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Article 18
Pharmacokinetic characterization of [ F]UCBH PET radiopharmaceutical in the rat brain Guillaume Becker, Corentin Warnier, Maria Elisa Serrano, Mohamed Ali Bahri, Joël Mercier, Christian Lemaire, Eric Salmon, André Luxen, and Alain Plenevaux Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00235 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017
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Pharmacokinetic characterization of [18F]UCB-H PET radiopharmaceutical in the rat brain Guillaume Becker,*§† Corentin Warnier,§† Maria Elisa Serrano,† Mohamed Ali Bahri,† Joël Mercier,‡ Christian Lemaire,† Eric Salmon,† André Luxen† and Alain Plenevaux.†
†
GIGA Cyclotron Research Centre In Vivo Imaging, University of Liege, 4000 Liege, Belgium ‡
*
Corresponding author:
UCB BioPharma, 1420 Braine-l’Alleud, Belgium
University of Liege Allee du six Aout, 8, B.30, 4000 Liege, Belgium Tel: +32 4 366 23 28
Fax: +32 4 366 29 46
Email:
[email protected]
Keywords: SV2A, [18F]UCB-H, PET, chirality, population-based input function
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Abstract
The synaptic vesicle glycoprotein 2A (SV2A), a protein essential to the proper nervous system function, is found in presynaptic vesicles. Thus, SV2A targeting, using dedicated radiotracers combined with positron emission tomography (PET), allows the assessment of synaptic density in the living brain. The first-in-class fluorinated SV2A specific radioligand, [18F]UCB-H, is now available at high-activity through an efficient radiosynthesis compliant with the current good manufacturing practices (cGMP). We report here a non-invasive method to quantify [18F]UCB-H binding in rat brain with microPET. Validation study in rats confirmed the need of high enantiomeric purity to target SV2A in vivo. We demonstrated the reliability of a populationbased input function to quantify SV2A in preclinical microPET setting. Finally, we investigated the in vivo metabolism of [18F]UCB-H and confirmed the negligible amount of radiometabolites in the rat brain. Hence, the in vivo quantification of SV2A using [18F]UCB-H microPET seems a promising tool for the assessment of the synaptic density in the rat brain, and opens the way for longitudinal follow-up in neurodegenerative diseases rodents’ models.
Introduction Neurotransmitter release from a presynaptic nerve terminal is triggered by action potentials. The latter induces the opening of Ca2+ channel, resulting in the stimulation of synaptic vesicles (SV) exocytosis.1 To support rapid and repeated rounds of release, SV undergo a trafficking cycle. Important SV proteins in this process have been studied in great details and their function are now well established.2 However, some SV proteins still require further investigations. In this
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context, the SV glycoprotein 2 (SV2) isoforms are experiencing renewed interest due to their implication in the regulation of neurotransmitter release and their potential value as drug targets.3,4 SV2 is a component of SV and neuroendocrine secretory vesicles, highly conserved in all vertebrates. Three SV2 genes encode highly homologous protein isoforms referred to as SV2A, SV2B and SV2C. SV2 proteins contain 12 transmembrane domains with cytoplasmic Nand C-termini and are structurally close to carbohydrate transporters in eukaryotes and bacteria.5 Recently, it has been shown that SV2A acts as galactose transporter in yeast.6 SV2A is ubiquitously distributed in the rodent brain, whereas SV2B exhibits a more restricted distribution, and SV2C is present only in a small subset of neurons in the basal forebrain and striatal brain regions.7,8 SV2A has been demonstrated to be critical for proper nervous system function as mice lacking SV2A exhibit seizures at post-natal day 7 leading to death around post-natal day 15.9,10 Interestingly, SV2A has been implicated in the pathophysiology of epilepsy since the discovery of SV2A as the binding site of the antiepileptic drug levetiracetam (LEV).11 However, it is still unknown how LEV interacts with SV2A to prevent partial onset seizures. There is currently a growing interest for the in vivo study of SV2A thanks to the visualization of SV2A in the living brain with positron emission tomography (PET).12,13 The radiotracer [18F]UCB-H was the first radiopharmaceutical developed with nanomolar affinity for SV2A.14 More recently, Finnema and colleagues showed that [11C]UCB-J PET radiotracer can be used to assess brain synaptic density in vivo.15 Despite the undoubtedly valuable properties of [11C]UCB-J, the limited availability and short half-life of carbon-11 limits the use of [11C]radiopharmaceuticals in clinical investigations.16 Regarding [18F]UCB-H, preclinical results and dosimetry, and first-in-man studies confirmed that [18F]UCB-H possesses all suitable
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characteristics to measure SV2A density in vivo, making it a key element to understand the physiological role of SV2A and to reveal its fundamental implication in various neurological diseases.12,17 However, few limitations remained due to the complexity and inefficiency of the radiosynthesis. Those are now lifted thanks to a new one step synthesis achieved by the radiolabeling of an enantiomerically pure N-heteroaryliodonium precursor.18 [18F]UCB-H is a chiral molecule ((R)-[18F]UCB-H is the most active form, and is referred as [18F]UCB-H thereafter), with a difference of 1 log unit in the target affinity between both enantiomers. Here we evaluated the impact of enantio-selectivity on SV2A in vivo targeting using microPET imaging.19 Then, we quantified the [18F]UCB-H binding in the rat brain using kinetic modeling and a population-based input function (PBIF). Finally, we investigated the in vivo metabolism of the radiotracer, and its impact on image quality, through the radiosynthesis of the main detected metabolite.
