral artery, and arterial blood was collected continuously for 3 min using an automated blood sampling system (ABSS) (Allogg, Mariefred, Sweden) at a speed of 3.0 mL/min. Blood sampling (1.0–3.0 ml) was performed manually for the measurement of radioactivity and metabolism at 1, 1.5, 2, 2.5, 3, 5, 15, 30, 60, 90 and 120 min after the injection.

Each NHP had a baseline scan and a variable number of pretreatment scans, not necessarily on the same day. Baseline and the first post treatment scan were, in most cases, performed on the same day. All the pretreatment drugs (SB 242084, MDL 100,907 and pimavanserin were administered at the same fixed time points relative to the radioligand injection (−30 min, duration 15 minutes). Additionally, venous blood samples were taken at several time points to measure pimavanserin exposure (see details of the analytical methods below). The times relative to administration of radioligand were −1 min, +30 min (not obtained for all subjects), +60 min and +120 min. The amount for blood withdrawn from an animal was restricted to not more than 0.6% of body animals body weight on any scanning day (i.e. 6 mL of blood for every kilogram of body weight). For subsequent treatment scans there was a minimum of a 6 week rest period between treatment scans.

Study design

First, four cynomolgus animals underwent a total of 16 [¹¹C]CIMBI-36 dynamic PET scans, at baseline and post-administration of various doses of pimavanserin (from 0.001 mg/kg to 1.0 mg/kg), SB 242084 (selective 5-HT_2C antagonist, dose 1.0 mg/kg) and MDL 100,907 (selective 5-HT_2A antagonist/inverse agonist, dose 0.1 mg/kg).

Then, three cynomolgus monkeys (one of which was also analysed with [¹¹C]CIMBI-36) underwent 10 [¹¹C]AC1332 PET scans at baseline and post-administration of various doses of pimavanserin (from 0.01 mg/kg to 1.0 mg/kg), SB 242084 (dose 1.0 mg/kg) and MDL 100,907 (dose 0.05 mg/kg).

A list of individual scans and doses, animal weight on scanning day, with associated activity levels (MBq/mCi) and pimavanserin plasma concentrations are listed in supplementary Table S1.

Pimavanserin was supplied by Acadia Pharmaceuticals. The 5-HT_2CR selective antagonist SB 242084 and 5-HT_2AR selective antagonist/inverse agonist MDL 100,907 were obtained from (TOCRIS, Bio-Techne Ltd). SB 242084 for pre-treatment (1.0 mg/ml as free base) was formulated in the mixture of 10:20:70 of EtOH/PEG400/25% Captisol in saline respectively. MDL 100907 for pre-treatment (0.1 and 0.05 mg/ml) was formulated in the mixture of 3:13:80 of DMSO/PEG200/10% Captisol in saline respectively. Pimavanserin for pre-treatment (range of concentrations from 0.003 to 1.0 mg/ml as free base) was formulated in sterile saline. In all cases, formulated compounds were vortexed and if necessary, sonicated to ensure complete dissolution, and filtered through Millex-GV sterile filters prior to administration to the monkeys. Samples of the filtered drug solutions were HPLC analysed to confirm the drug concentrations.

Image analysis

Each subject’s PET image was initially cropped to focus the analysis on the cerebral area. Structural MRI underwent brain extraction followed by a registration on the subject’s PET image. The CNC-CNRS atlas¹⁷ was non-linearly warped to the subject’s MR image to enable automated definition of regions of interest (ROIs).

The set of ROIs were selected a priori and included: cerebellum, frontal cortex, occipital cortex, temporal cortex, parietal cortex, cingulate cortex, thalamus, striatum, amygdala and insula. Hippocampus and choroid plexus were manually delineated by an expert analyst by using the MRI and summed PET (software used: ITKSnap®,¹⁸). The region for the choroid plexus was defined around the radioactivity in the fourth ventricle using fused PET and MRI images, as described previously.¹⁰ The hippocampus was defined by the radioactivity in the head and tail of the hippocampus using fused PET and MRI images, since the automatic atlas segmentation was deemed too variable and large in the region definition. ROIs defined on the MRI images were applied to the dynamic PET data to derive regional time-activity curves (TACs, see example in supplementary Figure S3 and S4), with activity concentrations expressed as standardised uptake values (SUV), and calculated as radioactivity concentration [kBq/cm³]/(radioactivity injected [MBq]/body weight [kg]). The cerebellum was considered the reference region.

Radiometabolite analysis

The quantitative metabolite analysis of [¹¹C]CIMBI-36 and its radioactive metabolites in the NHP plasma performed using a simple protein precipitation sample preparation method coupling with reversed-phase radio-HPLC.¹⁹ Chromatographic separation was performed with ACE C18 column, (250 mm × 10 mm I.D). A gradient elution of acetonitrile (A) and ammonium formate (AMF) 0.1 M (B) were used as the mobile phase at 5.0 mL/min, with the following gradient; 0–4.0 min, (A/B) 60:40 → 90:10 v/v; 4.0–6.0 min, (A/B) 90:10 v/v for [¹¹C]CIMBI-36 and 0–4.0 min, (A/B) 65:35 → 90:10 v/v; with an Agilent binary pump (Agilent 1200 series) coupled to a manual injection valve (7725i, Rheodyne), 5.0 mL loop and a radiation detector (Oyokoken, S-2493Z) housed in a shield of 50 mm thick lead measurements with 10 second radio-detector accumulation. In addition, a ChemStation Rev. B.04.03; Agilent, was used to aid the collecting and controlling the data.

