Evaluation of the α-synuclein PET Radiotracer (d3)-[11C]MODAG-001 in Pigs

Evaluation of the α-synuclein PET Radiotracer (d3)-[11C]MODAG-001 in Pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the α-synuclein PET Radiotracer (d 3 )-[ 11 C]MODAG-001 in Pigs Nakul Ravi Raval, Clara Aabye Madsen, Vladimir Shalgunov, Arafat Nasser, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1268531/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: A positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels is a histopathological hallmark. The radiotracer (d 3 )-[ 11 C]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 µg α-PFF, and in the other hemisphere, either 75 µg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer’s disease (AD) was injected. All pigs underwent one or two (d 3 )-[ 11 C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region. Results: The α-PFF and AD homogenate injected brain regions had high uptake of (d 3 )-[ 11 C]MODAG-001 compared to the occipital cortex or cerebellum. BP ND values in 150 µg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BP ND values in the 150 µg and 75 µg injected region of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001. Conclusion: We find that both α-PFF and AD brain homogenates give rise to increased binding of (d 3 )-[ 11 C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d 3 )-[ 11 C]MODAG-001 shows promise as a lead tracer for future radiotracer development. Alpha-synuclein PET tracer Positron emission tomography intracerebral protein injection amyloid-beta brain imaging larger animal PET pig model Figures Figure 1 Figure 2 Figure 3 Background Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are histopathologically characterized by progressive nigrostriatal, limbic and neocortical neurodegeneration and aggregation of the intracellular presynaptic protein α-synuclein [ 1 – 3 ]. These diseases are collectively known as α-synucleinopathies [ 4 ]. Patients with PD or DLB have α-synuclein-rich neuronal inclusions called Lewy bodies and Lewy neurites, predominantly in the substantia nigra in PD and throughout the cerebral cortex in DLB [ 5 ]. On the other hand, patients with MSA show filamentous aggregates in oligodendrocytes and neurons [ 6 ]. However, it is yet unknown to which extent α-synuclein aggregates contribute to neurodegeneration (Wong and Krainc 2017), and further, the clinical diagnosis of a PD or PD+ disorder is difficult, particularly in the early phases [ 7 ]. In drug-naive patients with subtle clinical parkinsonian motor symptoms, dopamine transporter neuroimaging has high sensitivity and specificity in distinguishing between patients with and without striatal neurodegeneration [ 8 ] but access to a neuroimaging tool to specifically assess α-synuclein aggregates would be a highly valuable addition. Positron emission tomography (PET) has proven valuable for the detection of amyloid-β and tau protein aggregates and is used for differential diagnosis and drug development evaluation for neurodegenerative conditions such as Alzheimer’s disease (AD) [ 9 ]. PET imaging of α-synuclein would be advantageous for, e.g., early disease detection, differential diagnosis, and monitoring disease progression of synucleinopathies. In addition, the field is moving towards early eradication of α-synuclein aggregates as a promising therapeutic strategy in α-synucleinopathies, an approach that would require in vivo imaging for clinical application. As of today, no clinically validated PET radioligand exists for imaging α-synuclein [ 10 , 11 ]. Several attempts to develop a suitable radioligand for α-synuclein have been made, and some tracers looked promising in rodents [ 12 – 16 ]. One of these is the diphenylpyrazole derivative [ 11 C]MODAG-001/(d 3 )-[ 11 C]MODAG-001 [ 12 ]. It was developed from the lead structure anle138b, a compound with therapeutic properties in PD and MSA rodent models due to its binding characteristics to α-synuclein aggregates [ 17 , 18 ]. Anle138b and its derivatives, like [ 3 H]/[ 11 C]MODAG-001, have undergone extensive in vitro and rodent biodistribution experiments [ 12 , 19 ]. (d 3 )-[ 11 C]MODAG-001 showed the most promise as a candidate radioligand for detecting α-synuclein aggregates due to its high affinity, good brain penetration, and ability to detect α-synuclein pre-formed fibril (α-PFF) in a protein deposition rat model [ 12 ]. In the present study, we test and characterize (d 3 )-[ 11 C]MODAG-001 in a large animal model. To evaluate binding characteristics to α-synuclein aggregates, we use a pig model where α-PFF is intracerebrally injected (ICI) immediately before scanning, creating an artificial target brain region [ 20 ]. We assess the sensitivity of the radioligand to identify α-synucleinopathy DLB human brain homogenate injected in the pig brain. The binding selectivity to α-synuclein is assessed by comparison to a brain region where amyloid and tau pathology-rich AD human brain homogenate is injected. Methods Radiochemistry Precursor and reference compound for (d 3 )-[ 11 C]MODAG-001 were prepared as previously described [ 12 ]; see supplementary information for more details. (d 3 )-[ 11 C]MODAG-001 was obtained by reductive amination of desmethyl precursor with [ 11 C]CH 2 O. The radioactivity yield was 650±297 MBq (mean±SD) (n=9, range 250-1214) after 70 min of synthesis time. The radiochemical purity of the formulated tracer was >95%. Radiochemical conversion from trapped [11C]CH3I was 45±2% (n=6). Molar activity at the end of the radioligand synthesis was on average 28.14±5.3 GBq/µmol. Animals We included four female domestic pigs (crossbreed of Landrace, Yorkshire, and Duroc) weighing 27±1 kg and aged 10-11 weeks (Table 1 ). Before any experiments, pigs were sourced from a local farm and acclimatized for 7-10 days in an enriched environment. Preparation and surgical procedure A detailed description of the preparation, anesthesia, surgery, and transport has previously been described [ 21 , 22 ]. Briefly, anesthesia was induced with an intramuscular (IM) injection of Zoletil mixture and maintained with 10-15 mg/kg/h propofol intravenous (IV) infusion. Analgesia was achieved with 5 µg/kg/h fentanyl IV infusion. Endotracheal intubation allowed for ventilation with 34% oxygen in normal air at 10-12 mL/kg. The left and right superficial mammary veins, ear veins, and femoral arteries were catheterized for venous and arterial access. The animals' heart rate, blood pressure, peripheral oxygen saturation (SpO 2 ), end-tidal CO 2 (EtCO 2 ), blood glucose, and temperature were monitored throughout the scan. Using a modified stereotactic approach [ 20 ], pigs were intracerebrally injected into the medial prefrontal cortex (mPFC) with 25 µL of 3 or 6 mg/mL α-PFF (molecular weight of monomer: 14,460 Da, corresponding to 208 µM or 415 µM) (produced at H. Lundbeck A/S, Copenhagen, Denmark), AD human brain homogenate (10% homogenate in saline [α-synuclein aggregates -ve]) or DLB human brain homogenate (10% homogenate in saline [amyloid-β and tau aggregates -ve]), as outlined for each pig in Table 1 . The post-mortem human brain homogenates used in this study are the same as described previously [ 20 ], namely a homogenate mixture of two regions (frontal and temporal) from 2 different patients with each disease (i.e., AD and DLB). In a previous study, the injection target point in the mPFC: 8, 25, 14 mm in X, Y, Z coordinates relative to bregma was validated [ 20 ]. After the surgical procedure, the animals were transported to the scanner facilities. Table 1 Pig characteristics: Body weight, injectate in the PFC, injected dose/mass of (d 3 )-[ 11 C]MODAG-001, and availability of blocking and test-retest scans. Pig no. Weight (kg) Injection in the right PFC Injection in the left PFC (d 3 )-[ 11 C]MODAG-001 MODAG-001 blocking study Test- retest Scan 1 : Injected dose (MBq) & mass (µg) Scan 2 : Injected dose (MBq) & mass (µg) 1 28 DLB homogenate 150µg ⍺-PFF 181 MBq (3.18 µg) - - - 2 25 AD homogenate 150µg ⍺-PFF 334 MBq (7.43 µg) - - - 3 26 75µg ⍺-PFF 150µg ⍺-PFF 359 MBq (6.75 µg) 436 MBq (8 µg) - ✓ 4 29 75µg ⍺-PFF 150µg ⍺-PFF 322 MBq (6.64 µg) 302 MBq (4.58 µg) ✓ - 150µg α-PFF : α-synuclein preformed fibrils (150µg/25µL, 415 µM) 75µg α- PFF : α-synuclein preformed fibrils (75µg/25µL, 208 µM) DLB homogenate : Dementia with Lewy bodies human brain homogenate (10%, 25µL) [Braak stage II, n=2 x2 regions, Aβ and tau -ve] AD homogenate : Alzheimer’s disease human brain homogenate (10%, 25µL) [Braak stage IV, n=2 x2 regions, α-syn -ve] MODAG-001 block : 1 mg/kg dissolved in 19% dimethyl sulfoxide in saline PET scanning protocol Pigs were PET-scanned either once or twice (same day) in a Siemens high-resolution research tomograph (HRRT) scanner (CTI /Siemens, Malvern, PA, USA). (d 3 )-[ 11 C]MODAG-001 was injected as a rapid bolus (~20 seconds) through one of the superficial mammary veins (IV), and PET data were acquired