[18F]Atorvastatin Pharmacokinetics and Biodistribution in Healthy Female and Male Rats.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that are widely used to prevent cardiovascular diseases. However, a series of pleiotropic mechanisms have been associated with statins, particularly with atorvastatin. Therefore, the assessment of [18F]atorvastatin kinetics with positron emission tomography (PET) may elucidate the mechanism of action of statins and the impact of sexual dimorphism, which is one of the most debated interindividual variations influencing the therapeutic efficacy. [18F]Atorvastatin was synthesized via a previously optimized 18F-deoxyfluorination strategy, used for preclinical PET studies in female and male Wistar rats (n = 7 for both groups), and for subsequent ex vivo biodistribution assessment. PET data were fitted to several pharmacokinetic models, which allowed for estimating relevant kinetic parameters. Both PET imaging and biodistribution studies showed negligible uptake of [18F]atorvastatin in all tissues compared with the primary target organ (liver), excretory pathways (kidneys and small intestine), and stomach. Uptake of [18F]atorvastatin was 38 ± 3% higher in the female liver than in the male liver. The irreversible 2-tissue compartment model showed the best fit to describe [18F]atorvastatin kinetics in the liver. A strong correlation (R2 > 0.93) between quantitative Ki (the radiotracer's unidirectional net rate of influx between compartments) and semi-quantitative liver's SUV (standard uptake value), measured between 40 to 90 min, showed potential to use the latter parameter, which circumvents the need for blood sampling as a surrogate of Ki for monitoring [18F]atorvastatin uptake. Preclinical assays showed faster uptake and clearance for female rats compared to males, seemingly related to a higher efficiency for exchanges between the arterial input and the hepatic tissue. Due to the slow [18F]atorvastatin kinetics, equilibrium between the liver and plasma concentration was not reached during the time frame studied, making it difficult to obtain sufficient and accurate kinetic information to quantitatively characterize the radiotracer pharmacokinetics over time. Nevertheless, the reported results suggest that the SUV can potentially be used as a simplified measure, provided all scans are performed at the same time point. Preclinical PET-studies with [18F]atorvastatin showed faster uptake and clearance in female compared to male rats, apparently related to higher efficiency for exchange between arterial blood and hepatic tissue.

Modular Medical Imaging Agents Based on Azide-Alkyne Huisgen Cycloadditions: Synthesis and Pre-Clinical Evaluation of 18 F-Labeled PSMA-Tracers for Prostate Cancer Imaging.

Since the seminal contribution of Rolf Huisgen to develop the [3+2] cycloaddition of 1,3-dipolar compounds, its azide-alkyne variant has established itself as the key step in numerous organic syntheses and bioorthogonal processes in materials science and chemical biology. In the present study, the copper(I)-catalyzed azide-alkyne cycloaddition was applied for the development of a modular molecular platform for medical imaging of the prostate-specific membrane antigen (PSMA), using positron emission tomography. This process is shown from molecular design, through synthesis automation and in vitro studies, all the way to pre-clinical in vivo evaluation of fluorine-18- labeled PSMA-targeting 'F-PSMA-MIC' radiotracers (t1/2 =109.7 min). Pre-clinical data indicate that the modular PSMA-scaffold has similar binding affinity and imaging properties to the clinically used [68 Ga]PSMA-11. Furthermore, we demonstrated that targeting the arene-binding in PSMA, facilitated through the [3+2]cycloaddition, can improve binding affinity, which was rationalized by molecular modeling. The here presented PSMA-binding scaffold potentially facilitates easy coupling to other medical imaging moieties, enabling future developments of new modular imaging agents.

Structure-activity relationship study towards non-peptidic positron emission tomography (PET) radiotracer for gastrin releasing peptide receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide.

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [18F](S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.

Synthesis and preclinical evaluation of [18F]AlF-NODA-MP-C6-CTHRSSVVC as a PET tracer for CD163-positive tumor-infiltrating macrophages.

