Quantification of metabotropic glutamate receptor 5 availability with both [11C]ABP688 and [18F]FPEB positron emission tomography in the Sapap3 knockout mouse model for obsessive compulsive-like behavior

Open AccessPublished:November 28, 2021DOI:https://doi.org/10.1016/j.bpsc.2021.11.010

      ABSTRACT

      This study provides a first direct comparison between positron emission tomography (PET) radioligands targeting the allosteric site of the metabotropic glutamate receptor 5 (mGluR5): [11C]ABP688 and [18F]FPEB. A blocking paradigm was set up to substantiate the common binding site of both radioligands. Second, both radioligands were applied in Sapap3 knockouts showing compulsive-like behavior characterized by a lower in vivo mGluR5 availability.

      Methods

      First, wildtype mice (n=7) received four μPET/CT scans: an [11C]ABP688 and [18F]FPEB scan, and two blocking scans using cold FPEB and cold ABP688, respectively. A second experiment compared both radioligands in wildtype (n=7) and Sapap3 knockout mice (n=10). The simplified reference tissue method was used to calculate the nondisplaceable binding potential (BPND) representing the in vivo availability of the mGluR5 in the brain.

      Results

      Using cold FPEB as a blocking compound for [11C]ABP688 μPET and vice versa, we observed averaged global reductions in mGluR5 availability of circa 98% for [11C]ABP688 and 82%-96% for [18F]FPEB. For knockouts, the [11C]ABP688 BPND was on average 25% lower compared to wildtype controls (p<0.0001-0.001), while this was about 17% for [18F]FPEB (p<0.05).

      Conclusion(s)

      The current findings substantiate a common binding site and suggest a strong relationship between mGluR5 availability levels measured with both radioligands. In Sapap3 knockouts, a reduced mGluR5 availability could therefore be demonstrated with both radioligands. With [11C]ABP688, higher significance levels were achieved in more brain regions. These findings may imply [11C]ABP688 as a preferable radiotracer to quantify mGluR5 availability, as exemplified here in a model for compulsive-like behavior.

      INTRODUCTION

      Various neurological and psychiatric disorders were shown to be associated with a dysfunctional glutamate transmission such as obsessive compulsive disorder (OCD)(
      • Esterlis I.
      • Holmes S.E.
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      Metabotropic Glutamatergic Receptor 5 and Stress Disorders: Knowledge Gained From Receptor Imaging Studies.
      ,
      • Pillai R.L.I.
      • Tipre D.N.
      Metabotropic glutamate receptor 5 – a promising target in drug development and neuroimaging.
      ).
      OCD is a severe psychiatric disorder with a lifetime prevalence of 1-3%(
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      The epidemiology of obsessive-compulsive disorder in the National Comorbidity Survey Replication.
      ) and is characterized by recurrent and persistent thoughts/urges/images (obsessions) and/or by time-consuming repetitive thoughts/actions (compulsions) to neutralize these obsessions(4,5). The first-line pharmacological treatment consists of selective serotonin reuptake inhibitors. Unfortunately, a considerable amount of patients remains refractory(6), emphasizing the need for new superior treatment options. The past two decades, the glutamate system emerged as an important player in the pathophysiology of OCD based on preclinical, genetic and imaging studies(7–14). There may be a role for the mainly peri- and postsynaptic G-protein coupled metabotropic glutamate receptor 5 (mGluR5), which modulates glutamate neurotransmission and neuronal excitability(15). Both suppression and induction of compulsive-like behavior was possible via the administration of mGluR5 allosteric modulators(16). Also, multiple promising glutamate modulators were tested(17–19). Although promising, overall results remain inconclusive. A common conclusion comprises the need for larger study populations, considering symptom dimensions and comorbidities(20).
      Different validated positron emission tomography (PET) tracers (
      • Kim J.H.
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      A review of molecular imaging of glutamate receptors.
      ) enable the in vivo visualization of mGluR5 distribution, both in the preclinical and the clinical setting with a high translational potential. Moreover, this highly sensitive, non-invasive imaging modality allows regional and longitudinal quantification of mGluR5 availability in health and disease. This can provide valuable insights in underlying disease mechanisms and could facilitate the search of novel and superior therapeutics. Currently, there are two commonly used radioligands to image mGluR5 with PET: [3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-0-11C-methyloxime] ([11C]ABP688)(
      • Ametamey S.M.
      • Kessler L.J.
      • Honer M.
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      • et al.
      Radiosynthesis and preclinical evaluation of 11C-ABP688 as a probe for imaging the metabotropic glutamate receptor subtype 5.
      ) and 3-[18F]Fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB)(