over 121 min. Molar activity at the time of injection was 19.0±2.1 GBq/µmol (injected dose and mass in Table 1 ). Pig 3 received a test-retest on the same day. In Pig 4, we perform a self-blocking study with 1 mg/kg non-deuterated unlabelled MODAG-001. Unlabelled MODAG-001 (29.1 mg) was dissolved in 40 mL of saline with 19% dimethyl sulfoxide to ensure full solubility and injected IV over 15 min starting ~6 min before the injection of (d 3 )-[ 11 C]MODAG-001. Blood sampling and radio-HPLC analysis Radio-HPLC analysis of plasma samples were performed in Pig 4 for both baseline and block scans. Manual arterial blood samples were drawn at 1.5, 5, 20, 40, and 60 min after injection. Samples were also drawn at 90 and 120 min, but data is not shown due to low and noisy radioactivity counts. Pig 4 also received a third injection of (d 3 )-[ 11 C]MODAG-001 (180 MBq, 3.74 µg) to assess radiometabolites crossing the blood-brain barrier for which a blood and brain sample was acquired at 15 min and 22 min post tracer injection. A blood sample was drawn before injection of 20 mL pentobarbital/lidocaine for euthanasia. Immediately after, the skull was exposed, and the occipital bone was sawed open. A small brain sample from the occipital cortex was excised and rinsed in saline to remove excessive blood. Radiolabeled parent and metabolite fractions were determined in plasma and brain tissue using isocratic elution, as previously described [ 12 ], but with some modifications (details in Supplementary information). In vitro methodologies After the last scanning, the animals were euthanized by IV injection of 20 mL pentobarbital and lidocaine. After euthanasia, the brains were removed, snap-frozen with powdered dry-ice, and stored at −20°C until further use. Intracerebrally injections were confirmed using fluorescence immunohistochemistry; procedure and results are available in the supplementary data. PET data reconstruction and preprocessing PET scans were reconstructed using ordinary Poisson 3D ordered subsets expectation-maximization, including modeling the point-spread function, using 16 subsets, ten iterations, and standard corrections [ 23 ]. Attenuation correction was performed using the MAP-TR µ-map [ 24 ]. Emission data were binned into time frames of increasing lengths: 6 × 10 s, 6 × 20 s, 4 × 30 s, 9 × 60 s, 2 × 180 s, 8 × 300 s, and 3 × 600 s. Each frame consisted of 207 planes of 256 × 256 voxels of 1.22 × 1.22 × 1.22 mm in size. Brain parcellation was performed according to our previously published automatic PET-MR pig brain atlas method [ 25 ]. The input for the methodology was frame-length weighted, summed PET images of the total scan time (0–120 min). Time-activity curves (TACs) from the neocortex, occipital cortex, temporal cortex, cerebellum (here defined as without vermis), and injection regions were extracted for the present study. The regions of the injection sites were delineated as described in our previous study [ 20 ], while all other regions were part of the Saikali atlas [ 26 ] modified for PET [ 25 ]. Pharmacokinetic modeling For image quantification, we used the non-invasive Logan graphical analysis [ 27 ] with the occipital cortex and cerebellum as reference regions. In order to estimate the average k 2 over R 1 ratio (k 2 ’), we applied the simplified reference tissue model (SRTM) [ 28 ] to high binding regions (i.e., α-PFF injected regions) and calculated k 2 ’. For the non-invasive Logan plot, we chose the threshold time, t*, of 23 min (last 15 frames) since it showed the lowest average maximum percentage of variance. BP ND values estimated using the occipital cortex as a reference region were more stable than those derived using the cerebellum. These are therefore presented in the results section below. All kinetic modeling was performed using the “kinfitr” package (v. 0.6.1) (Matheson, 2019; Tjerkaski et al., 2020) in R (v. 4.0.2; “Taking Off Again,” R core team, Vienna, Austria). For the pig that received a test-retest scan, we calculated the % test-rest change using Equation 1. For the pig that received a baseline-block scan, we calculated the % blocking in the α-PFF injected regions using Equation 2. Regional radioactivity concentration (kBq/mL) was normalized to injected dose (MBq) and corrected for the animal weight (kg) to provide standardized uptake values (SUV, g/mL) used in graphical plots in Figures 1 and 3 . PMOD 3.7 (PMOD Technologies, Zürich, Switzerland) was used to visualize and create all representative PET images (Figure 1 and 3 ), which are summed images over the entire period of the scan (0-121 min) with the “Triangle” PMOD pixel interpolation function; for more details see “ https://www.pmod.com/files/download/v31/doc/pbas/4145.htm ”. Graph-Pad Prism (v. 9.2.0; GraphPad Software, San Diego, CA, USA) was used for data visualization. Results Brain uptake and kinetics of (d 3 )-[ 11 C]MODAG-001 We observed high brain uptake (~ 2.5 SUV) and a relatively quick radioligand wash-out after (d 3 )-[ 11 C]MODAG-001 injection. The plasma kinetics of (d 3 )-[ 11 C]MODAG-001 were relatively fast, with approximately 10% of the parent radioligand remaining in plasma after 20 min (Supplementary Figure 1). Regions with either 150 µg (n = 4) or 75 µg (n = 2) α-PFF and AD homogenate (n = 1) had higher radioactivity retention (Figure 1 A-C and Figure 2 A) compared to the occipital cortex and cerebellum. By contrast, the DLB homogenate region (n = 1) TAC behaved essentially as background tissue radiotracer retention (Figure 1 A). Almost identical TACs were seen in the pig with test-retest scans (Supplementary Figure 2). In a pig euthanized 15 min after tracer injection, 10.8% of (d 3 )-[ 11 C]MODAG-001 parent compound remained in the plasma while 56.1% parent compound remained in brain homogenate from the occipital cortex (Supplementary Table 1). The remaining signal from the plasma and brain came from polar and non-polar radiometabolites (Supplementary Table 1). Blocking experiment using MODAG-001 Pretreatment with 1 mg/kg MODAG-001 shortly before the injection of (d 3 )-[ 11 C]MODAG-001 significantly reduced the radioactive signal in the 150 µg and 75 µg α-PFF regions, which showed substantially faster radioligand kinetics than the regional baseline TACs (Figure 2 B); the TACs became comparable to those in the occipital cortex and cerebellum (Figure 3 B). Kinetic modeling of (d 3 )-[ 11 C]MODAG-001 BP ND in different brain regions are shown in Figure 3 A. BP ND in the 150 µg α-PFF regions was 0.78 ± 0.1 (mean±SD, n = 4) while in the 75 µg regions, BP ND α-PFF injected regions was 0.29 (n = 2), showing a dose-dependent effect of (d 3 )-[ 11 C]MODAG-001 binding to the α-PFF. BP ND in the AD homogenate region was 0.73, in the same order as the 150 µg α-PFF. The DLB homogenate region, cerebellum, and temporal cortex had BP ND values close to zero (Figure 3 A). The (d 3 )-[ 11 C]MODAG-001 test-retest scan on the same day showed a -6.2% change in BP ND (Supplementary figure 2). Pretreatment with MODAG-001 resulted in a reduction in regional binding levels such that they became comparable to the reference regions. In the pig that underwent a baseline-block study, we observed >100% occupancy in the α-PFF injected regions. A modest reduction in binding was also observed in the temporal cortex and cerebellum (Figure 3 B). Discussion PET neuroimaging of aggregated protein has proved critical for diagnosing and monitoring disease progression and treatment evaluation in neurodegenerative diseases with amyloid-ꞵ and tau pathology [ 29 , 30 ]. The ability to detect and quantify α-synuclein aggregates in the living human brain would be a milestone achievement for the research of PD and other α-synucleinopathies [ 10 , 31 ]. Due to its high affinity to α-synuclein and favorable binding in rodent models, [ 11 C]MODAG-001 and its analogs are currently some of the most promising radioligands for α-synuclein neuroimaging [ 12 , 19 ]. To the best of our knowledge, this is the first time (d 3 )-[ 11 C]MODAG-001 has been tested in a higher species and shown promising translational results. We evaluated (d 3 )-[ 11 C]MODAG-001 in a pig model of intracerebral injection of α-PFF and postmortem human AD and DLB brain homogenates. We see high brain uptake and quick-wash out of the radioligand in the brain. The pharmacokinetics in healthy mice and the α-PFF rat model were comparable to that in pigs [ 12 ]. We saw a relatively high uptake of the radioligand in the α-PFF regions at micromolar concentrations, with a dose-dependent response with 150 µg (415 µM) and 75 µg (208 µM) injections (Figure 1 - 3 ). Kuebler et al. tested both [ 11 C]MODAG-001 and the deuterium incorporated (d 3 )-[ 11 C]MODAG-001 [ 12 ]; deuterium incorporation was meant to improve the pharmacokinetic and metabolic profile of the radioligand [ 32 ]. Notably, we observed faster metabolism in the pigs than what was observed in the mice, which are much