μPET Imaging Research Articles

Preclinical Pharmacokinetics and Dosimetry of an 89Zr Labelled Anti-PDL1 in an Orthotopic Lung Cancer Murine Model.

Anti-PDL1 is a monoclonal antibody targeting the programmed death-cell ligand (PD-L1) by blocking the programmed death-cell (PD-1)/PD-L1 axis. It restores the immune system response in several tumours, such as non-small cell lung cancer (NSCLC). Anti-PDL1 or anti-PD1 treatments rely on PD-L1 tumoural expression assessed by immunohistochemistry on biopsy tissue. However, depending on the biopsy extraction site, PD-L1 expression can vary greatly. Non-invasive imaging enables whole-body mapping of PD-L1 sites and could improve the assessment of tumoural PD-L1 expression.MethodsPharmacokinetics (PK), biodistribution and dosimetry of a murine anti-PDL1 radiolabelled with zirconium-89, were evaluated in both healthy mice and immunocompetent mice with lung cancer. Preclinical PET (μPET) imaging was used to analyse [89Zr]DFO-Anti-PDL1 distribution in both groups of mice. Non-compartmental (NCA) and compartmental (CA) PK analyses were performed in order to describe PK parameters and assess area under the concentration-time curve (AUC) for dosimetry evaluation in humans.ResultsOrgan distribution was correctly estimated using PK modelling in both healthy mice and mice with lung cancer. Tumoural uptake occurred within 24 h post-injection of [89Zr]DFO-Anti-PDL1, and the best imaging time was at 48 h according to the signal-to-noise ratio (SNR) and image quality. An in vivo blocking study confirmed that [89Zr]DFO-anti-PDL1 specifically targeted PD-L1 in CMT167 lung tumours in mice. AUC in organs was estimated using a 1-compartment PK model and extrapolated to human (using allometric scaling) in order to estimate the radiation exposure in human. Human-estimated effective dose was 131 μSv/MBq.ConclusionThe predicted dosimetry was similar or lower than other antibodies radiolabelled with zirconium-89 for immunoPET imaging.

18 F]-labeled positron emission tomography ligand for the histamine H4 receptor.

We synthesized 5-[18 F]-fluoro-1H-indol-2-yl)(4-methyl-1-piperazinyl)methanone ([18 F]5) via a Suzuki approach starting from a protected pinacol borane precursor followed by acidic hydrolysis of the t-Boc protecting group. The non-optimized radiochemical yield was 5.7 ± 1.35%, radiochemical purity was over 99%, and molar activity was 100.7 ± 34.5 GBq/μmol (n = 3). [18 F]5 was stable in rat plasma for at least 4 h and was evaluated by μPET imaging and biodistribution using a unilateral quinolinic acid rat model of neuroinflammation. The time-activity curve showed that [18 F]5 entered the brain immediately after intravenous injection and then left it progressively with a very low level reached from 30 min after injection. The biodistribution study showed no difference in the accumulation of [18 F]5 between the lesioned and intact side of the brain and between control rats and animals pretreated with a saturating dose of JNJ-7777120 as a specific H4R antagonist. Hence, despite its in vitro nanomolar affinity for H4R, and its ability to cross the blood-brain barrier in rats, [18 F]5 does not appear suitable to image in vivo the receptor by PET.

Preclinical evaluation of [18F]cabozantinib as a PET imaging agent in a prostate cancer mouse model

IntroductionCabozantinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of medullary thyroid cancer, renal cell carcinoma and hepatocellular carcinoma, and is currently in clinical trials for the treatment of prostate cancer and others. It exerts its therapeutic effect mainly through inhibition of the tyrosine kinases MET (hepatocyte growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2), in addition to several other kinases involved in cancer. PET imaging with TKIs such as [18F]cabozantinib could potentially aid in cancer diagnosis and guide treatment. This study aims to evaluate the utility of [18F]cabozantinib as a PET imaging probe in PC3 tumor xenografted mice. Methods[18F]cabozantinib was evaluated in non-tumor and tumor bearing (PC3 xenografted) male mice by ex vivo biodistribution studies and in vivo μPET imaging. Pretreatment studies were performed in the tumor bearing mice with the MET inhibitor PF04217903. Mouse plasma was analyzed with HPLC to quantify radiometabolites. To further evaluate the binding specificity of [18F]cabozantinib, in vitro autoradiography studies on heart and PC3 tumor sections were performed in the presence of authentic cabozantinib or specific MET and VEGFR2 inhibitors. ResultsTissue distribution studies in non-tumor bearing mice revealed slow blood clearance, absence of brain uptake and a high myocardial uptake. In the tumor bearing mice, tumor uptake was low (0.58 ± 0.20% ID/g at 30 min post tracer injection), which was confirmed by μPET imaging. No differences in tissue distribution and kinetics were observed in both biodistributions and μPET studies after pretreatment with the MET inhibitor PF04217903. At 30 min post tracer injection, 60 ± 3% of the recovered radioactivity in plasma in non-tumor bearing mice was present as intact tracer. [18F]cabozantinib binding in vitro to heart and tumor tissues was partly blocked in the presence of selective MET and VEGFR2 inhibitors (up to 40% block). The fraction of non-specific binding was relatively high for both tissues (66% for heart and 39% for tumor). Conclusion[18F]cabozantinib exhibits non-favorable properties as a PET imaging probe, demonstrated by slow excretion kinetics along with low tumor uptake and high non-specific binding in tumor and heart tissue. The results reflect cabozantinibs multi-kinase activity, making PET imaging of tumor specific kinase expression with [18F]cabozantinib challenging.

Preclinical Evaluation of a Novel 18F-Labeled dTCO-Amide Derivative for Bioorthogonal Pretargeted Positron Emission Tomography Imaging.

Pretargeted positron emission tomography (PET) imaging based on the bioorthogonal inverse-electron-demand Diels–Alder reaction between tetrazines (Tz) and trans-cyclooctenes (TCO) has emerged as a promising tool for solid tumor imaging, allowing the use of short-lived radionuclides in immune-PET applications. With this strategy, it became possible to achieve desirable target-to-background ratios and at the same time to decrease the radiation burden to nontargeted tissues because of the fast clearance of small PET probes. Here, we show the synthesis of novel 18F-labeled dTCO-amide probes for pretargeted immuno-PET imaging. The PET probes were evaluated regarding their stability, reactivity toward tetrazine, and pharmacokinetic profile. [18F]MICA-213 showed an extremely fast kinetic rate (10,553 M–1 s–1 in 50:50 MeOH/water), good stability in saline and plasma up to 4 h at 37 °C with no isomerization observed, and the biodistribution in healthy mice revealed a mixed hepatobiliary and renal clearance with no defluorination and low background in other tissues. [18F]MICA-213 was further used for in vivo pretargeted immune-PET imaging carried out in nude mice bearing LS174T colorectal tumors that were previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49). Pretargeted μPET imaging results showed clear visualization of the tumor tissue with a significantly higher uptake when compared to the control.

18F-Labeled Cyclized \u03b1-Melanocyte-Stimulating Hormone Derivatives for Imaging Human Melanoma Xenograft with Positron Emission Tomography

Since metastatic melanoma is deadly, early diagnosis thereof is crucial for managing the disease. We recently developed α-melanocyte-stimulating hormone (αMSH) derivatives, [68Ga]Ga-CCZ01048 and [18F]CCZ01064, that target the melanocortin 1 receptor (MC1R) for mouse melanoma imaging. In this study, we aim to evaluate [18F]CCZ01064 as well as a novel dual-ammoniomethyl-trifluoroborate (AmBF3) derivative, [18F]CCZ01096, for targeting human melanoma xenograft using μPET imaging. The peptides were synthesized on solid phase using Fmoc chemistry. Radiolabeling was achieved in a one-step 18F-19F isotope-exchange reaction. μPET imaging and biodistribution studies were performed in NSG mice bearing SK-MEL-1 melanoma xenografts. The MC1R density on the SK-MEL-1 cell line was determined to be 972 ± 154 receptors/cell (n = 4) via saturation assays. Using [18F]CCZ01064, moderate tumor uptake (3.05 ± 0.47%ID/g) and image contrast were observed at 2 h post-injection. Molar activity was determined to play a key role. CCZ01096 with two AmBF3 motifs showed comparable sub-nanomolar binding affinity to MC1R and much higher molar activity. This resulted in improved tumor uptake (6.46 ± 1.42%ID/g) and image contrast (tumor-to-blood and tumor-to-muscle ratios were 30.6 ± 5.7 and 85.7 ± 11.3, respectively) at 2 h post-injection. [18F]CCZ01096 represents a promising αMSH-based μPET imaging agent for human melanoma and warrants further investigation for potential clinical translation.