      Wang JQ, Tueckmantel W, Zhu A, Pellegrino D, Brownell AL (2007): Synthesis and preliminary biological evaluation of 3-[18F] fluoro-5-(2-pyridinylethynyl)benzonitrile as a PET radiotracer for imaging metabotropic glutamate receptor subtype 5. Synapse. https://doi.org/10.1002/syn.20445

      ). Both are negative allosteric modulators (NAM), hence they do not compete with endogenous glutamate for binding at the orthosteric site(24,25). Also, they seem to target solely cell surface receptors, not internalized receptors(26). Both tracers are characterized by adequate blood-brain-barrier penetration, a highly specific and reversible binding to mGluR5, and favourable brain kinetics, in the absence of troublesome metabolites(27,28). In theory, [18F]FPEB offers different advantages. In vitro, it has a higher affinity for mGluR5 (KD=0.04±0.02 nM-0.15±0.11 nM)(
      • Patel S.
      • Hamill T.G.
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      • Jagoda E.
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      Species differences in mGluR5 binding sites in mammalian central nervous system determined using in vitro binding with [18F]F-PEB.
      ) compared to [11C]ABP688 (KD in vitro=1.7±0.2 nM)(
      • Ametamey S.M.
      • Kessler L.J.
      • Honer M.
      • Wyss M.T.
      • Buck A.
      • Hintermann S.
      • et al.
      Radiosynthesis and preclinical evaluation of 11C-ABP688 as a probe for imaging the metabotropic glutamate receptor subtype 5.
      ) and is thought to have a higher specificity. Also, the F-18 isotope has longer half-life, which contributes to a higher cost-effectiveness as a larger number of subjects can be imaged with a single radiotracer production. The short half-life of C-11 requires an on-site cyclotron. However, for F-18 possible defluorination in the skull could hamper quantification. Both radiotracers already made important contributions to the understanding of the role of this receptor in health and disease(1,2,21). For OCD, one study demonstrated significant correlations between the [11C]ABP688 distribution volume ratio and the Yale-Brown Obsessive Compulsive Scale obsessions subscore within the cortico-striato-thalamocortical circuit(30).
      Both [11C]ABP688 and [18F]FPEB can be blocked by the administration of other mGluR5 NAMs, such as MPEP and MTEP(27,31–33). These findings provide indirect evidence that both tracers bind the same, or close to the same, site of the receptor(34). To date, it remains unclear whether either of these two probes proves to be preferable. (i) A first step is to further verify their common binding site via the quantification of mGluR5 with both [11C]ABP688 and [18F]FPEB within one animal, followed by a paradigm in which [11C]ABP688 is blocked with unlabelled “cold” FPEB and vice versa (Figure S1). To date, no direct within-subject comparison between both radiotracers has been reported for both humans and animals. (ii) A second aim is to verify whether [18F]FPEB, like [11C]ABP688, is able to capture a significantly lower mGluR5 availability(35) in the Sapap3 knockout mouse model(12). These mice lack the SAP90/PSD-95 associated protein (Sapap3) which belongs to the group of scaffolding proteins that link different types of glutamate receptors at the level of the postsynaptic density(36,37). They show excessive pathological grooming, increased anxiety-like behavior and an imbalanced striatal output and were proposed as a model for OCD(16). (iii) Taken together the two previous study goals, as a common endpoint, we consider the correlation between the [11C]ABP668 and the [18F]FPEB regional BPND outcome parameter, which represents receptor availability, for separate animals.

      METHODS

       Mice

      Mice were bred in-house at the University of Antwerp from heterozygous Sapap3+/- breeder pairs (kindly obtained from Prof. Dr. Feng, Massachusetts Institute of Technology). Genotypes were determined via PCR on mouse ear DNA. Both the animals of experiment 1 (male C57BL/6J: wt n=7) and experiment 2 (male C57BL/6J: wt n=7; Sapap3-/- ko n=10) were kept under the same circumstances. They were co-housed in individually ventilated cages under controlled conditions (12h normal light–dark cycles, 20–23°C, and 50–55% relative humidity) with water and rodent food pellets ad libitum. All animals were scored biweekly for skin lesions, based on the severity. Figure S1 provides an overview of all experimental procedures. All animal procedures were performed in accordance with the European Ethics Committee (decree 86/609/CEE). The study protocol was approved by the Animal Experimental Ethical Committee of the University of Antwerp, Antwerp, Belgium.