Positron emission tomography (PET) can provide information about tumor-associated macrophage (TAM) infiltration, as long as a suitable tracer is available. This study aimed to evaluate the radiolabeled peptide [18F]AlF-NODA-MP-C6-CTHRSSVVC as a potential PET tracer for imaging of the CD163 receptor, which is expressed on M2-type tumor-associated macrophages. The conjugated peptide NODA-MP-C6-CTHRSSVVC was labeled with aluminum [18F]fluoride. Tracer binding and its biodistribution were evaluated in an in vitro binding assay and in healthy BALB/c mice, respectively. In addition, different treatments with cyclophosphamide in tumor-bearing mice were used to assess whether the tracer could detect differences in CD163 expression caused by differential TAM infiltration. After 7 days of treatment, animals were injected with [18F]AlF-NODA-MP-C6-CTHRSSVVC, and a 60-min dynamic PET scan was performed, followed by an ex vivo biodistribution study. [18F]AlF-NODA-MP-C6-CTHRSSVVC was prepared in 23 ± 6 % radiochemical yield and showed approximately 50 % of specific receptor-mediated binding in an in vitro binding assay on human CD163-expressing tissue homogenates. No CD163-mediated binding of [18F]AlF-NODA-MP-C6-CTHRSSVVC was detected by PET under normal physiological conditions in healthy BALB/c mice. On the other hand, CD163-positive xenograft tumors were clearly visualized with PET and a positive correlation was found between CD163 levels and the [18F]AlF-NODA-MP-C6-CTHRSSVVC tumor-to-muscle ratio (TMR) obtained from the PET images (Pearson r = 0.76, p = 0.002). No significant differences in the CD163 protein level and in the tracer uptake between treatment groups were found in the tumors. Taken together, [18F]AlF-NODA-MP-C6-CTHRSSVVC appears a promising candidate PET tracer for M2-type TAM, as it binds specifically to CD163 in vitro and its tumor uptake correlates well with CD163 expression in vivo.

In vivo evaluation of 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-ß-D-glucopyronuronate: a positron emission tomographic tracer for imaging ß-glucuronidase activity in a tumor/inflammation rodent model.

ß-Glucuronidase (ß-GUS) plays an important role in inflammation and degenerative processes. The enzyme has also been investigated as a target in prodrug therapy for cancer. To investigate the role of ß-GUS in pathologies and to optimize ß-GUS-based prodrug therapies, we recently developed a positron emission tomographic (PET) tracer, 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-ß-D-glucopyronuronate ([18F]FEAnGA), which proved to be selectively cleaved by ß-GUS. Here we present the in vivo evaluation of [18F]FEAnGA for imaging of ß-GUS in a tumor/inflammation model. Ex vivo biodistribution of [18F]FEAnGA was conducted in healthy rats. PET imaging and pharmacokinetic modeling were performed in Wistar rats bearing C6 tumors of different sizes and sterile inflammation. The biodistribution studies of [18F]FEAnGA indicated low uptake in major organs and rapid excretion through the renal pathway. MicroPET studies revealed three times higher uptake in the viable part of larger C6 gliomas than in smaller C6 gliomas. Uptake in inflamed muscle was significantly higher than in control muscle. The distribution volume of [18F]FEAnGA in the viable part of the tumor correlated well with the cleavage of the tracer to [18F]fluoroethylamine and the spacer 4-hydroxy-3-nitrobenzyl alcohol. [18F]FEAnGA is a PET tracer able to detect increased activity of ß-GUS in large solid tumors and in inflamed tissues.

Induction of ß-glucuronidase release by cytostatic agents in small tumors.