Effect of genotype and age on cerebral [18F]FDG uptake varies between transgenic APPSwe-PS1dE9 and Tg2576 mouse models of Alzheimer\u2019s disease

Back-translation of clinical imaging biomarkers of Alzheimer’s disease (AD), such as alterations in cerebral glucose metabolism detected by [18F]FDG positron emission tomography (PET), would be valuable for preclinical studies evaluating new disease-modifying drugs for AD. However, previous confounding results have been difficult to interpret due to differences in mouse models and imaging protocols between studies. We used an equivalent study design and [18F]FDG µPET imaging protocol to compare changes in cerebral glucose metabolism in commercial transgenic APPSwe-PS1dE9 (n = 12), Tg2576 (n = 15), and wild-type mice (n = 15 and 9). Dynamic [18F]FDG scans were performed in young (6 months) and aged (12 or 17 months) mice and the results verified by ex vivo methods (i.e., tissue counting, digital autoradiography, and beta-amyloid and Iba-1 immunohistochemistry). [18F]FDG uptake exhibited significant regional differences between genotypes (TG < WT) and ages (6 months <12 months) in the APPSwe-PS1dE9 model, whereas similar differences were not present in Tg2576 mice. In both models, only weak correlations were detected between regional beta-amyloid deposition or microgliosis and [18F]FDG uptake. By using equivalent methodology, this study demonstrated differences in cerebral glucose metabolism dysfunction detected with [18F]FDG PET between two widely used commercial AD mouse models.

Abstract 3028: PET imaging with the bispecific 89Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse models

Abstract BACKGROUND: ERY974, a modified monoclonal IgG4 bispecific antibody directed against human CD3 on T cells and glypican 3 (GPC3) on tumor cell, is currently in phase I clinical trial. The oncofetal protein GPC3 is overexpressed in several tumor types. Radiolabeling ERY974 with positron emission tomography (PET) isotope zirconium-89 (89Zr) enables non-invasive molecular imaging of tumor targeting and whole-body distribution. We aimed to evaluate 89Zr-ERY974 tumor targeting and effect of T cells on tumor uptake in mouse models, including a humanized mouse model. METHODS: ERY974 and two control molecules namely bispecific CD3xkeyhole limpet hemocyanin (KLH) and KLHxKLH antibodies were radiolabeled with 89Zr. Studies were performed in immunodeficient NOD/Shi-SCID/IL-2Rgnull (NOG) as well as human CD34+ hematopoietic stem cell engrafted NOG mice (huNOG), all subcutaneously inoculated with GPC3 overexpressing human hepatocellular carcinoma HepG2 cells. Mice received 10 µg 89Zr-ERY974, 89Zr-CD3xKLH or 89Zr-KLHxKLH intravenously, with subsequent µPET scanning at 24, 72, 120 and 168 h followed by ex vivo biodistribution. Organs of interest were quantified on µPET scans as mean standardized uptake value (SUVmean) and with ex vivo biodistribution as % injected dose/gram of tissue (%ID/g). Tumor, spleen and lymph nodes were analyzed with autoradiography and immunohistochemical CD3 staining. RESULTS: µPET imaging revealed increased tumor-to-blood ratio (TBR) of 89Zr-ERY974 in NOG over time with maximal TBR of 2.2±0.3 at 168 h post tracer injection (pi). At 168 h, tumor uptake was specific as 89Zr-CD3xKLH and 89Zr-KLHxKLH showed a TBR of only 0.6±0.2 and 0.8±0.3, respectively. In huNOG mice human CD3+ T cells were present in tumor, spleen and lymph nodes. In huNOG mice tumor uptake of 89Zr-ERY974 was higher than in NOG mice as measured on µPET scans (SUVmean at 168 h pi 6.9±2.6 vs 2.9±0.2; P&amp;lt;0.01) and with ex vivo biodistribution (60.9±26.2 %ID/g vs 16.7±2.3 %ID/g; P&amp;lt;0.001), whereas 89Zr-CD3xKLH tumor uptake in both mouse models was lower (P&amp;lt;0.05) but were similar in these mouse models. Autoradiography 168 h following 89Zr-ERY974 administration to huNOG mice showed 89Zr in extensive T cell infiltrate areas in the tumors of huNOG mice, whereas T cell infiltrate was lower in tumors of 89Zr-CD3xKLH and 89Zr-KLHxKLH injected huNOG mice. Spleens of huNOG mice showed CD3+ specific uptake as 89Zr-ERY974 and 89Zr-CD3xKLH uptake were higher than 89Zr-KLHxKLH uptake(P&amp;lt;0.05), whereas spleen uptake in NOG mice of the 3 tracers was similar. Moreover, in huNOG CD3+ mesenteric lymph nodes 89Zr-ERY974 uptake was higher than 89Zr-KLHxKLH uptake (P&amp;lt;0.05) CONCLUSION: 89Zr-ERY974 demonstrates specific tumor uptake in NOG and huNOG mice, while in huNOG mice tumor uptake colocalized with T cell rich infiltrate and also uptake in in spleen and lymph nodes was observed. Citation Format: Stijn J. Waaijer, Danique Giesen, Takahiro Ishiguro, Yuji Sano, Norihisa Ohishi, Athos Gianella-Borradori, Carolien P. Schröder, Elisabeth G. de Vries, Marjolijn N. Lub-de Hooge. PET imaging with the bispecific 89Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3028.