Experimental section In vivo metabolism in rats To qualitatively analyze the major metabolites of the reference product UCB-H, terminal blood samples were taken following 5mg/kg i.v. dosing at 1 of 3 time points (10, 30, and 60 minutes). Rats were sacrificed and whole brains extracted. Brains were homogenized in water and assayed together with plasma samples using a UPLC-MS/MS system (SI 1).
Chemistry
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Solvents and chemicals were purchased from VWR and Sigma Aldrich and used without further purification, unless otherwise stated. The cold reagent UCB-H was provided by UCB Pharma S.A. Using the synthesis and chiral separation previously described for UCB-H and (R,S)-UCB-H pyridyliodonium
precursor
salts,
the
enantiomerically
pure
compound
(S)-UCB-H
pyridyliodonium precursor was synthesized.18 Its synthesis yield and characterization data were entirely compliant with our previous work.
[18F]UCB-H enantiomers radiosynthesis The radiolabeling of enantiomerically pure [18F]UCB-H and (S)-[18F]UCB-H, and the racemic (R,S)-[18F]UCB-H, was also performed according to the procedure reported in the above cited reference, briefly summarized in figure 1, and yielded the expected 34±2% of (S)-[18F]UCB-H. As previously described, the HPLC analyses for the measurement of the radiotracer’s enantiomeric excess were carried out with an analytical Daicel Chiralpak® AD-RH column, using a 35/65 MeCN/pH 8 phosphate buffer as eluent at 0.3 mL/min (SI 2).18
Figure 1. One-step, fully automated and cGMP compliant radiosynthesis of [18F]UCB-H.
[18F]UCB-H-N-oxide radiosynthesis
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In order to achieve the radiosynthesis of [18F]UCB-H-N-oxide (the main metabolite of [18F]UCB-H) in the most time-efficient way, the strategy was to radiolabel the UCB-H pyridyliodonium salt precursor to afford [18F]UCB-H according to the above described method (Figure 1) followed by direct oxidation with m-CPBA (Figure 2). It rapidly turned out that this strategy was viable, with the oxidation of [18F]UCB-H to [18F]UCB-H-N-oxide in the presence of a large excess of pure m-CPBA in Et2O, providing 40% of radiochemical yield decay corrected (RCYd.c.). Therefore, an automated radiosynthesis was developed to allow the production of large doses of [18F]UCB-H-N-oxide (SI 3). The mean specific activities of [18F]UCB-H (S, R and racemic) and of [18F]UCB-H-N-oxide produced with the reported method were 815±185 GBq/µmol at the end of synthesis.
Figure 2. Main metabolization pathway of [18F]UCB-H in vivo (upper panel). The developed synthetic pathway for the radiolabeling of [18F]UCB-H-N-oxide (lower panel).