       Tracer synthesis

      Based on a previous description of the radiosynthesis(22), [11C]ABP688-E was prepared as such(35). The injected cold mass was aimed <5.00 nmol/kg to avoid cold mass effects(38)(Table S1). For the blocking experiment, the mean molar activity (Am) at the time of injection was 43.19±6.66 MBq/nmol for the test scans and 29.93±3.48 MBq/nmol for the blocking scans. In experiment 2 comparing genotypes, the mean Am was 59.35±12.22 MBq/nmol for the wt and 53.84±10.63 MBq/nmol for the ko group.
      • [18F]FPEB was synthesized using 3-chloro-5-[(pyridine-2-yl)]ethynyl]benzonitrile at high MA and RCY on an automated synthesis module (Comecer, The Netherlands). [18F]Fluoride, produced by irradiation of [18O]H2O via 18O(p, n)18F nuclear reaction (cyclotron, Siemens Eclipse), was trapped on a Sep-Pak® QMA cartridge and eluted into a V-shaped reaction vial with a solution of Kryptofix 222 (12 mM) and K2CO3 (6 mM) in MeCN/H2O (96/4, 1.5 mL). The solvents were azeotropically distilled at 90°C under a stream of helium. After that, anhydrous acetonitrile (1 mL) was added and distillation was continued. This process was repeated once more until a dry white residue was obtained. The reaction vial was heated to 180-190°C and the FPEB precursor (5-6 mg), dissolved in anhydrous DMSO (0.5 mL), was added. The resulting mixture was refluxed for 20 min and after cooling, it was quenched by addition of sterile water (1.5 mL) and purified using isocratic semi-preparative reverse phase HPLC (Phenomenex® Kinetex 5 μm EVO C18 100 Å 10×150 mm, λ = 254 nm; mobile phase: NaOAc 0.05 M pH 5.5/EtOH 60/40 v/v, 3 mL/min). The collected radioactive product was diluted with 15 mL of sterile water and passed through Sep-Pak® Alumina N and Sep-Pak® tC18 cartridges assembled together in this order. The cartridges were washed with sterile water (5 mL) and the pure [18F]FPEB was released with 96% EtOH (0.5 mL), diluted with sterile saline (5 mL) and filtered in a sterile manner. Radiochemical purity was determined. The total synthesis time, calculated from the end of bombardment and including HPLC purification, was approximately 70 minutes. Typical irradiation of the target lasted 20 minutes with a beam current of 60 μA. The injected cold mass was aimed <3.00 nmol/kg to avoid cold mass effects(38)(Table S1). For the blocking experiment, the mean molar activity (Am) at the time of injection was 144.8±41.21 for the test scans and 90.02±46.83 MBq/nmol for the blocking scans. In experiment 2 comparing genotypes, the mean Am was 142.60±53.98 MBq/nmol for the wt and 140.80±64.70 MBq/nmol for the ko group.

       Behavioral evaluation

      For experiment 2 (Figure S1), prior to μPET scanning, each animal was monitored by video recording to assess grooming behavior. All thirty-minute recordings and manual scorings were performed according to a previously described protocol(35).

       Cross-sectional [11C]ABP688 and [18F]FPEB dynamic (blocking) μPET/CT scans

      Both the cohorts from experiment 1 and 2 were subjected to a baseline dynamic μPET/CT acquisition with both [11C]ABP688 and [18F]FPEB (Figure S1). Mice were anesthetized using isoflurane (IsoFlo®, Zoetis, USA) mixed with medical oxygen (induction 5%, maintenance 2%) and placed on a heated blanket (37.5°C). A catheter (tubing: P10, Instech Solomon, USA; needle: BD Microlance™ 30G, BD, USA) was placed in the tail vein for later iv bolus administration of the tracer. Afterwards, the animals were positioned on the heated bed of the scanner. Parallel to the start of the 60-minute dynamic μPET acquisition, a 0.2 mL bolus of the radioligand was administered using an automated syringe pump at a rate of 1 mL/min (model 11 Elite, Harvard Apparatus, USA). Subsequently, a 10-minute 80kV/500μA CT scan was acquired for attenuation and scatter correction. Acquisitions (60+10 min; frames: 12x10s, 3x20s, 3x30s, 3x60s, 3x150s and 9x300s) were performed on two Siemens Inveon μPET/CT scanners (Siemens Preclinical Solutions, USA). During scanning procedures, both the respiratory and heart rate were monitored and body temperature was kept at 37.0°C with a feedback air flow system (Minerve, France). For the blocking scans, a 0.1 mL iv 5% DMSO in saline bolus containing either unlabeled ABP688 (11.9 mg/mL) or FPEB (0.2 mg/mL) was administered five minutes prior to the radiotracer injection. The animal and the scan parameters are represented in Table S1.