Radiolabeling and Preclinical Evaluation of a New S-Alkylated Cysteine Derivative Conjugated to C-Substituted Macrocycle for Positron Emission Tomography.

A new S-alkylated cysteine-derivatized tumor targeting agent, 2,2′-(12-(2-((2-acetamido-2-carboxyethyl)thio)acetamido)-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid was developed for positron emission tomography (PET) imaging. N-Acetyl cysteine (NAC) was conjugated to ATRIDAT as a specific targeting agent toward L-type and ASC amino acid transporter systems in the oncogenic cells. NAC was attached via S-alkylation to prevent its incorporation at undesired recognition sites affecting the signal-to-noise ratio. NAC-ATRIDAT was subjected to gallium-68 complexation with >75% radiolabeling yield. The radiocomplex was purified through the tc18 cartridge to obtain 99.89% radiochemical yield. IC-50 of the NAC-ATRIDAT conjugate was 0.8 mM in A549 cells as evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide assay. Binding affinity experiments on A549 cells showed noteworthy binding with KD in the nanomolar range. A time course study showed a Km value of 0.19 μM and Vmax value of 0.49 pmol/μg protein/min showing reasonable tumor kinetics. Efflux studies showed that the synthesized radioligand is transported majorly by LAT followed by the ASC system. Clearance was found to be renal with 7.67 ± 1.48% ID/g uptake at 30 min which substantially declined to 0.52 ± 0.% ID/g at 4 h. A significant uptake of 10.06 ± 1.056% ID/g was observed at the tumor site in mice at 1 h. μPET images revealed a high contrast with a tumor-to-kidney ratio of 4.8 and a tumor-to-liver ratio of 35.85 at 1 h after injection. These preclinical in vitro and in vivo evaluation supports its potential on the way of becoming a successful 68Ga-radiolabeled amino acid-based PET imaging agent.

A new method measuring the interaction of radiotracers with the human P-glycoprotein (P-gp) transporter

In drug development, biomarkers for cerebral applications have a lower success rate compared to cardiovascular drugs or tumor therapeutics. One reason is the missing blood brain barrier penetration, caused by the tracer's interaction with efflux transporters such as the P-gp (MDR1 or ABCB1). Aim of this study was the development of a reliable model to measure the interaction of radiotracers with the human efflux transporter P-gp in parallel to the radiolabeling process. LigandTracer® Technology was used with the wildtype cell line MDCKII and the equivalent cell line overexpressing human P-gp (MDCKII-hMDR1). The method was evaluated based on established PET tracers with known interaction with the human P-gp transporter and in nanomolar concentration (15 nM). [11C]SNAP-7941 and [18F]FE@SNAP were used as P-gp substrates by comparing the real-time model with an uptake assay and μPET images. [11C]DASB [11C]Harmine, [18F]FMeNER,[18F]FE@SUPPY and [11C]Me@HAPTHI were used as tracers without interactions with P-gp in vitro. However, [11C]Me@HAPTHI shows a significant increase in SUV levels after blocking with Tariquidar. The developed real-time kinetic model uses directly PET tracers in a compound concentration, which is reflecting the in vivo situation. This method may be used at an early stage of radiopharmaceutical development to measure interactions to P-gp before conducting animal experiments.

