An In Vivo Study of a Rat Fluid-Percussion-Induced Traumatic Brain Injury Model with ¹¹C-PBR28 and ¹⁸F-flumazenil PET Imaging

Krishna Kanta Ghosh¹, Parasuraman Padmanabhan^1,2, Chang-Tong Yang^1,3,4, Zhimin Wang¹, Mathangi Palanivel¹ , Kian Chye Ng⁵, Jia Lu⁵, Jan Carlstedt-Duke⁶, Christer Halldin^1,7 and Balázs Gulyás^1,2,7

1 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

2 Cognitive Neuroimaging Centre, Nanyang Technological University, Singapore

3 Department of Nuclear Medicine and Molecular Imaging, Radiological Sciences Division, Singapore General Hospital, Singapore

4 Duke-NUS Medical School, Singapore

5 DSO National Laboratories (Kent Ridge), Singapore;

6 President’s Office, Nanyang Technological University, Singapore;

7 Department of Clinical Neuroscience, Karolinska Institute, Sweden

https://doi.org/10.3390/ijms22020951

Summary

Traumatic brain injury (TBI) modelled by lateral fluid percussion-induction (LFPI) in rats is a widely used experimental rodent model to explore and understand the underlying cellular and molecular alterations in the brain caused by TBI in humans. The present study aims to investigate whether the two adioligands, ¹¹C-PBR28 and ¹⁸F-flumazenil, are able to accurately quantify in vivo molecular-cellular changes in a rodent TBI-model for two different biochemical targets of the processes. As ¹¹C-PBR28 is a radioligand of the 18 kD translocator protein (TSPO), the up-regulation of which is coupled to the level of neuroinflammation in the brain, and ¹⁸F-flumazenil is a radioligand for GABAA-benzodiazepine receptors, whose level capable of indicating at a high precision the neuronal loss that ensues in various brain disorders and injuries, the use of the two radioligands may reveal two critical features of TBI. An up-regulation in the ¹¹C-PBR28 uptake triggered by the LFP in the injured (right) hemisphere was noted on day 14, while the uptake of ¹⁸F-flumazenil was down-regulated on day 14. When comparing the left (contralateral) and right (LFPI) hemispheres, the differences between the two in neuroinflammation were obvious. In vitro immunohistochemical analyses on the corpus callosum and hippocampal sections of the cerebrum were done to validate the results obtained in the PET imaging Results demonstrate a potential way to measure the molecular alterations in a rodent-based TBI model using PET imaging with ¹¹C-PBR28 and ¹⁸F-flumazenil. These radioligands are promising options that can be eventually used in exploring the complex in vivo pharmacokinetics and delivery mechanisms of nanoparticles in TBI treatment.

Results from nanoScan PET/MRI

According to a standard LFP procedure, a combination of focal and diffuse injury was inflicted on the cerebral cortex and hippocampus of the right hemisphere of rats to create a TBI model for the study. The left hemisphere served as an internal control for the study. Following the LFP procedure on the brain’s right hemisphere of 12 Sprague Dawley rats, day 2 post operation 3D dynamic PET scans were performed using the nanoScan PET/MRI scanner. After injecting approximately 30±4MBq ¹¹C-PBR28 or 18±4MBq ¹⁸F-flumazenil to the tail vein, dynamic 63min PET scan was performed with ¹¹C-PBR28. The second PET radioligand ¹⁸F-flumazenil was injected 80min later, and the animals were scanned for a duration of 90min. The detailed time frames for the respective scan protocols were as follows: 8x15s, 4x30s, 2x1min, 2x2min, 4x5min, 3x10min for ¹¹C-PBR28; 8x15s, 4x30s, 2x1min, 2x2min, 4x5min and 6x10min for ¹⁸F-flumazenil.

Results show:

• Compared to day 2 post-op, there is an increase in the uptake of ¹¹C-PBR28 on day 14 due to the LFP in the right hemisphere (injured)
• ¹⁸F-flumazenil uptake was down-regulated on day 14, compared to day 2
• The time activity curves (TACs) of the whole brain also clearly demonstrate that there was a higher ¹¹C-PBR28 and lower ¹⁸F-flumazenil uptake in day 14 as compared to day 2

• When juxtaposing the right and left hemispheres using an area-under-the-curve (AUC) measure, the discrepancies between the two hemispheres for the ¹¹C-PBR28 radiotracer were apparent. This is an indication that local increases in neuroinflammation due to the physical impact can be observed in the LFPI TBI rodent model. On the other hand, while ¹⁸F-flumazenil uptake is slightly higher in the left hemisphere, the lack of marked changes between the two hemispheres may reflect either the lack of neuron density alterations or the inappropriateness of the radioligand in indicating neuron density changes. This is the first LFPI TBI rat study to evaluate neuroinflammation and loss of neuronal density using two radioligands subsequently on the same day.

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging

Isabella Silins¹, Anders Sundin¹, Patrik Nordeman², Mahabuba Jahan², Sergio Estrada², Azita Monazzam³, Mark Lubberink¹, Franklin Aigbirhio⁴, Per Hellman¹, Gunnar Antoni²

¹ Department of Surgical Sciences, Uppsala University

² Medicinal Chemistry and Uppsala University

³ Medical Sciences at Uppsala University

⁴ Wolfson Brain Imaging Centre, University of Cambridge

https://www.medsci.org/v18p2187.htm

Summary

¹¹C-Metomidate (¹¹C-MTO) was developed as a PET radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). However, the use of ¹¹C-MTO is somewhat hampered by considerable accumulation in the liver, which, because of its close proximity to the right adrenal gland, may obscure adrenal pathology and make PET measurements unreliable. Moreover, increased ¹¹C-MTO uptake has been found in various liver lesions, such as adenoma, hepatocellular cancer and focal nodular hyperplasia, with the risk of false positive imaging results. Another disadventage of the tracer is that the clinical availability is restricted because of the short half-life of carbon-11 (T1/2= 20.4 min), which limits its use to PET centres with an in-house cyclotron and radiopharmacy.