The arterial blood samples were taken 2, 5, 15, 30, 60, 90 and 120 minutes after administration of [¹¹C]CIMBI-36 into the NHP. The plasma samples were separated using centrifuged and mixed with 1.4 times volume of acetonitrile and again centrifuged (2000 rpm × 4 min) to precipitate the plasma protein. Finally, the supernatants were diluted with water and injected into a reversed phased radio-HPLC. Peaks for radioactive compounds eluting from the column were integrated and their areas were expressed as a percentage of the sum of the areas of all detected radioactive compounds considering the time decay correction.

The quantitative metabolite analysis of [¹¹C]AC1332 and its radioactive metabolites in the NHP plasma performed as in [¹¹C]CIMBI-36 (see above), using a simple protein precipitation sample preparation method coupling with reversed-phase radio-HPLC. The gradient elution of acetonitrile (A) and ammonium formate (AMF) 0.1 M (B) used as the mobile phase at 5.0 mL/min, were the follows for [¹¹C]AC1332; 0–4.0 min, (A/B) 60:40 → 90:10 v/v; 4.0–6.0 min, (A/B) 90:10 v/v with an Agilent binary pump (Agilent 1200 series) coupled to a manual injection valve (7725i, Rheodyne), 5.0 mL loop and a radiation detector (Oyokoken, S-2493Z) housed in a shield of 50 mm thick lead measurements with 10 second radio-detector accumulation.

Pharmacokinetic estimation of pimavanserin in plasma

Study samples were received on dry ice and stored at −70°C until the analysis. HiStock solutions and spiking solutions were performed in acetonitirle: DMOS (95:5). Solutions were stored at −20°C when not used. Fresh Stock and spiking solutions were prepared for each analytial instance. Pimavanserin was extracted from the plasma by protein precipitation and the samples were then analyzed using reversed phase LC-MS/MS. The method calibration range in the plasma samples ranged from 500 to 0.1 ng/mL. Human blank plasma was used as anticoagulant for the calibration samples.

Kinetic modelling

[¹¹C]CIMBI-36 PET tissue data were analysed using Multilinear Analysis 1 (MA1), with t* = 30 minutes), as previously described in^20,21 to derive the total volume of distribution V_T and the non-displaceable binding potential (BP_ND) using the cerebellum as reference region, as in ${B P}_{N D} = \frac{V_{T}}{V_{ref}} - 1$ , with V_ref being the volume of distribution of the reference region. The parent plasma concentration of the tracer was derived by fitting the plasma-over-blood data to an exponential approaching to a constant model and the metabolite data to a sigmoid model, and then used as input to the kinetic modelling.

The percentage reduction in binding potential in an ROI following drug administration was quantified as ${Δ B P}_{N D} = 100 \times (1 - \frac{{B P}_{N D}^{post - dose}}{{B P}_{N D}^{baseline}})$ and used to build the relationship between plasma concentration of pimavanserin and occupancy. The Emax model defined as:

{Δ B P}_{N D} = E_{\max} \frac{C^{}}{C^{} + {E C}_{50}^{}}

(1)

was fitted to the data to derive the

{E C}_{50}

(the plasma concentration of pimavanserin C that corresponds to 50% receptor occupancy at the time point being studied) and

E_{\max}

(the maximal attainable signal change).

[¹¹C]AC1332 PET tissue time-activity curves were analysed using kinetic modelling methods that employ a metabolite-corrected plasma input function (calculated as for the [¹¹C]CIMBI-36 tracer) such as the one or two tissue compartmental models for reversible and irreversible binding.²² We also tested a simple blood contribution model and the MA1 approach. The optimal model was identified using goodness of fit and precision of estimates. Since the tracer was identified as reversible, the non-displaceable binding potential BP_ND was calculated as above using the cerebellum as reference region. The percentage reduction in binding potential following drug administration was also calculated as above and used to build the relationship between plasma concentration of pimavanserin and occupancy. The Emax model (equation (1)) was then fitted to the data to derive the ${E C}_{50}$ and $E_{\max}$ .

Estimation of 5-HT_2A and 5-HT_2C fractions using [¹¹C]CIMBI-36

Since [¹¹C]CIMBI-36 has comparable affinity for 5-HT₂Rs (Ki (nM): 5-HT_2A = 0.5–0.8; 5-HT_2B = 0.5; 5-HT_2C = 1.7),²³ and the brain contains regionally varying proportions of 5-HT_2A and 5-HT_2CRs, ΔBP_ND would be expected to change in different ROIs. In particular, it is known that the cortical regions mainly express 5-HT_2ARs,²⁴ the choroid plexus is dominated by 5-HT_2CRs,²⁵ and the hippocampus contains a mixture of 5-HT_2A and 5-HT_2CRs.¹⁰

A number of methods for calculating 5-HT_2A and 5-HT_2C receptor occupancy following single doses of drugs have been examined previously.²⁶ Under the assumption that specific binding in the cortex constitutes mainly 5-HT_2A binding and that pimavanserin has lower affinity for 5-HT_2CRs, occupancy in the occipital cortex (Occ_CORT) and pimavanserin concentration in plasma (C) were used to estimate EC_50,2A as:

{OCC}_{CORT} = E_{\max 2 A} \frac{C}{C + {EC}_{50, 2 A}}

(2)

Occupancy in the choroid plexus (5-HT_2C-rich region) Occ_CP and pimavanserin concentration in plasma (C) were used to estimate EC_50,2C as:

{OCC}_{C P} = E_{\max 2 C} \frac{C}{C + {E C}_{50, 2 C}}

(3)

$E_{\max}$ is the maximal attainable signal change, ${E C}_{50, 2 A}$ and ${E C}_{50, 2 C}$ are the plasma concentrations that correspond to 50% of the maximal signal change for 5-HT_2A and 5-HT_2C respectively.

