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Evaluation of [18F]FNM biodistribution and dosimetry based on whole-body PET imaging of rats
Accepted Manuscript Evaluation of [18F]FNM biodistribution and dosimetry based on whole-body PET imaging of rats
Anne-Sophie Salabert, Erick Mora-Ramirez, Marie Beaurain, Mathieu Alonso, Charlotte Fontan, Hafid Belhadj Tahar, Marie Laure Boizeau, M. Tafani, Manuel Bardiès, Pierre Payoux PII: DOI: Reference:
S0969-8051(17)30363-3 https://doi.org/10.1016/j.nucmedbio.2017.12.003 NMB 7986
To appear in: Received date: Revised date: Accepted date:
25 October 2017 5 December 2017 17 December 2017
Please cite this article as: Anne-Sophie Salabert, Erick Mora-Ramirez, Marie Beaurain, Mathieu Alonso, Charlotte Fontan, Hafid Belhadj Tahar, Marie Laure Boizeau, M. Tafani, Manuel Bardiès, Pierre Payoux , Evaluation of [18F]FNM biodistribution and dosimetry based on whole-body PET imaging of rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Nmb(2017), https://doi.org/10.1016/j.nucmedbio.2017.12.003
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ACCEPTED MANUSCRIPT Evaluation of [18F]FNM biodistribution and dosimetry based on whole-body PET imaging of rats Salabert A S 1,2 ,Mora-Ramirez E3,5,6, Beaurain M 1,2, Alonso M 2, Fontan C2, Belhadj Tahar H 7 , Boizeau
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Marie Laure 8 , Tafani , M 1,2, Bardiès M 3,5, Payoux P1,4
ToNIC, Toulouse NeuroImaging Center, Université de Toulouse, Inserm, UPS, France
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University Hospital, Radiopharmacy Unit, Toulouse, France
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Inserm, UMR1037 CRCT, F-31000 Toulouse, France
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University Hospital, Nuclear Medicine Unit, Toulouse, France
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Université Toulouse III-Paul Sabatier, UMR1037 CRCT, F-31000 Toulouse, France
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Universidad de Costa Rica, CICANUM-Escuela de Física, San José, Costa Rica
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Research and Expertise Group, French Association for the Promotion of Medical Research (AFPREMED), Toulouse, France 8
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Advanced Technology Institute in Life Sciences (ITAV), Centre National de la Recherche Scientifique-Université Paul Sabatier Toulouse III (CNRS-UPS), Université Paul Sabatier Toulouse III (UPS), Université de ToulouseToulouse, France.
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Corresponding author: Corresponding author: Anne-Sophie Salabert UMR 1214 Inserm TONIC (Toulouse Neuro-imaging Center) Université Toulouse III - Paul Sabatier CHU PURPAN - Pavillon BAUDOT Place du Dr Joseph Baylac 31024 TOULOUSE CEDEX 3 France Tel: 05 62 74 61 66
Email addresses: Anne-Sophie Salabert: [email protected]; Mora-Ramirez Erick : [email protected]; Marie Beaurain : [email protected], Mathieu Alonso: [email protected]; Charlotte Fontan : [email protected]; Hafid Belhadj-Tahar : [email protected] ; Marie Laure Boizeau : [email protected]; Manuel Bardiès : [email protected]; [email protected]; Pierre Payoux: [email protected].
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENT The authors would like to thank all members of the PET Center and CREFRE noninvasive exploration ( Inserm UMS 006 ) for their availability. This work has been supported in part by a grant from the French National Agency for Research called “Investissements d’Avenir” n°ANR-11-LABX-0018-01.
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ABSTRACT
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Introduction: The aim of this work was to study the biodistribution, metabolism and radiation dosimetry of rats injected with [18F]FNM using PET/CT images. This novel radiotracer targeting NMDA receptor has potential for
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investigation for neurological and psychiatric diseases.
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Methods: Free fraction and stability in fresh human plasma were determined in vitro. PET/CT was performed on anesthetized rats. Organs were identified and 3D volumes of interest (VOIs) were manually drawn on the CT in the
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center of each organ. Time activity curves (TACs) were created with these VOIs, enabling the calculation of residence times. To confirm these values, ex vivo measurements of organs were performed. Plasma and urine were
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also collected to study in vivo metabolism. Data was extrapolated to humans, effective doses were estimated using ICRP-60 and ICRP-89 dosimetric models and absorbed doses were estimated using OLINDA/EXM V1.0 and
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OLINDA/EXM V2.0 (which use weighting factors from ICRP-103 to do the calculations).
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Results: The [18F]FNM was stable in human plasma and the diffusible free fraction was 53%. As with memantine, this tracer is poorly metabolized in vivo. Ex vivo distributions validated PET/CT data as well as demonstrating a
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decrease of radiotracer uptake in the brain due to anesthesia. Total effective dose was around 6.11µSv/MBq and
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4.65µSv/MBq for female and male human dosimetric models, respectively. Conclusions: This study shows that the presented compound exhibits stability in plasma and plasma protein binding very similar to memantine. Its dosimetry shows that it is suitable for use in humans due to a low total effective dose compared to other PET radiotracers. Keywords (4 to 6): Dosimetry; [18F]FNM; Position emission tomography (PET); NMDA receptor; OLINDA
ACCEPTED MANUSCRIPT INTRODUCTION
The pathophysiological mechanisms associated with neurodegenerative diseases remain largely unknown. A process appears to be noticeably present in the phenomena of neurodegeneration: neurotoxicity induced by a massive influx of calcium following excessive activation of NMDA receptors (GluN) [1]. With regards to Alzheimer’s disease, for
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example, the amyloid peptide appears to induce an over-activation of NMDA receptors, resulting in their
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internalization [2]. Moreover, deregulation of calcium homeostasis affects the metabolism of amyloid precursor proteins [3], which leads to increased amyloid-β formation and contributes to the evolution of Alzheimer's disease. A
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recent study has established the role of selective NMDA extra-synaptic receptors in this pathology [4].
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With molecular imaging via positron emission tomography (PET) and radiotracers, it is now possible to detect and quantify these phenomena in vivo. We have recently developed a new radioligand, derived from memantine, which
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targets the open channel, GluN [5]. The [18F]FNM (Fluoroethylnormemantine) crosses the blood-brain barrier and its distribution within the brain has been found to correlate well with the location of GluN. First experiences (in vivo, in
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rats) using this product on animals demonstrated that its kinetics properties are suitable for PET/CT, due to an uptake
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in the brain that is constant after 40 minutes of injection [5].The highest uptake was found in the cortex and cerebellum, whilst the lowest was found in white matter. -6
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The promising characteristics of [18F]FNM (Affinity : IC50 = 6.1 10 M (compared with [3H]TCP) , logD : 1.93, ex-vivo specificity : brain distribution of [18F]-FNM closely matched that of NMDAr1 in immunohistochemistry
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study) make this tracer a good candidate for human use in order to better understand neurological and psychiatric
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diseases. The objective of this article is to perform biological evaluation of [ 18F]FNM and estimate human radiation dosimetry of this tracer using preclinical studies. This will permit us to better estimate allowable [18F]FNM activity for use in human PET imaging.
METHODS Radiopharmaceutical production [18F]FNM was synthesized according to a previously described method [5]. In brief, [18F]FNM is produced by nucleophilic substitution using 1-[N-(tert-butyloxy)carbamoyl]-3-(tosyl)ethyl-adamantane as the precursor on a Raytest® module]. After complete removal of the solvent by azeotropic drying, the precursor is added to the reaction
ACCEPTED MANUSCRIPT vial and is heated for 20 min at 125°C. The reaction mixture is then cooled and added to the hydrolysis solution and heated for 10 min at 110°C, causing hydrolysis of the BOC (tert-butoxycarbonyle) group. The reaction mixture is then neutralized by adding 6 N NaOH and 0.5 M trisodium citrate solutions. Pre-purification is achieved using a SepPak cartridge (waters C18 Plus). The lipophilic compound trapped in the cartridge is eluted with 2 mL of ethanol. HPLC purification is carried out in a semipreparative column (Cluzeau Info Labo Stability Basic C-18 CIL; 250 x 10
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mm, particle size 5 µm), with a mobile phase consisting of ethanol absolute /sodium acetate (0.1M) mixture (45/55;
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v/v). The [18F]FNM retention time was 15 min, with a flow rate of 2 ml/min. At the end of synthesis, volumetric
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activity was measured. For animal experimentation post-purification was performed in order to obtain a solution containing less than 10% alcohol. This solution, referred to as the mother solution, was then diluted in sodium
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chloride (0.9%) to obtain a volumetric activity of 200 MBq/ml.
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Subject/experimental animals This study was conducted under protocols approved by French animal Ethics Committee (n°2016021711398144). 16 Sprague Dawley female rats were used for this study 320.42 ± 23.28 g. The animals were housed in a climate-
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controlled room with a 12/12-hour light cycle. The subjects had free access to food and water during housing.
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Ex vivo biodistribution investigation Initially, all rats were anesthetized with an intraperitoneal injection of pentobarbital (55 mg/kg). After a single
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intravenous administration of [18F]FNM: 44 ± 11MBq, rats were sacrificed at 30 min (n=2); 60 min (n=3) and 90 min (n=2) post injection (p.i) with a lethal dose of pentobarbital (50 mg). Blood samples and organs of interest
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(whole brain, heart, lung, spleen, kidney, stomach and liver) were removed, blotted dry, and weighed. Radioactivity in each organ was measured on a Gamma wizard counter. The organ-to-blood ratio (activity of blood and organ were
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expressed in cpm/g) was calculated to avoid the need for decay correction and to improve reproducibility in case of injection issues and diffusion of [18F]FNM into the tail compartment. To explore a depressive effect of pentobarbital on brain uptake, the same experiment was performed with nonanesthetized rats. Rats were sacrificed by decapitation at 60 min (n= 3) after a tail-vein injection of [18F]FNM. To explore the depressive effect of the volatile anesthetic, isoflurane, on brain uptake, the same experiment was performed with isoflurane anesthesia with an induction of 4% and anesthesia during 60 min at 2%, the rats were sacrificed with a lethal amount of pentobarbital (n=3).
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In vitro plasma stability test Four samples of 500 µL fresh human plasma (prepared by centrifugation (15 min, 4000 g) at room temperature) were incubated with 296 kBq [18F]FNM at 37°C for 10 min, 20 min, 30 min, 1 hr and 2 hr. After incubation, the proteins
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were precipitated by the addition of 500 µL acetonitrile. Tubes were agitated for 5 min and centrifuged for 10 min at 4000 g. 20 µL of supernatant was then injected into an HPLC column (250*4.6 ProntoSil C18). The isocratic mobile
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phase was a mixture of acetonitrile and sodium acetate 0.1M (60/40) with a 3 mL/min flow rate. The retention time of the radioactive mother solution [18F]FNM in these conditions was 4-5 min. Each incubation process was repeated
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three times.