The aim of this study was to evaluate the binding properties and in vivo behaviour of the previously published 18F-labeled (halflife 109.5min) etomidate analogue, para-chloro-2-¹⁸F-fluoroethyl etomidate; (¹⁸F-CETO), as an adrenal PET tracer. Comparative studies were also performed with ¹¹C-MTO and with ¹⁸F-FETO, another adrenocortical imaging agent, which has not reached widespread clinical use, partial due to its two-stage radiosynthesis.

Autoradiography on human and cynomolgus monkey tissues show specific, high ¹⁸F-CETO uptake in normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas.

Following in vitro binding kinetic analysis and the evaluation of ex vivo biodistribution, in vivo imaging studies revealed high specificity of ¹⁸F-CETO accumulation in the adrenal cortex qualitatively surpassing those of ¹¹C-MTO. Non-specific binding to the liver was significantly lower than that of ¹¹C-MTO. ¹⁸F-CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.

Results from nanoScan PET/MRI

¹⁸F-CETO imaging in rats and mice:

Eight female C57BL/6 mice and two male Sprague Dawley rats were were injected with of ¹⁸F-CETO (1.6±1MBq or 4-5MBq, respectively) and the 1h long dynamic PET imaging was started immediately. Four of the mice and one of the rats were co-injected with metomidate (1μmol/kg). The PET examination was followed by an MRI acquisition.

• ¹⁸F-CETO accumulated predominantly in the liver and in the adrenal glands, thus obfuscating the view of the adrenal glands in mice (Figure 1A: baseline; B: after blockage with metomidate)
• in contrast in rats the uptake was concentrated mainly in the adrenal glands with 120min p.i. peak adrenal uptake (Figure 2A: baseline; B: after blockage with metomidate)

¹⁸F-FETO imaging in rats:

Rats were were iv. injected with 4.0-4.8 MBq of ¹⁸F-FETO, one of the rats was given metomidate (1μmol/kg) and 1h long PET scan was started immediately.

• ¹⁸F-FETO in rats was also concentrated mainly in the adrenal glands. However, it was unable to block the uptake with metomidate, thus, re-evaluation of ¹⁸F-FETO has to be discontinued (Figure 2C: baseline; D: after blockage with metomidate)

Simultaneous Visualization of ¹⁶¹Tb- and ¹⁷⁷Lu-Labeled Somatostatin Analogues Using Dual-Isotope SPECT Imaging

Francesca Borgna¹, Patrick Barritt¹, Pascal V. Grundler¹, Zeynep Talip¹, Susan Cohrs¹, Jan Rijn Zeevaart², Ulli Köster³, Roger Schibli^1,4, Nicholas P. van der Meulen^1,5 and Cristina Müller^1,4

¹ Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland

² Radiochemistry, South African Nuclear Energy Corporation (Necsa), Brits 0240, South Africa

³ Institut Laue-Langevin, 38042 Grenoble, France

⁴ Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland

⁵ Laboratory of Radiochemistry, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland

https://doi.org/10.3390/pharmaceutics13040536

Summary

¹⁷⁷Lu (half-life 6.7d) is currently the most often applied radiometal for therapeutic purposes, as it has a particulate emission (β− or Auger electron) for effecting therapy and emits several accompanying γ-photons of 208 keV (11%) and 113 keV (6.4%), which are used for diagnostic evaluation and dosimetry.

¹⁶¹Tb is a more recently introduced radiolanthanide for therapeutic applications. ¹⁶¹Tb decays with a half-life of 6.89 days to stable ¹⁶¹Dy, while emitting β¯-particles (Eβ͞av = 154 keV) suitable for therapeutic purposes and γ-radiation (Eγ = 49 keV, I = 17.0%; Eγ = 75 keV, I = 10.2%) useful for SPECT imaging. ¹⁶¹Tb also emits a substantial number of low-energy conversion and Auger electrons, which makes this radionuclide exceptionally interesting for the treatment of disseminated cancers with multiple metastases ranging from a single cell (diameter: ~10μm) to micro cell clusters (diameter: < 1mm). Monte Carlo simulations assessed the dose delivered to 10μm spheres revealed a 3.5-fold increased value when using ¹⁶¹Tb as compared to ¹⁷⁷Lu. In larger tumors (diameter > 10mm), the emitted electron energy from ¹⁶¹Tb and ¹⁷⁷Lu respectively is almost entirely absorbed, resulting in a 1.3-fold higher absorbed electron energy fraction per decay for ¹⁶¹Tb compared to ¹⁷⁷Lu, making ¹⁶¹Tb more potent than ¹⁷⁷Lu.

The aim of the present study was to use dual-isotope SPECT imaging in order to demonstrate that ¹⁶¹Tb and ¹⁷⁷Lu are interchangeable without compromising the pharmacokinetic profile of the radiopharmaceutical.

After in vitro characterization, ¹⁶¹Tb- and ¹⁷⁷Lu-labeled somatostatin (SST) analogues DOTATOC (agonist) and DOTA-LM3 (antagonist) were injected to AR42J tumor-bearing nude mice. In vivo disptribution profiles were investigatd by dual-isotope SPECT/CT imaging. Results revealed identical pharmacokinetic profiles of the two peptides, irrespective of whether it was labeled with ¹⁶¹Tb or ¹⁷⁷Lu. Moreover, the visualization of the sub-organ distribution confirmed similar behavior of ¹⁶¹Tb- and ¹⁷⁷Lu-labeled SST analogues. These and previous findings suggest that any future (pre)clinical studies with ¹⁶¹Tb can be based on preclinical data obtained with its ¹⁷⁷Lu-labeled counterpart. This will allow the focusing of future investigations directly on the therapeutic efficacy of ¹⁶¹Tb, which is likely to be superior to the effect obtained with ¹⁷⁷Lu.

Results from nanoSPECT/CT

Five-week-old female CD1 nude mice were subcutaneously inoculated with AR42J tumor cells (5x106 cells in 100µl PBS). The scans were performed 10–14 days after tumor cell inoculation when the tumor size reached a volume of ~250mm3.