For this study, we followed the approach proposed previously²⁶ to derive the fraction of [¹¹C]CIMBI-36 signal in the hippocampus attributable to 5-HT_2CRs_. Occupancy values in the hippocampus (Occ_HIPP) were used to estimate the f_{2C_HIPP} (fraction of [¹¹C]CIMBI-36 signal in the hippocampus attributable to the 5-HT_2C) as:

{OCC}_{HIPP} = {OCC}_{\max 2 A} (1 - f_{2 C_HIPP}) \frac{C}{C + {E C}_{50, 2 A}} + {OCC}_{\max 2 C} f_{2 C_HIPP} \frac{C}{C + {E C}_{50, 2 A}}

(4)

Note that in hippocampus, $f_{2 A} + f_{2 C} = 1$ , with $f_{2 A}$ fraction of [¹¹C]CIMBI-36 signal in the hippocampus attributable to 5-HT_2A.

Equations (2) to (4) were solved simultaneously using a simultaneous estimation approach (SIME),²⁷ where all the parameters are determined together at the same time. $E_{\max}$ was shared across all three regions (based on individual fitting results showing very close estimates for Occ_max2A and OCC_max2C).

To note, since with [¹¹C]AC1332 only 5-HT_2C regions presented displaceable binding, no SIME approach could be carried out to estimate the fraction of 5-HT_2A and 5-HT_2CRs.

Results

A total of 26 PET scans (16 after administration of [¹¹C]CIMBI-36 and 10 after administration of [¹¹C]AC1332) were successfully acquired in 6 different animals.

There was a relatively slow decline of the pimavanserin plasma concentration throughout the duration of the scan (see supplementary Figure S2 and supplementary table S1). Concentrations at 60 minutes post-dose were used to estimate plasma concentration for each PET scan.

Figures 1 and 2 show examples of distribution of [¹¹C]CIMBI-36 and [¹¹C]AC1332 signals in the brain, at baseline and after administration of the 5-HT_2C selective antagonist SB 242084, the 5-HT_2A selective antagonist/inverse agonist MDL 100,907 and the 5-HT_2A/2C antagonist/inverse agonist pimavanserin. Binding patterns of the 5-HT_2A/2C ligand [¹¹C]CIMBI-36 at baseline showed strong signals throughout the brain including highest in the cortex, slightly lower in hippocampus, and lower again in choroid plexus and amygdala as indicated in Figures 1 and 2. Baseline binding patterns of the 5-HT_2C selective ligand [¹¹C]AC1332 were much less widespread than [¹¹C]CIMBI-36, mainly restricted to a high signal in choroid plexus, with a smaller, but still clear signal in the hippocampus, and very little binding in cortex or other subcortical regions (see Figures 1 and 2).

Figure 1.

Coronal cross sections of [¹¹C]CIMBI-36 (left) and [¹¹C]AC1332 (right) SUV PET scans. SUV images are summed from 0–90 minutes from a representative subject at baseline, post-1 mg/kg SB 242084 (5-HT_2C specific), post- 0.5 and 1 mg/kg MDL 100,907 (5-HT_A specific) and post-1 mg/kg pimavanserin (5-HT_AC and 5-HT_2C specific) administration (left panel). Images show the expected distribution of [¹¹C]CIMBI-36 signal. Images show high uptake in 5-HT_2C regions (hippocampus and choroid plexus).

Figure 2.

(a). BP_ND of [¹¹C]CIMBI-36 in NHP at baseline and following administration of SB 242084 (5-HT_2C specific), MDL 100,907 (5-HT_2A specific) or Pimavanserin (5-HT_{2A & 2C} specific). [¹¹C]CIMBI-36 binding was measured at baseline (n = 4; mean ± SD), after pretreatment with SB 242084 (SB) (n = 2; mean ± SD), MDL 100,907 (MDL) (n = 1) and Pimavanserin (Pim) (n = 2; mean ± SD). Error bars represent one standard deviation. (b) and BP_ND of [¹¹C]AC1332 in NHP at baseline and following administration of SB 242084 (5-HT_2C specific), MDL 100,907 (5-HT_2A specific) or Pimavanserin (5-HT_{2A & 2 C} specific). Baseline data from n = 3, SB data from n = 2, MDL data from n = 1 and Pim data from n = 2. Error bars represent one standard deviation.

For [¹¹C]CIMBI-36 and [¹¹C]AC1332, kinetic modelling with MA1 produced acceptable model fits to tissue time-activity curve data in all cases (an example for each tracer is reported in supplementary Figure S3 and S4 respectively) and the V_T were well determined (see supplementary Tables S2 and Table S3). BP_ND calculated using the cerebellum as reference region and selected regions are reported are reported in supplementary Tables S4 and S5.