smaller mammals [ 33 ]. The results showed that ~10% parent fraction remained 15 min post-injection in the pigs, compared to ~30% parent fraction in mice. Radio-HPLC on brain homogenate (non-perfused) from a pig euthanized at 15 min showed ~50% parent fraction; in contrast, mice showed on average ~90% parent fraction after 15 min (Supplementary Figure 1, Supplementary Table 1). We performed the non-invasive kinetic modeling with the occipital cortex as a reference region since we previously have shown in our pig model that the occipital cortex has similar tissue properties as saline-injected target regions and that these are not affected by the intracerebral injection [ 22 ]. Due to the lack of other high-affinity molecules, an unlabelled MODAG-001 block scan was our best option to examine the signal specificity. Pretreatment of 1 mg/kg of MODAG-001 leads to complete blocking of the specific (d 3 )-[ 11 C]MODAG-001 binding in the α-PFF injected region. We observe a very high blocking percentage with values above 100% (using BP ND values from reference modeling), although these estimates are based on only one pig and likely prone to noise. We also see high uptake in the amyloid-ꞵ and tau-rich AD homogenates but no significant uptake in the DLB homogenate region (Figure 1 and 3 ); this is remarkable since DLB is considered to have a pure α-synuclein pathology. Ideally, a radioligand should have high α-synuclein selectivity for it to distinguish α-synuclein aggregates from amyloid-ꞵ and tau aggregates [ 10 , 34 ]. Several things make us less enthusiastic about the prospect of (d 3 )-[ 11 C]MODAG-001 as a radioligand in human studies: (d 3 )-[ 11 C]MODAG-001 did not display high binding in the DLB homogenate region; this could be due to low concentrations of aggregated α-synuclein, as is most often seen in human pathology, especially at early disease stages. This null-finding could also be due to the hypothesized difference in pathological morphology in pure α-synuclein DLB subjects [ 35 ]. (d 3 )-[ 11 C]MODAG-001 was also not very selective for α-synuclein and had significant binding to the AD homogenate region. This observation is also on par with previous autoradiography studies where the highest uptake was noted in human brain sections with AD [ 12 ]. Improving the signal-to-background ratio and selectivity will be critical for the further development of the tracer, and this work is currently ongoing [ 12 ]. The intracerebral protein injection model used in the current study also comes with a set of limitations. Since the intracerebral injections are done a few hours prior to scanning, it is unlikely that protein aggregates enter into the brain cells, which does not mimic the intracellular inclusions seen in α-synucleinopathies well [ 3 , 5 ]. The concentration of the α-PFF in the model is much higher than that of diseased brains, where α-synuclein is found to be at nanomolar concentration [ 12 , 36 ]. This particular setup allowed us to show proof of concept for α-synuclein aggregate detecting radioligands. The signal-to-background ratio of (d 3 )-[ 11 C]MODAG-001 makes it challenging to detect pathologically relevant α-synuclein, i.e., at nanomolar concentrations. In spite of the poor specificity and relatively modest signal-to-background ratio, we believe that (d 3 )-[ 11 C]MODAG-001 with its high affinity for α-synuclein is a suitable lead molecule for further radioligand development and evaluation. Conclusions We demonstrate in vivo detection of α-PFF in pigs using (d 3 )-[ 11 C]MODAG-001, which has previously only been shown using a similar α-PFF injection rat model. The radioligand shows excellent brain kinetics and test-retest variability. Although (d 3 )-[ 11 C]MODAG-001 displays low specificity towards α-synuclein and a potential passage of radiometabolites through the blood-brain barrier, it shows promise as a lead tracer for further radiotracer development. Abbreviations α-PFF: α-synuclein preformed fibrils AD: Alzheimer’s disease BP ND : binding potential non-displaceable DLB: dementia with Lewy bodies HRRT: high-resolution research tomograph IV: intra-venous IM: intra-muscular mPFC: medial prefrontal cortex MSA: multiple system atrophy PD: Parkinson’s disease PET: positron emission tomography R-HPLC: radio-high performance liquid chromatography SRTM: simplified reference tissue model SUV: standardized uptake values TAC: time-activity curve Declarations Ethics approval All animal procedures were performed in accordance with the European Commission's Directive 2010/63/EU, as well as the ARRIVE guidelines, and were approved by the Danish Council of Animal Ethics (Journal no. 2017-15-0201-01375). Consent for publication Not applicable Availability of data and material All data, including R scripts, is available at a GitHub repository (https://github.com/nakulrrraval/Protien_inj_pig_model_MODAG001). All other requests are directed to this article's corresponding or first author. Competing interests Lundbeck A/S, Denmark provided the α-synuclein preformed fibrils as part of the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 813528. However, they had no other financial interests in the project. GMK received honoraria as a speaker and consultant for Sage Pharmaceuticals/Biogen and Sanos A/S. All other authors declare no conflict of interest. Funding This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 813528. This project also received funding from Parkinsonforeningen, Denmark (R16-A247). Pontus Plavén Sigray was supported by the Lundbeck Foundation (R303-2018-3263). Vladimir Shalgunov was supported by BRIDGE – Translational Excellence Program at the Faculty of Health and Medical Sciences, University of Copenhagen, funded by the Novo Nordisk Foundation (grant agreement no. NNF18SA0034956). Authors’ contribution NRR, MMH, HDH, PPS, GMK: conceptualization and design. NRR, CAM, EEB, LMJ, HDH: surgical setup and PET scanning. VS, AN, UMB: compound synthesis, radiochemistry, and HPLC analysis. NRR, VS, AN, UMB, MJ, PPS: analysis and software. NRR, MMH, HDH, GMK: resources. NR, HDH, PPS, GMK: data curation. LMJ, MMH, HDH, PPS, GMK: supervision. NRR: preparation of manuscript draft including figures. NRR, CAM, VS, AN, UMB, EEB, MJ, LMJ, MMH, HDH, PPS, GMK: manuscript review and editing. NRR, MMH, GMK: funding acquisition. All authors have read and agreed to the current version of the manuscript. Acknowledgments We want to thank Lundbeck A/S, Valby, Denmark, for providing the α-synuclein preformed fibrils. 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Research Square. https://doi.org/10.21203/rs.3.rs-313183/v1 Raval NR, Nasser A, Madsen CA, et al (2022) An in vivo pig model for testing novel PET radioligands targeting cerebral protein aggregates. bioRxiv 2021.12.31.473908 Sureau FC, Reader AJ, Comtat C, Leroy C, Ribeiro MJ, Buvat I, Trébossen R (2008) Impact of image-space resolution modeling for studies with the high-resolution research tomograph. J Nucl Med 49:1000–1008 Keller SH, Svarer C, Sibomana M (2013) Attenuation correction for the HRRT PET-scanner using transmission scatter correction and total variation regularization. IEEE Trans Med Imaging 32:1611–1621 Villadsen J, Hansen HD, Jørgensen LM, Keller SH, Andersen FL, Petersen IN, Knudsen GM, Svarer C (2017) Automatic delineation of brain regions on MRI and PET images from the pig. J Neurosci Methods 294:51–58 Saikali S, Meurice P, Sauleau P, Eliat P-A, Bellaud P, Randuineau G, Vérin M, Malbert C-H (2010) A three-dimensional digital segmented and deformable brain atlas of the domestic pig. J Neurosci Methods 192:102–109 Logan J, Fowler JS, Volkow ND, Wang GJ, Ding YS, Alexoff DL (1996) Distribution volume ratios without blood sampling from graphical analysis of PET data. J Cereb Blood Flow Metab 16:834–840 Lammertsma AA, Hume SP (1996) Simplified reference tissue model for PET receptor studies. Neuroimage 4:153–158 Barthel H, Sabri O (2017) Clinical Use and Utility of Amyloid Imaging. J Nucl Med 58:1711–1717 Shah M, Catafau AM (2014) Molecular Imaging Insights into Neurodegeneration: Focus on Tau PET Radiotracers. J Nucl Med 55:871–874 Eberling JL, Dave KD, Frasier MA (2013) α-synuclein imaging: a critical need for Parkinson’s disease research. J Parkinsons Dis 3:565–567 Klenner MA, Pascali G, Fraser BH, Darwish TA (2021) Kinetic isotope effects and synthetic strategies for deuterated carbon-11 and fluorine-18 labelled PET radiopharmaceuticals. Nucl Med Biol 96-97:112–147 Tang H, Mayersohn M (2018) Porcine prediction of pharmacokinetic parameters in people: A pig in a poke? Drug Metab Dispos 46:1712–1724 Alpha-Synuclein Imaging Prize. https://www.michaeljfox.org/news/alpha-synuclein-imaging-prize. Accessed 24 Dec 2021 Peng C, Gathagan RJ, Lee VM-Y (2018) Distinct α-Synuclein strains and implications for heterogeneity among α-Synucleinopathies. Neurobiol Dis 109:209–218 Morgan SA, Lavenir I, Fan J, Masuda-Suzukake M, Passarella D, DeTure