Mice were i.v. injected with a mixture of ¹⁶¹Tb-DOTATOC (~15MBq) and ¹⁷⁷Lu-DOTATOC (~15MBq) or a mixture of ¹⁶¹Tb-DOTA-LM3 and ¹⁷⁷Lu-DOTA-LM3 (~30MBq) at a ¹⁶¹Tb/¹⁷⁷Lu activity ratio of 1:1. For specificity test, blocking studies were performed under the same experimental conditions; however, in this case, an excess of unlabeled DOTATOC or DOTA-LM3 was added to the injection solution. SPECT/CT scans were acquired 2h, 4h, and 24h after injection of the radiopeptides using the dual-isotope SPECT acquisition protocol with a frame time of 60s resulting in a scan time of 45min.

For the SPECT/CT scan, simultaneous acquisition of counts stemming from ¹⁶¹Tb and ¹⁷⁷Lu, respectively, was performed by the selection of distinct energy windows for the two radionuclides. The two energy windows chosen for ¹⁶¹Tb were set at 47.7keV±10%, which enabled the detection of X-rays and γ-rays (46.0keV, 48.9keV and 52.0keV), and at 74.6keV±10%, enabling the detection of the γ-rays at 74.6keV. For ¹⁷⁷Lu, the windows were set at 112.9keV±10% and 208.4±10% to detect the γ-rays. Prior to animal studies, phantom scans were carried out in order to verify of the dual-isotope SPECT imaging protocol using Eppendorf vials filled with ¹⁶¹Tb or ¹⁷⁷Lu or both: analysis revealed no interference between ¹⁶¹Tb and ¹⁷⁷Lu in the acquired scans; each radionuclide was visualized independently of the other with high accuracy.

Analysis of the SPECT/CT images revealed:

• Equal in vivo distribution of simultaneously injected ¹⁶¹Tb-DOTATOC and ¹⁷⁷Lu-DOTATOC. The same observation was made for ¹⁶¹Tb-DOTA-LM3 and ¹⁷⁷Lu-DOTA-LM3. Images reconstructed using the energies of either radiolanthanide (red-to-yellow scale and green-to-yellow scale for ¹⁶¹Tb and ¹⁷⁷Lu, respectively), provided the distribution for each radiopeptide separately in the same mouse.

• Experiments performed by co-injection of excess unlabeled peptide resulted in SSTR blockade and, hence, accumulation of the radiopeptides in AR42J tumors was not observed. These additional studies proved that the uptake of the SST analogues in AR42J tumor xenografts was SSTR-specific.
• Quantification of the accumulated activity in AR42J tumors and kidneys confirmed equal distribution of the ¹⁶¹Tb- and ¹⁷⁷Lu-labeled counterparts. This was the ultimate proof that the chosen radiolanthanide did not have an impact on the tissue distribution profile of the radiopeptides.
• The SPECT/CT images showed activity accumulation in the AR42J xenografts, which was higher for the antagonist than for the agonist. In agreement with quantitative data from biodistribution studies, the activity was efficiently cleared through the kidneys over time and almost entirely excreted after 24h. Due to the favorable uptake of radiolabeled DOTA-LM3 in the tumor tissue, the tumor-to-kidney ratio was higher as compared to the ratio obtained after injection of radiolabeled DOTATOC.
• Dual-isotope SPECT image sections enabled, for the first time, visualization of the ¹⁶¹Tb- and ¹⁷⁷Lu-labeled peptide distribution at a sub-organ level in the same animal. Most important to note is that the pattern of activity distribution in tumors and kidneys was the same, irrespective of whether ¹⁶¹Tb or ¹⁷⁷Lu was used. The uptake in the tumor was quite homogenous, which can be ascribed to the well-vascularized AR42J xenograft. Accumulation of activity in the kidneys was more prominent in the cortex where the megalin-mediated reabsorption of radiopeptides occurs, and where various SSTR subtypes are known to be expressed

Iodine‑124 PET quantification of organ‑specific delivery and expression of NIS‑encoding RNA

Matthias Miederer¹, Stefanie Pektor¹, Isabelle Miederer¹, Nicole Bausbacher¹, Isabell Sofia Keil²,

Hossam Hefesha³, Heinrich Haas³, Ugur Sahin^2,3 and Mustafa Diken^2,3

¹ Department of Nuclear Medicine, University Medical Center of Johannes Gutenberg University, Mainz, Germany

² TRON - Translational Oncology at the University Medical Center, Johannes Gutenberg University Mainz GmbH, Mainz, Germany

³ Biopharmaceutical New Technologies (BioNTech) SE, Mainz, Germany

https://doi.org/10.1186/s13550-021-00753-2

Summary

There has been increased interest in the development of mRNA-based vaccines for protection against various infectious diseases and also for cancer immunotherapies since lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. In the present study, RNA-lipoplex nanoparticles were assembled by complexing sodium-iodide-symporter (NIS) coding mRNA with liposomes at different charge ratios. Two kinds of RNA-lipoplex systems were used: one system with net anionic charge mediating translation primarily within the spleen, and the other with net positive charge yielding translation primarily within the lungs. After in vitro analysis of the expression kinetics, mice were iv. injected with the mRNA-lipoplexes then 6h later with ¹²⁴Iodine. Functional NIS protein translation was investigated by PET/MRI imaging. Results revealed rapid increase of ¹²⁴Iodine uptake in the spleen or lung compared to control-RNA-lipoplexes (containing non-coding RNA) with minimal background in other organs except from thyroid, stomach and salivary gland (where NIS is physiologically expressed). The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.

Results from nanoScan PET/MRI

Female BALB/c mice were intravenously injected with RNA-lipoplexes containing 20μg NIS RNA. Six hours later 6.64±0.66MBq ¹²⁴Iodine was injected intravenously. Three hours after ¹²⁴Iodine injection, mice were anesthetized and static imaging was performed over 20min by nanoScan PET/MRI. Additionally, one animal per group was imaged dynamically for one hour.