For [¹¹C]CIMBI-36 scans, post administration of SB 242084 (in both subjects), there was a significant increase in the V_T (see Figure 1 and Table S2) which was associated with increased ligand by a higher peak in both scans (see representative TAC Figure S3). Despite the increases in V_T following SB 242084 for both subjects (see Table S2), the BP_ND ratio [target/reference region] was reduced following the SB 242084 administration. This result is consistent with those seen previously.¹⁰

For [¹¹C]CIMBI-36 scans post administration of the 5-HT_2C selective antagonist SB 242084, the highest reduction in BP_ND was in the choroid plexus (74%), the smallest change was in cortical regions (5–27%) and there was a 38% reduction in hippocampal BP_ND. On the other hand, 0.1 mg/kg of MDL 100,907 (5-HT_2A selective antagonist/inverse agonist) led to 77–92% reductions in cortical BP_ND, but substantially lower reductions in the 5-HT_2C rich areas (hippocampus and choroid plexus, 33% and 26% respectively). See Table 2 for selected regions and supplementary Table S4 for all BP_ND estimates.

Table 2.

Summary Non-displaceable binding potential (BP_ND, unitless) (ΔBP_ND %) values obtained from Multilinear Analysis 1 (MA1) after the injection of [¹¹C]AC1332 and [¹¹C]CIMBI in NHP during conditions and after pretreatment.

	[¹¹C]AC1332 BP_ND (Std Dev) ΔBP_ND%						[¹¹C]CIMBI-36 BP_ND (Std Dev) ΔBP_ND%												Plasma conc. Pim. ng/ml
Dose	Choroid plexus			Hippocampus			Choroid plexus			Hippocampus			Frontal Cortex			Occipital Cortex			Plasma conc. Pim. ng/ml
Baseline	2.99	(1.02)		1.53	(0.38)		0.75	(0.24)		1.01	(0.22)		1.86	(0.54)		1.42	(0.16)
SB 1.0 mg/kg	0.05		98%	0.23		83%	0.2	(0.02)	74%	0.82	(0.01)	38%	1.43	(0.11)	27%	1.4	(0.27)	5%
MDL 0.05/0.1 mg/kg	2.14		5%	1.02		20%	0.58		26%	0.99		33%	0.33		86%	0.18		87%
Pim. 0.001 mg/kg							0.84		−12%	0.84		17%	1.34		−21%	1.18		17%	0.13
Pim. 0.003 mg/kg							0.86	(0.15)	8%	1.01	(0.01)	15%	1.21	(0.06)	16%	1.07	(0.35)	21%	0.25	(0.02)
Pim. 0.011 mg/kg	1.42		44%	0.96		29%	0.9		21%	0.83		43%	0.69		67%	0.54		58%
Pim. 0.033 mg/kg	1.64		36%	1.04		24%	0.6	(0.04)	35%	0.6	(0.07)	49%	0.43	(0.15)	73%	0.41	(0.08)	70%	3.13	(2.49)
Pim. 0.1 mg/kg	0.91		64%	0.86		37%	0.35	(0.08)	63%	0.46	(0.15)	69%	0.47	(0.23)	80%	0.29	(0.06)	79%	12.8	(11.29)
Pim. 0.3 mg/kg	0.85		80%	0.75		62%													13.1
Pim. 1.0 mg/kg	0.40		90%	0.47		76%	0.16		86%	0.34		77%	0.18		92%	0.16		88%	117.8	(45.54)

Pretreatments: 5-HT_2C specific SB 242084 (SB), 5-HT_2A specific MDL 100,907 (MDL) and 5-HT_{2A & 2 C} specific Pimavanserin (Pim). Pimavanserin plasma concentration (ng/mL) from sample obtained at 60 minutes post the start of scanning. Hippocampus and choroid plexus were manually derived, Subj: Subject number. ΔBP_ND: change in binding potential (%). BP_ND measured uses cerebellum as reference tissue.

After pimavanserin administration, there was a generally robust dose dependent displacement of [¹¹C]CIMBI-36, with displacement occurring at lower doses and plasma levels in the cortex, and higher doses and plasma levels in the choroid plexus, while the hippocampus results were more variable, with displacement of the tracer occurring at doses and exposures between frontal cortex and choroid plexus values (see Table 2 and supplementary Figure S3). Given that pimavanserin has higher affinity for 5-HT_2A over 5-HT_2C receptors, these results are consistent with the observations that the cortex expressed mainly 5-HT_2A receptors, the choroid plexus expresses mainly 5-HT_2C receptors, and the hippocampus has mixed expression of both 5-HT_2A and 5-HT_2C receptors.

The relationship between the measured plasma concentrations of pimavanserin and resulting ΔBP_ND values was determined by fitting the Emax model of equation (1). [¹¹C]CIMBI-36 data and model fit are plotted in Figure 3. To note, PK values for subject 002 (pimavanserin dose 0.011 mg/kg) and subject 003 (pimavanserin dose 0.1 mg/kg) were excluded from results as the samples were believed to be contaminated. Using [¹¹C]CIMBI-36 the EC₅₀ was estimated as 0.6 ng/ml for occipital cortex, 6.0 ng/ml for choroid plexus and 0.8 ng/ml for hippocampus. E_max was estimated between 70% and 85% for the regions considered (see Figure 3).

Figure 3.

Comparison of Pimavanserin plasma concentration versus occupancy curves for [¹¹C]CIMBI-36 in the occipital cortex, a 5-HT_2A-rich region (a), or in the choroid plexus, a 5-HT_2C-rich region (b), and then [¹¹C]AC1332 in choroid plexus (c), determined by using the Emax model. ■ 1.0 mg/kg, ▸ 0.3 mg/kg, ● 0.1 mg/kg, ▲ 0.033 mg/kg, « 0.011 mg/kg, ♦ 0.0033 mg/kg and ★ 0.001 mg/kg pimavanserin.