MA, Dickson DW, Ghetti B, Goedert M (2020) α-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species. J Biol Chem 295:6652–6664 Supplementary Files 202201MODAG001supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1268531","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":88574757,"identity":"0e2e7ce4-abfe-4774-a909-ea5531c44d1e","order_by":0,"name":"Nakul Ravi Raval","email":"","orcid":"https://orcid.org/0000-0001-5637-7219","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Nakul","middleName":"Ravi","lastName":"Raval","suffix":""},{"id":88574758,"identity":"1053e395-e848-48de-97bc-1a738ceef949","order_by":1,"name":"Clara Aabye Madsen","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Clara","middleName":"Aabye","lastName":"Madsen","suffix":""},{"id":88574759,"identity":"3a3db25e-2d63-420b-81ab-48c02ab43e8d","order_by":2,"name":"Vladimir Shalgunov","email":"","orcid":"","institution":"Kobenhavns Universitet","correspondingAuthor":false,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Shalgunov","suffix":""},{"id":88574760,"identity":"71ee1544-f79f-40d4-b1c9-1d6a2ca73f9f","order_by":3,"name":"Arafat Nasser","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Arafat","middleName":"","lastName":"Nasser","suffix":""},{"id":88574761,"identity":"74acc4f1-4af9-43b8-a6fe-03601f087777","order_by":4,"name":"Umberto Maria Battisti","email":"","orcid":"","institution":"Kobenhavns Universitet","correspondingAuthor":false,"prefix":"","firstName":"Umberto","middleName":"Maria","lastName":"Battisti","suffix":""},{"id":88574762,"identity":"0c4b48dc-bb33-42a5-beb4-6f9b6c87d1b6","order_by":5,"name":"Emily Eufaula Beaman","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"Eufaula","lastName":"Beaman","suffix":""},{"id":88574763,"identity":"7190cc96-5fd1-4512-86c2-d5b1f94c35ec","order_by":6,"name":"Morten Juhl","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Morten","middleName":"","lastName":"Juhl","suffix":""},{"id":88574764,"identity":"e081a7d9-c9cb-41c0-b6a1-8b68de2695a6","order_by":7,"name":"Louise Møller Jørgensen","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Louise","middleName":"Møller","lastName":"Jørgensen","suffix":""},{"id":88574765,"identity":"1f849d16-c9e4-4bb4-a673-294a7e0106bd","order_by":8,"name":"Matthias Manfred Herth","email":"","orcid":"","institution":"Kobenhavns Universitet","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"Manfred","lastName":"Herth","suffix":""},{"id":88574766,"identity":"195c2109-60f8-4c8b-bcee-747f4a48ff60","order_by":9,"name":"Hanne Demant Hansen","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Hanne","middleName":"Demant","lastName":"Hansen","suffix":""},{"id":88574767,"identity":"92a7d358-be74-45df-a381-fa325782dbc2","order_by":10,"name":"Pontus Plavén-Sigray","email":"","orcid":"","institution":"Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Pontus","middleName":"","lastName":"Plavén-Sigray","suffix":""},{"id":88574768,"identity":"9fcd3942-a698-4127-b75b-480bb1da1093","order_by":11,"name":"Gitte Moos Knudsen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAiklEQVRIiWNgGAWjYFACNsYHYPoMCVqYDUjWwiZBmhb5sGNp1YV7DjPwnTlApBbD22nHbs94dphB8mwDsVpmp7fd5jlwmMHgPLEOA2kpJk2LvHTaMWawFqIdZiCdlizNcyCdR5Jo78vPTjP8zHPAWo7vTAKxtkDN5iFSPciWBuLVjoJRMApGwUgFADEcKKsgc1h/AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1508-6866","institution":"Rigshospitalet","correspondingAuthor":true,"prefix":"","firstName":"Gitte","middleName":"Moos","lastName":"Knudsen","suffix":""}],"badges":[],"createdAt":"2022-01-17 11:56:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1268531/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1268531/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18986701,"identity":"e9b9bb92-0f6d-47ce-8aef-d1cbfa03650d","added_by":"auto","created_at":"2022-03-08 15:41:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9993400,"visible":true,"origin":"","legend":"\u003cp\u003eRegional TACs of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 in pigs injected with 150 µg α-PFF and A) DLB homogenate, B) AD homogenate and C)\u0026nbsp;75 µg α-PFF. TACs for the two reference regions, ie, the occipital cortex and cerebellum, are also shown. D) SUV-scaled PET images from representative TACs.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1268531/v1/fd5dba4932dea37660af900a.jpg"},{"id":18986702,"identity":"fb00dded-c27e-4f44-a073-1f971bebc2ad","added_by":"auto","created_at":"2022-03-08 15:41:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5631618,"visible":true,"origin":"","legend":"\u003cp\u003e(d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 baseline and block.\u003cstrong\u003e \u003c/strong\u003eTACs and SUV scaled PET images A) (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 baseline and B) (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001+ MODAG-001 (1 mg/kg) block scan from a pig with 150 µg and 75 µg α-PFF.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1268531/v1/f001ebda21fa1158740779e6.jpg"},{"id":18986699,"identity":"3973425b-04ba-4880-adee-9355865c2bbf","added_by":"auto","created_at":"2022-03-08 15:41:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2474158,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic modeling outcomes of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001.\u003cstrong\u003e \u003c/strong\u003eA) BP\u003csub\u003eND\u003c/sub\u003e as determined with the non-invasive Logan graphical analysis using the occipital cortex as a reference region, in the injected brain regions, temporal cortex, and cerebellum. Retest and block are not included. B) BP\u003csub\u003eND\u003c/sub\u003e at baseline after (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 blocking.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1268531/v1/7332e190d4d0a2c93ae1541a.jpg"},{"id":21944662,"identity":"eaebf665-cc92-4309-9df0-70135e32e9d0","added_by":"auto","created_at":"2022-05-27 04:31:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":711677,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1268531/v1/8ba0f85f-c697-4a9a-aea0-3fea7789f7fa.pdf"},{"id":18986879,"identity":"a9c62c10-c2ef-482b-b947-2cfefb0bc7c5","added_by":"auto","created_at":"2022-03-08 15:44:30","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":625764,"visible":true,"origin":"","legend":"","description":"","filename":"202201MODAG001supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-1268531/v1/14eb4b4c6d1c16d3f1207bad.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEvaluation of the α-synuclein PET Radiotracer (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 in Pigs\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are histopathologically characterized by progressive nigrostriatal, limbic and neocortical neurodegeneration and aggregation of the intracellular presynaptic protein \u0026alpha;-synuclein [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. These diseases are collectively known as \u0026alpha;-synucleinopathies [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Patients with PD or DLB have \u0026alpha;-synuclein-rich neuronal inclusions called Lewy bodies and Lewy neurites, predominantly in the substantia nigra in PD and throughout the cerebral cortex in DLB [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. On the other hand, patients with MSA show filamentous aggregates in oligodendrocytes and neurons [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, it is yet unknown to which extent \u0026alpha;-synuclein aggregates contribute to neurodegeneration (Wong and Krainc 2017), and further, the clinical diagnosis of a PD or PD+ disorder is difficult, particularly in the early phases [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. In drug-naive patients with subtle clinical parkinsonian motor symptoms, dopamine transporter neuroimaging has high sensitivity and specificity in distinguishing between patients with and without striatal neurodegeneration [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e] but access to a neuroimaging tool to specifically assess \u0026alpha;-synuclein aggregates would be a highly valuable addition.