Extracellular ß-glucuronidase (ß-GUS) in tumors has been investigated as a target enzyme for prodrug therapy. However, despite encouraging preclinical results, animal studies also indicate that the success of prodrug therapy might be limited by the insufficient prodrug-converting enzyme activity, especially in small tumors. We hypothesized that a single dose of a cytostatic drug might induce the release of ß-GUS in small tumors, resulting in increased levels of extracellular ß-GUS and consequently a higher efficacy of the prodrug treatment. Here we examine the extent of ß-GUS release in small C6 glioma tumors after a single treatment of doxorubicin (DOX), carmustine (BCNU) and tumor necrosis factor α (TNF-α) with positron emission tomography (PET) and the tracer 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-ß-d-glucopyronuronate, [(18)F]FEAnGA, which has been proven to be selective for extracellular ß-GUS. Induction of ß-GUS release was first investigated in cultured C6 glioma cells. In addition, a [(18)F]FEAnGA PET study was performed in C6 tumor-bearing rats 48 h after a single treatment with different cytostatics to evaluate the extent of ß-glucuronidase release. The cleavage of [(18)F]FEAnGA by ß-GUS was analyzed in tumor homogenates. The induction of tumor necrosis and leukocyte infiltration was confirmed by histochemical analysis and flow cytometry. The in vitro studies indicated that all treatments resulted in a decline of viable cells and an increase of extracellular ß-GUS activity. PET studies confirmed that ß-GUS was released in vivo and the distribution volume of the PET tracer [(18)F]FEAnGA in C6 gliomas was increased significantly by 15-70%, depending on the treatment. Histochemical analysis of the tumors indicated that carmustine and TNF-α treatment caused a larger necrotic area with the absence of infiltrating immune cells, whereas doxorubicin induced an increase in leukocyte infiltration. These results were confirmed by flow cytometry. In conclusion, the present study demonstrates that a single dose of a cytostatic agent is able to increase the release of ß-GUS. The release in ß-GUS can be monitored by [(18)F]FEAnGA PET in a noninvasive manner. This study may open the way to a two-step chemotherapy-prodrug approach, in which tumors are treated with a single dose of a cytostatic drug prior to prodrug treatment.

Patient-derived parathyroid organoids as a tracer and drug-screening application model.

Parathyroid diseases are characterized by dysregulation of calcium homeostasis and alterations in parathyroid hormone (PTH) excretion. The development of parathyroid-targeted treatment and imaging tracers could benefit from in vitro models. Therefore, we aim to establish a patient-derived parathyroid organoid model representing human parathyroid tissue. Hyperplastic parathyroid tissue was dispersed, and parathyroid organoids (PTOs) were cultured and characterized. PTO-derived cells exhibited self-renewal over several passages, indicative of the presence of putative stem cells. Immunofluorescence and RNA sequencing confirmed that PTOs phenocopy hyperplastic parathyroid tissue. Exposure of PTOs to increasing calcium concentrations and PTH-lowering drugs resulted in significantly reduced PTH excretion. PTOs showed specific binding of the imaging tracers 11C-methionine and 99mTc-sestamibi. These data show the functionality of PTOs resembling the parathyroid. This PTO model recapitulates the originating tissue on gene and protein expression and functionality, paving the way for future physiology studies and therapeutic target and tracer discovery.

18F-BMS986192 PET Imaging of PD-L1 in Metastatic Melanoma Patients with Brain Metastases Treated with Immune Checkpoint Inhibitors: A Pilot Study.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) frequently induces tumor response in metastatic melanoma patients. However, tumor response often takes months and may be heterogeneous. Consequently, additional local treatment for nonresponsive metastases may be needed, especially in the case of brain metastases. Noninvasive imaging may allow the characterization of (brain) metastases to predict response. This pilot study uses 18F-BMS986192 PET for PD-L1 expression to explore the variability in metastatic tracer uptake and its relation to tumor response, with a special focus on brain metastases. Methods: Metastatic melanoma patients underwent whole-body 18F-BMS986192 PET/CT scanning before and 6 wk after starting ICI therapy. 18F-BMS986192 uptake was measured in healthy tissues, organs, and tumor lesions. Tumor response was evaluated at 12 wk using CT of the thorax/abdomen and MRI of the brain. RECIST, version 1.1, was used to define therapy response per patient. Response per lesion was measured by the percentage change in lesion diameter. Toxicity was assessed according to Common Terminology Criteria for Adverse Events, version 4.0. Results: Baseline 18F-BMS986192 PET/CT was performed in 8 patients, with follow-up scans in 4 patients. The highest tracer uptake was observed in the spleen, bone marrow, kidneys, and liver. Tracer uptake in tumor lesions was heterogeneous. In total, 42 tumor lesions were identified at baseline, with most lesions in the lungs (n = 21) and brain (n = 14). Tracer uptake was similar between tumor locations. 18F-BMS986192 uptake in lesions at baseline, corrected for blood-pool activity, was negatively correlated with the change lesion diameter at response evaluation (r = -0.49, P = 0.005), both in intra- and extracerebral lesions. Receiver-operating-characteristic analysis demonstrated that 18F-BMS986192 uptake can discriminate between responding and nonresponding lesions with an area under the curve of 0.82. At the follow-up scan, an increased 18F-BMS986192 uptake compared with baseline scan was correlated with an increased lesion diameter at response evaluation. In the follow-up 18F-BMS986192 PET scan of 2 patients, ICI-related toxicity (thyroiditis and colitis) was detected. Conclusion: In this pilot study, 18F-BMS986192 PET showed heterogeneous uptake in intra- and extracerebral metastatic lesions in melanoma patients. Baseline 18F-BMS986192 uptake was able to predict an ICI treatment-induced reduction in lesion volume, whereas the follow-up PET scan allowed the detection of treatment-induced toxicity.