In vitro imaging of bacteria using 18F-fluorodeoxyglucose micro positron emission tomography

Positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (18F-FDG) can be applied to detect infection and inflammation. However, it was so far not known to what extent bacterial pathogens may contribute to the PET signal. Therefore, we investigated whether clinical isolates of frequently encountered bacterial pathogens take up 18F-FDG in vitro, and whether FDG inhibits bacterial growth as previously shown for 2-deoxy-glucose. 22 isolates of Gram-positive and Gram-negative bacterial pathogens implicated in fever and inflammation were incubated with 18F-FDG and uptake of 18F-FDG was assessed by gamma-counting and µPET imaging. Possible growth inhibition by FDG was assayed with Staphylococcus aureus and the Gram-positive model bacterium Bacillus subtilis. The results show that all tested isolates accumulated 18F-FDG actively. Further, 18F-FDG uptake was hampered in B. subtilis pts mutants impaired in glucose uptake. FDG inhibited growth of S. aureus and B. subtilis only to minor extents, and this effect was abrogated by pts mutations in B. subtilis. These observations imply that bacteria may contribute to the signals observed in FDG-PET infection imaging in vivo. Active bacterial FDG uptake is corroborated by the fact that the B. subtilis phosphotransferase system is needed for 18F-FDG uptake, while pts mutations protect against growth inhibition by FDG.

Small Animal micro-PET imaging: an overview

Molecular imaging has become an interesting and growing clinical practice and valuable research field. It permits one to understand the molecular pathways and biochemical changes associated with disease development. It also allows researchers to interrogate particular expression of key molecules that play essential role in diagnostic or therapeutic processes. Small animals remain instrumental in deciphering many biological aspects of human diseases. The implementation of modern imaging technologies in preclinical models provides excellent tools for investigating intrinsic molecular and biochemical changes associated with a given disease model. The role of micro-positron emission tomography (µPET) in this context is quite unique and has potential to prove its utilities in drug discovery and development. One of the challenges associated with this technology is selection of appropriate animal models, how far it can represent a human disorder, and how the experimental outcome is predictive of future clinical trials. Animal anaesthesia, which if not taken into consideration could have an adverse impact on data interpretation. Animal handling and monitoring protocols are key during preclinical imaging. There are also some questions regarding the performance of the µPET scanner used to acquire the data and how this information can be integrated with other modalities or correlated with histopathological finding to reveal valuable and reliable outcome. The applications of µPET imaging are numerous and have been extensively reported in the literature with special focus on cardiology, neurology and oncology.

Abstract 498: In vivo Assessment of Murine Valvular and Vascular Calcification Using 18 F Sodium Fluoride Micro Positron-Emission Tomography and Computed Tomography

Calcific aortic valvular and vascular disease (CAVVD) is associated with increased morbidity and mortality. In addition to the commonly practiced computed tomography (CT) to evaluate for the presence of CAVVD, recent work has demonstrated the use of 18 F-sodium fluoride positron-emission tomography ( 18 F-PET) to assess both valvular and vascular calcification in humans. In this pilot study, we combined 18 F μPET and μCT to assess for aortic valvular and aortic arch calcification in vivo . Aged Apoe -/- mice (n=5) were injected with ~200 μCi 18 F-sodium fluoride and, one hour later, were imaged with fused μPET-μCT (Figure A,C). Intravenous contrast was used for the μCT studies to assist with anatomic localization (Figure B,D). To assess valvular hemodynamics, direct cardiac catheterization was performed on the mice to determine the peak-to-peak pressure gradient (PPG) across the aortic valve, between the left ventricle and aorta (Figure E,F). All mice were found to have aortic arch calcification present on both μPET and μCT imaging. In mice with aortic valve calcification specifically identified on μPET-μCT (Figure A-D, red arrows), there was increased 18 F uptake in the heart and aorta (58.8 ± 7.7 %ID/cc) compared to the mouse without aortic valve calcification (16.2 %ID/cc). Additionally, in the mice with aortic valve calcification, the mean transvalvular PPG was 9.7 ± 2.5 mmHg, and in the mouse without valvular calcification, the PPG was 3.3 mmHg. Alizarin red staining of histological sections from the aortic valves and aortic roots from these mice was performed to assess for the presence of calcium mineral. In conclusion, these findings suggest that the use of 18 F μPET-μCT in small animals provides a method to determine the presence of CAVVD in vivo . Future studies will determine whether changes in 18 F μPET-μCT signal reliably correlate with meaningful changes in CAVVD.