\u003c/p\u003e\n\u003cp\u003ePositron emission tomography (PET) has proven valuable for the detection of amyloid-\u0026beta; and tau protein aggregates and is used for differential diagnosis and drug development evaluation for neurodegenerative conditions such as Alzheimer\u0026rsquo;s disease (AD) [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. PET imaging of \u0026alpha;-synuclein would be advantageous for, e.g., early disease detection, differential diagnosis, and monitoring disease progression of synucleinopathies. In addition, the field is moving towards early eradication of \u0026alpha;-synuclein aggregates as a promising therapeutic strategy in \u0026alpha;-synucleinopathies, an approach that would require in vivo imaging for clinical application. As of today, no clinically validated PET radioligand exists for imaging \u0026alpha;-synuclein [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eSeveral attempts to develop a suitable radioligand for \u0026alpha;-synuclein have been made, and some tracers looked promising in rodents [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. One of these is the diphenylpyrazole derivative [\u003csup\u003e11\u003c/sup\u003eC]MODAG-001/(d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. It was developed from the lead structure anle138b, a compound with therapeutic properties in PD and MSA rodent models due to its binding characteristics to \u0026alpha;-synuclein aggregates [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Anle138b and its derivatives, like [\u003csup\u003e3\u003c/sup\u003eH]/[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001, have undergone extensive in vitro and rodent biodistribution experiments [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 showed the most promise as a candidate radioligand for detecting \u0026alpha;-synuclein aggregates due to its high affinity, good brain penetration, and ability to detect \u0026alpha;-synuclein pre-formed fibril (\u0026alpha;-PFF) in a protein deposition rat model [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn the present study, we test and characterize (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 in a large animal model. To evaluate binding characteristics to \u0026alpha;-synuclein aggregates, we use a pig model where \u0026alpha;-PFF is intracerebrally injected (ICI) immediately before scanning, creating an artificial target brain region [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. We assess the sensitivity of the radioligand to identify \u0026alpha;-synucleinopathy DLB human brain homogenate injected in the pig brain. The binding selectivity to \u0026alpha;-synuclein is assessed by comparison to a brain region where amyloid and tau pathology-rich AD human brain homogenate is injected.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eRadiochemistry\u003c/h2\u003e\n\u003cp\u003ePrecursor and reference compound for (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 were prepared as previously described [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]; see supplementary information for more details. (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 was obtained by reductive amination of desmethyl precursor with [\u003csup\u003e11\u003c/sup\u003eC]CH\u003csub\u003e2\u003c/sub\u003eO. The radioactivity yield was 650\u0026plusmn;297 MBq (mean\u0026plusmn;SD) (n=9, range 250-1214) after 70 min of synthesis time. The radiochemical purity of the formulated tracer was \u0026gt;95%. Radiochemical conversion from trapped [11C]CH3I was 45\u0026plusmn;2% (n=6). Molar activity at the end of the radioligand synthesis was on average 28.14\u0026plusmn;5.3 GBq/\u0026micro;mol.\u003c/p\u003e\n\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003eWe included four female domestic pigs (crossbreed of Landrace, Yorkshire, and Duroc) weighing 27\u0026plusmn;1 kg and aged 10-11 weeks (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Before any experiments, pigs were sourced from a local farm and acclimatized for 7-10 days in an enriched environment.\u003c/p\u003e\n\u003ch2\u003ePreparation and surgical procedure\u003c/h2\u003e\n\u003cp\u003eA detailed description of the preparation, anesthesia, surgery, and transport has previously been described [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, anesthesia was induced with an intramuscular (IM) injection of Zoletil mixture and maintained with 10-15 mg/kg/h propofol intravenous (IV) infusion. Analgesia was achieved with 5 \u0026micro;g/kg/h fentanyl IV infusion. Endotracheal intubation allowed for ventilation with 34% oxygen in normal air at 10-12 mL/kg. The left and right superficial mammary veins, ear veins, and femoral arteries were catheterized for venous and arterial access. The animals\u0026apos; heart rate, blood pressure, peripheral oxygen saturation (SpO\u003csub\u003e2\u003c/sub\u003e), end-tidal CO\u003csub\u003e2\u003c/sub\u003e (EtCO\u003csub\u003e2\u003c/sub\u003e), blood glucose, and temperature were monitored throughout the scan. Using a modified stereotactic approach [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], pigs were intracerebrally injected into the medial prefrontal cortex (mPFC) with 25 \u0026micro;L of 3 or 6 mg/mL \u0026alpha;-PFF (molecular weight of monomer: 14,460 Da, corresponding to 208 \u0026micro;M or 415 \u0026micro;M) (produced at H. Lundbeck A/S, Copenhagen, Denmark), AD human brain homogenate (10% homogenate in saline [\u0026alpha;-synuclein aggregates -ve]) or DLB human brain homogenate (10% homogenate in saline [amyloid-\u0026beta; and tau aggregates -ve]), as outlined for each pig in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The post-mortem human brain homogenates used in this study are the same as described previously [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], namely a homogenate mixture of two regions (frontal and temporal) from 2 different patients with each disease (i.e., AD and DLB). In a previous study, the injection target point in the mPFC: 8, 25, 14 mm in X, Y, Z coordinates relative to bregma was validated [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. After the surgical procedure, the animals were transported to the scanner facilities.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePig characteristics: Body weight, injectate in the PFC, injected dose/mass of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001, and availability of blocking and test-retest scans.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePig no.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eWeight (kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eInjection in the right PFC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eInjection in the left PFC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e(d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMODAG-001\u003c/p\u003e\n \u003cp\u003eblocking study\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTest-\u003c/p\u003e\n \u003cp\u003eretest\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eScan 1\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eInjected dose (MBq) \u0026amp; mass (\u0026micro;g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eScan 2\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eInjected dose (MBq) \u0026amp; mass (\u0026micro;g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003cp\u003ehomogenate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u0026micro;g\u003c/p\u003e\n \u003cp\u003e⍺-PFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e181 MBq\u003c/p\u003e\n \u003cp\u003e(3.18 \u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAD\u003c/p\u003e\n \u003cp\u003ehomogenate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u0026micro;g\u003c/p\u003e\n \u003cp\u003e⍺-PFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e334 MBq\u003c/p\u003e\n \u003cp\u003e(7.43 \u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u0026micro;g\u003c/p\u003e\n \u003cp\u003e⍺-PFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u0026micro;g\u003c/p\u003e\n \u003cp\u003e⍺-PFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e359 MBq\u003c/p\u003e\n \u003cp\u003e(6.75 \u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e436 MBq\u003c/p\u003e\n \u003cp\u003e(8 \u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u0026micro;g\u003c/p\u003e\n \u003cp\u003e⍺-PFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u0026micro;g\u003c/p\u003e\n \u003cp\u003e⍺-PFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e322 MBq\u003c/p\u003e\n \u003cp\u003e(6.64 \u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e302 MBq\u003c/p\u003e\n \u003cp\u003e(4.58 \u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003e\u003cstrong\u003e150\u0026micro;g \u0026alpha;-PFF\u003c/strong\u003e: \u0026alpha;-synuclein preformed fibrils (150\u0026micro;g/25\u0026micro;L, 415 \u0026micro;M)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e75\u0026micro;g \u0026alpha;- PFF\u003c/strong\u003e: \u0026alpha;-synuclein preformed fibrils (75\u0026micro;g/25\u0026micro;L, 208 \u0026micro;M)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDLB homogenate\u003c/strong\u003e: Dementia with Lewy bodies human brain homogenate (10%, 25\u0026micro;L) [Braak stage II, n=2 x2 regions, A\u0026beta; and tau -ve]\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAD homogenate\u003c/strong\u003e: Alzheimer\u0026rsquo;s disease human brain homogenate (10%, 25\u0026micro;L) [Braak stage IV, n=2 x2 regions, \u0026alpha;-syn -ve]\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMODAG-001 block\u003c/strong\u003e: 1 mg/kg dissolved in 19% dimethyl sulfoxide in saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePET scanning protocol\u003c/h2\u003e\n\u003cp\u003ePigs were PET-scanned either once or twice (same day) in a Siemens high-resolution research tomograph (HRRT) scanner (CTI /Siemens, Malvern, PA, USA). (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 was injected as a rapid bolus (~20 seconds) through one of the superficial mammary veins (IV), and PET data were acquired over 121 min. Molar activity at the time of injection was 19.0\u0026plusmn;2.1 GBq/\u0026micro;mol (injected dose and mass in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Pig 3 received a test-retest on the same day. In Pig 4, we perform a self-blocking study with 1 mg/kg non-deuterated unlabelled MODAG-001. Unlabelled MODAG-001 (29.1 mg) was dissolved in 40 mL of saline with 19% dimethyl sulfoxide to ensure full solubility and injected IV over 15 min starting ~6 min before the injection of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001.