A proof-of-concept study on the use of a fluorescein-based 18F-tracer for pretargeted PET.

BACKGROUND: Pretargeted immuno-PET tumor imaging has emerged as a valuable diagnostic strategy that combines the high specificity of antibody-antigen interaction with the high signal and image resolution offered by short-lived PET isotopes, while reducing the irradiation dose caused by traditional 89Zr-labelled antibodies. In this work, we demonstrate proof of concept of a novel 'two-step' immuno-PET pretargeting approach, based on bispecific antibodies (bsAbs) engineered to feature dual high-affinity binding activity for a fluorescein-based 18F-PET tracer and tumor markers. RESULTS: A copper(I)-catalysed click reaction-based radiolabeling protocol was developed for the synthesis of fluorescein-derived molecule [18F]TPF. Binding of [18F]TPF on FITC-bearing bsAbs was confirmed. An in vitro autoradiography assay demonstrated that [18F]TPF could be used for selective imaging of EpCAM-expressing OVCAR3 cells, when pretargeted with EpCAMxFITC bsAb. The versatility of the pretargeting approach was showcased in vitro using a series of fluorescein-binding bsAbs directed at various established cancer-associated targets, including the pan-carcinoma cell surface marker EpCAM, EGFR, melanoma marker MCSP (aka CSPG4), and immune checkpoint PD-L1, offering a range of potential future applications for this pretargeting platform. CONCLUSION: A versatile pretargeting platform for PET imaging, which combines bispecific antibodies and a fluorescein-based 18F-tracer, is presented. It is shown to selectively target EpCAM-expressing cells in vitro and its further evaluation with different bispecific antibodies demonstrates the versatility of the approach.

Potential PET tracers for imaging of tumor-associated macrophages.

The increasing incidence of cancer over the years is one of the most challenging problems in healthcare. As cancer progresses, the recruitment of several immune cells is triggered. Infiltration of tumor-associated macrophages (TAMs) is correlated with poor patient prognosis. Since TAMs constitute a big portion of the tumor mass, targeting these cells seems to be an attractive approach for cancer immunotherapy. Additionally, TAM assessment using non-invasive imaging techniques, such as positron emission tomography (PET), might provide a better understanding of the role of TAMs in cancer, and a means for tumor profile characterization, patient selection for individualized immunotherapy and treatment monitoring. Imaging of TAMs using PET tracers is still in its infancy. TAMs have several characteristics that could be exploited as potential targets for imaging. Various PET tracers for these TAM biomarkers have been developed, although often in the context of (neuro)inflammatory diseases rather than cancer. Since macrophages in inflammatory diseases express similar biomarkers as TAMs, these PET tracers could potentially also be applied for the assessment of TAMs in the tumor microenvironment. Therefore, the present review provides an overview of the TAM biomarkers, for which potential PET tracers are available and discusses the status of these tracers.