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Plasma protein binding Plasma protein binding of [18F]FNM was measured in plasma of heparinized blood samples. Plasma was prepared from fresh human blood by centrifugation (15 min, 4000 g) at room temperature and thereafter handled at 37°C. An
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800 µL sample of fresh human plasma was incubated at 37°C for 2 hr with 100 µL of a solution containing
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[18F]FNM (296 kBq). The plasma was mixed with 200 μL of 1-octanol, agitated for 30 s, and centrifuged for 10 min at 4115 g. The 1-octanol layer was then removed and counted. The amount of radioactivity in the 1-octanol was
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regarded as the lipoprotein-bound fraction of [18F]-FNM. The remaining plasma was then mixed with 200 μL acetonitrile, agitated for 30 s, and centrifuged as before. The acetonitrile layer was collected and counted. The
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amount of radioactivity in the acetonitrile fraction was regarded as the protein-bound fraction of [18F]-FNM. The remaining plasma was counted and the value obtained was regarded as the unbound or free fraction. Three samples
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were tested for each incubation time. In total, 12 samples were sampled. The percentage of each fraction was calculated using equation 1:
% 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑥 =
𝑐𝑝𝑚 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑥 𝑐𝑝𝑚 𝑠𝑢𝑚 ( 𝑠𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑠)
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In vivo metabolism Plasma (500 µL) and urine (500 µL) samples of 2 rats anaesthetized (pentobarbital), sacrificed at 60 min after injection, were removed. After centrifugation (5 min, 4000g) and protein precipitation in acetonitrile (800 µL), an
ACCEPTED MANUSCRIPT HPLC injection of supernatant (20 µL) in phenylhexyl luna phenomex column (4.6*150 mm) at 1.5 mL/min with a mobile phase consisting of sodium acetate 0.1M /acetonitrile (70/30, v/v) mixture was performed. The retention time of the radioactive mother solution [18F]FNM in those conditions was 3 min. Image data acquisition Sprague Dawley rats were anesthetized with 55mg/kg of pentobarbital and then a CT scan was performed.
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Intravenous injections 67.8 ± 10 MBq of [18F]FNM in the tail were performed at the beginning of PET acquisition.
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Acquisition was performed in list mode on PET/CT and lasted 70 min (image size 256*300, 2-mm FWHM Gaussian
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filter, 6 iterations, 16 subsets). The PET list mode data was used to create 16 3D-sinograms such as dynamic histograms (3*10 s; 5*30 s; 4*105 s; 2*600 s; 2*1200 s). All dynamic images were automatically corrected for
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radioactive decay during acquisition as per manufacturer software settings. Following the reconstruction, the CT images were spatially aligned to match the PET images. In addition to being reconstructed into a single image, the
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CT data was used for attenuation correction of PET images.
Processing of reconstructed images was performed with an in-house piece of software[6]. Organs were identified and
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3D volumes of interest (VOIs) were manually drawn on the CT in the center of each organ. All VOIs were
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transferred to dynamic PET images and the decay corrected mean time activity curves (TACs) were extracted for each target organ. Eight VOIs were selected: brain, heart, lung, spleen, kidney, stomach, liver and whole body.
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Residence time and absorbed dose calculation In total, three animals were used for dosimetric purposes. All data used for residence time estimations and TACs
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generation was non-decay corrected. For each rat, an average of three measurements of the same region of interest
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was done in order to obtain the values of activity as a function of time for each organ. All mathematical processing was carried out using Wolfram Mathematica[7]. A mathematical model was used to fit time-activity data. Measure time points were at t = 0.17, 0.33, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.75, 6.50, 8.25, 10.00, 20.00, 30.00, 50.00, 70.00 min. The mathematical model (Equation 2) takes into account the uptake and the clearance phases of the radiopharmaceutical for each organ [8]. 𝑓(𝑡) = 𝑎 ∗ (𝑒 −𝑏𝑡 − 𝑒 −𝑐𝑡 )
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ACCEPTED MANUSCRIPT For some organs, the fitting model did not match experimental data, this happens especially for heart, lung and brain. For these particular cases, fitting was carried out by segments, using two or three data points (and respectively a first or second-degree polynomial). In order to estimate the cumulated activity, integration was done by computing the area under the polynomials. After the last experimental data point, mono-exponential decay (considering only physical decay) was assumed and integration from this point to infinity was estimated. To take into account the
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contribution of the whole body, a remainder was estimated, this means for each rat summing all organs and subtract
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them to the whole body.
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Cumulated activity and residence time for each organ of each rat were calculated and the average of these results was estimated in order to extrapolate to humans. The cumulated activity for humans was calculated using mass scaling
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between mouse and human for whole body and organs. This was done according to equation 3 [8][9]:
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𝑚𝑅𝑎𝑡 𝑚𝐻𝑢𝑚𝑎𝑛 𝐴̃𝑂𝑟𝑔𝑎𝑛,𝐻𝑢𝑚𝑎𝑛 = ( ) ∗( ) ∗ 𝐴̃𝑂𝑟𝑔𝑎𝑛,𝑅𝑎𝑡 𝑚𝐻𝑢𝑚𝑎𝑛 𝑊𝐵 𝑚𝑅𝑎𝑡 𝑂𝑟𝑔
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Rat organ masses were obtained after sacrificing the rats, according to the protocol mentioned in the section, ex vivo biodistribution. Using information provided by ICRP-60[10] and ICRP-89 [11], whole body and organ masses for
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human males and females were chosen in order to calculate the absorbed doses for a selected group of organs using OLINDA/EXM V1.0 [12] and OLINDA/EXM V2.0 [13]. OLINDA/EXM V1.0 considers the information of the
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RESULTS Radiosynthesis
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dosimetric phantom model of ICRP-60 and OLINDA/EXM V2.0 from ICRP-89.
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7 Syntheses were successfully performed. The radiochemical yield was optimized and was 27 ± 7 %, with an average activity of [18F]FNM of 3940 ± 1600 MBq. The specific activity of [ 18F]FNM was always above 355 GBq/µmol. Ex vivo examination An ex vivo diagram (figure 1) confirms that the kidney is a significant clearance pathway. Signal from the lung was elevated but not very homogenous between animals. The brain-to-blood ratio was compared for pentobarbital anesthetized rats, isoflurane anesthetized rats and non-anesthetized rats at 60 min after injection of radiotracer (figure 2). There is a difference between non-anesthetized rats (ratio: 6.2), pentobarbital anesthetized rats (ratio was 1.67) and isoflurane rats (ratio was 2.7).
ACCEPTED MANUSCRIPT In vitro plasma stability test The [18F]FNM was stable in human plasma, regardless of the incubation time. No other peak than [18F]FNM was detected (figure 3). The peak of free fluoride on the solvent front was not observed. Using a Geiger counter, it was verified that there was no more activity attached to the HPLC column.
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Plasma protein binding [18F]FNM seemed to bind strongly to lipoprotein 42.81 ± 0.5%. This compound also bound a little with protein 4.16
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± 0.9%. This result is not surprising due to its lipophilic characteristic (logD= 1.93). Furthermore, due to the fact that this molecule is lipophilic, it will tend to preferentially bind to plasma lipoproteins. On average, the diffusible free
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fraction is 53%. It has been noted that there is little variability between the results obtained for three samples. Total
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plasma protein binding was 46.97%.
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In vivo metabolism 60 min after injection of [18F]FNM, no metabolite was distinguished in plasma samples of rats but a small amount of hydrophilic metabolite (18%) was observed in the urine. This is consistent with literature data of memantine.
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In vivo PET/CT imaging TACs show a rapid decrease in cardiac uptake this could be due to a rapid blood clearance (figure 4). There is a little
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uptake in spleen, liver, stomach and lung. The kidney seems to be the significant clearance pathway. Uptake in the lung is very fast and remains high. Uptake in the liver, spleen and stomach seems to be more gradual. Uptake by the
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retina and pituitaryPx ( schiffer atlas) was also observed for all animals (figure 5). Organ-to-heart ratios at 60 min with PET/CT data and ex vivo data were also compared. T test was performed on ratio (7 organs per rats) in order to
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compare the two methodologies (t =0.84; n=28 ratio). No difference was observed between all in vivo and all ex vivo
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ratio. Figure 5 demonstrates the same cross-sectional evolution of [18F]FNM biodistribution (LUT is the same for all
Absorbed dose estimation Table 1 shows the results for cumulated activities and residence times for each rat. For the majority of the organs, the cumulated activities and residence times are of the same order of magnitude. In the case of Rat 3, measurements of the stomach contents were not available and therefore not included in the average.
ACCEPTED MANUSCRIPT Table 2 shows parameters and results obtained after mass scaling; the differences in masses between human models and measured rat organs are between two or three orders of magnitude. It can be seen in this table that the organs with the highest residence times for the human models are the brain and the lungs, both for ICRP-60 and ICRP-89. From table 3, results for the two versions of OLINDA show the highest total absorbed dose were for spleen, liver,
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lungs and kidneys.
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OLINDA/EXM V2.0 generated results for effective dose according to ICRP-103 recommendations. Using this
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method, the mean effective doses were 4.65 x 10-3 mSv/MBq and 6.11 x 10-3 mSv/MBq for male and female models, respectively. This would correspond to effective doses of 1.63 mSv (male) and 2.14 mSv (female) for an
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administrated activity of 350 MBq. On the other hand, OLINDA 1 generated results for effective dose according to ICRP-60 recommendations, in this case the mean effective dose was 5.09 x 10-3 mSv/MBq for the adult male model,
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this would correspond to effective dose of 1.78 mSv, for the same administrated activity.
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DISCUSSION A dosimetric and biodistribution study, such as this one, has to be performed before human use of experimental
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radiotracer in order to estimate the absorbed doses potentially delivered to human organs, thereby assessing the safety of the diagnostic procedure [14].