       Image processing

      μPET images were reconstructed using two-dimensional ordered subset expectation maximization (2D-OSEM) with four iterations and 16 subsets after Fourier rebinning. The images were reconstructed on a 128×128×159 grid with a voxel size of 0.776×0.776×0.796 mm. Normalization, dead time correction, random subtraction, CT-based attenuation and simulated single-scatter corrections, as well as detector response modeling for parallax errors were applied(39). Reconstructed images were processed in PMOD v3.6 (PMOD Technologies, Switzerland). For all test images, a static image corresponding to the time-averaged frames of each dynamic acquisition was spatially transformed to a mouse brain [11C]ABP688 or [18F]FPEB PET template (in-house), respectively. This PET template (via CT) already corresponded to a standardized MR template space (Waxholm MR)(
      • Johnson G.A.
      • Badea A.
      • Brandenburg J.
      • Cofer G.
      • Fubara B.
      • Liu S.
      • Nissanov J.
      Waxholm Space: An image-based reference for coordinating mouse brain research.
      ) with the corresponding volume-of-interest (VOI) definitions. The obtained matrix from the aforementioned brain normalization step was applied to transform all dynamic scans to that [11C]ABP688 template space. For all blocking images, the CT of the blocking scan was manually matched to the CT of the test scan of the same animal. This transformation was applied to the dynamic blocking image. The resulting dynamic blocking image was then subjected to an identical image processing pipeline as its corresponding test scan. The regional time-activity curves (TACs) were extracted from the resulting raw non-smoothed images via the superimposition of the VOI template. For both radioligands, these extracted TACs served as input for the simplified reference tissue model (SRTM)(
      • Lammertsma A.A.
      • Hume S.P.
      Simplified reference tissue model for PET receptor studies.
      ). Based on this method (implemented in PMOD), the regional non-displaceable binding potential (BPND) was calculated with the cerebellum as an earlier validated reference region(27,28). Based on the spill-in effect of anterior brain structures to the cerebellum and the skull defluorination present with [18F]FPEB, the cerebellar VOI was concentrically reduced. Pixelwise kinetic modeling, using SRTM2(42), was applied to generate a parametric BPND image for each animal. Subsequently, averaged BPND images were calculated for all groups. For visualization purposes, these images were smoothed using an isotropic Gaussian filter (FWHM=0.5 mm).

       Data and statistical analysis

      All statistical analysis was performed in GraphPad Prism 8 (GraphPad Software, USA), and a significance level of p<0.05 was imposed. The analysis of the behavioral data was performed according to a previously described protocol(35). To compare behavioral parameters between both genotypes, a Mann-Whitney test was performed. All behavioral data is expressed as the averaged parameter-of-interest ± standard error (SE) for both wt and ko groups.
      A Shapiro-Wilk test confirmed normality for the VOI-based μPET data for both experiment 1 and 2. A two-way analysis of variance (ANOVA) with post-hoc Bonferroni correction for multiple comparisons was applied to investigate whether significant regional differences were present between the test and blocking conditions (with repeated measures) and between the genotypes (without repeated measures). For experiment 2, comparing two genotypes, the analysis includes Cohen’s d effect size. All imaging data is represented as the averaged value ± standard deviation (SD).
      A voxel-based analysis was performed on the filtered BPND images using Statistical Parametric Mapping (SPM) 12 (Wellcome Department of Imaging Neuroscience, UK) in Matlab (R2016a, The Mathworks Inc, USA). Statistical T-maps were calculated at a significance level of p=0.01 and a cluster threshold of 100 voxels (voxel size: 0.09x0.09x0.09 mm).
      A Pearson’s r correlation was used to determine the relationship between the outcome [11C]ABP688 and [18F]FPEB regional BPND values (cortex, striatum, hippocampus, and amygdala) of individual animals.