A new approach to produce [18F]MC225 via one-step synthesis, a PET radiotracer for measuring P-gp function.

BACKGROUND: [18F]MC225 is a radiotracer for imaging P-glycoprotein (P-gp) function at the blood-brain barrier. The P-gp function can be altered due to different factors, for instance, decreased P-gp function has been described in patients with Alzheimer's or Parkinson's Disease. The current applied radiosynthesis of [18F]MC225 involves 2 steps, including the distillation of the [18F] fluoroethylbromide intermediate. To develop a more robust synthetic procedure, it is of interest to produce the radiotracer via a 1-step synthesis. The present study describes a new synthetic approach to produce [18F]MC225 via direct 18F-fluorination. Moreover, we also provide the appropriate conditions for the automation of the synthesis. A mesylate precursor was synthesized via a multi-step synthetic route and used for the radiolabeling. The nucleophilic substitution of the mesylate group by [18F] Fluoride was automated in two different synthesis modules: IBA Synthera and Eckert and Ziegler PharmTracer (E&Z). RESULTS: The mesylate precursor was synthesized in 7 steps starting with 5-hydroxy-1-tetralone (commercially available) in practical yields. The stability of the precursor was improved via mesylate salt formation method. The radiolabeling was done by adding the mesylate precursor dissolved in DMF to the dried [18F]KF/K2.2.2 complex and heating at 140 °C for 30 min. Quality control by UPLC confirmed the production of [18F]MC225 with a molar activity (Am) higher than 100 GBq/micromole. The synthesis time in Synthera was 106 min and the product was obtained with a radiochemical purity higher than 95% and RCY of 6.5%, while the production in E&Z lasted 120 min and the product had a lower radiochemical purity (91%) and RCY (3.8%). CONCLUSIONS: [18F]MC225 was successfully produced via a 1-step reaction. The procedure is suitable for automation using commercially available synthesis modules. The automation of the radiosynthesis in the Synthera module allows the production of the [18F]MC225 by a reliable and simple method.

[18F]-sodium fluoride autoradiography imaging of nephrocalcinosis in donor kidneys and explanted kidney allografts.

Nephrocalcinosis is present in up to 43% of kidney allograft biopsies at one-year after transplantation and is associated with inferior graft function and poor graft survival. We studied [18F]-sodium fluoride ([18F]-NaF) imaging of microcalcifications in donor kidneys (n = 7) and explanted kidney allografts (n = 13). Three µm paraffin-embedded serial sections were used for histological evaluation of calcification (Alizarin Red; Von Kossa staining) and ex-vivo [18F]-NaF autoradiography. The images were fused to evaluate if microcalcification areas corresponded with [18F]-NaF uptake areas. Based on histological analyses, tubulointerstitial and glomerular microcalcifications were present in 19/20 and 7/20 samples, respectively. Using autoradiography, [18F]-NaF uptake was found in 19/20 samples, with significantly more tracer activity in kidney allograft compared to deceased donor kidney samples (p = 0.019). Alizarin Red staining of active microcalcifications demonstrated good correlation (Spearman's rho of 0.81, p < 0.001) and Von Kossa staining of consolidated calcifications demonstrated significant but weak correlation (0.62, p = 0.003) with [18F]-NaF activity. This correlation between ex-vivo [18F]-NaF uptake and histology-proven microcalcifications, is the first step towards an imaging method to identify microcalcifications in active nephrocalcinosis. This may lead to better understanding of the etiology of microcalcifications and its impact on kidney transplant function.

Synthesis and Evaluation of 18F-Enzalutamide, a New Radioligand for PET Imaging of Androgen Receptors: A Comparison with 16ß-18F-Fluoro-5α-Dihydrotestosterone.