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For peripheral organs there was an important uptake in kidney because of its role in radiotracer elimination. Moreover, literature data suggests that there are peripheral NMDA receptors in this organ. In the lung, large inter-
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individual variability in [18F]FNM uptake was observed (figure 4). In the pulmonary tract, there are a very large number of NMDA receptors that are heavily involved in the phenomena of fibrosis and edema; the pulmonary state
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of each different rat could explain variability found in the presented results. The variability of the lung and kidney uptake could also be due the variable amount of ethanol in injected solution because lung and kidney are organs excreting ethanol from the body [15].There are also NMDA receptors in the liver, which are involved in liver inflammation [16]. Finally, an important uptake was noticed in the retina that is in agreement with literature data [17]. Difference was observed between uptake in anesthetized and non-anaesthetized rats, showing that, globally, pentobarbital inhibits the excitatory nervous system. Brain to blood ratio results on non-anesthetized rats was
ACCEPTED MANUSCRIPT identical than data mentioned in Salabert et Al [18] in their in vivo biodistribution study and was 6.2, 0.35 % of injected dose/g. There is a difference between non-anesthetized rats, pentobarbital anesthetized rats (ratio was 1.67, 0.061% of injected dose/g) and isoflurane rats (ratio was 2.7) . There is a decrease of FNM uptake with anesthetized rats. Difference was found between the brain-to-blood ratio PET data (pentobarbital anesthetised rats) and ex vivo data (non-anesthetised rats). This seems to show that use of pentobarbital for euthanizing animals induces a decrease
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of [18F]FNM uptake in the brain. Study on awoken rats would be very useful [19]. It would be interesting to test this
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compound on non-anesthetized human, since these studies suggest depressive role of the anesthetic compound. The
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use of anesthesia in some of the rats used in this research could have caused an under-estimate of absorbed dose calculations in the brain. Anesthetic drugs selected in this study are drugs, which have no known affinity for the
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NMDA receptor (pentobarbital or isoflurane), in contrast to the most used anesthetic in rodents, ketamine (a wellknown antagonist of NMDAr and therefore would have had a direct impact on tracer uptake). However, despite the
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fact that they have no affinity for NMDAr, it has been shown in numerous articles ([20]–[22] that halogenated derivatives such as isoflurane inhibit NMDA activity. The mechanism of action does not seem to be elucidated.
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Ogata and all had measured a depression of 22-30% of NMDAr using isoflurane on xenopus oocytes, which really does not reflect a whole animal. An ex vivo study comparing the activity of the organs with tracer of activated
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NMDAr on anaesthetized or non-anesthetized animals could provide evidence of this decrease. This has been done in
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this article only for the brain and it is possible that this phenomenon of NMDA inhibition may also have affected the peripheral receptors and thus the estimated whole body dose.
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Concerning biological properties, it has been shown that this tracer is very stable in human plasma, and it is
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minimally metabolized (in vivo). This is in concordance with memantine’s stability data [23] [24]. We determined free [18F]FNM fraction because it is the only fraction able to diffuse into tissues. To be able to carry out quantification studies, it is important to have free fraction plasma data. This may not be an issue as long as the free fraction is constant throughout the experiment, therefore its correction can be disregarded, however, it becomes a serious issue, when the free fraction of the ligand in plasma is subject to change in pathological conditions [25]. [18F]FNM lipoprotein plasma binding (47%) is very close to memantine’s 45% (ANSM data for market authorization, http://agence-prd.ansm.sante.fr/php/ecodex/rcp/R0218846.htm). The large free fraction (53 %) allows
ACCEPTED MANUSCRIPT a rapid elimination of the compound because it is more readily accessible to renal filtration. This may explain the low residence time of [18F]FNM in the body. Dynamic PET imaging and ex vivo organ counting were performed on rats. Animal pharmacokinetics were extrapolated to human’s models, in order to estimate dosimetry of the compound on a reference anthropomorphic model. Absorbed dose per injected activity (Gy/Bq) was computed to calculate the absorbed doses potentially
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delivered to human organs, thereby assessing the safety of the diagnostic procedure, this will enable a better selection
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of [18F]FNM activity for use in human PET imaging.
For rats, the kidney has the highest cumulated activity and residence time, implying the highest absorbed dose.
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Conversely, for brain the cumulated activity and residence time were amongst the lowest of all considered organs, showing low irradiation, and therefore low absorbed dose, for this organ.
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Recently, Boschi et al. 2016 [26] conducted a pre-clinical study using
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F-PSMA-11, for dosimetric analysis and
calculations using NUKFIT and OLINDA/EXM V1.0. The effective dose results in our study are in the same order
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of magnitude than theirs. This may be due to the fact that in this study, the whole body of the rat was measured. On
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the other hand, their results for humans were based on ICRP-60 recommendations, whereas we tried to implement ICRP-103 recommendations that propose a separate assessment of the absorbed dose for males and females.
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Additionally results for ICRP were estimated. Since we had no specific assessment of male/female pharmacokinetics from animal experiments, the gender specific estimate was obtained during the extrapolation phase, by scaling
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differently (according to different organ masses) the same pharmacokinetics.
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In the case of the human models (ICRP-60 and ICRP-89), differences in the residence times for each organ can be explained by the differences in relative organ masses (table 2) between models. [18F]FNM has very fast pharmacokinetics, thereby delivering an irradiation of 6.11 µSv/MBq and 4.65 µSv/MBq for females and males, respectively, considering results of ICRP-103 and 5.09 µSv/MBq for male considering results of ICRP-60. This is lower when compared to other PET tracers ( 18F tracers) that are used in neurology. For example, [18F] DPA714 is around 17.2 µSv/MBq [27], [18F]FEDDA is around 36 µSv/MBq [28] and the [18F] amyloid tracer, flutemetamol, is around 25-30 µSv/MBq [29]. Some of the potential explanations were already discussed above. Due
ACCEPTED MANUSCRIPT to a high free fraction; [18F]FNM is quickly eliminate by renal pathway that allow to have a low residence time of this compound in the body and a low dosimetry impact for the patient.
CONCLUSIONS
This study shows that [18F]FNM present good biological properties for imaging :stability in vitro in human plasma,
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weak metabolism in rat , plasma protein binding close to memantine. Its dosimetry shows that it is suitable for use in
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neurology due to an estimated effective dose of around 6.11µSv/MBq and 4.65µSv/MBq for female and male human
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dosimetric models, respectively considering ICRP-103. These values are lower when compared with other PET tracers for the same purposes. Anesthetic seems to play a major role in brain depression and can decrease NMDAr
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activity therefore the use of anesthesia in animal studies may include a bias in uptake measurements, and so in dosimetry estimation. Human studies using [18F]FNM seem to be safe in terms of dosimetry and this compound
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could be used to gain a better understanding of the glutamate excitatory system and NMDA receptor implication in
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neurological and psychiatric diseases.
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COMPLIANCE WITH ETHICAL STANDARDS
All applicable international guidelines for the care and use of animals were followed. This protocol was approved by
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the local competent authorities (authorization no 2016021711398144). All procedures performed in studies involving
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conducted.
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animals were in accordance with the ethical standards of the institution or practice at which the studies were
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REFERENCES [1] C. M. Wroge, J. Hogins, L. Eisenman, S. Mennerick, "Synaptic NMDA receptors mediate hypoxic excitotoxic death", J. Neurosci., Vol. 32, No 19, p. 6732-6742, May 2012. [2] W. Danysz, C. G. Parsons, "Alzheimer’s disease, β-amyloid, glutamate, NMDA receptors and memantine – searching for the connections", Br. J. Pharmacol., Vol. 167, No 2, p. 324-352, Sept. 2012. [3] K. Bordji, J. Becerril-Ortega, A. Buisson, "Synapses, NMDA receptor activity and neuronal Aβ production in Alzheimer’s disease", Rev. Neurosci., Vol. 22, No 3, Jan. 2011. [4] K. Bordji, J. Becerril-Ortega, O. Nicole, A. Buisson, "Activation of Extrasynaptic, But Not Synaptic, NMDA Receptors Modifies Amyloid Precursor Protein Expression Pattern and Increases Amyloid- Production", J. Neurosci., Vol. 30, No 47, p. 15927-15942, Nov. 2010. [5] A. S. Salabert et al., "Radiolabeling of [(18)F]-fluoroethylnormemantine and initial in vivo evaluation of this innovative PET tracer for imaging the PCP sites of NMDA receptors", Nucl. Med. Biol., Vol. 42, No 8, p. 643653, Aug. 2015. [6] F. Tensaouti, J. A. Lotterie, "Sysiphe-Neuroimaging software toolbox", in European Society for Magnetic Resonance Medicine and Biology 2008 Congress Oct, 2008, p. 2–4. [7] Wolfram Research, Inc., Mathematica, version 10.4. Champaign, Illinois, 2015. [8] B. J. McParland, Nuclear Medicine Radiation Dosimetry: Advanced Theoretical Principles. Springer Science & Business Media, 2010. [9] T. Tolvanen et al., "Biodistribution and radiation dosimetry of [(11)C]choline: a comparison between rat and human data", Eur. J. Nucl. Med. Mol. Imaging, Vol. 37, No 5, p. 874-883, May 2010. [10] ICRP, "1990 Recommendations of the International Commission on Radiological Protection. ICRP Publication 60" ICRP21(1-3), ICRP-1991. [11] "Basic anatomical and physiological data for use in radiological protection: reference values. A report of ageand gender-related differences in the anatomical and physiological characteristics of reference individuals. ICRP Publication 89", Ann. ICRP, Vol. 32, No 3-4, p. 5-265, 2002. [12] M. G. Stabin, R. B. Sparks, E. Crowe, "OLINDA/EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine", J. Nucl. Med., Vol. 46, No 6, p. 1023-1027, June 2005. [13] M. G. Stabin, X. G. Xu, M. A. Emmons, W. P. Segars, C. Shi, M. J. Fernald, "RADAR reference adult, pediatric, and pregnant female phantom series for internal and external dosimetry", J. Nucl. Med., Vol. 53, No 11, p. 1807-1813, Nov. 2012. [14] C. C. Constantinescu, A. Garcia, M. R. Mirbolooki, M. L. Pan, J. Mukherjee, "Evaluation of [18F]Nifene biodistribution and dosimetry based on whole-body PET imaging of mice", Nucl. Med. Biol., Vol. 40, No 2, p. 289-294, Feb. 2013. [15] H. Kalant, "Absorption, Diffusion, Distribution, and Elimination of Ethanol: Effects on Biological Membranes", in The Biology of Alcoholism, Springer, Boston, MA, 1971, p. 1-62. [16] J. Du, X. H. Li, Y. J. Li, "Glutamate in peripheral organs: Biology and pharmacology", Eur. J. Pharmacol., Vol. 784, p. 42-48, Aug. 2016. [17] A. A. M. Abdel-Hamid, A. E. D. L. Firgany, E. M. T. Ali, "Effect of memantine: A NMDA receptor blocker, on ethambutol-induced retinal injury", Ann. Anat., Vol. 204, p. 86-92, Mar. 2016. [18] A. S. Salabert et al., "Radiolabeling of [18F]-fluoroethylnormemantine and initial in vivo evaluation of this innovative PET tracer for imaging the PCP sites of NMDA receptors", Nucl. Med. Biol., Vol. 42, No 8, p. 643653, Aug. 2015. [19] D. Schulz et al., "Simultaneous assessment of rodent behavior and neurochemistry using a miniature positron emission tomograph", Nat. Methods, Vol. 8, No 4, p. 347-352, Apr. 2011. [20] M. W. Hollmann, H. T. Liu, C. W. Hoenemann, W. H. Liu, M. E. Durieux, "Modulation of Nmda receptor function by ketamine and magnesium. Part Ii: interactions with volatile anesthetics", Anesth. Analg., Vol. 92, No 5, p. 1182-1191, May 2001. [21] J. Ogata, M. Shiraishi, T. Namba, C. T. Smothers, J. J. Woodward, R. A. Harris, "Effects of anesthetics on mutant N-methyl-D-aspartate receptors expressed in Xenopus oocytes", J. Pharmacol. Exp. Ther., Vol. 318, No 1, p. 434-443, July. 2006. [22] T. Yamakura, R. A. Harris, "Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol", Anesthesiology, Vol. 93, No 4, p. 1095-1101, Oct. 2000. [23] M. G. Beconi et al., "Pharmacokinetics of memantine in rats and mice", PLoS Curr., Vol. 3, p. RRN1291, 2011. [24] S. Lam, C. Smith, I. H. Gomolin, "Memantine Standard Tablet and Extended-Release Dosing Considerations: A Pharmacokinetic Analysis", J. Am. Geriatr. Soc., Vol. 63, No 2, p. 383-384, Feb. 2015.