      RESULTS

      Significant reductions in the nondisplaceable binding potential (BPND) of high binding brain regions upon blocking of [11C]ABP688 or [18F]FPEB supports their common binding site in vivo
      To directly verify the common binding site of ABP6888 and FPEB in vivo, a blocking study was performed (Figure S1 – experiment 1). Based on the standard uptake value (SUV) TACs (Figure 1A), both tracers show a rapid brain uptake within the first 10 minutes in different brain regions. Moreover, both blocking conditions using unlabeled ABP688 and unlabeled FPEB respectively resulted in a decrease of the SUV TACs of high binding brain regions towards the level of the cerebellar reference region. Correspondingly, both the [11C]ABP688 and the [18F]FPEB BPND were significantly reduced in all investigated regions following the FPEB and the ABP688 pre-treatment, respectively. All regional BPND estimates ± SD are reported in Table 1 (Two-way repeated measures ANOVA: [11C]ABP688 main effect of brain region F(3,24)=2.906, p=0.0554; main effect of condition F(1,24)=1209, p<0.0001; main effect of subject F(24,24)=1.087, p=0.4202; main effect of brain region x condition F(3,24)=3.424, p=0.0332 - [18F]FPEB main effect of brain region F(3,24)=2.447, p=0.0883; main effect of condition F(1,24)=863.1, p<0.0001; main effect of subject F(24,24)=2.214, p=0.0286; main effect of brain region x condition F(3,24)=11.86, p<0.0001).
      Figure thumbnail gr1
      Figure 1A. Target engagement of mGluR5 radioligands in the wildtype mouse brain. (A) Average regional [11C]ABP688 and [18F]FPEB time-activity curves (60-min acquisition) expressed in standardized uptake values with (block) and without (test) a pre-treatment with cold FPEB or cold ABP688. All the time-activity curves of the high binding regions decrease to the level of the cerebellar reference curve for both radioligands. (B) The corresponding BPND (SRTM) values for the blocking condition are significantly reduced in all investigated brain regions, when compared to the test condition (). (****p<0.0001; ctx, cortex; str, striatum; hc, hippocampus; amd, amygdala; BPND, nondisplaceable binding potential; SUV, standard uptake value) FIGURE B. Validation of compulsive-like grooming in 9-mo old Sapap3 ko mice versus wildtype controls reflected by behavioral parameters (A) grooming frequency and (B) % duration grooming for wt and Sapap3 ko mice. (mo = months; wt = wild-types; ko = knockouts; **p<0.01, **p<0.01, ***p<0.001) FIGURE C. μPET imaging of mGluR5 radioligands in the wildtype versus the Sapap3 ko mouse brain (A) Average [11C]ABP688 and [18F]FPEB μPET BPND (SRTM2) parametric images superimposed on a mouse MR template (red: cortex; yellow: striatum; purple: hippocampus; green: amygdala; pink: cerebellum reference region) (B) combined with the corresponding averaged [11C]ABP688 and [18F]FPEB PET BPND values ± SD (). (*p<0.05, ***p<0.001; ****p<0.0001; ctx, cortex; str, striatum; hc, hippocampus; amd, amygdala; BPND, nondisplaceable binding potential; SUV, standard uptake value; wt, wildtype; ko, knockout) FIGURE D. Voxel-based analysis via statistical parametric mapping of μPET imaging of mGluR5 radioligands in the wildtype versus the Sapap3 ko mouse brain. Hypo T-maps (voxel-based SPM analysis) superimposed on a mouse MR template (red: cortex; yellow: striatum; purple: hippocampus; green: amygdala; pink: cerebellum reference region) showing clusters of voxels (threshold = 100 voxels) with a significantly lower BPND in the ko group versus the wt group for [11C]ABP688 (p<0.01) and [18F]FPEB (p<0.001), respectively. (MR = magnetic resonance)
      TABLE 1The blocking effect of cold FPEB and cold ABP688 on [11C]ABP688 and [18F]FPEB binding respectively via the SRTM quantification of the regional BPND values for a 60-min dynamic μPET acquisition in wildtype mice.
      [11C]ABP688 test[11C]ABP688 block[18F]FPEB test[18F]FPEB block
      BPNDBPNDBPNDBPND
      RegionMean ± SDCOV (%)Mean ± SDDiff (-%)Mean ± SDCOV (%)Mean ± SDDiff (-%)
      Ctx1.26 ± 0.1814.10.02 ± 0.0298.4§3.43 ± 0.5315.30.94 ± 0.3372.7§