\u003c/p\u003e\n\u003ch2\u003eBlood sampling and radio-HPLC analysis\u003c/h2\u003e\n\u003cp\u003eRadio-HPLC analysis of plasma samples were performed in Pig 4 for both baseline and block scans. Manual arterial blood samples were drawn at 1.5, 5, 20, 40, and 60 min after injection. Samples were also drawn at 90 and 120 min, but data is not shown due to low and noisy radioactivity counts. Pig 4 also received a third injection of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 (180 MBq, 3.74 \u0026micro;g) to assess radiometabolites crossing the blood-brain barrier for which a blood and brain sample was acquired at 15 min and 22 min post tracer injection. A blood sample was drawn before injection of 20 mL pentobarbital/lidocaine for euthanasia. Immediately after, the skull was exposed, and the occipital bone was sawed open. A small brain sample from the occipital cortex was excised and rinsed in saline to remove excessive blood. Radiolabeled parent and metabolite fractions were determined in plasma and brain tissue using isocratic elution, as previously described [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e], but with some modifications (details in Supplementary information).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vitro\u003c/em\u003e methodologies\u003c/h2\u003e\n\u003cp\u003eAfter the last scanning, the animals were euthanized by IV injection of 20 mL pentobarbital and lidocaine. After euthanasia, the brains were removed, snap-frozen with powdered dry-ice, and stored at \u0026minus;20\u0026deg;C until further use. Intracerebrally injections were confirmed using fluorescence immunohistochemistry; procedure and results are available in the supplementary data.\u003c/p\u003e\n\u003ch2\u003ePET data reconstruction and preprocessing\u003c/h2\u003e\n\u003cp\u003ePET scans were reconstructed using ordinary Poisson 3D ordered subsets expectation-maximization, including modeling the point-spread function, using 16 subsets, ten iterations, and standard corrections [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Attenuation correction was performed using the MAP-TR \u0026micro;-map [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Emission data were binned into time frames of increasing lengths: 6 \u0026times; 10 s, 6 \u0026times; 20 s, 4 \u0026times; 30 s, 9 \u0026times; 60 s, 2 \u0026times; 180 s, 8 \u0026times; 300 s, and 3 \u0026times; 600 s. Each frame consisted of 207 planes of 256 \u0026times; 256 voxels of 1.22 \u0026times; 1.22 \u0026times; 1.22 mm in size. Brain parcellation was performed according to our previously published automatic PET-MR pig brain atlas method [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. The input for the methodology was frame-length weighted, summed PET images of the total scan time (0\u0026ndash;120 min). Time-activity curves (TACs) from the neocortex, occipital cortex, temporal cortex, cerebellum (here defined as without vermis), and injection regions were extracted for the present study. The regions of the injection sites were delineated as described in our previous study [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], while all other regions were part of the Saikali atlas [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] modified for PET [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch2\u003ePharmacokinetic modeling\u003c/h2\u003e\n\u003cp\u003eFor image quantification, we used the non-invasive Logan graphical analysis [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] with the occipital cortex and cerebellum as reference regions. In order to estimate the average k\u003csub\u003e2\u003c/sub\u003e over R\u003csub\u003e1\u003c/sub\u003e ratio (k\u003csub\u003e2\u003c/sub\u003e\u0026rsquo;), we applied the simplified reference tissue model (SRTM) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] to high binding regions (i.e., \u0026alpha;-PFF injected regions) and calculated k\u003csub\u003e2\u003c/sub\u003e\u0026rsquo;. For the non-invasive Logan plot, we chose the threshold time, t*, of 23 min (last 15 frames) since it showed the lowest average maximum percentage of variance. BP\u003csub\u003eND\u003c/sub\u003e values estimated using the occipital cortex as a reference region were more stable than those derived using the cerebellum. These are therefore presented in the \u003cspan class=\"InternalRef\"\u003eresults\u003c/span\u003e section below.\u003c/p\u003e\n\u003cp\u003eAll kinetic modeling was performed using the \u003cem\u003e\u0026ldquo;kinfitr\u0026rdquo; package\u003c/em\u003e (v. 0.6.1) (Matheson, 2019; Tjerkaski et al., 2020) in R (v. 4.0.2; \u0026ldquo;Taking Off Again,\u0026rdquo; R core team, Vienna, Austria).\u003c/p\u003e\n\u003cp\u003eFor the pig that received a test-retest scan, we calculated the % test-rest change using Equation 1. For the pig that received a baseline-block scan, we calculated the % blocking in the \u0026alpha;-PFF injected regions using Equation 2.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eRegional radioactivity concentration (kBq/mL) was normalized to injected dose (MBq) and corrected for the animal weight (kg) to provide standardized uptake values (SUV, g/mL) used in graphical plots in Figures \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. PMOD 3.7 (PMOD Technologies, Z\u0026uuml;rich, Switzerland) was used to visualize and create all representative PET images (Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), which are summed images over the entire period of the scan (0-121 min) with the \u0026ldquo;Triangle\u0026rdquo; PMOD pixel interpolation function; for more details see \u0026ldquo;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pmod.com/files/download/v31/doc/pbas/4145.htm\u003c/span\u003e\u003c/span\u003e\u0026rdquo;. Graph-Pad Prism (v. 9.2.0; GraphPad Software, San Diego, CA, USA) was used for data visualization.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eBrain uptake and kinetics of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001\u003c/h2\u003e\n\u003cp\u003eWe observed high brain uptake (~ 2.5 SUV) and a relatively quick radioligand wash-out after (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 injection. The plasma kinetics of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 were relatively fast, with approximately 10% of the parent radioligand remaining in plasma after 20 min (Supplementary Figure 1). Regions with either 150 \u0026micro;g (n = 4) or 75 \u0026micro;g (n = 2) \u0026alpha;-PFF and AD homogenate (n = 1) had higher radioactivity retention (Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-C and Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) compared to the occipital cortex and cerebellum. By contrast, the DLB homogenate region (n = 1) TAC behaved essentially as background tissue radiotracer retention (Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Almost identical TACs were seen in the pig with test-retest scans (Supplementary Figure 2). In a pig euthanized 15 min after tracer injection, 10.8% of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 parent compound remained in the plasma while 56.1% parent compound remained in brain homogenate from the occipital cortex (Supplementary Table 1). The remaining signal from the plasma and brain came from polar and non-polar radiometabolites (Supplementary Table 1).