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[25] F. E. Turkheimer et al., "The methodology of TSPO imaging with positron emission tomography", Biochem. Soc. Trans., Vol. 43, No 4, p. 586-592, Aug. 2015. [26] S. Boschi et al., "Synthesis and preclinical evaluation of an Al(18)F radiofluorinated GLU-UREALYS(AHX)-HBED-CC PSMA ligand", Eur. J. Nucl. Med. Mol. Imaging, Vol. 43, No 12, p. 2122-2130, Nov. 2016. [27] N. Arlicot et al., "Initial evaluation in healthy humans of [18F]DPA-714, a potential PET biomarker for neuroinflammation", Nucl. Med. Biol., Vol. 39, No 4, p. 570-578, May 2012. [28] A. Takano et al., "Biodistribution and radiation dosimetry of the 18 kDa translocator protein (TSPO) radioligand [18F]FEDAA1106: a human whole-body PET study", Eur. J. Nucl. Med. Mol. Imaging, Vol. 38, No 11, p. 2058-2065, Nov. 2011. [29] M. Koole et al., "Whole-Body Biodistribution and Radiation Dosimetry of 18F-GE067: A Radioligand for In Vivo Brain Amyloid Imaging", J. Nucl. Med., Vol. 50, No 5, p. 818-822, May 2009.
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40.0 rats 30 min 35.0 rats 60 min 30.0
rats 90 min
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25.0 20.0 15.0
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(Activity/g)organ /(Activity/g)blood
45.0
10.0
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5.0 spleen
kidney
heart
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Liver
brain
stomach
lung
Organs
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Figure 1. Ex vivo biodistribution of [18F]FNM 30 min after injection (n=2); 60 min after injection (n=3) and 90 min after injection (n=3). Ordinate value is the ratio of activity/g of each organ by activity/g of blood sample. (error bars = SD)
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(cpm/g)brain /(cpm/g)blood
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1 0
Pentobrabital
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No anesthesic
Isoflurane
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Figure 2. Ordinate value is the Brain to blood ratio of [ 18F]FNM uptake using different kind of anesthesia. (error bars = SD), n= 3 for all each experiment.
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Figure 3: Figure 3. Metabolization study. HPLC diagrams (detection gamma ) ABCD show [ F]FNM peak after
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incubation in human plasma( respectively during 10,20,30,120 min). Figure E represent HPLC diagram (gamma detection) of rat urinary fraction ( 60 min after injection of [18F]FNM ) . Figure E represent HPLC diagram ( gamma detection) of rat plasma ( 60 min after injection of [18F]FNM ) .
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Figure 4. Left: Sagittal whole body PET-CT of a sprague dawley rat. Right-up: Percentage of injected activity for each organ after an intravenous injection of [18F]FNM, for rat 1 (Ao= 61.63 MBq), with the remainder. Right-down: Percentage of injected activity for each organ after an intravenous injection of [ 18F]FNM, for rat 1, without the remainder.
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Figure 5. Left: Whole body biodistribution of [ F]FNM after injection of 60 MBq in tail vein.
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Rigth: Arrow shows uptake in pituitary PX ( schiffer atlas). .
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Rat 2, Ao = 18.00 MBq
Rat 3, Ao = 111.00 MBq
Organs
A (Bq*min)
T (h)
A (Bq*min)
T (h)
A (Bq*min)
T (h)
T (h)
Brain
2.09E+07
5.64E-03
4.01E+06
3.71E-03
6.30E+07
9.46E-03
6.27E-03
Heart
1.62E+07
4.37E-03
3.50E+06
3.24E-03
4.57E+07
Kidney
1.67E+08
4.51E-02
4.54E+07
4.20E-02
3.02E+08
4.54E-02
4.42E-02
Liver
1.68E+08
4.55E-02
6.35E+07
5.88E-02
3.80E+08
5.71E-02
5.38E-02
Lung
3.90E+07
1.06E-02
9.71E+06
8.99E-03
1.09E+08
1.64E-02
1.20E-02
Spleen
1.48E+07
3.99E-03
1.18E+06
1.09E-03
7.38E+07
1.11E-02
5.39E-03
Stomach
2.25E+07
6.09E-03
8.89E+06
8.23E-03
-
-
7.16E-03
Whole body
3.05E+09
8.26E-01
4.40E+08
4.07E-01
7.25E+09
1.09E+00
7.74E-01
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Rat 1, Ao = 61.63 MBq
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Table 1. Summary of results for cumulative activity and residence times for each rat.
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6.87E-03
ICRP-89 Mfemale (g)
Brain
1450
1300
Heart
510
370
Kidney
310
Liver
1800
Lung
1200
Spleen Stomach
ICRP-60
Mrat (g)
Tmale (h)
Tfemale (h)
Thuman (h)
1420
1.99
2.07E-02
2.25E-02
2.00E-02
316
1.22
9.12E-03
8.05E-03
5.60E-03
275
299
2.61
2.37E-02
2.56E-02
2.27E-02
1400
1910
8.20
5.34E-02
5.05E-02
5.61E-02
950
1000
1.65
3.94E-02
3.80E-02
3.25E-02
150
130
183
0.65
5.62E-03
5.93E-03
6.79E-03
250
230
158
1.34
6.04E-03
6.76E-03
3.78E-03
73000
60000
73700
330
7.74E-01
7.74E-01
7.74E-01
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Total Body
ICRP-89
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ICRP-60
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Table 2. Masses values used for mass scaling and male/female residence times
Average
4.83E-03
ACCEPTED MANUSCRIPT Table 3. Absorbed dose and effective dose coefficients results for adult male from Olinda 1, male and female phantoms from Olinda 2 Male – OLINDA 1
Male – OLINDA 2
Target organ
Total (mGy/MBq)
Adrenals
5.30E-03
1.33E-05
Adrenals
6.80E-03
6.28E-05
8.17E-03
7.54E-05
Brain
4.19E-03
1.05E-05
Brain
4.22E-03
4.22E-05
5.16E-03
5.16E-05
Breasts
3.41E-03
1.71E-04
Breast
-
-
4.25E-03
5.10E-04
Gallbladder Wall
5.59E-03
-
4.84E-03
1.94E-04
5.56E-03
2.22E-04
LLI Wall
4.57E-03
5.49E-04
Eyes
3.52E-03
0.00E00
4.35E-03
0.00E00
Small Intetine
4.84E-03
1.21E-05
Gallbladder Wall
6.07E-03
5.61E-05
6.69E-03
6.17E-05
Stomach Wall
5.83E-03
7.00E-04
Left colon
2.44E-04
6.09E-03
2.95E-04
4.81E-03
1.20E-05
Small Intestine
4.56E-05
5.59E-03
5.16E-05
5.66E-03
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Stomach Wall
7.19E-03
8.63E-04
8.69E-03
1.04E-03
Kidneys
1.64E-02
4.10E-05
Right colon
5.03E-03
2.44E-04
6.02E-03
2.92E-04
Liver
8.78E-03
4.39E-04
Rectum
4.62E-03
1.06E-04
5.62E-03
1.29E-04
Lungs
7.72E-03
9.27E-04
Heart Wall
6.66E-03
6.15E-05
8.00E-03
7.38E-05
Muscle
3.94E-03
9.86E-06
Kidneys
1.67E-02
1.54E-04
2.01E-02
1.86E-04
Ovaries
4.74E-03
9.49E-04
Liver
9.04E-03
3.61E-04
1.09E-02
4.37E-04
Pancreas
5.45E-03
1.36E-05
Lungs
7.87E-03
9.45E-04
9.58E-03
1.15E-03
Red Marrow
3.89E-03
4.66E-04
Ovaries
-
-
5.72E-03
2.29E-04
Osteogenic Cells
6.08E-03
6.08E-05
Pancreas
5.51E-03
5.08E-05
6.93E-03
6.39E-05
Skin
3.09E-03
3.09E-05
Prostate
4.60E-03
2.12E-05
-
-
9.59E-03
2.40E-04
Salivary Glands
4.07E-03
4.07E-05
4.63E-03
4.63E-05
3.76E-03
-
Red Marrow
4.01E-03
4.82E-04
4.89E-03
5.87E-04
4.19E-03
1.05E-05
Osteogenic Cells
3.56E-03
3.56E-05
4.19E-03
4.19E-05
Thyroid
4.02E-03
2.01E-04
Spleen
9.63E-03
8.89E-05
1.20E-02
1.10E-04
Urinary Bladder Wall
4.50E-03
2.25E-04
Testes
3.78E-03
1.51E-04
-
-
Uterus
4.78E-03
1.20E-05
Thymus
4.56E-03
4.21E-05
5.78E-03
5.34E-05
Total Body
4.28E-03
-
Thyroid
4.33E-03
1.73E-04
4.89E-03
1.96E-04
Spleen Testes Thymus
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5.03E-03
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4.94E-03
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Esophagus
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Total ICRP-103 Total ICRP-103 (mGy/MBq) (mSv/MBq) (mGy/MBq) (mSv/MBq)
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Heart Wall
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ULI Wall
Effective Target organ Dose (mSv/MBq)
Female– OLINDA 2
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-
5.09E-03
Urinary Bladder Wall
4.54E-03
1.82E-04
4.66E-03
1.86E-04
-
-
5.67E-03
2.62E-05
Total Body
4.23E-03
-
5.30E-03
-
Total effective dose (mSv/MBq)
-
4.65E-03
-
6.11E-03
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Uterus
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Total effective dose (mSv/MBq)
Anne-Sophie Salabert, Erick Mora-Ramirez, Marie Beaurain, Mathieu Alonso, Charlotte Fontan, Hafid Belhadj Tahar, Marie Laure Boizeau, M. Tafani, Manuel Bardiès, Pierre Payoux PII: DOI: Reference:
S0969-8051(17)30363-3 https://doi.org/10.1016/j.nucmedbio.2017.12.003 NMB 7986
To appear in: Received date: Revised date: Accepted date:
25 October 2017 5 December 2017 17 December 2017
Please cite this article as: Anne-Sophie Salabert, Erick Mora-Ramirez, Marie Beaurain, Mathieu Alonso, Charlotte Fontan, Hafid Belhadj Tahar, Marie Laure Boizeau, M. Tafani, Manuel Bardiès, Pierre Payoux , Evaluation of [18F]FNM biodistribution and dosimetry based on whole-body PET imaging of rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Nmb(2017), https://doi.org/10.1016/j.nucmedbio.2017.12.003
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ACCEPTED MANUSCRIPT Evaluation of [18F]FNM biodistribution and dosimetry based on whole-body PET imaging of rats Salabert A S 1,2 ,Mora-Ramirez E3,5,6, Beaurain M 1,2, Alonso M 2, Fontan C2, Belhadj Tahar H 7 , Boizeau
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Marie Laure 8 , Tafani , M 1,2, Bardiès M 3,5, Payoux P1,4
ToNIC, Toulouse NeuroImaging Center, Université de Toulouse, Inserm, UPS, France
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University Hospital, Radiopharmacy Unit, Toulouse, France
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Inserm, UMR1037 CRCT, F-31000 Toulouse, France
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University Hospital, Nuclear Medicine Unit, Toulouse, France
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Université Toulouse III-Paul Sabatier, UMR1037 CRCT, F-31000 Toulouse, France
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Universidad de Costa Rica, CICANUM-Escuela de Física, San José, Costa Rica
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Research and Expertise Group, French Association for the Promotion of Medical Research (AFPREMED), Toulouse, France 8
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Advanced Technology Institute in Life Sciences (ITAV), Centre National de la Recherche Scientifique-Université Paul Sabatier Toulouse III (CNRS-UPS), Université Paul Sabatier Toulouse III (UPS), Université de ToulouseToulouse, France.