      Str1.54 ± 0.2314.90.04 ± 0.0197.6§4.72 ± 0.5015.40.56 ± 0.5088.1§
      Hipp1.54 ± 0.2516.10.01 ± 0.0199.6§5.01 ± 0.8516.90.69 ± 0.4886.2§
      Amy1.31 ± 0.1914.80.04 ± 0.0397.2§4.24 ± 0.7016.40.84 ± 0.3380.3§
      Ctx, cortex; Str, striatum; Hipp, hippocampus; Amy, amygdala; BPND, nondisplaceable binding potential; SD, standard deviation; COV, coefficient of variation; Diff, difference; §p<0.0001
      The presence of compulsive-like grooming behavior is confirmed in the Sapap3 knockout mice
      In line with a previous and more extensive longitudinal characterization of compulsive-like grooming behavior in Sapap3 ko mice(35), the grooming frequency (wt: 10.57±1.53, ko: 46.50±7.49, p<0.001) and % duration grooming (wt: 5.71±1.24%, ko: 32.71±7.50%, p=0.0046) were significantly higher in the ko group, when compared to wt controls (Figure B). Moreover, these values were of the same magnitude.
      Both [11C]ABP688 and [18F]FPEB measure lower mGluR5 availability in Sapap3 knockout mice, however with different statistical significance compared to wildtypes
      The resulting (A) BPND parametric images with the corresponding (B) VOI-based BPND values for both genotypes and radioligands are represented in Figure C. For [11C]ABP688, a cross-sectional comparison between genotypes revealed a significantly lower BPND in the cortex (-23.12%, p=0.0003), the striatum (-26.46%, p<0.0001), the hippocampus (-25.90%, p<0.0001), and the amygdala (-24.38%, p=0.0001) of ko mice. On the other hand, for [18F]FPEB, a more subtle BPND decline was significant for the striatum (-18.34%, p=0.0132), the hippocampus (-16.55%, p=0.0192), and the amygdala (-17.72%, p=0.0442). All regional BPND estimates ± SD are reported in Table 2 (Two-way ANOVA: [11C]ABP688 main effect of brain region F(3,60)=11.03, p<0.0001; main effect of genotype F(1,60)=101.2, p<0.0001; main effect of brain region x genotype F(3,60)=0.6404, p=0.5920 - [18F]FPEB main effect of brain region F(3,60)=21.70, p<0.0001; main effect of genotype F(1,60)=27.59, p<0.0001; main effect of brain region x genotype F(3,60)=0.2725, p=0.8450).
      TABLE 2Overview of the average [11C]ABP688 and [18F]FPEB μPET BPND values of wildtype versus Sapap3 knockout mice.
      [11C]ABP6888 wt[11C]ABP688 ko[18F]FPEB wt[18F]FPEB ko
      BPNDBPNDBPNDBPND
      RegionMean ± SDCOV (%)Mean ± SDCOV (%)Diff (-%)Eff. dMean ± SDCOV (%)Mean ± SDCOV (%)Diff (-%)Eff. d
      Ctx1.24 ± 0.1411.70.95 ± 0.099.223.12.463.28 ± 0.298.82.76 ± 0.5118.416.01.25
      Str1.49 ± 0.1812.41.10 ± 0.1412.326.5§2.424.63 ± 0.408.63.78 ± 0.7018.518.31.49
      Hipp1.47 ± 0.1611.01.09 ± 0.1311.525.9§2.924.91 ± 0.448.94.10 ± 0.7418.116.61.33
      Amy1.24 ± 0.1513.40.94 ± 0.1313.524.42.144.11 ± 0.389.23.38 ± 0.6619.617.71.36
      Ctx, cortex; Str, striatum; Hipp, hippocampus; Amy, amygdala; BPND, nondisplaceable binding potential; SD, standard deviation; COV, coefficient of variation; Diff, difference; p<0.05, p<0.001, §p<0.0001; Eff. d, effect size Cohen’s d.
      A more sensitive voxel-based analysis confirmed the established VOI-based significant differences in [11C]ABP688 and [18F]FPEB BPND between the genotypes. Figure D shows a significantly lower [11C]ABP688 BPND in 80.37% of all voxels within the total striatal volume (p<0.001; cluster threshold = 100) of the ko group. This significant BPND decrease was also present in a substantial part of the voxels from other regions (cortex: 40.00%, hippocampus: 54.75%, and amygdala 50.38%). In comparison, the [18F]FPEB BPND was significantly lower in 77.88% of all voxels within the striatal volume (p<0.01; cluster threshold = 100). For the cortex, the hippocampus, and the amygdala a significantly lower BPND in ko mice was found in 28.81%, 49.24%, and 47.72% of all voxels respectively. Notably, when a more strict significance level of p<0.001 was imposed on the [18F]FPEB BPND data, similarly to the level imposed on [11C]ABP688 BPND data, the percentage of significant voxel declined to less than 0.6% for all of the aforementioned regions.