16ß-18F-fluoro-5α-dihydrotestosterone (18F-FDHT) is a radiopharmaceutical that has been investigated as a diagnostic agent for the assessment of androgen receptor (AR) density in prostate cancer using PET. However, 18F-FDHT is rapidly metabolized in humans and excreted via the kidneys into the urine, potentially compromising the detection of tumor lesions close to the prostate. Enzalutamide is an AR signaling inhibitor currently used in different stages of prostate cancer. Enzalutamide and its primary metabolite N-desmethylenzalutamide have an AR affinity comparable to that of FDHT but are excreted mainly via the hepatic route. Radiolabeled enzalutamide could thus be a suitable candidate PET tracer for AR imaging. Here, we describe the radiolabeling of enzalutamide with 18F. Moreover, the in vitro and in vivo behavior of 18F-enzalutamide was evaluated and compared with the current standard, 18F-FDHT. Methods:18F-enzalutamide was obtained by fluorination of the nitro precursor. In vitro cellular uptake studies with 18F-enzalutamide and 18F-FDHT were performed in LNCaP (AR-positive) and HEK293 (AR-negative) cells. Competition assays with both tracers were conducted on the LNCaP (AR-positive) cell line. In vivo PET imaging, ex vivo biodistribution, and metabolite studies with 18F-enzalutamide and 18F-FDHT were conducted on athymic nude male mice bearing an LNCaP xenograft in the shoulder. Results:18F-enzalutamide was obtained in 1.4% ± 0.9% radiochemical yield with an apparent molar activity of 6.2 ± 10.3 GBq/µmol. 18F-FDHT was obtained in 1.5% ± 0.8% yield with a molar activity of more than 25 GBq/µmol. Coincubation with an excess of 5α-dihydrotestosterone or enzalutamide significantly reduced the cellular uptake of 18F-enzalutamide and 18F-FDHT to about 50% in AR-positive LNCaP cells but not in AR-negative HEK293 cells. PET and biodistribution studies on male mice bearing a LnCaP xenograft showed about 3 times higher tumor uptake for 18F-enzalutamide than for 18F-FDHT. Sixty minutes after tracer injection, 93% of 18F-enzalutamide in plasma was still intact, compared with only 3% of 18F-FDHT. Conclusion: Despite its lower apparent molar activity, 18F-enzalutamide shows higher tumor uptake and better metabolic stability than 18F-FDHT and thus seems to have more favorable properties for imaging of AR with PET. However, further evaluation in other oncologic animal models and patients is warranted to confirm these results.

GMP Compliant Synthesis of [18F]Canagliflozin, a Novel PET Tracer for the Sodium-Glucose Cotransporter 2.

Inhibition of the sodium-glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mellitus results in large between-patient variability in clinical response. To better understand this variability, the positron emission tomography (PET) tracer [18F]canagliflozin was developed via a Cu-mediated 18F-fluorination of its boronic ester precursor with a radiochemical yield of 2.0 ± 1.9% and a purity of >95%. The GMP automated synthesis originated [18F]canagliflozin with a yield of 0.5-3% (n = 4) and a purity of >95%. Autoradiography showed [18F]canagliflozin binding in human kidney sections containing SGLT2. Since [18F]canagliflozin is the isotopologue of the extensively characterized drug canagliflozin and thus shares its toxicological and pharmacological characteristics, it enables its immediate use in patients.

Mapping Arginase Expression with 18F-Fluorinated Late-Generation Arginase Inhibitors Derived from Quaternary α-Amino Acids.