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Corresponding author: Corresponding author: Anne-Sophie Salabert UMR 1214 Inserm TONIC (Toulouse Neuro-imaging Center) Université Toulouse III - Paul Sabatier CHU PURPAN - Pavillon BAUDOT Place du Dr Joseph Baylac 31024 TOULOUSE CEDEX 3 France Tel: 05 62 74 61 66
Email addresses: Anne-Sophie Salabert: [email protected]; Mora-Ramirez Erick : [email protected]; Marie Beaurain : [email protected], Mathieu Alonso: [email protected]; Charlotte Fontan : [email protected]; Hafid Belhadj-Tahar : [email protected] ; Marie Laure Boizeau : [email protected]; Manuel Bardiès : [email protected]; [email protected]; Pierre Payoux: [email protected].
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENT The authors would like to thank all members of the PET Center and CREFRE noninvasive exploration ( Inserm UMS 006 ) for their availability. This work has been supported in part by a grant from the French National Agency for Research called “Investissements d’Avenir” n°ANR-11-LABX-0018-01.
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ABSTRACT
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Introduction: The aim of this work was to study the biodistribution, metabolism and radiation dosimetry of rats injected with [18F]FNM using PET/CT images. This novel radiotracer targeting NMDA receptor has potential for
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investigation for neurological and psychiatric diseases.
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Methods: Free fraction and stability in fresh human plasma were determined in vitro. PET/CT was performed on anesthetized rats. Organs were identified and 3D volumes of interest (VOIs) were manually drawn on the CT in the
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center of each organ. Time activity curves (TACs) were created with these VOIs, enabling the calculation of residence times. To confirm these values, ex vivo measurements of organs were performed. Plasma and urine were
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also collected to study in vivo metabolism. Data was extrapolated to humans, effective doses were estimated using ICRP-60 and ICRP-89 dosimetric models and absorbed doses were estimated using OLINDA/EXM V1.0 and
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OLINDA/EXM V2.0 (which use weighting factors from ICRP-103 to do the calculations).
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Results: The [18F]FNM was stable in human plasma and the diffusible free fraction was 53%. As with memantine, this tracer is poorly metabolized in vivo. Ex vivo distributions validated PET/CT data as well as demonstrating a
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decrease of radiotracer uptake in the brain due to anesthesia. Total effective dose was around 6.11µSv/MBq and
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4.65µSv/MBq for female and male human dosimetric models, respectively. Conclusions: This study shows that the presented compound exhibits stability in plasma and plasma protein binding very similar to memantine. Its dosimetry shows that it is suitable for use in humans due to a low total effective dose compared to other PET radiotracers. Keywords (4 to 6): Dosimetry; [18F]FNM; Position emission tomography (PET); NMDA receptor; OLINDA
ACCEPTED MANUSCRIPT INTRODUCTION
The pathophysiological mechanisms associated with neurodegenerative diseases remain largely unknown. A process appears to be noticeably present in the phenomena of neurodegeneration: neurotoxicity induced by a massive influx of calcium following excessive activation of NMDA receptors (GluN) [1]. With regards to Alzheimer’s disease, for
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example, the amyloid peptide appears to induce an over-activation of NMDA receptors, resulting in their
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internalization [2]. Moreover, deregulation of calcium homeostasis affects the metabolism of amyloid precursor proteins [3], which leads to increased amyloid-β formation and contributes to the evolution of Alzheimer's disease. A
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recent study has established the role of selective NMDA extra-synaptic receptors in this pathology [4].
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With molecular imaging via positron emission tomography (PET) and radiotracers, it is now possible to detect and quantify these phenomena in vivo. We have recently developed a new radioligand, derived from memantine, which
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targets the open channel, GluN [5]. The [18F]FNM (Fluoroethylnormemantine) crosses the blood-brain barrier and its distribution within the brain has been found to correlate well with the location of GluN. First experiences (in vivo, in
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rats) using this product on animals demonstrated that its kinetics properties are suitable for PET/CT, due to an uptake
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in the brain that is constant after 40 minutes of injection [5].The highest uptake was found in the cortex and cerebellum, whilst the lowest was found in white matter. -6
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The promising characteristics of [18F]FNM (Affinity : IC50 = 6.1 10 M (compared with [3H]TCP) , logD : 1.93, ex-vivo specificity : brain distribution of [18F]-FNM closely matched that of NMDAr1 in immunohistochemistry
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study) make this tracer a good candidate for human use in order to better understand neurological and psychiatric
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diseases. The objective of this article is to perform biological evaluation of [ 18F]FNM and estimate human radiation dosimetry of this tracer using preclinical studies. This will permit us to better estimate allowable [18F]FNM activity for use in human PET imaging.
METHODS Radiopharmaceutical production [18F]FNM was synthesized according to a previously described method [5]. In brief, [18F]FNM is produced by nucleophilic substitution using 1-[N-(tert-butyloxy)carbamoyl]-3-(tosyl)ethyl-adamantane as the precursor on a Raytest® module]. After complete removal of the solvent by azeotropic drying, the precursor is added to the reaction
ACCEPTED MANUSCRIPT vial and is heated for 20 min at 125°C. The reaction mixture is then cooled and added to the hydrolysis solution and heated for 10 min at 110°C, causing hydrolysis of the BOC (tert-butoxycarbonyle) group. The reaction mixture is then neutralized by adding 6 N NaOH and 0.5 M trisodium citrate solutions. Pre-purification is achieved using a SepPak cartridge (waters C18 Plus). The lipophilic compound trapped in the cartridge is eluted with 2 mL of ethanol. HPLC purification is carried out in a semipreparative column (Cluzeau Info Labo Stability Basic C-18 CIL; 250 x 10
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mm, particle size 5 µm), with a mobile phase consisting of ethanol absolute /sodium acetate (0.1M) mixture (45/55;
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v/v). The [18F]FNM retention time was 15 min, with a flow rate of 2 ml/min. At the end of synthesis, volumetric
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activity was measured. For animal experimentation post-purification was performed in order to obtain a solution containing less than 10% alcohol. This solution, referred to as the mother solution, was then diluted in sodium
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chloride (0.9%) to obtain a volumetric activity of 200 MBq/ml.
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Subject/experimental animals This study was conducted under protocols approved by French animal Ethics Committee (n°2016021711398144). 16 Sprague Dawley female rats were used for this study 320.42 ± 23.28 g. The animals were housed in a climate-
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controlled room with a 12/12-hour light cycle. The subjects had free access to food and water during housing.
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Ex vivo biodistribution investigation Initially, all rats were anesthetized with an intraperitoneal injection of pentobarbital (55 mg/kg). After a single
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intravenous administration of [18F]FNM: 44 ± 11MBq, rats were sacrificed at 30 min (n=2); 60 min (n=3) and 90 min (n=2) post injection (p.i) with a lethal dose of pentobarbital (50 mg). Blood samples and organs of interest
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(whole brain, heart, lung, spleen, kidney, stomach and liver) were removed, blotted dry, and weighed. Radioactivity in each organ was measured on a Gamma wizard counter. The organ-to-blood ratio (activity of blood and organ were
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expressed in cpm/g) was calculated to avoid the need for decay correction and to improve reproducibility in case of injection issues and diffusion of [18F]FNM into the tail compartment. To explore a depressive effect of pentobarbital on brain uptake, the same experiment was performed with nonanesthetized rats. Rats were sacrificed by decapitation at 60 min (n= 3) after a tail-vein injection of [18F]FNM. To explore the depressive effect of the volatile anesthetic, isoflurane, on brain uptake, the same experiment was performed with isoflurane anesthesia with an induction of 4% and anesthesia during 60 min at 2%, the rats were sacrificed with a lethal amount of pentobarbital (n=3).
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In vitro plasma stability test Four samples of 500 µL fresh human plasma (prepared by centrifugation (15 min, 4000 g) at room temperature) were incubated with 296 kBq [18F]FNM at 37°C for 10 min, 20 min, 30 min, 1 hr and 2 hr. After incubation, the proteins
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were precipitated by the addition of 500 µL acetonitrile. Tubes were agitated for 5 min and centrifuged for 10 min at 4000 g. 20 µL of supernatant was then injected into an HPLC column (250*4.6 ProntoSil C18). The isocratic mobile
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phase was a mixture of acetonitrile and sodium acetate 0.1M (60/40) with a 3 mL/min flow rate. The retention time of the radioactive mother solution [18F]FNM in these conditions was 4-5 min. Each incubation process was repeated
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three times.