       The intra-animal relationship between [11C]ABP688 and [18F]FPEB

      A comparison between the [11C]ABP688 and the [18F]FPEB PET BPND values across all regions of interest (cortex, striatum, hippocampus, amygdala) per animal resulted in a strong correlation (r=0.8282, p<0.0001) for experiment 1 and a moderate correlation (r=0.5642, p<0.001) for experiment 2 including both wt and ko animals. When combining BPND values for the wildtype animals from both experiments, again a strong correlation (all regions: r=0.7187, p<0.0001 – regional: cortex r=0.5939, p=0.0323; striatum r=0.6208, p=0.0236; hippocampus r=0.6473, p=0.0168; amygdala r=0.5023, p=0.0803) between the two mGluR5 radioligands could be demonstrated (Figure S2 – panel A). When only the ko animals from experiment 2 were considered, a similar comparison resulted in a moderate correlation (all regions: r=0.4042, p=0.0097 – regional: cortex r=0.0608, p=0.8674; striatum r=0.2615, p=0.4655; hippocampus r=0.1057, p=0.7714; amygdala r=0.2480, p=0.4987) (Figure S2 – panel B).

      DISCUSSION

      The study goals included: (i) setting up a blocking μPET paradigm to verify the common binding site for both [11C]ABP688 and [18F]FPEB at the mGluR5 allosteric site in vivo and in a direct manner; (ii) providing a direct comparison between both radioligands in the robust Sapap3 ko animal OCD model, which was earlier characterized by a lowered in vivo mGluR5 availability; (iii) generate direct intra-animal comparisons between mGluR5 availability measurements with both radioligands. Taken together, this setup allows to further investigate the lower regional mGluR5 availability in the Sapap3 ko mouse model exhibiting compulsive-like behavior(35). Through this preclinical step, we seek to contribute to future applications of mGluR5 radioligands in a clinical setting, allowing to gather additional information regarding the role of mGluR5 and the glutamate system in the pathophysiology of OCD and as a target for potential novel drug candidates.
      In the blocking paradigm, we observed global reductions in mGluR5 availability (BPND) of approximately 98% for [11C]ABP688 and 82% for [18F]FPEB, respectively. It might appear that unlabeled ABP688 is only able to partially block [18F]FPEB suggesting the possibility of a second binding site. For this reason, the [18F]FPEB blocking paradigm of experiment 1 was repeated in a satellite cohort of wildtype mice (Table S2) with a double dose of unlabeled ABP688 solution and this in 10% instead of 5% DMSO saline solution to further increase ABP688 solubility. With this adjusted set-up, an average blocking percentage of 96% was achieved (Table S3; Figure S3). For all blocking experiments, the obtained regional high-binding TACs decreased to a similar level as the cerebellar reference curve, thereby further substantiating the specificity of both radioligands for their target. This supports the current body of literature(34,43,44) which already provided strong, but indirect, arguments for the same or closely to the same binding site for both [11C]ABP688 and [18F]FPEB. First, previous preclinical PET studies showed blocking of the binding of these radioligands using other mGluR5 NAMs (MPEP and MTEP) in different species for both [11C]ABP688 (MPEP in rats(22,33), MPEP in mice(45), and MTEP in baboons(32)) and [18F]FPEB (MTEP in rats(23,27) and rhesus monkeys(46)). Moreover, clinical studies already explored whether [18F]FPEB was able to reproduce [11C]ABP688 results obtained in healthy controls. DeLorenzo and colleagues provided an indirect comparison between same day test-retest [11C]ABP688 and [18F]FPEB PET measurements in healthy subjects(34). They show a significantly higher estimated BPND’ for both [11C]ABP688 and [18F]FPEB in the retest scan, indicating consistency between the findings for both radioligands.
      Secondly, both radioligands were directly compared in the Sapap3 ko mice, previously shown to possess a decreased mGluR5 availability for [11C]ABP688(35) parallel to worsening of OCD-like grooming behavior(16). Possibly, this reduced mGluR5 availability within the cortico-striato-thalamocortical “OCD” circuitry is associated with excessive mGluR5 signalling in this model via constitutive activation mechanisms(16,47). As the BPND parameter is proportional to the affinity and the availability (and possibly expression) of the receptor(48), changes in these parameters such as receptor internalization(49) could also explain the decreased binding of both radioligands in Sapap3 ko mice. Future studies should seek to clarify the mechanisms underlying such alterations in mGluR5 availability. As anticipated, [18F]FPEB was also able to pick up group differences between ko mice and wt controls, but this to a lesser extent. The significance levels were lower and limited to a smaller number of regions when compared to [11C]ABP688. Moreover, parallels exist with