PET imaging studies
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All animal experiments were performed according to the Helsinki declaration and conducted in accordance with the European guidelines for care of laboratory animals (2010/63/EU). All procedures were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the University of Liège, Belgium (LA1610005). Animals were housed in individually ventilated cages (2 per cages, room maintained at 22°C and at humidity of approximately 50%, 12-h day-night cycle). Eighteen male Sprague-Dawley rats (Janvier, France) were used for all imaging experiments. Standard pellet food and tap water were provided ad libidum. Population-based input function [18F]UCB-H PET data with arterial input functions (AIF) were obtained for 8 rats (mean body mass ± SD = 421.8±32.4 g) injected with [18F]UCB-H (mean injected activity ± SD = 140.8±10.2 MBq, produced by the former radiosynthesis18) according to the procedure described by Warnock and colleagues.14 Arterial blood analysis, image processing and kinetic modeling were conducted to obtain the distribution volume (VT) as mentioned in the above cited reference. Individual arterial input functions (AIF), corrected for metabolites, were normalized by body mass (BM) and injected activity (IA), and then expressed as Standardized Uptake Value (SUV). The population-based input function (PBIF) was obtained by averaging the eight individual AIF. To validate the PBIF, we compared VT obtained using the individual AIF to the ones we obtained using PBIF (i.e. individual AIF vs PBIF)
[18F]UCB-H enantiomers and [18F]UCB-H-N-oxide PET imaging Anesthesia was induced with a mixture of 4% isoflurane gas in air delivered at a flow rate of 1 L/min in a dedicated box. Afterward, animals were placed prone in a specific rat holder
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(Minerve, France) and anesthesia was maintained using 1 to 2 % isoflurane in air at 0.6 l/min. Respiration rate and rectal temperature were permanently measured using a physiological monitoring system (Minerve, France). Temperature was maintained at 37±0.5°C using an air warming system. All individuals (n = 5, mean BM ± SD at testing = 354.8±13.1 g) underwent 3 PET scans on a Siemens FOCUS 120 microPET (Siemens, Knoxville, TN) on 3 experimental sessions (mean interval = 4±2 days). PET data acquisition started with a 10-min
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transmission scan, with single event acquisition mode, following which [18F]UCB-H, (S)[18F]UCB-H or (R,S)-[18F]UCB-H was injected in the lateral tail vein. Mean IA were 36.9±2.7 MBq, 31.3±7.1 MBq and 41.6±2.3 MBq respectively (the corresponding injected mass of compound were 14,7±1.1 ng, 12,5±2.8 ng and 16,5±0.9 ng respectively). Emission data were then recorded as previously described for 60 min.18 Immediately after PET acquisition, the anesthetized rats were transferred (without having been awaked or moved) into a 9.4 Tesla MRI horizontal bore system with a shielded gradient system (Agilent Technologies, Palo Alto, CA) and a 72 mm inner diameter volumetric coil (Rapid Biomedical GmbH, Würzurg, Germany). Anatomical T2-weighted brain images were acquired with a fast spin echo multislice sequence using the following parameters: TR/TEeff = 2000/40 ms, matrix = 256 x 256, FOV = 45 x 45 mm, 30 contiguous slices (thickness = 0.80 mm, inplane voxel size: 0.176 x 0.176 mm). PMOD software version 3.6 (PMOD Technologies Ltd., Zurich, Switzerland) was used to process the image data, to extract time activity curves (TACs) in volume of interests (VOIs) positioned over individual set of PET images and finally to compute the kinetic analysis. The VOIs were obtained using PMOD rat brain atlas. The structural MRI images were coregistered to the corresponding PET images using the rigid matching methods. The coregistered MRI images were then spatially normalized onto the PMOD
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structural MRI image template. The inverse spatial normalization parameter estimates were applied to the PMOD rat brain atlas in order to bring it into the native dynamic PET space. Individual TACs were normalized by BM and IA, and then expressed as SUV. [18F]UCB-H binding was estimated with Logan graphic analysis, both individual TACs and PBIF, were used to compute the regional distribution volume VT.20 In separate experiments, the above described protocol was followed for [18F]UCB-H-N-oxide PET acquisitions and processing (n = 4, mean BM: 207.6±40.2 g, mean IA: 29.1±2.9 MBq, mean injected mass: 11,6±1.2 ng). During the PET acquisition (which last for 60 min.), blood samples were taken at 10 min, via a catheter inserted in the lateral tail vein, and assayed as previously described. Blood and Brain metabolite analyses In a separate study to determine the unchanged parent fraction of [18F]UCB-H, 5 rats (mean BM ± SD = 378.2±33.3 g) were injected in the tail vein with 116.1±25.6 MBq of [18F]UCB-H. Blood samples were harvested via a catheter inserted in the contralateral tail vein at 5 and 10 minutes. At 20 minutes the rats were sacrificed, terminal blood samples were collected and whole brains extracted. Brains were homogenized in water and assayed together with plasma samples as previously described.14 We provided a summary of all the in vivo protocols used in our study in the SI 7.