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Plasma protein binding Plasma protein binding of [18F]FNM was measured in plasma of heparinized blood samples. Plasma was prepared from fresh human blood by centrifugation (15 min, 4000 g) at room temperature and thereafter handled at 37°C. An
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800 µL sample of fresh human plasma was incubated at 37°C for 2 hr with 100 µL of a solution containing
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[18F]FNM (296 kBq). The plasma was mixed with 200 μL of 1-octanol, agitated for 30 s, and centrifuged for 10 min at 4115 g. The 1-octanol layer was then removed and counted. The amount of radioactivity in the 1-octanol was
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regarded as the lipoprotein-bound fraction of [18F]-FNM. The remaining plasma was then mixed with 200 μL acetonitrile, agitated for 30 s, and centrifuged as before. The acetonitrile layer was collected and counted. The
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amount of radioactivity in the acetonitrile fraction was regarded as the protein-bound fraction of [18F]-FNM. The remaining plasma was counted and the value obtained was regarded as the unbound or free fraction. Three samples
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were tested for each incubation time. In total, 12 samples were sampled. The percentage of each fraction was calculated using equation 1:
% 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑥 =
𝑐𝑝𝑚 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑥 𝑐𝑝𝑚 𝑠𝑢𝑚 ( 𝑠𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑠)
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In vivo metabolism Plasma (500 µL) and urine (500 µL) samples of 2 rats anaesthetized (pentobarbital), sacrificed at 60 min after injection, were removed. After centrifugation (5 min, 4000g) and protein precipitation in acetonitrile (800 µL), an
ACCEPTED MANUSCRIPT HPLC injection of supernatant (20 µL) in phenylhexyl luna phenomex column (4.6*150 mm) at 1.5 mL/min with a mobile phase consisting of sodium acetate 0.1M /acetonitrile (70/30, v/v) mixture was performed. The retention time of the radioactive mother solution [18F]FNM in those conditions was 3 min. Image data acquisition Sprague Dawley rats were anesthetized with 55mg/kg of pentobarbital and then a CT scan was performed.
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Intravenous injections 67.8 ± 10 MBq of [18F]FNM in the tail were performed at the beginning of PET acquisition.
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Acquisition was performed in list mode on PET/CT and lasted 70 min (image size 256*300, 2-mm FWHM Gaussian
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filter, 6 iterations, 16 subsets). The PET list mode data was used to create 16 3D-sinograms such as dynamic histograms (3*10 s; 5*30 s; 4*105 s; 2*600 s; 2*1200 s). All dynamic images were automatically corrected for
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radioactive decay during acquisition as per manufacturer software settings. Following the reconstruction, the CT images were spatially aligned to match the PET images. In addition to being reconstructed into a single image, the
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CT data was used for attenuation correction of PET images.
Processing of reconstructed images was performed with an in-house piece of software[6]. Organs were identified and
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3D volumes of interest (VOIs) were manually drawn on the CT in the center of each organ. All VOIs were
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transferred to dynamic PET images and the decay corrected mean time activity curves (TACs) were extracted for each target organ. Eight VOIs were selected: brain, heart, lung, spleen, kidney, stomach, liver and whole body.
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Residence time and absorbed dose calculation In total, three animals were used for dosimetric purposes. All data used for residence time estimations and TACs
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generation was non-decay corrected. For each rat, an average of three measurements of the same region of interest
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was done in order to obtain the values of activity as a function of time for each organ. All mathematical processing was carried out using Wolfram Mathematica[7]. A mathematical model was used to fit time-activity data. Measure time points were at t = 0.17, 0.33, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.75, 6.50, 8.25, 10.00, 20.00, 30.00, 50.00, 70.00 min. The mathematical model (Equation 2) takes into account the uptake and the clearance phases of the radiopharmaceutical for each organ [8]. 𝑓(𝑡) = 𝑎 ∗ (𝑒 −𝑏𝑡 − 𝑒 −𝑐𝑡 )
2
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contribution of the whole body, a remainder was estimated, this means for each rat summing all organs and subtract
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them to the whole body.
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Cumulated activity and residence time for each organ of each rat were calculated and the average of these results was estimated in order to extrapolate to humans. The cumulated activity for humans was calculated using mass scaling
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between mouse and human for whole body and organs. This was done according to equation 3 [8][9]:
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𝑚𝑅𝑎𝑡 𝑚𝐻𝑢𝑚𝑎𝑛 𝐴̃𝑂𝑟𝑔𝑎𝑛,𝐻𝑢𝑚𝑎𝑛 = ( ) ∗( ) ∗ 𝐴̃𝑂𝑟𝑔𝑎𝑛,𝑅𝑎𝑡 𝑚𝐻𝑢𝑚𝑎𝑛 𝑊𝐵 𝑚𝑅𝑎𝑡 𝑂𝑟𝑔
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Rat organ masses were obtained after sacrificing the rats, according to the protocol mentioned in the section, ex vivo biodistribution. Using information provided by ICRP-60[10] and ICRP-89 [11], whole body and organ masses for
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human males and females were chosen in order to calculate the absorbed doses for a selected group of organs using OLINDA/EXM V1.0 [12] and OLINDA/EXM V2.0 [13]. OLINDA/EXM V1.0 considers the information of the
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RESULTS Radiosynthesis
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dosimetric phantom model of ICRP-60 and OLINDA/EXM V2.0 from ICRP-89.
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7 Syntheses were successfully performed. The radiochemical yield was optimized and was 27 ± 7 %, with an average activity of [18F]FNM of 3940 ± 1600 MBq. The specific activity of [ 18F]FNM was always above 355 GBq/µmol. Ex vivo examination An ex vivo diagram (figure 1) confirms that the kidney is a significant clearance pathway. Signal from the lung was elevated but not very homogenous between animals. The brain-to-blood ratio was compared for pentobarbital anesthetized rats, isoflurane anesthetized rats and non-anesthetized rats at 60 min after injection of radiotracer (figure 2). There is a difference between non-anesthetized rats (ratio: 6.2), pentobarbital anesthetized rats (ratio was 1.67) and isoflurane rats (ratio was 2.7).
ACCEPTED MANUSCRIPT In vitro plasma stability test The [18F]FNM was stable in human plasma, regardless of the incubation time. No other peak than [18F]FNM was detected (figure 3). The peak of free fluoride on the solvent front was not observed. Using a Geiger counter, it was verified that there was no more activity attached to the HPLC column.
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Plasma protein binding [18F]FNM seemed to bind strongly to lipoprotein 42.81 ± 0.5%. This compound also bound a little with protein 4.16
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± 0.9%. This result is not surprising due to its lipophilic characteristic (logD= 1.93). Furthermore, due to the fact that this molecule is lipophilic, it will tend to preferentially bind to plasma lipoproteins. On average, the diffusible free
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fraction is 53%. It has been noted that there is little variability between the results obtained for three samples. Total
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plasma protein binding was 46.97%.
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In vivo metabolism 60 min after injection of [18F]FNM, no metabolite was distinguished in plasma samples of rats but a small amount of hydrophilic metabolite (18%) was observed in the urine. This is consistent with literature data of memantine.
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In vivo PET/CT imaging TACs show a rapid decrease in cardiac uptake this could be due to a rapid blood clearance (figure 4). There is a little
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uptake in spleen, liver, stomach and lung. The kidney seems to be the significant clearance pathway. Uptake in the lung is very fast and remains high. Uptake in the liver, spleen and stomach seems to be more gradual. Uptake by the
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retina and pituitaryPx ( schiffer atlas) was also observed for all animals (figure 5). Organ-to-heart ratios at 60 min with PET/CT data and ex vivo data were also compared. T test was performed on ratio (7 organs per rats) in order to
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compare the two methodologies (t =0.84; n=28 ratio). No difference was observed between all in vivo and all ex vivo
images).
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ratio. Figure 5 demonstrates the same cross-sectional evolution of [18F]FNM biodistribution (LUT is the same for all
Absorbed dose estimation Table 1 shows the results for cumulated activities and residence times for each rat. For the majority of the organs, the cumulated activities and residence times are of the same order of magnitude. In the case of Rat 3, measurements of the stomach contents were not available and therefore not included in the average.
ACCEPTED MANUSCRIPT Table 2 shows parameters and results obtained after mass scaling; the differences in masses between human models and measured rat organs are between two or three orders of magnitude. It can be seen in this table that the organs with the highest residence times for the human models are the brain and the lungs, both for ICRP-60 and ICRP-89. From table 3, results for the two versions of OLINDA show the highest total absorbed dose were for spleen, liver,
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lungs and kidneys.
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OLINDA/EXM V2.0 generated results for effective dose according to ICRP-103 recommendations. Using this
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method, the mean effective doses were 4.65 x 10-3 mSv/MBq and 6.11 x 10-3 mSv/MBq for male and female models, respectively. This would correspond to effective doses of 1.63 mSv (male) and 2.14 mSv (female) for an
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administrated activity of 350 MBq. On the other hand, OLINDA 1 generated results for effective dose according to ICRP-60 recommendations, in this case the mean effective dose was 5.09 x 10-3 mSv/MBq for the adult male model,
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this would correspond to effective dose of 1.78 mSv, for the same administrated activity.
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DISCUSSION A dosimetric and biodistribution study, such as this one, has to be performed before human use of experimental
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radiotracer in order to estimate the absorbed doses potentially delivered to human organs, thereby assessing the safety of the diagnostic procedure [14].