When using the SIME approach to estimate the fraction of 5-HT_2C receptors in the hippocampus using the E_max model, consistent estimates of both the ED₅₀ and EC₅₀ for the 5-HT_2A and 5-HT_2C subtypes were found. Results from the both the individual fits of the Emax model using the pimavanserin concentration (PK-RO model) and the Emax model using the pimavanserin administered doses (dose-RO model) supported the use of a shared E_max for the SIME model. The model fits are reported in Figure 5(a) and (b). The estimated EC₅₀ was 0.6 ng/ml for 2 A subtype, 6.0 ng/ml for 2 C subtype and E_max was estimated 82%. The estimated fraction of 2 C subtype from PK-RO in the hippocampus f_2c was 0.5. The estimated ED₅₀ was 0.007 mg/kg for 2 A subtype, 0.041 mg/kg for 2 C subtype, and E_max was estimated 85%. The estimated fraction of 2 C receptor subtype from dose-RO curves was 0.57(f_2c) in the hippocampus (see Figure 5(a) and (b)).

[¹¹C]AC1332 demonstrated a binding pattern in the NHP brain consistent with a selective 5-HT_2C PET ligand, with quantifiable binding in hippocampus and choroid plexus, and minimal binding in the cortex (see Figures 1, 2(b) and Table 2). Post administration of SB 242084 (5-HT_2C blocker), the highest reduction was in the choroid plexus (98%), and there was an 83% reduction in hippocampal BP_ND. On the other hand, 0.05 mg/kg of MDL 100,907 (5-HT_2A blocker) led to 20% reduction in hippocampus and 5% in the choroid plexus (the latest being within baseline variability). Both compounds demonstrated the expected change in [¹¹C]AC1332 binding consistent with its selectivity for 5-HT_2C receptors.

After pimavanserin administration, there were generally robust, dose-dependent reductions in the binding of [¹¹C]AC1332 in the hippocampus and choroid plexus (see Figures 1, 2(b) and Table 2). The reductions were consistent with the 5-HT_2C related changes in [¹¹C]CIMBI-36 binding identified in the first part of the study.

Figure 3 shows a summary of the pimavanserin exposure vs receptor occupancy curves for [¹¹C]CIMBI-36 (Figure 3(a) and (b)) and [¹¹C]AC1332 (Figure 3(c)), for 5-HT_2C-rich regions (i.e. choroid plexus, 3B and 3C) and 5-HT_2A-rich regions (i.e. occipital cortex, 3A). Figure 4 shows a summary of the pimavanserin dose vs receptor occupancy curves for [¹¹C]CIMBI-36 (Figure 4(a) and (b)) and [¹¹C]AC1332 (Figure 4(c)), for 5-HT_2C-rich regions (i.e. choroid plexus, 4B and 4C) and 5-HT_2A-rich regions (i.e. occipital cortex, 4A).

Figure 4.

Comparison of Pimavanserin dose versus occupancy curves for [¹¹C]CIMBI-36 in the occipital cortex, a 5-HT_2A-rich region (A), or in the choroid plexus, a 5-HT_2C-rich region (B), and then [¹¹C]AC1332 in choroid plexus (C), determined by using the Emax model. ■ 1.0 mg/kg, ▸ 0.3 mg/kg, ● 0.1 mg/kg, ▲ 0.033 mg/kg, « 0.011 mg/kg, ♦ 0.0033 mg/kg and ★ 0.001 mg/kg pimavanserin.

Figure 5.

(a) [¹¹C]CIMBI-36 Pimavanserin PK-occupancy E_max model solved with SIME allowing to determine the fraction of 5-HT_2C receptors in the hippocampus (f_2C) and (b) [¹¹C]CIMBI-36 Pimavanserin dose-occupancy E_max model solved with SIME allowing to determine the fraction of 5-HT_2C receptors in the hippocampus (f_2C).

The occupancy of 5-HT_2C receptors by pimavanserin quantified using [¹¹C]AC1332 (ED₅₀ 0.062 mg/kg: with a 95% confidence interval of −0.039–0.162 mg/kg) provided estimates consistent with those generated using [¹¹C]CIMBI-36 (ED₅₀ 0.046 mg/kg: with a 95% confidence interval of 0.010–0.083 mg/kg). The estimated 5-HT_2A:5-HT_2C affinity ratio for pimavanserin was approximately 6–10 fold.

Discussion

We evaluated two complimentary methods to estimate drug occupancy at the 5-HT_2CRs in the non-human primate brain in vivo. The first used [¹¹C]CIMBI-36 binding in discrete anatomical regions known to contain high proportions of the 5-HT_2A (cortical regions) and 5-HT_2C (choroid plexus) receptor sub-types. [¹¹C]CIMBI-36 is a high affinity 5-HT₂ selective radioligand with similar affinity for 5-HT_2A and 5-HT_2C receptors in vitro (K_i (nM): 5-HT_2A = 0.5–0.8; 5-HT_2C = 1.7,^10,23) The second approach used a novel, 5-HT_2C selective radioligand, [¹¹C]AC1332. Pimavanserin, an FDA approved drug with known affinity for the 5-HT_2A and 5-HT_2C receptors was used to evaluate the two approaches above. The contribution of the 5-HT_2A and 5-HT_2C components to the regional [¹¹C]CIMBI-36 V_T values was confirmed by the administration of blocking compounds selective for 5-HT_2A (MDL 100,907) and 5-HT_2C (SB 242084). Overall, the results obtained via the two methods were internally consistent and in the case of [¹¹C]CIMBI, in line with previous literature.¹⁰