Results In vivo metabolism in rats On the basis of mass spectrometry response, the parent compound UCB-H was the major detected component in rat plasma and brain (SI 1a and 1b). The metabolization of [18F]UCB-H
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occurred by 2 major pathways: Oxidation, leading to an N-oxide (the major detected metabolite, Figure 2) and hydroxylated metabolites, and N-dealkylation of the pyrrolidinone ring (SI 4.1). The integration of the MS response area revealed that the N-oxide represents 90.2% of the formed metabolites. The other formed metabolites account for 9.8%. Population-based input function After normalization with IA and BM, the time-activity curve of the AIF showed a high level of similarity across measurements in different rats (Figure 3). The peak time of averaged AIFs was 31.1 seconds and it ranged between 26 and 34 seconds. PBIF-computed VT are strongly correlated with the VT measured with individual IF (SI 5.1), and VT values in all brain regions are very consistent between both methods (SI 5.2).
Figure 3. In the main graph, the black time-activity curves represent the eight individual arterial input functions (AIFs), corrected for metabolites, normalized by injected activity and body mass and the red curve represents the mean of the normalized AIF. The insert graph shows the same data focusing around the peak of the AIFs.
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[18F]UCB-H enantiomers PET image analyses and kinetic modeling Examples of [18F]UCB-H, (R,S)-[18F]UCB-H or (S)-[18F]UCB-H scans are shown in Figure 4. Those results clearly highlight that [18F]UCB-H displayed the highest brain uptake. (R,S)[18F]UCB-H also showed a substantial brain uptake but with a highest background noise. (S)[18F]UCB-H displayed the lowest brain uptake and the higher background noise. The mean TACs extracted from thalamus, striatum and cerebellum confirmed that [18F]UCB-H provided the highest SUV values. Moreover (S)-[18F]UCB-H, and to a lesser extent (R,S)-[18F]UCB-H, underwent a rapid and extensive brain clearance, with respectively 32.2% and 55.3% of the peak SUV remaining at 600 seconds (values extracted from the striatum). Regarding [18F]UCB-H in the same region, the SUV at 600 seconds represented 68.9% of the SUV peak (Figure 4).
Figure 4. Representative images of [18F]UCB-H, (R,S)-[18F]UCB-H and (S)-[18F]UCB-H and the corresponding TACs after normalization by the injected activities and the body mass (mean ± SD, n = 5). Upon each TAC is drawn the chemical structure of the corresponding compound
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(along with the affinity at SV2A protein). Grey circles: thalamus; black squares: striatum; open diamonds: cerebellum.
By using the PBIF and the Logan graphical analysis, we obtained reliable VT estimates with relatively low coefficient of variance (CV) ranging between 9.8% and 12.5%. The Figure 5 displays the regional VT values (mean ± SD), with higher values in the thalamus and the caudateputamen (13.9±1.4 and 12.4±1.5 ml/cm3 respectively), and lower values in the cerebellum and the medulla (8.7±0.9 and 8.2±0.7 ml/cm3 respectively). An example of an individual parametric VT map, along with the corresponding individual MRI is shown in Figure 5.
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Figure 5. Example of an individual parametric VT map of [18F]UCB-H binding in rat, along with the corresponding individual MRI (upper panel). Mean regional VT values (± SD, n = 5) obtained with the PBIF (lower panel).
Blood and Brain metabolite analyses [18F]UCB-H displayed fairly fast metabolism in rats. The parent fraction in the plasma accounted for 64.3±8.2%, 43.3±6.6% and 20±3.5% of the radioactivity at 5, 10 and 20 min after injection (n = 5), respectively. The parent fraction was also assessed in the rat brains at 20 min, and the parent compound [18F]UCB-H represented 92.6±3.7% of the radioactivity.
[18F]UCB-H-N-oxide PET image analysis The Figure 6 shows a representative example of [18F]UCB-H-N-oxide PET image. This clearly indicates the absence of any specific radioactivity in the brain. The mean TAC extracted from the whole brain reached a plateau around 0.6 SUV at 46±15 seconds after the injection. The SUV remained stable until the end of the acquisition with a SUV of 0.7 at 1 hour, compared to the residual activity of 1.2±0.1 SUV (at the same time point) of the mean TAC extracted from the whole brain of [18F]UCB-H experiments (Figure 6). Blood analyses showed that, at 10 min, about 99% of the radioactivity is supported by the [18F]UCB-H-N-oxide.