Arginase hydrolyzes L-arginine and influences levels of polyamines and nitric oxide. Arginase overexpression is associated with inflammation and tumorigenesis. Thus, radiolabeled arginase inhibitors may be suitable PET tracers for staging arginase-related pathophysiologies. We report the synthesis and evaluation of 2 radiolabeled arginase inhibitors, 18F-FMARS and 18F-FBMARS, developed from α-substituted-2-amino-6-boronohexanoic acid derivatives. Methods: Arylboronic ester-derived precursors were radiolabeled via copper-mediated fluorodeboronation. Binding assays using arginase-expressing PC3 and LNCaP cells were performed. Autoradiography of lung sections from a guinea pig model of asthma overexpressing arginase and dynamic small-animal PET imaging with PC3-xenografted mice evaluated the radiotracers' specific binding and pharmacokinetics. Results:18F-fluorinated compounds were obtained with radiochemical yields of up to 5% (decay-corrected) and an average molar activity of 53 GBq⋅µmol-1 Cell and lung section experiments indicated specific binding that was blocked up to 75% after pretreatment with arginase inhibitors. Small-animal PET studies indicated fast clearance of the radiotracers (7.3 ± 0.6 min), arginase-mediated uptake, and a selective tumor accumulation (SUV, 3.0 ± 0.7). Conclusion: The new 18F-fluorinated arginase inhibitors have the potential to map increased arginase expression related to inflammatory and tumorigenic processes. 18F-FBMARS showed the highest arginase-mediated uptake in PET imaging and a significant difference between uptake in control and arginase-inhibited PC3 xenografted mice. These results encourage further research to examine the suitability of 18F-FBMARS for selecting patients for treatments with arginase inhibitors.

[18F]FDG Uptake in Adipose Tissue Is Not Related to Inflammation in Type 2 Diabetes Mellitus.

PURPOSE: 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake is a marker of metabolic activity and is therefore used to measure the inflammatory state of several tissues. This radionuclide marker is transported through the cell membrane via glucose transport proteins (GLUTs). The aim of this study is to investigate whether insulin resistance (IR) or inflammation plays a role in [18F]FDG uptake in adipose tissue (AT). PROCEDURES: This study consisted of an in vivo clinical part and an ex vivo mechanistic part. In the clinical part, [18F]FDG uptake in abdominal visceral AT (VAT) and subcutaneous AT (SAT) was determined using PET/CT imaging in 44 patients with early type 2 diabetes mellitus (T2DM) (age 63 [54-66] years, HbA1c [6.3 ± 0.4 %], HOMA-IR 5.1[3.1-8.5]). Plasma levels were measured with ELISA. In the mechanistic part, AT biopsies obtained from 8 patients were ex vivo incubated with [18F]FDG followed by autoradiography. Next, a qRT-PCR analysis was performed to determine GLUT and cytokine mRNA expression levels. Immunohistochemistry was performed to determine CD68+ macrophage infiltration and GLUT4 protein expression in AT. RESULTS: In vivo VAT [18F]FDG uptake in patients with T2DM was inversely correlated with HOMA-IR (r = - 0.32, p = 0.034), and positively related to adiponectin plasma levels (r = 0.43, p = 0.003). Ex vivo [18F]FDG uptake in VAT was not related to CD68+ macrophage infiltration, and IL-1ß and IL-6 mRNA expression levels. Ex vivo VAT [18F]FDG uptake was positively related to GLUT4 (r = 0.83, p = 0.042), inversely to GLUT3 (r = - 0.83, p = 0.042) and not related to GLUT1 mRNA expression levels. CONCLUSIONS: In vivo [18F]FDG uptake in VAT from patients with T2DM is positively correlated with adiponectin levels and inversely with IR. Ex vivo [18F]FDG uptake in AT is associated with GLUT4 expression but not with pro-inflammatory markers. The effect of IR should be taken into account when interpreting data of [18F]FDG uptake as a marker for AT inflammation.

Highlight selection of radiochemistry and radiopharmacy developments by editorial board.

BACKGROUND: The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highlight commentary to update the readership on trends in the field of radiopharmaceutical development. RESULTS: This commentary of highlights has resulted in 21 different topics selected by each member of the Editorial Board addressing a variety of aspects ranging from novel radiochemistry to first in man application of novel radiopharmaceuticals. Also the first contribution in relation to MRI-agents is included. CONCLUSIONS: Trends in (radio)chemistry and radiopharmacy are highlighted demonstrating the progress in the research field being the scope of EJNMMI Radiopharmacy and Chemistry.

Synthesis and evaluation of [18F]-FEAnGA as a PET Tracer for beta-glucuronidase activity.