The in vivo affinity of pimavanserin at the 5-HT_2CR has not been previously examined. The estimates obtained in this study were similar using either [¹¹C]CIMBI-36 or [¹¹C]AC1332 (ED₅₀ values of 0.046 vs 0.062 mg/kg; EC₅₀ values of 6.0 vs 2.5 ng/ml respectively). The in vivo affinity of pimavanserin at the 5-HT_2AR was previously examined in a human PET study using the radioligand [¹¹C]NMSP, which displays high binding potential in the cortex, accounting for the 5-HT_2AR binding of this radioligand.²⁸ Although the pimavanserin EC₅₀ for cortical regions was not calculated in that prior study, visual examination of the occupancy plots indicates a figure between 0.5 to 1 ng/ml, in good agreement with the estimate of 0.6 ng/ml obtained from the cortical occupancy estimates obtained with [¹¹C]CIMBI-36 scans in our study. Comparison of the 5-HT_2AR and 5-HT_2CR affinity of pimavanserin obtained from regional occupancy estimates of [¹¹C]CIMBI-36 PET scans provides a selectivity ratio of approximately 10-fold higher affinity for the 5-HT_2AR over the 5-HT_2CR. This affinity ratio is broadly consistent with the literature results obtained in vitro, of 3-to-5 fold higher radioligand binding affinity for the 5-HT_2AR over the 5-HT_2CR.^13,14 Pimavanserin occupancy of 5-HT_2A receptors is likely over 97% at the therapeutic dose of 34 mg given that the C_max is estimated to be 50 ng/ml at steady state.²⁹ Therefore, assuming the in vivo selectivity ratio of approximately 10-fold that we observed in this study also occurs in humans, pimavanserin occupancy of 5-HT_2C receptors is likely to be close to 90% at that same dose.

[¹¹C]CIMBI-36 regional binding in the NHP brain was broadly consistent with previous reports,¹⁰ with highest V_T values in the cortical regions, lower ones in the hippocampus, choroid plexus and the amygdala, very low V_T values in the striatum and thalamus and lowest ones in the cerebellum.¹⁰ The baseline [¹¹C]CIMBI-36 scans demonstrated a regional binding pattern very similar to that reported previously in primates and in humans.^10,30 Previous studies have used the 2TCM model to quantify the [¹¹C]CIMBI-36 data.^10,23,30 In our study the MA1 model performed slightly better than the 2TCM and the study results and conclusions are unchanged whether the 2TCM or MA1 model is used. [¹¹C]AC1332 regional binding was highest in the choroid plexus, followed by the hippocampus and negligible displaceable binding in all other regions, consistent with the pilot scan previously presented.¹² The regional binding pattern for [¹¹C]CIMBI-36 and [¹¹C]AC1332 is consistent with expectations based on the affinity of the two radioligands for the 5-HT_2CRs and 5-HT_2ARs and the distribution of the two receptors seen using autoradiography in post-mortem samples. 5-HT_2CRs are expressed in many regions of the monkey brain including cortex, substantia nigra, basal ganglia, amygdala, hippocampus and hypothalamus, and have considerable overlap with 5-HT_2ARs, but the density of 5-HT_2ARs is significantly higher in most brain regions.³¹ An exception is the choroid plexus which primarily expresses 5-HT_2CRs in monkeys.

We have used the BP_ND as the preferred outcome parameter to quantify [¹¹C]CIMBI-36 PET scans (Table 2, S4 and S5). This decision was based on availability of the cerebellum as a reference region with negligible 5-HT₂ receptor expression, as well as the previous findings that reported increases in regional V_T values following the administration of the 5-HT_2C antagonist SB 242084¹⁰. V_T values of [¹¹C]CIMBI-36 increased approximately 60% after the administration of SB 242084 in the current study. A global increase in V_T of 100% was also reported previously.¹⁰ Finnema et al attributed these changes to increases in the plasma free fraction (fp) following the administration of SB 242084 (fp = 0.10 vs. 0.05 ± 0.01 during baseline conditions).¹⁰ In contrast to the V_T, the BP_ND changes (supplementary table S4) demonstrate the expected direction of change, although we did see some paradoxical increases in BP_ND in subject 4 when they were administered very low levels of Pim. We believe these changes are due to experimental variability at very low occupancy levels. We did not measure fp in our experiments as it is irrelevant for the estimation of BP_ND. If future studies intend to use V_T as the outcome parameter we would recommend monitoring changes in fp.

In conclusion, both [¹¹C]CIMBI-36 and [¹¹C]AC1332 enable the estimation of brain 5-HT_2CR occupancy by pharmaceutical agents. While [¹¹C]CIMBI-36 allows estimation of occupancy at both 5-HT_2A and 5-HT_2C receptors, estimating the 5-HT_2C binding component of this radioligand is more complex than it is for [¹¹C]AC1332, because in most brain regions such as the hippocampus, the contribution of 5-HT_2CRs to binding must be separated from the 5-HT_2AR component. In choroid plexus however, [¹¹C]CIMBI-36 binding is primarily due to 5-HT_2CRs. [¹¹C]AC1332 demonstrated a binding pattern in the NHP brain consistent with a selective 5-HT_2C PET ligand, with quantifiable in hippocampus and choroid plexus, and minimal binding in the cortex. The choice of either ligand for future studies will depend on the exact parameters that are to be investigated, though for solely measuring 5-HT_2CR occupancy, [¹¹C]AC1332 may be a more strainght forward option. These studies illustrate the feasibility of estimating drug binding to 5-HT_2ARs and 5-HT_2CRs in vivo using [¹¹C]CIMBI-36 as well as the potential utility of [¹¹C]AC1332 for quantifying drug occupancy of 5-HT_2CRs in vivo.