previous clinical findings, despite species differences. A study in healthy controls showed that the [11C]ABP688 regional VT decreased on average by 21.3±21.4% upon infusion with a subanesthetic dose of ketamine, when compared to a baseline scan on the same day(50). A second study with a similar set-up combining [11C]ABP688 PET with a ketamine challenge in major depressive disorder (MDD) patients and healthy controls(51) also reported a significant ketamine-induced decrease in [11C]ABP688 binding of the same magnitude as the previous study for the healthy controls (-19±22%). The MDD patients, which already had a significantly lower baseline binding levels, also showed a significant decrease in binding with the ketamine challenge (-14±9%). In a third [18F]FPEB study, again with a similar set-up, the ketamine-induced changes in mGluR5 availability were of smaller magnitude (circa 10%)(
      • Holmes S.E.
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      • Davis M.T.
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      Measuring the effects of ketamine on mGluR5 using [18F]FPEB and PET.
      ). These findings suggest possible superiority of [11C]ABP688 to detect mGluR5 changes in drug challenge paradigms. The current findings in Sapap3 ko mice characterized by intrinsic mGluR5 abnormalities further support these findings: [11C]ABP688 BPND decreased on average with circa 25%, while [18F]FPEB BPND decreased on average with 17% compared to wt controls. Especially since herein, the limitations of possible dynamic effects of a glutamate modulating compound such as ketamine on tracer delivery and washout were circumvented. Combining these findings renders [11C]ABP688 more suitable to pick up mGluR5 differences in subjects possessing intrinsic mGluR5 aberrations. Still, extrapolation of results to other models/species should be done with caution, keeping in mind the presence of (non-)physiological sources of variability and the possible existence of the aforementioned diverse mechanisms underlying changes in mGluR5 availability. Also, methodological and technical differences between studies could hamper the generalization of findings. For example for [18F]FPEB, tracer dose conditions were assumed based on the absence of a negative correlation between the injected cold mass and the obtained BPND per animal. However, this has not yet been more rigorously demonstrated based on a wider range of unlabeled FPEB doses(52).
      Multiple studies have shown that the timing of two consecutive PET scans for mGluR5 availability evaluation should be considered. Here, the test scans were separated by four or more days (experiment 1: 4.0±0.0 days; experiment 2: wt 9.0±2.8 days, ko 9.2±2.3 days). Both human test-retest studies using either [11C]ABP688(53–55) or [18F]FPEB(56,57) as rodent studies(27,28,52,58), with days to weeks between consecutive scans reported sufficient test-retest stability. Based on these findings, we assume a low variability in mGluR5 availability within one subject between consecutive PET scans.
      (iii) The current study design allowed a first within-subject, and consequently direct, comparison between the regional BPND values for both studied radioligands to verify possible radioligand superiority. In an unperturbed glutamate system, only considering the wt controls, we found a strong correlation between the [11C]ABP688 and the [18F]FPEB regional BPND values. These findings again suggest that a similar pool of mGluR5 receptors is targeted by both ligands. In addition, when only considering subjects with a perturbed and especially lower mGluR5 availability (Sapap3 ko mice) the correlation loses its strength.
      In conclusion, the current study provides a first direct comparison between [11C]ABP688 and [18F]FPEB, which will continue to play a significant role in the understanding of disease mechanisms underlying psychiatric and neurological disorders and their treatment. This study directly implies a competition between both radioligands for a common binding site at the level of the allosteric pocket of the receptor. Furthermore, both [11C]ABP688 and [18F]FPEB were able to quantify a lower brain mGluR5 availability in Sapap3 ko mice showing OCD-like behavior. Considering individual wt mice, our results suggest a strong relationship between the BPND parameter for mGluR5 availability for both radioligands. Notwithstanding, compared to [18F]FPEB, our data suggest that [11C]ABP688 may be a more sensitive radiotracer for measuring mGluR5 availability in (models of) disease with intrinsic mGluR5 abnormalities such as the Sapap3 ko mouse model. In summary, the current finding of a higher variability in the regional BPND for [18F]FPEB compared to [11C]ABP688 in the brain of Sapap3 ko mice, suggests the use of [11C]ABP688 when designing a PET study for mGluR5 quantification.

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