MicroPET Outperforms Beta-Microprobes in Determining Neuroreceptor Availability under Pharmacological Restriction for Cold Mass Occupancy.

Both non-invasive micro-positron emission tomography (μPET) and in situ beta-microprobes have the ability to determine radiotracer kinetics and neuroreceptor availability in vivo. Beta-microprobes were proposed as a cost-effective alternative to μPET, but literature revealed conflicting results most likely due to methodological differences and inflicted tissue damage. The current study has three main objectives: (i) evaluate the theoretical advantages of beta-microprobes; (ii) perform μPET imaging to assess the impact of (beta-micro)probe implantation on relative tracer delivery (R1) and receptor occupancy (non-displaceable binding potential, BPND) in the rat brain; and (iii) investigate whether beta-microprobe recordings produce robust results when a pharmacological restriction for cold mass dose (tracer dose condition) is imposed. We performed acquisitions (n = 61) in naive animals, dummy probe implanted animals (outer diameter: 0.75 and 1.00 mm) and beta-microprobe implanted animals (outer diameter: 0.75 mm) using two different radiotracers with high affinity for the striatum: [11C]raclopride (n = 29) and [11C]ABP688 (n = 32). In addition, acquisitions were completed with or without an imposed restriction for cold mass occupancy. We estimated BPND and R1 values using the simplified reference tissue method (SRTM). [11C]raclopride dummy μPET BPND (0.75 mm: −13.01 ± 0.94%; 1.00 mm: −13.89 ± 1.20%) and R1 values (0.75 mm: −29.67 ± 4.94%; 1.00 mm: −39.07 ± 3.17%) significantly decreased at the implant side vs. the contralateral intact side. A similar comparison for [11C]ABP688 dummy μPET, demonstrated significantly (p < 0.05) decreased BPND (−19.09 ± 2.45%) and R1 values (−38.12 ± 6.58%) in the striatum with a 1.00 mm implant, but not with a 0.75 mm implant. Particularly in tracer dose conditions, despite lower impact of partial volume effects, beta-microprobes proved unfit to produce representative results due to tissue destruction associated with probe insertion. We advise to use tracer dose μPET to obtain accurate results concerning receptor availability and tracer delivery, keeping in mind associated partial volume effects for which it is possible to correct.

Evaluation of [18 F]BR420 and [18 F]BR351 as radiotracers for MMP-9 imaging in colorectal cancer.

MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [18 F]BR420 and [18 F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [18 F]BR420 and [18 F]BR351 were synthesized using an automated synthesis module. For [18 F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. μPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [18 F]BR420 (3.15%±0.03% ID/g vs 0.94%±0.18% ID/g for [18 F]BR351 at 2hours pi) but slower blood clearance compared with [18 F]BR351. [18 F]BR351 was quickly metabolized in plasma with 20.28%±5.41% of intact tracer remaining at 15minutes postinjection (PI). By contrast, [18 F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [18 F]BR420 displayed a higher tumor uptake and higher stability compared with [18 F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.

Biotinidase Resistant 68Gallium-Radioligand Based on Biotin/Avidin Interaction for Pretargeting: Synthesis and Preclinical Evaluation.

A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly 68Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled with 68Ga(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC50 of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. μPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

Combined [18F]DPA-714 micro-positron emission tomography and autoradiography imaging of microglia activation after closed head injury in mice