• PET/MRI of anionic NIS-RNA lipoplexes showed a visually detectable increase of ¹²⁴Iodine uptake in the spleen compared to control-RNA lipoplexes. Due to the high physiological NIS expression in the adjacent gastric wall, this increase was only visually clear with anatomical correlation by MRI. On PET imaging, spleen uptake appeared as an irregularity of the gastric wall which is not detected in control animals
• Lung uptake of NIS-RNA transported by cationic RNA-lipoplexes was depicted more clearly due to larger organ size and no adjacent physiological NIS uptake
• The quantified radioactivity from imaging matched well with the extent of uptake as measured in organs ex vivo, showing enhanced uptake of NIS-RNA and expression of functional NIS-protein in lung or spleen compared to the control RNA
• The uptake in lung was rapid and remained high over the first hour of dynamic acquisition

SPECT imaging evaluation of ¹¹¹indium-chelated cetuximab for diagnosing EGFR-positive tumor in an HCT-15-induced colorectal xenograft

Bin-Bin Shih^a, Yi-Fang Chang^b, Chun-Chia Cheng^b, Hao-Jhih Yang^c, Kang-Wei Chang^c, Ai-Sheng Ho^a, Hua-Ching Lin^d, Chun Yeh^a, Chun-Chao Chang^e,f

^a Division of Gastroenterology, Cheng Hsin General Hospital, Taipei, Taiwan, ROC

^b Hematology and Oncology, Mackay Memorial Hospital, Taipei, Taiwan, ROC

^c Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, Taiwan, ROC

^d Division of Proctology, Cheng Hsin General Hospital, Taipei, Taiwan, ROC

^e Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan, ROC

^f Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC

http://dx.doi.org/10.1016/j.jcma.2017.02.010

Summary

Colorectal cancer (CRC) occurs with high incidence worldwide, but is usually diagnosed in late stage with metastasis by the conventional methods. Epidermal growth factor receptor (EGFR) is overexpressed in 97% of CRC cells, serving a promising diagnostic candidate. In the present study, Cetuximab, an anti-EGFR monoclonal antibody was conjugated with an isotope chelator, diethylene triamine penta acetic acid (DTPA), labeled with ¹¹¹indium (¹¹¹In) and injected to tumor bearing mice. Biological distrubution was investigated by SPECT/CT imaging.

Results revealed that ¹¹¹In-Cetuximab accumulated in the both small (50mm³) and large (250mm³) tumors, whereas the ratio of tumor to muscle in the large tumor was 7.5-fold. The biodistribution data indicated that the ¹¹¹In-cetuximab bound to tumor specifically that was higher than that in other organs. Consequently, ¹¹¹In-cetuximab is suggested to be suitable for early diagnosis and prognostic monitor of EGFR-positive CRC in further clinical practice.

Results from nanoSPECT/CT

• The tumor of the ¹¹¹In-Cetuximab group was apparently observed both in 24h and 48h and higher than that in the ¹¹¹In group.
• ¹¹¹In-Cetuximab majorly accumulated in liver and tumor, otherwise, ¹¹¹In accumulated only in the kidney.
• The tumor to muscle ratio of ¹¹¹In-Cetuximab was measured 7.5-fold, which was higher than that of ¹¹¹In group measured as 3.1-fold, indicating that ¹¹¹In-cetuximab specifically bound to EGFR-positive tumors as a reliable diagnosing agent.
• The result also indicated that ¹¹¹In labeled with Cetuximab through chelator DTPA was easily excreted out the mice better than free ¹¹¹In, suggesting that this labeling method may not lead to accumulation of ¹¹¹In metal in mice.

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1

Anzhelika Vorobyeva^1,2,† , Elena Konovalova^3,†, Tianqi Xu¹, Alexey Schulga^2,3, Mohamed Altai¹, Javad Garousi¹, Sara S. Rinne⁴, Anna Orlova^2,4,5, Vladimir Tolmachev^1,2 and Sergey Deyev^2,3,6,7 

¹ Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden

² Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, Tomsk, Russia

³ Molecular Immunology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

⁴ Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden

⁵ Science for Life Laboratory, Uppsala University, Uppsala, Sweden

⁶ Bio-Nanophotonic Lab, Institute of Engineering Physics for Biomedicine (PhysBio), National Research Nuclear University ‘MEPhI’, Moscow, Russia

⁷ Center of Biomedical Engineering, Sechenov University, Moscow, Russia

^† A.V. and E.K. contributed equally

https://doi.org/10.3390/ijms21093310

Summary

Up to 85% of ovarian cancer patients are diagnosed only at advanced stages, when cancer has already spread through the body. The present study aimed to find a more efficient way for diagnosis and treatment using mouse xenograft models.

As epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%–75% of ovarian carcinomas (OC), it might be a promising target. Designed ankyrin repeats protein (DARPin) Ec1 binds to EpCAM with subnanomolar affinity. In the present study, DARPin Ec1 was labeled with ¹²⁵I using N-succinimidyl-para-iodobenzoate (PIB) and injected to mice bearing SKOV-3 or OVCAR-3 xenografts. In vitro experiments showed highly specific binding to ovarian carcinoma cells, moreover, slow internalization, which is essential for in vivo imaging a few hours after injection. In vivo biodistribution analyses of SPECT/CT images suggest that EpCAM on ovarian cancer xenografts is sufficiently accessible to permit DARPin-mediated delivery of cytotoxic payload.

Results from nanoScan SPECT/CT

For establishment of xenografts, 107 of SKOV-3 and OVCAR-3 cells or 5x106 Ramos cells (EpCAM-negative lymphoma xenografts served as specificity control) in 100µl of media were subcutaneously injected in the right hind leg of female Balb/c nu/nu mice. The experiments in mice bearing SKOV-3 and Ramos xenografts were performed 2–3 weeks after implantation. The experiments in mice bearing OVCAR-3 xenografts were performed 7 weeks after implantation.