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Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was fully funded by ACADIA Pharmaceuticals Inc., United States, which markets pimavanserin. ESB is an employee of ACADIA.

Acknowledgements

Acknowledge that the precursor for AC1332 was produced by Red Glead Discovery, Medicon Village. Scheelevaegen 8. Lund, SE, 22363. Specifically Filip Paulsen.

Acknowledge that the standard of CIMBI-36 and its precursor CIMBI-37 were produced by the Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 160, Building 22, Floor 1, DK-2100 Copenhagen, Denmark.

Acknowledge that the pharmacokinetic analysis was performed by Lablytica Life Science, Virdings Allee 16, 754 50 Uppsala, Sweden. Led by David Pekar.

The authors thank Piotr Ochniewicz, Yasir Khani and the staff of the Radiopharmacy Core Facility and Brain Molecular Imaging Centre of Karolinska Institutet for excellent assistance in the conduction of the PET studies.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: KR, GR and EAR are employees of Invicro.

Authors’ contributions

KR made significant contribution to design of the study, interpretation of the data, drafted sections of the manuscript and approved the final version.

GR analysed the data, made a significant contribution to interpretation of the data, drafted sections of the article and approved the final version.

ESB came up with the concept and the design of the study, drafted sections of the manuscript and revised the manuscript and approved the final version.

AFM contributed significantly to the design and acquisition of the data and drafted sections of the manuscript and as approved the final version.

VS contributed significantly to the design and acquisition of the data and drafted sections of the manuscript and as approved the final version.

CH contributed significantly to the design and acquisition of the data and drafted sections of the manuscript and as approved the final version.

EAR made significant contribution to design of the study, interpretation of the data, drafted sections of the manuscript and approved the final version.

ORCID iDs

Khanum Ridler

Eugenii A Rabiner

Supplementary material

Supplemental material for this article is available online.

References

Wold

Wild

Cunningham

, et al. Targeting the 5-HT2C receptor in biological context and the current state of 5-HT2C receptor ligand development. Curr Top Med Chem 2019; 19: 1381–1398.

Somerville

Horwood

Lee

, et al. 5-HT(2C) receptor activation inhibits appetitive and consummatory components of feeding and increases brain c-fos immunoreactivity in mice. Eur J Neurosci 2007; 25: 3115–3124.

Kroeze

Roth

BL.

The molecular biology of serotonin receptors: therapeutic implications for the interface of mood and psychosis. Biol Psychiatry 1998; 44: 1128–1142.

Christianson

Ragole

Amat

, et al. 5-hydroxytryptamine 2C receptors in the basolateral amygdala are involved in the expression of anxiety after uncontrollable traumatic stress. Biol Psychiatry 2010; 67: 339–345.

Higgins

Sellers

Fletcher

PJ.

From obesity to substance abuse: therapeutic opportunities for 5-HT2C receptor agonists. Trends Pharmacol Sci 2013; 34: 560–570.

Di Giovanni

De Deurwaerdère

New therapeutic opportunities for 5-HT2C receptor ligands in neuropsychiatric disorders. Pharmacol Ther 2016; 157: 125–162.

Brashier

DBS

Sharma

Dahiya

, et al. Lorcaserin: a novel antiobesity drug. J Pharmacol Pharmacother 2014; 5: 175–178.

Hamprecht

Micheli

Tedesco

, et al. Isoindolone derivatives, a new class of 5-HT2C antagonists: synthesis and biological evaluation. Bioorg Med Chem Lett 2007; 17: 428–433.

Innis

Cunningham

Delforge

, et al. Consensus nomenclature for in vivo imaging of reversibly binding radioligands. J Cereb Blood Flow Metab Off J Tab 2007; 27: 1533–1539.

10.

Finnema

Stepanov

Ettrup

, et al. Characterization of [(11)C]cimbi-36 as an agonist PET radioligand for the 5-HT(2A) and 5-HT(2C) receptors in the nonhuman primate brain. NeuroImage 2014; 84: 342–353.

11.

Bromidge

Duckworth

Forbes

, et al. 6-Chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]- indoline (SB-242084): the first selective and brain penetrant 5-HT2C receptor antagonist. J Med Chem 1997; 40: 3494–3496.

12.

Goodman

Zeng

Nye

, et al. Evaluation of [C-11]7-Chloro-2-[4-Methoxy-3-(2-(4-Methylpiperidin-1-Yl)ethoxy)phenyl]isoindolin-1-One as a PET imaging agent for 5-HT2C receptors in the nonhuman primate brain ACNP 58th annual meeting: poster session III, poster W66. Neuropsychopharmacology 2019; (44): 385–538.

13.

Hacksell

Burstein

McFarland

, et al. On the discovery and development of pimavanserin: a novel drug candidate for Parkinson’s psychosis. Neurochem Res 2014; 39: 2008–2017.

14.

Vanover

Weiner

Makhay

, et al. Pharmacological and behavioral profile of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N’-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel 5-hydroxytryptamine(2A) receptor inverse agonist. J Pharmacol Exp Ther 2006; 317: 910–918.

15.