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For peripheral organs there was an important uptake in kidney because of its role in radiotracer elimination. Moreover, literature data suggests that there are peripheral NMDA receptors in this organ. In the lung, large inter-
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individual variability in [18F]FNM uptake was observed (figure 4). In the pulmonary tract, there are a very large number of NMDA receptors that are heavily involved in the phenomena of fibrosis and edema; the pulmonary state
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of each different rat could explain variability found in the presented results. The variability of the lung and kidney uptake could also be due the variable amount of ethanol in injected solution because lung and kidney are organs excreting ethanol from the body [15].There are also NMDA receptors in the liver, which are involved in liver inflammation [16]. Finally, an important uptake was noticed in the retina that is in agreement with literature data [17]. Difference was observed between uptake in anesthetized and non-anaesthetized rats, showing that, globally, pentobarbital inhibits the excitatory nervous system. Brain to blood ratio results on non-anesthetized rats was
ACCEPTED MANUSCRIPT identical than data mentioned in Salabert et Al [18] in their in vivo biodistribution study and was 6.2, 0.35 % of injected dose/g. There is a difference between non-anesthetized rats, pentobarbital anesthetized rats (ratio was 1.67, 0.061% of injected dose/g) and isoflurane rats (ratio was 2.7) . There is a decrease of FNM uptake with anesthetized rats. Difference was found between the brain-to-blood ratio PET data (pentobarbital anesthetised rats) and ex vivo data (non-anesthetised rats). This seems to show that use of pentobarbital for euthanizing animals induces a decrease
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of [18F]FNM uptake in the brain. Study on awoken rats would be very useful [19]. It would be interesting to test this
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compound on non-anesthetized human, since these studies suggest depressive role of the anesthetic compound. The
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use of anesthesia in some of the rats used in this research could have caused an under-estimate of absorbed dose calculations in the brain. Anesthetic drugs selected in this study are drugs, which have no known affinity for the
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NMDA receptor (pentobarbital or isoflurane), in contrast to the most used anesthetic in rodents, ketamine (a wellknown antagonist of NMDAr and therefore would have had a direct impact on tracer uptake). However, despite the
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fact that they have no affinity for NMDAr, it has been shown in numerous articles ([20]–[22] that halogenated derivatives such as isoflurane inhibit NMDA activity. The mechanism of action does not seem to be elucidated.
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Ogata and all had measured a depression of 22-30% of NMDAr using isoflurane on xenopus oocytes, which really does not reflect a whole animal. An ex vivo study comparing the activity of the organs with tracer of activated
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NMDAr on anaesthetized or non-anesthetized animals could provide evidence of this decrease. This has been done in
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this article only for the brain and it is possible that this phenomenon of NMDA inhibition may also have affected the peripheral receptors and thus the estimated whole body dose.
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Concerning biological properties, it has been shown that this tracer is very stable in human plasma, and it is
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minimally metabolized (in vivo). This is in concordance with memantine’s stability data [23] [24]. We determined free [18F]FNM fraction because it is the only fraction able to diffuse into tissues. To be able to carry out quantification studies, it is important to have free fraction plasma data. This may not be an issue as long as the free fraction is constant throughout the experiment, therefore its correction can be disregarded, however, it becomes a serious issue, when the free fraction of the ligand in plasma is subject to change in pathological conditions [25]. [18F]FNM lipoprotein plasma binding (47%) is very close to memantine’s 45% (ANSM data for market authorization, http://agence-prd.ansm.sante.fr/php/ecodex/rcp/R0218846.htm). The large free fraction (53 %) allows
ACCEPTED MANUSCRIPT a rapid elimination of the compound because it is more readily accessible to renal filtration. This may explain the low residence time of [18F]FNM in the body. Dynamic PET imaging and ex vivo organ counting were performed on rats. Animal pharmacokinetics were extrapolated to human’s models, in order to estimate dosimetry of the compound on a reference anthropomorphic model. Absorbed dose per injected activity (Gy/Bq) was computed to calculate the absorbed doses potentially
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delivered to human organs, thereby assessing the safety of the diagnostic procedure, this will enable a better selection
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of [18F]FNM activity for use in human PET imaging.
For rats, the kidney has the highest cumulated activity and residence time, implying the highest absorbed dose.
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Conversely, for brain the cumulated activity and residence time were amongst the lowest of all considered organs, showing low irradiation, and therefore low absorbed dose, for this organ.
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Recently, Boschi et al. 2016 [26] conducted a pre-clinical study using
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F-PSMA-11, for dosimetric analysis and
calculations using NUKFIT and OLINDA/EXM V1.0. The effective dose results in our study are in the same order
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of magnitude than theirs. This may be due to the fact that in this study, the whole body of the rat was measured. On
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the other hand, their results for humans were based on ICRP-60 recommendations, whereas we tried to implement ICRP-103 recommendations that propose a separate assessment of the absorbed dose for males and females.
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Additionally results for ICRP were estimated. Since we had no specific assessment of male/female pharmacokinetics from animal experiments, the gender specific estimate was obtained during the extrapolation phase, by scaling
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differently (according to different organ masses) the same pharmacokinetics.
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In the case of the human models (ICRP-60 and ICRP-89), differences in the residence times for each organ can be explained by the differences in relative organ masses (table 2) between models. [18F]FNM has very fast pharmacokinetics, thereby delivering an irradiation of 6.11 µSv/MBq and 4.65 µSv/MBq for females and males, respectively, considering results of ICRP-103 and 5.09 µSv/MBq for male considering results of ICRP-60. This is lower when compared to other PET tracers ( 18F tracers) that are used in neurology. For example, [18F] DPA714 is around 17.2 µSv/MBq [27], [18F]FEDDA is around 36 µSv/MBq [28] and the [18F] amyloid tracer, flutemetamol, is around 25-30 µSv/MBq [29]. Some of the potential explanations were already discussed above. Due
ACCEPTED MANUSCRIPT to a high free fraction; [18F]FNM is quickly eliminate by renal pathway that allow to have a low residence time of this compound in the body and a low dosimetry impact for the patient.
CONCLUSIONS
This study shows that [18F]FNM present good biological properties for imaging :stability in vitro in human plasma,
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weak metabolism in rat , plasma protein binding close to memantine. Its dosimetry shows that it is suitable for use in
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neurology due to an estimated effective dose of around 6.11µSv/MBq and 4.65µSv/MBq for female and male human
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dosimetric models, respectively considering ICRP-103. These values are lower when compared with other PET tracers for the same purposes. Anesthetic seems to play a major role in brain depression and can decrease NMDAr
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activity therefore the use of anesthesia in animal studies may include a bias in uptake measurements, and so in dosimetry estimation. Human studies using [18F]FNM seem to be safe in terms of dosimetry and this compound
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could be used to gain a better understanding of the glutamate excitatory system and NMDA receptor implication in
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neurological and psychiatric diseases.
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COMPLIANCE WITH ETHICAL STANDARDS
All applicable international guidelines for the care and use of animals were followed. This protocol was approved by
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the local competent authorities (authorization no 2016021711398144). All procedures performed in studies involving
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conducted.
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animals were in accordance with the ethical standards of the institution or practice at which the studies were
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REFERENCES [1] C. M. Wroge, J. Hogins, L. Eisenman, S. Mennerick, "Synaptic NMDA receptors mediate hypoxic excitotoxic death", J. Neurosci., Vol. 32, No 19, p. 6732-6742, May 2012. [2] W. Danysz, C. G. Parsons, "Alzheimer’s disease, β-amyloid, glutamate, NMDA receptors and memantine – searching for the connections", Br. J. Pharmacol., Vol. 167, No 2, p. 324-352, Sept. 2012. [3] K. Bordji, J. Becerril-Ortega, A. Buisson, "Synapses, NMDA receptor activity and neuronal Aβ production in Alzheimer’s disease", Rev. Neurosci., Vol. 22, No 3, Jan. 2011. [4] K. Bordji, J. Becerril-Ortega, O. Nicole, A. Buisson, "Activation of Extrasynaptic, But Not Synaptic, NMDA Receptors Modifies Amyloid Precursor Protein Expression Pattern and Increases Amyloid- Production", J. Neurosci., Vol. 30, No 47, p. 15927-15942, Nov. 2010. [5] A. S. Salabert et al., "Radiolabeling of [(18)F]-fluoroethylnormemantine and initial in vivo evaluation of this innovative PET tracer for imaging the PCP sites of NMDA receptors", Nucl. Med. Biol., Vol. 42, No 8, p. 643653, Aug. 2015. [6] F. Tensaouti, J. A. Lotterie, "Sysiphe-Neuroimaging software toolbox", in European Society for Magnetic Resonance Medicine and Biology 2008 Congress Oct, 2008, p. 2–4. [7] Wolfram Research, Inc., Mathematica, version 10.4. Champaign, Illinois, 2015. [8] B. J. McParland, Nuclear Medicine Radiation Dosimetry: Advanced Theoretical Principles. Springer Science & Business Media, 2010. [9] T. Tolvanen et al., "Biodistribution and radiation dosimetry of [(11)C]choline: a comparison between rat and human data", Eur. J. Nucl. Med. Mol. Imaging, Vol. 37, No 5, p. 874-883, May 2010. [10] ICRP, "1990 Recommendations of the International Commission on Radiological Protection. ICRP Publication 60" ICRP21(1-3), ICRP-1991. [11] "Basic anatomical and physiological data for use in radiological protection: reference values. A report of ageand gender-related differences in the anatomical and physiological characteristics of reference individuals. ICRP Publication 89", Ann. ICRP, Vol. 32, No 3-4, p. 5-265, 2002. [12] M. G. Stabin, R. B. Sparks, E. Crowe, "OLINDA/EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine", J. Nucl. Med., Vol. 46, No 6, p. 1023-1027, June 2005. [13] M. G. Stabin, X. G. Xu, M. A. Emmons, W. P. Segars, C. Shi, M. J. Fernald, "RADAR reference adult, pediatric, and pregnant female phantom series for internal and external dosimetry", J. Nucl. Med., Vol. 53, No 11, p. 1807-1813, Nov. 2012. [14] C. C. Constantinescu, A. Garcia, M. R. Mirbolooki, M. L. Pan, J. Mukherjee, "Evaluation of [18F]Nifene biodistribution and dosimetry based on whole-body PET imaging of mice", Nucl. Med. Biol., Vol. 40, No 2, p. 289-294, Feb. 2013. [15] H. Kalant, "Absorption, Diffusion, Distribution, and Elimination of Ethanol: Effects on Biological Membranes", in The Biology of Alcoholism, Springer, Boston, MA, 1971, p. 1-62. [16] J. Du, X. H. Li, Y. J. Li, "Glutamate in peripheral organs: Biology and pharmacology", Eur. J. Pharmacol., Vol. 784, p. 42-48, Aug. 2016. [17] A. A. M. Abdel-Hamid, A. E. D. L. Firgany, E. M. T. Ali, "Effect of memantine: A NMDA receptor blocker, on ethambutol-induced retinal injury", Ann. Anat., Vol. 204, p. 86-92, Mar. 2016. [18] A. S. Salabert et al., "Radiolabeling of [18F]-fluoroethylnormemantine and initial in vivo evaluation of this innovative PET tracer for imaging the PCP sites of NMDA receptors", Nucl. Med. Biol., Vol. 42, No 8, p. 643653, Aug. 2015. [19] D. Schulz et al., "Simultaneous assessment of rodent behavior and neurochemistry using a miniature positron emission tomograph", Nat. Methods, Vol. 8, No 4, p. 347-352, Apr. 2011. [20] M. W. Hollmann, H. T. Liu, C. W. Hoenemann, W. H. Liu, M. E. Durieux, "Modulation of Nmda receptor function by ketamine and magnesium. Part Ii: interactions with volatile anesthetics", Anesth. Analg., Vol. 92, No 5, p. 1182-1191, May 2001. [21] J. Ogata, M. Shiraishi, T. Namba, C. T. Smothers, J. J. Woodward, R. A. Harris, "Effects of anesthetics on mutant N-methyl-D-aspartate receptors expressed in Xenopus oocytes", J. Pharmacol. Exp. Ther., Vol. 318, No 1, p. 434-443, July. 2006. [22] T. Yamakura, R. A. Harris, "Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol", Anesthesiology, Vol. 93, No 4, p. 1095-1101, Oct. 2000. [23] M. G. Beconi et al., "Pharmacokinetics of memantine in rats and mice", PLoS Curr., Vol. 3, p. RRN1291, 2011. [24] S. Lam, C. Smith, I. H. Gomolin, "Memantine Standard Tablet and Extended-Release Dosing Considerations: A Pharmacokinetic Analysis", J. Am. Geriatr. Soc., Vol. 63, No 2, p. 383-384, Feb. 2015.