Mice were injected with ¹²⁵I-PIB-Ec1 (20µg, 1.2MBq for SKOV-3, and 6µg, 2.8MBq for OVCAR-3), SPECT/CT images were acquired 6h pi time later for 20min. with nanoScan SPECT/CT.

• In vitro studies revealed specific binding to SKOV-3 and OVCAR-3 cells; rapid binding and slow dissociation and internalization
• SPECT/CT imaging demonstrated that radiolabeled ¹²⁵I-PIB-Ec1 provided clear visualization of both EpCAM-expressing xenografts. In vivo biodistribution is characterized by high tumor-to-organ ratio, the only organ with noticeable activity were kidneys.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment

Jacinta Jacob¹, Suchita Nadkarni², Alessia Volpe^3,6, Qi Peng¹, Sim L. Tung¹, Rosalind F. Hannen²,

Yasmin R. Mohseni^1,3, Cristiano Scotta¹, Federica M. Marelli-Berg⁴, Robert I. Lechler¹, Lesley A. Smyth^1,5,7, Gilbert O. Fruhwirth^3,7, and Giovanna Lombardi^1,7

¹MRC Centre for Transplantation, Peter Gorer Department of Immunobiology, School of Immunology and Microbial Science, King’s College London, Guy’s Hospital, London SE1 9RT, UK;

²Centre for Cell Biology & Cutaneous Research, The Blizard Institute, Bart’s and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK;

³Imaging Therapies and Cancer Group, School of Biomedical Engineering and Imaging Sciences, King’s College London, London SE1 7EH, UK;

⁴William Harvey Research Institute, Bart’s and The London School of Medicine, Queen Mary University of London, London EC1M 6BQ, UK;

⁵School of Health, Sport and Bioscience, Stratford Campus, University of East London, London E16 2RD, UK

⁶Present address: Molecular Imaging Group, Department of Radiology, Memorial Sloan Kettering Cancer Center, 417 E 68th St., New York, NY 10065, USA

⁷Senior author

https://doi.org/10.1016/j.omtm.2020.12.003

Summary

Regulatory T cells (Tregs) have gained important role in mechanisms of transplantation tolerance and graft survival due to their putative capability to control immune responses. They can prolong the survival of allografts when they are administered after their purification from blood and further manipulated in vitro.

Despite an increase in the number of clinical trials using Treg therapy, important questions remain unclear such as their in vivo biodistribution over couple of weeks.

The aim of the present study was to investigate their long-term (for up to 40 days) biodistribution in vivo by SPECT imaging in mice bearing human skin transplants. As long-term tracking is challenging if radioactive dose has to be minimized, transplanted mice received genetically modified human Treg cells: they were lentivirally transduced with the human sodium iodide symporter (hNIS) and for the imaging, its alternative substrate, pertechnetate (99mTcO4¯) was injected iv. on the days of the scan. In one group of mice Gr-1+ cells (including neutrophils and certain monocytes) were depleted using an antibody raised against Gr-1 to achieve a high level of immunocompromise.

Results show that 99mTcO4¯ uptake was elevated much earlier in the presence of Gr-1+ cells, suggesting their active, accelerating role in influencing Treg recruitment to the graft.

Results from nanoScan SPECT/CT

Skin grafts were transplanted onto 10-12-week-old recipient BRG or NSG mice. 5–6 week later, 5x106 peripheral blood mononuclear cells (PBMCs) were then administered iv. with or without 5x106 Tregs. Some BRG mice received 100mg anti-mouse Gr-1 ip. every two days.

For SPECT/CT imaging 20 MBq 99mTcO4¯ was administered iv. and SPECT scans were acquired 40 min later with nanoScan SPECT/CT. Data were reconstructed using Tera-Tomo with corrections for attenuation, detector dead time, and radioisotope decay in place as needed. CT images were used to draw ROIs and provide the volumes required for standard uptake value calculations. The total activity in the whole animal (excluding the tail) at the time of tracer administration was defined as the injected dose (ID).

• Serial SPECT/CT imaging of mice received Cr-1 antibody revealed that radiotracer uptake in the human skin grafts did not differ from control animals in the first 2 weeks, but at late time points (ranging 30–40 days after administration) Treg presence was significantly elevated.
• In case of the presence of Gr-1+ cells, Tregs were detectable at the skin grafts as early as 3 days after administration. Their signals peaked at around 8 days and remained detectable in the transplants up to 40 days after administration. Early trafficking of Tregs to the skin in the presence of Gr-1+ cells suggested an active, accelerating role of these cells in influencing Treg recruitment to the graft.

Nuclear imaging-guided PD-L1blockade therapy increases effectiveness of cancer immunotherapy

Hannan Gao¹, Yue Wu ¹, Jiyun Shi², Xin Zhang¹, Tianyu Liu¹, Biao Hu¹, Bing Jia¹, Yakun Wan³, Zhaofei Liu¹, Fan Wang^1,2,4

¹Medical Isotopes Research Center and Department of Radiation Medicine, State Key Laboratory of Natural and Biomimetic Drugs, School of Basic Medical Sciences, Peking University, Beijing, China

²Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

³Shanghai Novamab Biopharmaceuticals Co., Ltd, Shanghai, China

⁴Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, China

doi:10.1136/jitc-2020-001156

Summary

The interaction between programmed death receptor-1 (PD-1) and its ligand (PD-L1) inhibits the function of effector T cells and the priming of naive T cells, leading to impaired antitumor immunity. Therefore the blockade of PD-1/PD-L1 signaling pathway has been a breakthrough in cancer therapy, but the response rate in solid tumors is only 20-30%. In this study, a new radiolabeled nanobody-based imaging probe 99mTc-MY1523 targeting PD-L1 was developed for the enhanced therapeutic efficacy of PD-L1 blockade immunotherapy.

Results show that the new probe has high binding affinity and specificity to PD-L1. SPECT/CT imaging revealed fast blood clearance, renal-route excretion and satisfactory tumor uptake.