BackgroundTraumatic brain injury (TBI) is a major cause of death and disability. Neuroinflammation contributes to acute damage after TBI and modulates long-term evolution of degenerative and regenerative responses to injury. The aim of the present study was to evaluate the relationship of microglia activation to trauma severity, brain energy metabolism, and cellular reactions to injury in a mouse closed head injury model using combined in vivo PET imaging, ex vivo autoradiography, and immunohistochemistry.MethodsA weight-drop closed head injury model was used to produce a mixed diffuse and focal TBI or a purely diffuse mild TBI (mTBI) in C57BL6 mice. Lesion severity was determined by evaluating histological damage and functional outcome using a standardized neuroscore (NSS), gliosis, and axonal injury by immunohistochemistry. Repeated intra-individual in vivo μPET imaging with the specific 18-kDa translocator protein (TSPO) radioligand [18F]DPA-714 was performed on day 1, 7, and 16 and [18F]FDG-μPET imaging for energy metabolism on days 2–5 after trauma using freshly synthesized radiotracers. Immediately after [18F]DPA-714-μPET imaging on days 7 and 16, cellular identity of the [18F]DPA-714 uptake was confirmed by exposing freshly cut cryosections to film autoradiography and successive immunostaining with antibodies against the microglia/macrophage marker IBA-1.ResultsFunctional outcome correlated with focal brain lesions, gliosis, and axonal injury. [18F]DPA-714-μPET showed increased radiotracer uptake in focal brain lesions on days 7 and 16 after TBI and correlated with reduced cerebral [18F]FDG uptake on days 2–5, with functional outcome and number of IBA-1 positive cells on day 7. In autoradiography, [18F]DPA-714 uptake co-localized with areas of IBA1-positive staining and correlated strongly with both NSS and the number of IBA1-positive cells, gliosis, and axonal injury. After mTBI, numbers of IBA-1 positive cells with microglial morphology increased in both brain hemispheres; however, uptake of [18F]DPA-714 was not increased in autoradiography or in μPET imaging.Conclusions[18F]DPA-714 uptake in μPET/autoradiography correlates with trauma severity, brain metabolic deficits, and microglia activation after closed head TBI.

μPET imaging of the pharmacokinetic behavior of medium and high molar mass 89Zr-labeled poly(2-ethyl-2-oxazoline) in comparison to poly(ethylene glycol)

Poly(2-oxazoline)s are a promising class of polymers for biomedical applications and a versatile alternative to poly(ethylene glycol)s (PEG). In this work, the pharmacokinetic behavior of well defined 89Zr-labeled poly(2-ethyl-2-oxazoline)s (PEtOx) was evaluated and compared to that of 89Zr-labeled PEG, both with varying molar mass. Amine-terminated PEtOx of low dispersity in a molar mass range of 20 to 110kDa and PEG of 20 and 40kDa were functionalized with a desferrioxamine chelator and radiolabeled with 89Zr. The tissue distribution of both radiolabeled PEtOx and PEG polymers was studied by means of micro Positron Emission Tomography (μPET) molecular imaging in mice longitudinally up to 1week post injection, followed by ex vivo biodistribution.As previously described for other classes of non-ionic polymers, the blood clearance of PEtOx decreased with molar mass. The cut off for glomerular filtration of PEtOx is likely to be around 40kDa. The head to head comparison of PEG and PEtOx revealed that the biodistribution is mostly dominated by polymer chain length and not polymer molar mass. This study constitutes an important addition to further establishing PEtOx as a promising polymer in biomedical applications.

In Vivo Amyloid-β Imaging in the APPPS1-21 Transgenic Mouse Model with a (89)Zr-Labeled Monoclonal Antibody.

Introduction: The accumulation of amyloid-β is a pathological hallmark of Alzheimer’s disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-β antibody (JRF/AβN/25) to non-invasively assess amyloid-β burden in aged transgenic mice (APPPS1–21) with μPET imaging.Methods: We investigated the antibody JRF/AβN/25 that binds to full-length Aβ. JRF/AβN/25 was radiolabeled with a [89Zr]-desferal chelate and intravenously injected into 12–13 month aged APPPS1–21 mice and their wild-type (WT) controls. Mice underwent in vivo μPET imaging at 2, 4, and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution, and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [89Zr]-labeled antibody (trastuzumab) as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/AβN/25 and finally we assessed the possible confounding effects of blood retention.Results: Voxel-wise analysis of μPET data demonstrated significant [89Zr]-Df-Bz-JRF/AβN/25 retention in APPPS1–21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [89Zr]-Df-Bz-JRF/AβN/25 was found at 4 and 7 days pi in APPPS1–21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/AβN/25 only partially blocked [89Zr]-Df-Bz-JRF/AβN/25 uptake indicative of a high contribution of non-specific binding.Conclusion: Amyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibody in addition to non-specific binding prevented an accurate estimation of plaque burden. However, it should be noted that [89Zr]-Df-Bz-JRF/AβN/25 nevertheless demonstrated in vivo binding and strategies to increase brain penetrance would likely achieve better results.