Varrone

Sjöholm

Eriksson

, et al. Advancement in PET quantification using 3D-OP-OSEM point spread function reconstruction with the HRRT. Eur J Nucl Med Mol Imaging 2009; 36: 1639–1650.

16.

Karlsson

Farde

Halldin

, et al. PET examination of [11C]NNC 687 and [11C]NNC 756 as new radioligands for the D1-dopamine receptor. Psychopharmacology (Berl) 1993; 113: 149–156.

17.

Ballanger

Tremblay

Sgambato-Faure

, et al. A multi-atlas based method for automated anatomical Macaca fascicularis brain MRI segmentation and PET kinetic extraction. NeuroImage 2013; 77: 26–43.

18.

Yushkevich

Piven

Hazlett

, et al. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. NeuroImage 2006; 31: 1116–1128.

19.

Moein

Nakao

Amini

, et al. Sample preparation techniques for radiometabolite analysis of positron emission tomography radioligands; trends, progress, limitations and future prospects. TrAC Trends Anal Chem 2019; 110: 1–7.

20.

Erritzoe

Ashok

Searle

, et al. Serotonin release measured in the human brain: a PET study with [11C]CIMBI-36 and d-amphetamine challenge. Neuropsychopharmacology 2020; 45: 804–810.

21.

Ichise

Toyama

Innis

, et al. Strategies to improve neuroreceptor parameter estimation by linear regression analysis. J Cereb Blood Flow Metab 2002; 22: 1271–1281.

22.

Gunn

Cunningham

VJ.

Positron emission tomography compartmental models. J Cereb Blood Flow Metab Off J Tab 2001; 21: 635–652.

23.

Ettrup

Hansen

Santini

, et al. Radiosynthesis and in vivo evaluation of a series of substituted 11C-phenethylamines as 5-HT (2A) agonist PET tracers. Eur J Nucl Med Mol Imaging 2011; 38: 681–693.

24.

Beliveau

Ganz

Feng

, et al. A High-Resolution in vivo atlas of the human brain’s serotonin system. J Neurosci 2017; 37: 120–128.

25.

Zeng

Nye

Voll

, et al. Synthesis and evaluation of pyridyloxypyridyl indole carboxamides as potential PET imaging agents for 5-HT2C receptors. ACS Med Chem Lett 2018; 9: 188–192.

26.

Searle

Beaver

Tziortzi

, et al. Mathematical modelling of [¹¹C]-(+)-PHNO human competition studies. NeuroImage 2013; 68: 119–132.

27.

Ogden

Zanderigo

Choy

, et al. Simultaneous estimation of input functions: an empirical study. J Cereb Blood Flow Metab Off J Tab 2010; 30: 816–826.

28.

Nordstrom

A-L

Mansson

Jovanovic

, et al. PET analysis of the 5-HT2A receptor inverse agonist ACP-103 in human brain. Int J Neuropsychopharmacol 2008; 11: 163–171.

29.

Darwish

Bugarski-Kirola

Jaworowicz

, et al. Population pharmacokinetic modeling and stochastic simulations to support pediatric dose selection of pimavanserin. J Clin Pharmacol 2023; 63: 1408–1416. Epub ahead of print 20 July DOI: 10.1002/jcph.2315.

30.

Ettrup

da Cunha-Bang

McMahon

, et al. Serotonin 2A receptor agonist binding in the human brain with [¹¹C]cimbi-36. J Cereb Blood Flow Metab Off J Tab 2014; 34: 1188–1196.

31.

Pompeiano

Palacios

Mengod

Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Brain Res Mol Brain Res 1994; 23: 163–178.

32.

Kehne

Baron

Carr

, et al. Preclinical characterization of the potential of the putative atypical antipsychotic MDL 100,907 as a potent 5-HT2A antagonist with a favorable CNS safety profile. J Pharmacol Exp Ther 1996; 277: 968–981.

33.

Kennett

Wood

Bright

, et al. SB 242084, a selective and brain penetrant 5-HT2C receptor antagonist. Neuropharmacology 1997; 36: 609–620.

Supplementary Material

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Imaging the 5-HT 2C receptor with PET: Evaluation of 5-HT 2C and 5-HT 2A affinity of pimavanserin in the primate brain

Abstract

Keywords

Introduction

Material and methods

Preparation of [11C]MDL 100,907 and [11C]CIMBI-36

Non-human primates

PET and MRI system

PET experimental procedure

Study design

Image analysis

Radiometabolite analysis

Pharmacokinetic estimation of pimavanserin in plasma

Kinetic modelling

Estimation of 5-HT2A and 5-HT2C fractions using [11C]CIMBI-36

Results

Discussion

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-2-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-3-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-4-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-5-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-6-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-7-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-8-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Supplemental Material

sj-pdf-9-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT2C receptor with PET: Evaluation of 5-HT2C and 5-HT2A affinity of pimavanserin in the primate brain

Footnotes

Funding

Acknowledgements

Declaration of conflicting interests

Authors’ contributions

ORCID iDs

Supplementary material

References

Supplementary Material

Preparation of [¹¹C]MDL 100,907 and [¹¹C]CIMBI-36

Estimation of 5-HT_2A and 5-HT_2C fractions using [¹¹C]CIMBI-36

sj-pdf-1-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-2-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-3-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-4-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-5-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-6-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-7-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-8-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain

sj-pdf-9-jcb-10.1177_0271678X241276312 - Supplemental material for Imaging the 5-HT_2C receptor with PET: Evaluation of 5-HT_2C and 5-HT_2A affinity of pimavanserin in the primate brain