As the timing for PD-L1 blockade therapy is crucial due to dynamic and heterogeneous expression of PD-L1 in tumors, it was essential to prove that SPECT/CT imaging is able to detect changes in the PD-L1 expression. Therefore PD-L1 expression was increased by interferon-γ (IFN-γ) treatment of tumor bearing mice and PD-L1 expression was determined by SPECT/CT imaging and verified by the flow cytometry. It proves that SPECT/CT imaging of 99mTc-MY1523 can be used to monitor PD-L1 expression in tumors in a real time, dynamic and quantitative manner.

The PD-L1 blockade therapy initiated during the therapeutic time window determined by ⁹⁹mTc-MY1523 SPECT/CT imaging significantly enhanced the therapeutic efficacy: the tumor growth was dramatically suppressed, and the survival time of mice was evidently prolonged.

Results from nanoScan SPECT/CT

Three types of tumor cells (4T1, A20 or MC-38) were inoculated subcutaneously into the right flank of BALB/c or C57/BL6 mice, respectively. Four days later they were injected i.t. with PBS or IFN-γ for 5 days, and then were subjected to SPECT/CT imaging.

Mice were injected intraveneously with 18 MBq ⁹⁹mTc-MY1523 and imaged at 2 hours p.i. (n=4) using the nanoScan SPECT/CT system with the following parameters: pinhole SPECT (peak: 140 keV, 20% width; frame time: 25 s), helical CT (50 kVp, 0.67 mA, rotation 210°, exposure time: 300 ms). SPECT and CT images were merged using the Nucline software V.2.0 (Mediso Ltd.). The regions of interest were drawn for the determination of tumor sizes (mm3) and radioactivity (Bq), then the tumor uptake was calculated as percentage injected dose per volume (%ID/cc).

• Results show increased tumor uptake of 99mTc-MY1523 compared to the corresponding control group in all animal models:

When imaging results showed the upregulated PD-L1 expression in tumors after IFN-γ intervention on day 8 and 12 after tumor cell inoculation, the mice were subjected to PD-L1 blockade therapy: they were ip. injected with 200μg αPD-L1 antibody twice with 4 days interval, while using PBS, IFN-γ and αPD-L1 antibody without IFN-γ intervention as controls. Tumor sizes were measured twice a week and calculated as volumes (mm³)=length×width×height/2.

• As shown on the figure below, although IFN-γ intervention expedited the tumor growth, the imaging-guided therapy dramatically improved the therapeutic efficacy. The tumor growth was significantly suppressed, and three of five tumors completely disappeared. Compared to control groups, the survival time of mice in the treated group was also remarkably prolonged.

Preclinical Evaluation of a Novel ^99mTc-Labeled CB86 for Rheumatoid Arthritis Imaging

Peng Liu¹, Tingting Wang¹, Rongshui Yang¹, Wentao Dong¹, Qiang Wang¹, Zhide Guo², Chao Ma¹, Weixing Wang¹, Huaibo Li¹, and Xinhui Su¹ 

¹Department of Nuclear Medicine, Zhongshan Hospital Xiamen University, Xiamen 361004, China

²Center for Molecular Imaging and Translational Medicine, Xiamen University, Xiamen 361102, China

https://doi.org/10.1021/acsomega.0c04066

Summary

Early diagnosis and therapy are crucial to control disease progression optimally and achieve a good prognosis in rheumatoid arthritis (RA). Moreover, therapeutic intervention should start as soon as the diagnosis has been established, with the aim of stopping inflammation before irreversible damage is caused, but unfortunately current diagnostic methods are still not very sensitive and specific to RA.

Early hallmark of RA is the increased number of activated macrophages in the synovium with strong increase of their translocator protein (TSPO) level.

In previous studies a 99mtechnetium-labeled TSPO ligand (99mTc-CB256) was used to image a TSPO-rich cancer cell in vitro; however, few 99mTc-CB256 in vivo evaluation has been reported so far probably due to the cytotoxicity of CB256 (75 times more than analogous CB86). Here, a novel TSPO targeting radiopharmaceutical consisting of CB86 and diethylenetriaminepentaacetic acid (DTPA) is described.

Cytotoxicity, binding affinity and specificity of 99mTc-DTPA-CB86 to TSPO were evaluated using RAW264.7 macrophage cells. Biodistribution and 99mTc-SPECT studies were conducted on RA rat models after the injection of 99mTc-DTPA-CB86 with or without co-injection of unlabeled DTPA-CB86.

The probe displayed good stability in vitro and binding specificity to RAW264.7 macrophage cells. In the biodistribution studies, 99mTc-DTPA-CB86 exhibited rapid inflammatory ankle accumulation. At 180 min after administration, 99mTc-DTPA-CB86 uptakes of the left inflammatory ankle were 2.35 ± 0.10 percentage of the injected radioactivity per gram of tissue (% ID/g), significantly higher than those of the normal tissues. 99mTc-SPECT imaging studies revealed that 99mTc-DTPA-CB86 could clearly identify the left inflammatory ankle with good contrast at 30−180 min after injection. Therefore, 99mTc-DTPA-CB86 may be a promising probe for arthritis 99mTc-SPECT imaging.

Results from nanoScan SPECT/CT

The RA rats (n = 4 for each group) were injected with 99mTc-DTPA-CB86 (0.37 MBq, 100 μL) with or without co-injection of unlabeled DTPA-CB86 (300 μg) through the tail vein. At 30, 90, and 180 min after injection, they were anesthetized with 2% isoflurane and placed on the SPECT bed. SPECT acquiring parameters were as follows: a 140 keV energy peak for 99mTc, window width of 20%, a matrix of 256 × 256 and time frame 30 s. Whole-body static images (200 000 counts) were acquired with a matrix of 218 × 218, and a zoom of 2.0. CT data were acquired using an X-ray voltage biased to 50 kVp with a 670 μA current, with #projections 720°. Regions of interest (ROI) were drawn over the left inflammatory ankle and normal muscle, and then the ratios of the left inflammatory ankle to muscle were calculated.