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[25] F. E. Turkheimer et al., "The methodology of TSPO imaging with positron emission tomography", Biochem. Soc. Trans., Vol. 43, No 4, p. 586-592, Aug. 2015. [26] S. Boschi et al., "Synthesis and preclinical evaluation of an Al(18)F radiofluorinated GLU-UREALYS(AHX)-HBED-CC PSMA ligand", Eur. J. Nucl. Med. Mol. Imaging, Vol. 43, No 12, p. 2122-2130, Nov. 2016. [27] N. Arlicot et al., "Initial evaluation in healthy humans of [18F]DPA-714, a potential PET biomarker for neuroinflammation", Nucl. Med. Biol., Vol. 39, No 4, p. 570-578, May 2012. [28] A. Takano et al., "Biodistribution and radiation dosimetry of the 18 kDa translocator protein (TSPO) radioligand [18F]FEDAA1106: a human whole-body PET study", Eur. J. Nucl. Med. Mol. Imaging, Vol. 38, No 11, p. 2058-2065, Nov. 2011. [29] M. Koole et al., "Whole-Body Biodistribution and Radiation Dosimetry of 18F-GE067: A Radioligand for In Vivo Brain Amyloid Imaging", J. Nucl. Med., Vol. 50, No 5, p. 818-822, May 2009.
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40.0 rats 30 min 35.0 rats 60 min 30.0
rats 90 min
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(Activity/g)organ /(Activity/g)blood
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5.0 spleen
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Figure 1. Ex vivo biodistribution of [18F]FNM 30 min after injection (n=2); 60 min after injection (n=3) and 90 min after injection (n=3). Ordinate value is the ratio of activity/g of each organ by activity/g of blood sample. (error bars = SD)
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(cpm/g)brain /(cpm/g)blood
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Figure 2. Ordinate value is the Brain to blood ratio of [ 18F]FNM uptake using different kind of anesthesia. (error bars = SD), n= 3 for all each experiment.
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Figure 3: Figure 3. Metabolization study. HPLC diagrams (detection gamma ) ABCD show [ F]FNM peak after
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incubation in human plasma( respectively during 10,20,30,120 min). Figure E represent HPLC diagram (gamma detection) of rat urinary fraction ( 60 min after injection of [18F]FNM ) . Figure E represent HPLC diagram ( gamma detection) of rat plasma ( 60 min after injection of [18F]FNM ) .
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Figure 4. Left: Sagittal whole body PET-CT of a sprague dawley rat. Right-up: Percentage of injected activity for each organ after an intravenous injection of [18F]FNM, for rat 1 (Ao= 61.63 MBq), with the remainder. Right-down: Percentage of injected activity for each organ after an intravenous injection of [ 18F]FNM, for rat 1, without the remainder.
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Figure 5. Left: Whole body biodistribution of [ F]FNM after injection of 60 MBq in tail vein.
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Rigth: Arrow shows uptake in pituitary PX ( schiffer atlas). .
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Rat 2, Ao = 18.00 MBq
Rat 3, Ao = 111.00 MBq
Organs
A (Bq*min)
T (h)
A (Bq*min)
T (h)
A (Bq*min)
T (h)
T (h)
Brain
2.09E+07
5.64E-03
4.01E+06
3.71E-03
6.30E+07
9.46E-03
6.27E-03
Heart
1.62E+07
4.37E-03
3.50E+06
3.24E-03
4.57E+07
Kidney
1.67E+08
4.51E-02
4.54E+07
4.20E-02
3.02E+08
4.54E-02
4.42E-02
Liver
1.68E+08
4.55E-02
6.35E+07
5.88E-02
3.80E+08
5.71E-02
5.38E-02
Lung
3.90E+07
1.06E-02
9.71E+06
8.99E-03
1.09E+08
1.64E-02
1.20E-02
Spleen
1.48E+07
3.99E-03
1.18E+06
1.09E-03
7.38E+07
1.11E-02
5.39E-03
Stomach
2.25E+07
6.09E-03
8.89E+06
8.23E-03
-
-
7.16E-03
Whole body
3.05E+09
8.26E-01
4.40E+08
4.07E-01
7.25E+09
1.09E+00
7.74E-01
T
Rat 1, Ao = 61.63 MBq
CR
Table 1. Summary of results for cumulative activity and residence times for each rat.
AN
US
IP
6.87E-03
ICRP-89 Mfemale (g)
Brain
1450
1300
Heart
510
370
Kidney
310
Liver
1800
Lung
1200
Spleen Stomach
ICRP-60
Mrat (g)
Tmale (h)
Tfemale (h)
Thuman (h)
1420
1.99
2.07E-02
2.25E-02
2.00E-02
316
1.22
9.12E-03
8.05E-03
5.60E-03
275
299
2.61
2.37E-02
2.56E-02
2.27E-02
1400
1910
8.20
5.34E-02
5.05E-02
5.61E-02
950
1000
1.65
3.94E-02
3.80E-02
3.25E-02
150
130
183
0.65
5.62E-03
5.93E-03
6.79E-03
250
230
158
1.34
6.04E-03
6.76E-03
3.78E-03
73000
60000
73700
330
7.74E-01
7.74E-01
7.74E-01
CE
AC
Total Body
ICRP-89
Mhuman (g)
PT
Mmale (g)
ICRP-60
ED
Organ
M
Table 2. Masses values used for mass scaling and male/female residence times
Average
4.83E-03
ACCEPTED MANUSCRIPT Table 3. Absorbed dose and effective dose coefficients results for adult male from Olinda 1, male and female phantoms from Olinda 2 Male – OLINDA 1
Male – OLINDA 2
Target organ
Total (mGy/MBq)
Adrenals
5.30E-03
1.33E-05
Adrenals
6.80E-03
6.28E-05
8.17E-03
7.54E-05
Brain
4.19E-03
1.05E-05
Brain
4.22E-03
4.22E-05
5.16E-03
5.16E-05
Breasts
3.41E-03
1.71E-04
Breast
-
-
4.25E-03
5.10E-04
Gallbladder Wall
5.59E-03
-
4.84E-03
1.94E-04
5.56E-03
2.22E-04
LLI Wall
4.57E-03
5.49E-04
Eyes
3.52E-03
0.00E00
4.35E-03
0.00E00
Small Intetine
4.84E-03
1.21E-05
Gallbladder Wall
6.07E-03
5.61E-05
6.69E-03
6.17E-05
Stomach Wall
5.83E-03
7.00E-04
Left colon
2.44E-04
6.09E-03
2.95E-04
4.81E-03
1.20E-05
Small Intestine
4.56E-05
5.59E-03
5.16E-05
5.66E-03
-
Stomach Wall
7.19E-03
8.63E-04
8.69E-03
1.04E-03
Kidneys
1.64E-02
4.10E-05
Right colon
5.03E-03
2.44E-04
6.02E-03
2.92E-04
Liver
8.78E-03
4.39E-04
Rectum
4.62E-03
1.06E-04
5.62E-03
1.29E-04
Lungs
7.72E-03
9.27E-04
Heart Wall
6.66E-03
6.15E-05
8.00E-03
7.38E-05
Muscle
3.94E-03
9.86E-06
Kidneys
1.67E-02
1.54E-04
2.01E-02
1.86E-04
Ovaries
4.74E-03
9.49E-04
Liver
9.04E-03
3.61E-04
1.09E-02
4.37E-04
Pancreas
5.45E-03
1.36E-05
Lungs
7.87E-03
9.45E-04
9.58E-03
1.15E-03
Red Marrow
3.89E-03
4.66E-04
Ovaries
-
-
5.72E-03
2.29E-04
Osteogenic Cells
6.08E-03
6.08E-05
Pancreas
5.51E-03
5.08E-05
6.93E-03
6.39E-05
Skin
3.09E-03
3.09E-05
Prostate
4.60E-03
2.12E-05
-
-
9.59E-03
2.40E-04
Salivary Glands
4.07E-03
4.07E-05
4.63E-03
4.63E-05
3.76E-03
-
Red Marrow
4.01E-03
4.82E-04
4.89E-03
5.87E-04
4.19E-03
1.05E-05
Osteogenic Cells
3.56E-03
3.56E-05
4.19E-03
4.19E-05
Thyroid
4.02E-03
2.01E-04
Spleen
9.63E-03
8.89E-05
1.20E-02
1.10E-04
Urinary Bladder Wall
4.50E-03
2.25E-04
Testes
3.78E-03
1.51E-04
-
-
Uterus
4.78E-03
1.20E-05
Thymus
4.56E-03
4.21E-05
5.78E-03
5.34E-05
Total Body
4.28E-03
-
Thyroid
4.33E-03
1.73E-04
4.89E-03
1.96E-04
Spleen Testes Thymus
ED
IP
CR
US
5.03E-03
AN
4.94E-03
M
Esophagus
T
Total ICRP-103 Total ICRP-103 (mGy/MBq) (mSv/MBq) (mGy/MBq) (mSv/MBq)
PT
CE
Heart Wall
AC
ULI Wall
Effective Target organ Dose (mSv/MBq)
Female– OLINDA 2
ACCEPTED MANUSCRIPT
-
5.09E-03
Urinary Bladder Wall
4.54E-03
1.82E-04
4.66E-03
1.86E-04
-
-
5.67E-03
2.62E-05
Total Body
4.23E-03
-
5.30E-03
-
Total effective dose (mSv/MBq)
-
4.65E-03
-
6.11E-03
CE
PT
ED
M
AN
US
CR
IP
T
Uterus
AC
Total effective dose (mSv/MBq)