\u003c/p\u003e\n\u003ch2\u003eBlocking experiment using MODAG-001\u003c/h2\u003e\n\u003cp\u003ePretreatment with 1 mg/kg MODAG-001 shortly before the injection of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 significantly reduced the radioactive signal in the 150 \u0026micro;g and 75 \u0026micro;g \u0026alpha;-PFF regions, which showed substantially faster radioligand kinetics than the regional baseline TACs (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB); the TACs became comparable to those in the occipital cortex and cerebellum (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003ch2\u003eKinetic modeling of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001\u003c/h2\u003e\n\u003cp\u003eBP\u003csub\u003eND\u003c/sub\u003e in different brain regions are shown in Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA. BP\u003csub\u003eND\u003c/sub\u003e in the 150 \u0026micro;g \u0026alpha;-PFF regions was 0.78 \u0026plusmn; 0.1 (mean\u0026plusmn;SD, n = 4) while in the 75 \u0026micro;g regions, BP\u003csub\u003eND\u003c/sub\u003e \u0026alpha;-PFF injected regions was 0.29 (n = 2), showing a dose-dependent effect of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 binding to the \u0026alpha;-PFF. BP\u003csub\u003eND\u003c/sub\u003e in the AD homogenate region was 0.73, in the same order as the 150 \u0026micro;g \u0026alpha;-PFF. The DLB homogenate region, cerebellum, and temporal cortex had BP\u003csub\u003eND\u003c/sub\u003e values close to zero (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 test-retest scan on the same day showed a -6.2% change in BP\u003csub\u003eND\u003c/sub\u003e (Supplementary figure 2). Pretreatment with MODAG-001 resulted in a reduction in regional binding levels such that they became comparable to the reference regions. In the pig that underwent a baseline-block study, we observed \u0026gt;100% occupancy in the \u0026alpha;-PFF injected regions. A modest reduction in binding was also observed in the temporal cortex and cerebellum (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePET neuroimaging of aggregated protein has proved critical for diagnosing and monitoring disease progression and treatment evaluation in neurodegenerative diseases with amyloid-ꞵ and tau pathology [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The ability to detect and quantify α-synuclein aggregates in the living human brain would be a milestone achievement for the research of PD and other α-synucleinopathies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Due to its high affinity to α-synuclein and favorable binding in rodent models, [\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 and its analogs are currently some of the most promising radioligands for α-synuclein neuroimaging [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is the first time (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 has been tested in a higher species and shown promising translational results. We evaluated (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 in a pig model of intracerebral injection of α-PFF and postmortem human AD and DLB brain homogenates. We see high brain uptake and quick-wash out of the radioligand in the brain. The pharmacokinetics in healthy mice and the α-PFF rat model were comparable to that in pigs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We saw a relatively high uptake of the radioligand in the α-PFF regions at micromolar concentrations, with a dose-dependent response with 150 \u0026micro;g (415 \u0026micro;M) and 75 \u0026micro;g (208 \u0026micro;M) injections (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKuebler et al. tested both [\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 and the deuterium incorporated (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; deuterium incorporation was meant to improve the pharmacokinetic and metabolic profile of the radioligand [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Notably, we observed faster metabolism in the pigs than what was observed in the mice, which are much smaller mammals [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The results showed that ~10% parent fraction remained 15 min post-injection in the pigs, compared to ~30% parent fraction in mice. Radio-HPLC on brain homogenate (non-perfused) from a pig euthanized at 15 min showed ~50% parent fraction; in contrast, mice showed on average ~90% parent fraction after 15 min (Supplementary Figure 1, Supplementary Table 1).\u003c/p\u003e \u003cp\u003eWe performed the non-invasive kinetic modeling with the occipital cortex as a reference region since we previously have shown in our pig model that the occipital cortex has similar tissue properties as saline-injected target regions and that these are not affected by the intracerebral injection [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to the lack of other high-affinity molecules, an unlabelled MODAG-001 block scan was our best option to examine the signal specificity. Pretreatment of 1 mg/kg of MODAG-001 leads to complete blocking of the specific (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 binding in the α-PFF injected region. We observe a very high blocking percentage with values above 100% (using BP\u003csub\u003eND\u003c/sub\u003e values from reference modeling), although these estimates are based on only one pig and likely prone to noise.\u003c/p\u003e \u003cp\u003eWe also see high uptake in the amyloid-ꞵ and tau-rich AD homogenates but no significant uptake in the DLB homogenate region (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); this is remarkable since DLB is considered to have a pure α-synuclein pathology. Ideally, a radioligand should have high α-synuclein selectivity for it to distinguish α-synuclein aggregates from amyloid-ꞵ and tau aggregates [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Several things make us less enthusiastic about the prospect of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 as a radioligand in human studies: (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 did not display high binding in the DLB homogenate region; this could be due to low concentrations of aggregated α-synuclein, as is most often seen in human pathology, especially at early disease stages. This null-finding could also be due to the hypothesized difference in pathological morphology in pure α-synuclein DLB subjects [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 was also not very selective for α-synuclein and had significant binding to the AD homogenate region. This observation is also on par with previous autoradiography studies where the highest uptake was noted in human brain sections with AD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Improving the signal-to-background ratio and selectivity will be critical for the further development of the tracer, and this work is currently ongoing [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe intracerebral protein injection model used in the current study also comes with a set of limitations. Since the intracerebral injections are done a few hours prior to scanning, it is unlikely that protein aggregates enter into the brain cells, which does not mimic the intracellular inclusions seen in α-synucleinopathies well [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The concentration of the α-PFF in the model is much higher than that of diseased brains, where α-synuclein is found to be at nanomolar concentration [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This particular setup allowed us to show proof of concept for α-synuclein aggregate detecting radioligands. The signal-to-background ratio of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 makes it challenging to detect pathologically relevant α-synuclein, i.e., at nanomolar concentrations.\u003c/p\u003e \u003cp\u003eIn spite of the poor specificity and relatively modest signal-to-background ratio, we believe that (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 with its high affinity for α-synuclein is a suitable lead molecule for further radioligand development and evaluation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe demonstrate in vivo detection of α-PFF in pigs using (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001, which has previously only been shown using a similar α-PFF injection rat model. The radioligand shows excellent brain kinetics and test-retest variability. Although (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 displays low specificity towards α-synuclein and a potential passage of radiometabolites through the blood-brain barrier, it shows promise as a lead tracer for further radiotracer development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026alpha;-PFF: \u0026alpha;-synuclein preformed fibrils\u003c/p\u003e\n\u003cp\u003eAD: Alzheimer\u0026rsquo;s disease\u003c/p\u003e\n\u003cp\u003eBP\u003csub\u003eND\u003c/sub\u003e: binding potential non-displaceable\u003c/p\u003e\n\u003cp\u003eDLB: dementia with Lewy bodies\u003c/p\u003e\n\u003cp\u003eHRRT: high-resolution research tomograph\u003c/p\u003e\n\u003cp\u003eIV: intra-venous\u003c/p\u003e\n\u003cp\u003eIM: intra-muscular\u003c/p\u003e\n\u003cp\u003emPFC: medial prefrontal cortex\u003c/p\u003e\n\u003cp\u003eMSA: multiple system atrophy\u003c/p\u003e\n\u003cp\u003ePD: Parkinson\u0026rsquo;s disease\u003c/p\u003e\n\u003cp\u003ePET: positron emission tomography\u003c/p\u003e\n\u003cp\u003eR-HPLC: radio-high performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eSRTM: simplified reference tissue model\u003c/p\u003e\n\u003cp\u003eSUV: standardized uptake values\u003c/p\u003e\n\u003cp\u003eTAC: time-activity curve\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eAll animal procedures were performed in accordance with the European Commission\u0026apos;s Directive 2010/63/EU, as well as the ARRIVE guidelines, and were approved by the Danish Council of Animal Ethics (Journal no. 2017-15-0201-01375).