• 99mTc-DTPA-CB86 accumulated in the left inflammatory ankles at 30 min and then showed a gradual increase of uptake. During 90−180 min after injection, the left inflammatory ankles were clearly visible, with good inflammatory to background contrast.
• When co-injected with unlabeled DTPA-CB86 (300 μg), the left inflammatory ankles were barely visible on SPECT images at 30−180 min after injection.
• Regions of interest (ROI) analysis of SPECT showed a high ratio of the left inflammatory ankle to muscle for RA rats injected unblocking dose compared to with 300 μg blocking dose at 30−180 min postinjection (P < 0.05).
• Evaluation of the probe in these RA rats demonstrated that 99mTc-DTPA-CB86 may be a promising agent for TSPO SPECT imaging.

Comparison of SARS-CoV-2 infection in two non-human primate species: rhesus and cynomolgus macaques

Kinga P. Boszormenyi¹, Marieke A. Stammes², Zahra C. Fagrouch¹, Gwendoline Kiemenyi-Kayere¹, Henk Niphuis¹, Daniella Mortier¹, Nikki van Driel³, Ivonne Nieuwenhuis², Ella Zuiderwijk-Sick⁴, Lisette Meijer², Petra Mooij¹, Ed J. Remarque¹, Gerrit Koopman¹, Alexis C. R. Hoste⁵, Patricia Sastre⁵, Bart L. Haagmans⁶, Ronald E. Bontrop^7,8, Jan A.M. Langermans^3,9, Willy M. Bogers¹, Ernst J. Verschoor¹, and Babs E. Verstrepen¹

 ¹Department of Virology, Biomedical Primate Research Centre (BPRC), Rijswijk, The Netherlands

²Department of Parasitology, BPRC, Rijswijk, The Netherlands

³Animal Science Department, BPRC, Rijswijk, The Netherlands

⁴Alternatives unit, BPRC, Rijswijk, The Netherlands

⁵Eurofins-Inmunologia y Genetica Aplicada (Eurofins-INGENASA), Madrid, Spain

⁶Department of Viroscience, Erasmus University Medical Center, Rotterdam, The Netherlands

⁷Department of Comparative Genetics and Refinement, BPRC, Rijswijk, The Netherlands

⁸Department of Biology, Theoretical Biology and Bioinformatics, Utrecht University, Utrecht, The Netherlands

⁹Department of Population Health Sciences, Unit Animals in Science and Society, Veterinary Faculty, Utrecht University, Utrecht, The Netherlands

https://doi.org/10.1101/2020.11.05.369413

Summary

SARS-CoV-2 is a coronavirus that sparked the current COVID-19 pandemic. To stop it, effective and safe vaccines, and antiviral therapies are urgently required. To facilitate the preclinical evaluation of intervention approaches, relevant animal models need to be developed and validated. Rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) are widely used in biomedical research and serve as models for SARS-CoV-2 infection, but this is the first controlled comparative study investigating which species of them is best suited to examine specific aspects of COVID-19. This study analysed replication and symptoms for three weeks after infection. Pulmonary lesions were detected on CT images acquired with MultiScan LFER PET/CT. Elevated body temperature and decreased in physical activity was also observed. Results show that both rhesus and cynomolgus macaques represent valid models for COVID-19 prophylactic and therapeutic treatments.

Results from MultiScan LFER PET/CT

CT imaging provides a valuable tool to specifically monitor the progression of COVID-19-related lung pathology during the entire course of the study. Respiratory-gated CT scans were performed on Day0, 2, 4, 6, 8, 10, 12, 14, 16, 22 post-infection to monitor lung pathology. A semi-quantitative scoring system for chest CT evaluation was used to estimate SARS-CoV-2-induced lung disease; maximum score of 35 could be reached per timepoint.

 Scans revealed that:

• All macaques show levels of pneumonia
• Detected different types of lesions: A) ground glass opacities, B) consolidations, and C) crazy paving patterns (Figure 1)
• Around days 8 and 10 pi., lesions were manifest in all animals, and in several macaques the coverage had increased
• Cumulative CT scores increased and no difference was observed between rhesus and cynomolgus macaques

Further results showed:

• Both groups of animals, the body temperature was significantly higher during the first two weeks after infection
• Significantly lower activity in all four rhesus macaques during the first period after infection, while this difference in cynomolgus macaques was less obvious
• Antibody response became evident between day 10 and 12 pi., IgG level continued to rise for several days (development of IgM titers was barely detected)
• Certain cytokines increased in the plasma of both macaque species

Heterodimeric Radiotracer Targeting PSMA and GRPR for Imaging of Prostate Cancer-Optimization of the Affnity towards PSMA by Linker Modification in Murine Model

Fanny Lundmark¹, Ayman Abouzayed¹, Bogdan Mitran^1,2, Sara S. Rinne¹, Zohreh Varasteh^1,3, Mats Larhed⁴ , Vladimir Tolmachev^5,6 , Ulrika Rosenström¹ and Anna Orlova ^1,4,6

¹ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden

² Department of Clinical Neuroscience, Centre for Psychiatry Research, Karolinska Institutet and  Stockholm County Council, 171 77 Stockholm, Sweden

³ Department of Nuclear Medicine, Klinikum rechts der Isar der TUM, 80802 Munich, Germany

⁴ Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, 751 23  Uppsala, Sweden

⁵ Department of Immunology, Genetics and Pathology, Uppsala University, 751 83 Uppsala, Sweden

⁶ Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, 634050 Tomsk, Russia

doi:10.3390/pharmaceutics12070614

Summary

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are overexpressed in prostate cancer (PCa) cells and are promising targets for molecular imaging methods used for diagnosis. Novel heterodimer - containing PSMA inhibitor and GRPR antagonist - has been demonstrated to bind specifically to both proteins with concomitant low uptake in normal tissues. In the current study, chemical structure of the heterodimer was modified in order to improve affinity towards PCa cells and binding characteristics were analysed. Tumor-bearing mice were injected with 111In-labeled heterodimer (BQ7812). In vivo biodistribution was investigated on harvested organs and also with SPECT/CT imaging. Quantitative analysis together with in vitro tests revealed that modifications in the molecular design resulted in 10-fold improved affnity towards PSMA and high activity uptake in tumors.