In vivo molecular neuroimaging of glucose utilization and its association with fibrillar amyloid-β load in aged APPPS1-21 mice.

IntroductionRadioligand imaging is a powerful in vivo method to assess the molecular basis of Alzheimer’s Disease. We therefore aimed to visualize the pathological deposition of fibrillar amyloid-β and neuronal dysfunction in aged double transgenic mice.MethodsUsing non-invasive positron emission tomography (PET) we assessed brain glucose utilization with [18F]FDG and fibrillar amyloidosis with [11C]PiB and [18F]AV45 in 12 month old APPPS1-21 (n = 10) mice and their age-matched wild-type controls (n = 15). PET scans were analyzed with statistical parametric mapping (SPM) to detect significant differences in tracer uptake between genotypes. After imaging, mice were sacrificed and ex vivo measures of amyloid-β burden with immunohistochemistry as well as glucose utilization with [14C]-2DG autoradiography were obtained as gold standards.ResultsVoxel-wise SPM analysis revealed significantly decreased [18F]FDG uptake in aged APPPS1-21 mice in comparison to WT with the thalamus (96.96 %, maxT = 3.35) and striatum (61.21 %, maxT = 3.29) demonstrating the most widespread reductions at the threshold of p < 0.01. [11C]PiB binding was significantly increased in APPPS1-21 mice, most notably in the hippocampus (87.84 %, maxT = 7.15) and cortex (69.08 %, maxT = 7.95), as detected by SPM voxel-wise analysis at the threshold of p < 0.01. Using the same threshold [18F]AV45 uptake was comparably lower with less significant differences. Compared to their respective ex vivo equivalents [18F]FDG demonstrated significant positive correlation to [14C]2-DG autoradiography (r = 0.67, p <0.0001) while [11C]PiB and [18F]AV45 binding did not correlate to ex vivo immunohistochemistry for amyloid-β (r = 0.25, p = 0.07 and r = 0.17, p = 0.26 respectively). Lastly no correlation was observed between regions of high amyloid burden and those with decreased glucose utilization (r = 0.001, p = 0.99).ConclusionsOur findings support that fibrillar amyloid-β deposition and reduced glucose utilization can be visualized and quantified with in vivo μPET imaging in aged APPPS1-21 mice. Therefore, the combined use of [18F]FDG and amyloid μPET imaging can shed light on the underlying relationship between fibrillar amyloid-β pathology and neuronal dysfunction.

Synthesis and preclinical evaluation of an 18F labeled PDE7 inhibitor for PET neuroimaging

IntroductionPhosphodiesterase 7 (PDE7) hydrolyzes selectively cyclic adenosine monophosphate (cAMP) which is an intracellular second messenger. PDE7 is expressed by 2 genes which are both present in the brain. To date there is no radiotracer for PDE7 imaging described and detection of PDE7 has only been performed by ex vivo techniques. In this report we describe the radiosynthesis of a novel fluorine-18 labeled radiotracer for PDE7 as well as the in vivo evaluation in mice to verify whether it has potential for imaging of PDE7 in the brain. MethodsWe have synthesized a potent fluorinated PDE7 inhibitor, [18F]MICA-003 (PDE7 IC50=17nM) and the corresponding tosylate precursor for radiolabeling. [18F]MICA-003 was injected in C57BL/6J mice (n=5) and in vivo images were acquired by μPET imaging. Radiometabolite analysis in plasma and brain was performed to determine the stability of the radioligand. Results[18F]MICA-003 was synthesized by direct fluorination of the tosylate and produced in high decay corrected radiochemical yield (40%), high radiochemical purity (>98%) and high specific activity (86–497GBq/μmol). μPET imaging revealed that [18F]MICA-003 crosses the blood brain barrier and has a homogenous distribution over the brain which washes out after the initial peak uptake. [18F]MICA-003 was quickly metabolized in plasma with 8.9%±0.59% of intact [18F]MICA-003 remaining at 5min post injection. We observed the formation of three distinct radiometabolites of which the main radiometabolite was also detected in the brain in a proportion of 25.7±2.57% at this same time point. ConclusionWe have described the synthesis and in vivo evaluation of a novel radioligand for PDE7 imaging. Despite high uptake in the brain and favorable kinetics in vivo, the presence of a brain penetrant radiometabolite makes [18F]MICA-003 unfavorable for the accurate quantification of PDE7 and more stable spiroquinazolinones analogs are in development.