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n\u003cp\u003eAll data, including R scripts, is available at a GitHub repository (https://github.com/nakulrrraval/Protien_inj_pig_model_MODAG001). All other requests are directed to this article\u0026apos;s corresponding or first author.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eLundbeck A/S, Denmark provided the \u0026alpha;-synuclein preformed fibrils as part of the European Union\u0026apos;s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 813528. However, they had no other financial interests in the project. GMK received honoraria as a speaker and consultant for Sage Pharmaceuticals/Biogen and Sanos A/S. All other authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis project has received funding from the European Union\u0026rsquo;s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 813528. This project also received funding from Parkinsonforeningen, Denmark (R16-A247). Pontus Plav\u0026eacute;n Sigray was supported by the Lundbeck Foundation (R303-2018-3263). Vladimir Shalgunov was supported by BRIDGE \u0026ndash; Translational Excellence Program at the Faculty of Health and Medical Sciences, University of Copenhagen, funded by the Novo Nordisk Foundation (grant agreement no. NNF18SA0034956).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contribution\u003c/h2\u003e\n\u003cp\u003eNRR, MMH, HDH, PPS, GMK: conceptualization and design. NRR, CAM, EEB, LMJ, HDH: surgical setup and PET scanning. VS, AN, UMB: compound synthesis, radiochemistry, and HPLC analysis. NRR, VS, AN, UMB, MJ, PPS: analysis and software. NRR, MMH, HDH, GMK: resources. NR, HDH, PPS, GMK: data curation. LMJ, MMH, HDH, PPS, GMK: supervision. NRR: preparation of manuscript draft including figures. NRR, CAM, VS, AN, UMB, EEB, MJ, LMJ, MMH, HDH, PPS, GMK: manuscript review and editing. NRR, MMH, GMK: funding acquisition. All authors have read and agreed to the current version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe want to thank Lundbeck A/S, Valby, Denmark, for providing the \u0026alpha;-synuclein preformed fibrils. This research project received human brain tissue from the Neuropathology Core of the Emory Center for Neurodegenerative Disease; we are grateful for their support. We would sincerely like to thank the staff and veterinarians at EMED, Panum, K\u0026oslash;benhavn University, and the PET and cyclotron unit at Rigshospitalet. Further, we would like to thank Ran Sing Saw, Marko Rosenholm, and Natalie Beschorner for their help during PET scans. We also extend our thanks to Ran Sing Saw for scientific discussions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBraak H, Braak E (2000) Pathoanatomy of Parkinson\u0026rsquo;s disease. 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ChemMedChem. https://doi.org/10.1002/cmdc.201900689\u003c/li\u003e\n \u003cli\u003eRaval NR, Nasser A, Madsen CA, et al (2022) An in vivo pig model for testing novel PET radioligands targeting cerebral protein aggregates. bioRxiv 2021.12.31.473908\u003c/li\u003e\n \u003cli\u003eJ\u0026oslash;rgensen LM, Baandrup AO, Mandeville J, Glud AN, S\u0026oslash;rensen JCH, Weikop P, Jespersen B, Hansen AE, Thomsen C, Knudsen GM (2021) An FMRI-compatible system for targeted electrical stimulation. Research Square. https://doi.org/10.21203/rs.3.rs-313183/v1\u003c/li\u003e\n \u003cli\u003eRaval NR, Nasser A, Madsen CA, et al (2022) An in vivo pig model for testing novel PET radioligands targeting cerebral protein aggregates. bioRxiv 2021.12.31.473908\u003c/li\u003e\n \u003cli\u003eSureau FC, Reader AJ, Comtat C, Leroy C, Ribeiro MJ, Buvat I, Tr\u0026eacute;bossen R (2008) Impact of image-space resolution modeling for studies with the high-resolution research tomograph. J Nucl Med 49:1000\u0026ndash;1008\u003c/li\u003e\n \u003cli\u003eKeller SH, Svarer C, Sibomana M (2013) Attenuation correction for the HRRT PET-scanner using transmission scatter correction and total variation regularization. IEEE Trans Med Imaging 32:1611\u0026ndash;1621\u003c/li\u003e\n \u003cli\u003eVilladsen J, Hansen HD, J\u0026oslash;rgensen LM, Keller SH, Andersen FL, Petersen IN, Knudsen GM, Svarer C (2017) Automatic delineation of brain regions on MRI and PET images from the pig. J Neurosci Methods 294:51\u0026ndash;58\u003c/li\u003e\n \u003cli\u003eSaikali S, Meurice P, Sauleau P, Eliat P-A, Bellaud P, Randuineau G, V\u0026eacute;rin M, Malbert C-H (2010) A three-dimensional digital segmented and deformable brain atlas of the domestic pig. J Neurosci Methods 192:102\u0026ndash;109\u003c/li\u003e\n \u003cli\u003eLogan J, Fowler JS, Volkow ND, Wang GJ, Ding YS, Alexoff DL (1996) Distribution volume ratios without blood sampling from graphical analysis of PET data. J Cereb Blood Flow Metab 16:834\u0026ndash;840\u003c/li\u003e\n \u003cli\u003eLammertsma AA, Hume SP (1996) Simplified reference tissue model for PET receptor studies. Neuroimage 4:153\u0026ndash;158\u003c/li\u003e\n \u003cli\u003eBarthel H, Sabri O (2017) Clinical Use and Utility of Amyloid Imaging. J Nucl Med 58:1711\u0026ndash;1717\u003c/li\u003e\n \u003cli\u003eShah M, Catafau AM (2014) Molecular Imaging Insights into Neurodegeneration: Focus on Tau PET Radiotracers. J Nucl Med 55:871\u0026ndash;874\u003c/li\u003e\n \u003cli\u003eEberling JL, Dave KD, Frasier MA (2013) \u0026alpha;-synuclein imaging: a critical need for Parkinson\u0026rsquo;s disease research. J Parkinsons Dis 3:565\u0026ndash;567\u003c/li\u003e\n \u003cli\u003eKlenner MA, Pascali G, Fraser BH, Darwish TA (2021) Kinetic isotope effects and synthetic strategies for deuterated carbon-11 and fluorine-18 labelled PET radiopharmaceuticals. Nucl Med Biol 96-97:112\u0026ndash;147\u003c/li\u003e\n \u003cli\u003eTang H, Mayersohn M (2018) Porcine prediction of pharmacokinetic parameters in people: A pig in a poke? Drug Metab Dispos 46:1712\u0026ndash;1724\u003c/li\u003e\n \u003cli\u003eAlpha-Synuclein Imaging Prize. https://www.michaeljfox.org/news/alpha-synuclein-imaging-prize. Accessed 24 Dec 2021\u003c/li\u003e\n \u003cli\u003ePeng C, Gathagan RJ, Lee VM-Y (2018) Distinct \u0026alpha;-Synuclein strains and implications for heterogeneity among \u0026alpha;-Synucleinopathies. Neurobiol Dis 109:209\u0026ndash;218\u003c/li\u003e\n \u003cli\u003eMorgan SA, Lavenir I, Fan J, Masuda-Suzukake M, Passarella D, DeTure MA, Dickson DW, Ghetti B, Goedert M (2020) \u0026alpha;-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species. J Biol Chem 295:6652\u0026ndash;6664\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alpha-synuclein, PET tracer, Positron emission tomography, intracerebral protein injection, amyloid-beta, brain imaging, larger animal PET, pig model","lastPublishedDoi":"10.21203/rs.3.rs-1268531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1268531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels is a histopathological hallmark. The radiotracer (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 µg α-PFF, and in the other hemisphere, either 75 µg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer’s disease (AD) was injected. All pigs underwent one or two (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe α-PFF and AD homogenate injected brain regions had high uptake of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 compared to the occipital cortex or cerebellum. BP\u003csub\u003eND\u003c/sub\u003e values in 150 µg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BP\u003csub\u003eND\u003c/sub\u003e values in the 150 µg and 75 µg injected region of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWe find that both α-PFF and AD brain homogenates give rise to increased binding of (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d\u003csub\u003e3\u003c/sub\u003e)-[\u003csup\u003e11\u003c/sup\u003eC]MODAG-001 shows promise as a lead tracer for future radiotracer development.\u003c/p\u003e","manuscriptTitle":"Evaluation of the α-synuclein PET Radiotracer (d3)-[11C]MODAG-001 in Pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-08 15:41:29","doi":"10.21203/rs.3.rs-1268531/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a2d68ff5-80fa-4ed3-95a6-ec8fe8da2b1b","owner":[],"postedDate":"March 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-27T04:31:08+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-08 15:41:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1268531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1268531","identity":"rs-1268531","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}