Results from nanoScan SPECT/CT

For the SPECT/CT studies, BALB/c nu/nu mice implanted with PC3-pip (isogenic human prostate carcinoma) cells were injected with 830kBq 111In-BQ7812. Groups were also co-injected with non-labeled GRPR antagonist and/or non-labeled PSMA-11 to block GRPR and/or PSMA to prove binding specificity. Imaging of the non-blocked group was performed at 1 and 3h pi and for the GRPR/PSMA-blocked group at 1h pi.

Result revealed that:

• images are in good agreement with the ex vivo analysis: tumor could be visualized already at 1h pi. and the only healthy organs with high activity uptake at this time point were the kidneys (Figure 1.A)
• Co‐injection of non-labeled PSMA-11 and NOTA-PEG4-RM26 resulted in a decreased kidney uptake and a negligible activity uptake in the tumor (Figure 1.B)
• activity cleared from healthy organs and blood with time, leading to an improved imaging contrast at 3h pi. (Figure 1.C)
• ∑ Together with the results from the in vitro and in vivo specificity tests, confirmed the specific binding of [111In]In-BQ7812 to both PSMA and GRPR

Preclinical evaluation of an 111In/225Ac theranostic targeting transformed MUC1 for triple negative breast cancer

Vanessa J Kelly¹, Shu-Ta Wu^2,3, Vijay Gottumukkala¹, Richard Coelho¹, Keryn Palmer¹, Surabhi Nair¹,

Timothy Erick², Rahul Puri³, Ohad Ilovich¹, Pinku Mukherjee^2,3

1. Invicro, LLC, Boston, MA, USA
2. Department of Biological Sciences, University of North Carolina, Charlotte, NC, USA
3. OncoTAb, Inc., Charlotte, NC, USA

 doi: 10.7150/thno.38236

Summary

Triple-negative breast cancers (TNBC) are associated with poor prognosis and high mortality rates following relapse. These cells do not express estrogen, progesterone or HER2/neu receptors, which means that receptor targeted therapies can’t be utilized. These cells overexpress transformed MUC1 (tMUC1) antigen, which is selectively bound by murine antibody TAB004. Once TAB004 binds to tMUC1 it has been shown to internalize, which makes them excellent therapeutic candidate for TNBC.

Current study aimed to evaluate humanized TAB004 (hTAB004) as a potential theranostic for TNBC. hTAB004 was labeled either with Indium-111 (for biodistribution analysis) or Actinium-225 (for alpha radiotherapy) and injected intravenously to orthotopic tumor bearing mice. Results demonstrate both high tumor concentrations and high tumor-to-blood ratios. Additionally, a single administration of ²²⁵Ac-DOTA-hTAB004 increased survival and resulted in consistently lower tumor volumes compared to the control group after 12 days. Together they are convincing proof-of-concept support for hTAB004 as a theranostic agent in triple negative breast cancer.

Results from nanoScan SPECT/CT

1)           In vivo biodistribution studies: NSG mice were inoculated with HCC70 tumors. 27 days later 7.5MBq ¹¹¹In-DOTAhTAB004 was intravenously injected via the tail vein. SPECT-CT imaging was performed at 4, 24, 48 and 120 h postinjection.

The tumor accumulation of ¹¹¹In-DOTA-hTAB004 increased over 120 h reaching a maximum of 65.4 ± 15.2 %ID/g. All other organs (blood, bone, kidneys, liver, lungs, muscle, pancreas, spleen) had <10% ID/g at this time.

2)           Efficacy studies: Athymic nude mice were inoculated with HCC70 tumor cells. 27 days later they were injected via the tail vein with either ²²⁵Ac-DOTA-hTAB004 (18.5 kBq) or DOTA-hTAB004 and monitored for tumor growth via caliper measurements. The ²²⁵Ac-DOTA-hTAB004 group had significantly smaller tumors and greater survival compared to the control group.

Electroacupuncture attenuates cognition impairment via anti-neuroinflammation in an Alzheimer’s disease animal model

Mudan Cai¹, Jun-Hwan Lee² and Eun Jin Yang²

¹Department of Herbal Medicine Research, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 305-811, Republic of Korea

²Department of Clinical Research, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 305-811, Republic of Korea

https://doi.org/10.1186/s12974-019-1665-3

Summary

Current study investigated the electroacupuncture (EA)-induced molecular mechanisms causing cognitive improvement and anti-inflammatory activity in 5XFAD mice, a transgenic animal model of Alzheimer’s Disease (AD). Mice were bilaterally treated with EA three times per week for 2 weeks, thereafter Y-maze tests, western blots, immunohistochemistry, and PET scans were performed. Results revealed that EA treatment significantly improved working memory and synaptic plasticity, activated cell metabolism in the frontal cortex and the hypothalamus, concomitantly reduced neuroinflammation, ultrastructural degradation of synapses, and the microglia-mediated amyloid β deposition.

Results from nanoScan PET/CT

For the PET/CT studies, 7.4MBq ¹⁸F-FDG was intravenously injected via the tail vein to the following treatment groups: a) non-transgenic (non-Tg), b) 5XFAD (Tg) and c) EA-treated 5XFAD (Tg+KI3). After 60min uptake period, 30min long PET acquisitions were performed. Data were reconstructed with Tera-Tomo 3D reconstruction method. For quantitative analysis regions of interest were drawn into the frontal cortex, cortex, hippocampus, and hypothalamus and standardized uptake values (SUV) were evaluated to determine the effect of EA treatment on brain metabolism. Result revealed that:

• EA stimulation caused a 1.1-fold increase in the mean glucose level of the frontal cortex, which is related to short-term and working memory showing that it improves cognitive functions

• The hypothalamus, which is related to energy metabolism, exhibited a 1.1-fold decreased glucose metabolism in Tg mice compared to non-Tg mice, and this effect was fully reversed in EA-treated Tg mice proving that it modulates the abnormal hypothalamic metabolism related to the amyloid pathology in AD mice