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Figure 6. TACs extracted from the whole brain as volume of interest, and normalized by the injected activities and the body mass (mean ± SD; [18F]UCB-H n = 5; [18F]UCB-H-N-oxide n = 4). Images are representative examples of [18F]UCB-H-N-oxide and [18F]UCB-H injections.
Discussion The main goal of this study was to characterize in vivo the [18F]UCB-H pharmacokinetic profile and to quantify the binding to SV2A protein using a non-invasive method with a population-based input function (PBIF). The [18F]UCB-H radiopharmaceutical was produced at high specific activity, through the radiolabeling of its pyridyliodonium precursor. Furthermore, this new and fully automated radiosynthesis method, compliant with the current good manufacturing practices (cGMP), allowed us to investigate the impact of chirality while aiming to target in vivo SV2A protein.18 The evaluation of metabolic pathways showed that the major circulating metabolite is the product of oxidation on the nitrogen atom of the pyridine ring, namely [18F]UCB-H-N-oxide. This metabolite is unlikely to cross the BBB, based on the polarity of such a product. Blood and brain metabolites analyses are consistent with an absence of BBB crossing. However, to rule out any participation of [18F]UCB-H-N-oxide in the rat brain PET signal, we decided to assess in
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vivo its biodistribution. Thus, the radiosynthesis of the labeled metabolite was developed and afforded a good radiochemical yield (SI 3). PET imaging with [18F]UCB-H-N-oxide confirmed the absence of BBB crossing for this compound (by passive diffusion or active transport) with TAC from the whole brain showing only background activity most likely coming from the cerebral blood flow (Figure 6). In addition, blood analyses realized 10 minutes after the injection indicated a slow metabolism, and thus attested that [18F]UCB-H-N-oxide is largely the dominant metabolite of [18F]UCB-H. We demonstrated here that [18F]UCB-H fulfils an important criterion for PET radiopharmaceuticals with the lack of troublesome brain radiometabolites.21 Furthermore, we investigated the inter-species metabolism of the reference product UCB-H in vitro using rat and human hepatocytes, and rat and human liver microsomes (data not shown). Based on MS response, the in vitro metabolic patterns of the reference product UCB-H appeared to be similar in the different species. Evaluation in rats of both [18F]UCB-H and (S)-[18F]UCB-H, and their racemic mixture indicated outstanding differences in brain signal intensity and signal-to-noise ratio. Although the difference between [18F]UCB-H and (S)-[18F]UCB-H was expected, based on their respective pIC50 at SV2A protein (Figure 5),19 our results highlight that 1 log unit of affinity dramatically impacts the brain kinetic of these PET tracers. As it has been demonstrated for radioligands in general, the lower affinity of (S)-[18F]UCB-H decreases the time needed to reach equilibrium (i.e. state where the rates of association and dissociation to and from the receptors are equal) with a faster washout, but also dramatically decreases the signal-to-noise ratio.22 Differences in isomers’s pharmacokinetics with optically active radioligand have already been reported.23 However, the difference reported here was not related to the brain distribution pattern, which was nearly the same between both enantiomers given the regional TACs pattern (Figure 5), but
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seemed to lie in the receptor-ligand interaction with a really fast washout for (S)-[18F]UCB-H assessed by the low percentage of SUV peak at 600 seconds. (R,S)-[18F]UCB-H PET imaging outcome in rats was interesting in that it emphasized the impact of (S)-[18F]UCB-H on the [18F]UCB-H kinetic. The radiosynthesis approach we choose for (R,S)-[18F]UCB-H, starting from the (R,S)-UCB-H pyridyliodonium precursor salt labelled with high RCY, guaranteed that both enantiomers were present at the same concentration at the target (given the same injected mass). Despite the 10-fold difference in the IC50 of [18F]UCB-H and (S)-[18F]UCB-H, the (R,S)[18F]UCB-H PET signal seemed to represent the average, with a faster washout and higher background noise as a consequence compared to the original [18F]UCB-H PET signal. Adverse effects of one enantiomer on the other has been described in the context of escitalopram-induced increase in extracellular serotonin levels, where co-injection of the non-active R-enantiomer counteracted this effect.24 Our results allowed the visualization of [18F]UCB-H pharmacokinetic modulation induced by the enantiomer (S)-[18F]UCB-H, and thus preclude the use of (R,S)[18F]UCB-H for SV2A PET imaging. Besides that, we investigated the metabolism rate of (S)[18F]UCB-H witch was very close to the metabolism rate of the [18F]UCB-H (data not shown). Thus, both compound are extremely difficult to identify in blood samples without a dedicated chiral HPLC system. Overall, our study highlights the important requirement of enantiomeric purity statement, as reported by Warnier and colleagues, while aiming to target in vivo SV2A with optically active radioligands.18 Quantification of [18F]UCB-H binding is of critical interest to investigate in vivo the pathophysiology of SV2A in rat models of epilepsy.25 According to Nabulsi and co-workers, white matter regions could be used as reference regions to quantify [11C]UCB-J binding in human and non-human primates.16 However, in rodent microPET studies, the poor spatial
