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Letter
Reaching out for sensitive evaluation of the mu opioid receptor in vivo: Positron emission tomography imaging of the agonist [11C]AH7921. Waqas Rafique, Shivashankar Khanapur, Mona Milde Spilhaug, and Patrick J Riss ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00075 • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 21, 2017
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ACS Chemical Neuroscience
Reaching out for sensitive evaluation of the mu opioid receptor in vivo: Positron emission tomography imaging of the agonist [11C]AH7921. Waqas Rafique1, Shivashankar Khanapur1,4, Mona M. Spilhaug1, Patrick J. Riss1,2,3,4* 1
realomics SFI, Kjemisk Institutt, Universitetet i Oslo, Sem Sælands vei 26, Kjemibygningen, 0371 Oslo, Norway Klinik for Kirurgi og Nevrofag, Oslo Universitets Sykehus HF–Rikshospitalet, Postboks 4950 Nydalen, 0424 Oslo 3 Norsk Medisinsk Syklotronsenter AS, Gaustad, Postboks 4950 Nydalen, 0424 Oslo 4 Radboud Translational Medicine BV, Geert Grooteplein 21, Postbus 9101 6500HB Nijmegen, Nederlande KEYWORDS:.opioid receptor, positron emission tomography, AH7921, carbon-11, carboxylation 2
ABSTRACT: Imaging of the mu opioid receptor (MOR) availability with PET is a pertinent challenge in Neuroscience. Both, regulation of receptor expression and occupancy by endogeneous opioids play into cognitive and behavioural phenotypes of healthy function and disease. Receptor expression in the active and inactive states can be measured using high affinity radioagonist and radioantagonist PET tracers, respectively. Occupancy assessment requires radioligands showing competitive and reversible binding with moderate affinity to the MOR, which may lead to physical extinction of the receptor specific signal in vivo. We investigated a moderately potent, selective MOR agonist in rat to test if a radiotracer design paradigm tailored to competition with endogeneous opioids leads to viable imaging results. The benzamide 3,4-dichlorobenzenecarboxylic acid (dimethylamino)cyclohexyl)methyl amide (AH-7921, 1) was synthesized and characterized in rat brain using autoradiography and positron emission tomography. Compound 1 was found to activate with low nanomolar potency the MOR and to a lesser extend KOR as a full agonist. Concentration dependent binding studies with agonist and antagonist radioligands were conducted to assess competition behavior and obtain inhibition constants. Kinetic analysis of 3,4-dichlorobenzene[11C]carboxylic acid (dimethylamino)cyclohexyl)methyl amide binding in rat brain resulted in low but reproducible binding potential in the thalamus (0.8±0.1). A radioactive metabolite was detected in brain (17%, after 15 min). Nonetheless, we conclude that quantitative imaging of MOR availability is possible when using a moderate affinity radiotracer.
INTRODUCTION The present study was motivated by a need for competitive mu (µ) opioid receptor (MOR) selective positron emission tomography (PET) radioligands to assess MOR availability or occupancy in the living brain.1 Such radioligands are useful in current research lines for example in the fields of addiction and pain processing.2-5 In particular, preclinical researchers demand optimised radiotracers to address pharmacological constraints inherent to OR PET studies.4-8 MOR is expressed in a relatively low receptor concentration in brain (Bmax (MOR, rat) ~ 100 – 102 fmol/mg protein; 10 -1 - 101 pmol/g tissue), but most validated radioligands provide a kd ≤ 10-1 nM.1 In consequence, OR occupancy reaches ~75% at a radioligand concentration as low as three times the kd.7-11 This causes a dilemma in opioid receptor occupancy studies with subnanomolar affinity radiotracers, because the required molar radioactivity to avoid significant occupancy of the receptor (97%. Seven male Harlan Sprague-Dawley rats (314±22 g) were injected with 12.3±1.28MBq and used for a PET study on a Siemens/CTI µPET Focus 120 small animal PET scanner under isoflurane anaesthesia.14,24 A tail vein was used for administration of the radiotracer. The protocol consisted of two different scanning sessions of 90 minutes length beginning with a bolus injection of [11C]1. The radiotracer was applied in either high specific activity (~200 MBq/nmol) or low specific activity (1.8-2.0 MBq/nmol) to test saturability of binding. Under baseline conditions, the injected activity reflects a dose of 0.24±0.1 nmol/kg. For partial saturation of the OR binding sites in brain, animals were injected with 1548 ± 57 nmol/kg. Two rats were exempt from imaging, instead these animals were sacrificed 15 minutes post injection to harvest blood and brain tissue samples for metabolite determination. At this timepoint, the intact parent compound [11C]1 accounted for 83% of the radioactivity in brain. However, only 40% of [11C]1 remained intact after 15 minutes in circulating blood. The N-desmethyl analogue was detected in metabolite blood samples. Each PET dataset was coregistered to an anatomical atlas of the rat brain to align the individual radioactivity distribution data with defined anatomical regions. The brain was segmented into regions of interest to derive curves of the regional activity concentration over time. A parametric map of the radiotracer distribution obtained via the kinetic analysis was generated and coregistered to an atlas of the Sprague-Dawley rat brain. A representative PET/MR-fusion image is shown in Figure 4.24-26 Since MOR binding sites amount for the majority of OR binding sites (70%) in a large volume of thalamic tissue, this region is well suited to analyze binding.6-15 Kinetic analysis was applied to regional data using Logan graphical analysis.24-26 This is not ideal considering detection of radiometabolites in the brain, but robust and reproducible outcomes where found when the cerebellum gray matter was chosen as the reference region. In baseline scans the activity concentration in the thalamus and the cerebellum peaked at 0.9% ID/ml, 8 minutes after administration of the radiotracer. Due to rapid washout of the radioactivity an equilibrium of binding is attained 15 minutes after venous injection of [11C]1. At higher dose, radioactivity concentrations reached 2% ID/ml within 5 minutes but binding in the thalamus was reduced by up to 47% compared to baseline, which is a reasonable effect with respect to the moderate dose. [11C]1
Figure 4. A coronal view of the radioactivity distribution (top left); The graph next to the image shows the average time-dependent course of activity concentration in the thalamus (THL), caudate putamen (CP), cingulate cortex (CC) and the cerebellum (CB) at baseline; the graph to the bottom right depicts a representative plot of the Logan analysis for the thalamus (10-50 min, DVR = 1.92). The respective outcomes for DVR and BPND as a function of scan duration are shown under the image. To address the impact of radiometabolite detection in brain, we investigated the effect of PET-scan duration on the binding potential (BPND = DVR-1) in Figure 4. The radiometabolite detected in brain appears to generate a nondisplaceable component of binding which influences quantitation in late timeframes. With 11C the effect (in our mass constrained protocol) is within the noise between 30 and 60 minutes p.i. After this, additional, non-specific binding seems to build up in cerebellum, however, it should be noted at this point the residual radioactivity in brain is very low. From an initial injected dose of 10 MBq, 1.25 MBq remain after three half-lives (60 minutes). Less than 0.5% of the activity is located within the brain, i.e.