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resolution (around 1.5 mm) prevents the use of white matter in the kinetic modeling process.26 In our previous work, [18F]UCB-H binding quantification was computed by means of an arterial input function measured with a β-microprobe system.14 The significant drawbacks of such a method are the degree of complexity introduced and its invasiveness, making it non-suitable for longitudinal studies. Standard input functions appear to be a noninvasive substitute for individual input function measurement.27 Therefore, we decided to use a PBIF, derived from measurements previously obtained with the β-microprobe system, and to implement it in the kinetic modeling of the individual rats scanned with [18F]UCB-H. We validated the use of the bi-exponential parent fraction curve described in our previous work by comparing the metabolism data. In this study, the measured parent fraction (64.3±8.2%, 43.3±6.6% and 20±3.5% at 5, 10 and 20 min respectively) matched the bi-exponential parent fraction curve fitted by Warnock and colleagues, where the value at the same time points were 62.1%, 41.2% and 23% respectively, ensuring us the possibility to use it for the correction of the whole-blood radioactivity (measured by the βmicroprobe).14 After averaging eight individual AIFs, we obtained the PBIF and plotted against the plasmatic radioactivity – metabolites corrected – measured in the blood samples collected at 5, 10 and 20 minutes (SI 6). Given the important matching between the PBIF and the activity measured in the blood samples, we didn’t scale the PBIF, as it is usually done in human studies to adapt the PBIF to the activity measure in the subjects28–30. Besides, VT values computed with both individual AIF are really close, and the variability with both methods is low (SI 5). Kinetic modeling analysis processed with this PBIF allowed us to quantify [18F]UCB-H binding, with VT values as outcomes, and our results are very consistent with previously reported results, despite slightly higher CV than in the study of Warnock and co-workers (ranged between 2.0% and 5.1%).14 More extensive test-retest reproducibility studies, as well as pharmacological
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competitions, should be performed to confirm the reliability of this method. Nevertheless, in [18F]UCB-H preclinical imaging setting, PBIF appears to be a very attractive and quantitative method for longitudinal follow-up in rodents.
ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge. 1 – 4: Supplemental Figures 1−4.2 and methods describing radiosynthesis and identification of compounds (PDF). 5 and 6: Supplemental Figures 5.1−6 and methods describing PBIF validation (PDF).
AUTHOR INFORMATION Corresponding Author * G.B. Office: +32 4 366 23 28. Fax: +32 4 366 29 46. Email:
[email protected] Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. § These authors contributed equally. Notes This study was funded by ULg grant 13/17-07, CWALity grant (Walloon Region, Belgium) and the European Regional Development Fund (Radiomed project). J. M. is an UCB Pharma
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employee, all the other authors declare no competing financial interest. A. P. is Research Director from FRS-FNRS, Belgium, and C. W. is a F.R.I.A. grantee (FRS-FNRS, Belgium).
ACKNOWLEDGEMENT We thank Kathleen Lambert and Florence Collard for technical assistance. The authors are grateful to Alain Seret for his contribution and valuable scientific discussions.
ABBREVIATION AIF, Arterial Input Function; BBB, Blood Brain Barrier; BM, Body Mass; cGMP: current Good Manufacturing Practice; CV, Coefficient of variance; Et2O: Diethyl ether; HPLC, High Performance Liquid Chromatography; IA, Injected Activity; IC50, half maximal inhibitory concentration; i.v., intravenous; LEV, Levetiracetam; MBq, Mega Becquerel; m-CPBA, mchloroperoxybenzoic acid; MRI, Magnetic Resonance Imaging; MS, Mass Spectrometry; PBIF, Population-based Input Function; PET, Positron Emission Tomography; RCYd.c., radiochemical yield decay corrected; SV, Synaptic Vesicle; SV2A, Synaptic Vesicle glycoprotein 2A; SUV, Standardized Uptake Value; TACs, Time Activity Curves; UPLC, Ultra Performance Liquid Chromatography; VOIs, Volumes of Interests; VT, Distribution Volume.
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