To increase the therapeutic index of chemotherapeutic drugs, prodrugs have been investigated as anticancer agents, as they may present fewer cytotoxic side effects than conventional cytotoxic drugs, while therapeutic efficacy is maintained or even increased. Extracellular beta-glucuronidase (beta-GUS) in the tumors has been investigated as a target enzyme for prodrug therapy, as it can convert nontoxic prodrugs into cytostatic drugs. To optimize beta-GUS-based prodrug therapies, PET imaging could be a useful tool by providing information regarding the localization and quantification of beta-GUS. Here, we describe our first PET tracer for extracellular beta-GUS, [(18)F]-FEAnGA, which consists of a 2-[(18)F]fluoroethylamine ([(18)F]-FEA) group bound to a glucuronic acid via a self-immolative nitrophenyl spacer. [(18)F]-FEAnGA was synthesized by alkylation of its imidazole carbamate precursor with [(18)F]-FEA, followed by deprotection of the sugar moiety with NaOH in 10-20% overall radiochemical yield. [(18)F]-FEAnGA is about 10-fold more hydrophilic than the cleavage product [(18)F]-FEA, and it is stable in PBS and rat plasma for at least 3 h. In the presence of either Escherichia coli beta-GUS or bovine liver beta-GUS, in vitro cleavage of [(18)F]-FEAnGA with complete release of [(18)F]-FEA was observed within 30 min. C6 glioma cells incubated with the tracer and Escherichia coli beta-GUS or bovine liver beta-GUS showed a 4- and 1.5-fold higher uptake of radioactivity, respectively, as compared to control C6 cells without beta-GUS. Incubation of CT26 murine colon adenocarcinoma cells or the genetically engineered CT26mbetaGUS cells, which expressed membrane-anchored GUS on the outer cell membrane, with the tracer, resulted in a 3-fold higher uptake into GUS-expressing cells as compared to control cells. In a preliminary microPET study in mice bearing both CT26 and CT26mbetaGUS tumors, [(18)F]-FEAnGA exhibited a 2-fold higher retention of radioactivity in the tumor expressing beta-GUS than in the control tumor. [(18)F]-FEA did not show any difference in tracer uptake between tumors. These results suggest that [(18)F]-FEAnGA may be a suitable PET tracer for evaluation of beta-GUS activity, since it is specifically cleaved by beta-GUS and the released [(18)F]-FEA remains attached to targeted cells.

[18F]Atorvastatin: synthesis of a potential molecular imaging tool for the assessment of statin-related mechanisms of action.

BACKGROUND: Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinically used in the primary and secondary prevention of cardiovascular diseases. However, a considerable group of patients does not respond to statin treatment, and the reason for this is still not completely understood. [18F]Atorvastatin, the 18F-labeled version of one of the most widely prescribed statins, may be a useful tool for statin-related research. RESULTS: [18F]Atorvastatin was synthesized via an optimized ruthenium-mediated late-stage 18F-deoxyfluorination. The defluoro-hydroxy precursor was produced via Paal-Knorr pyrrole synthesis and was followed by coordination of the phenol to a ruthenium complex, affording the labeling precursor in approximately 10% overall yield. Optimization and automation of the labeling procedure reliably yielded an injectable solution of [18F]atorvastatin in 19% ± 6% (d.c.) with a molar activity of 65 ± 32 GBq·µmol-1. Incubation of [18F]atorvastatin in human serum did not lead to decomposition. Furthermore, we have shown the ability of [18F]atorvastatin to cross the hepatic cell membrane to the cytosolic and microsomal fractions where HMG-CoA reductase is known to be highly expressed. Blocking assays using rat liver sections confirmed the specific binding to HMG-CoA reductase. Autoradiography on rat aorta stimulated to develop atherosclerotic plaques revealed that [18F]atorvastatin significantly accumulates in this tissue when compared to the healthy model. CONCLUSIONS: The improved ruthenium-mediated 18F-deoxyfluorination procedure overcomes previous hurdles such as the addition of salt additives, the drying steps, or the use of different solvent mixtures at different phases of the process, which increases its practical use, and may allow faster translation to clinical settings. Based on tissue uptake evaluations, [18F]atorvastatin showed the potential to be used as a tool for the understanding of the mechanism of action of statins. Further knowledge of the in vivo biodistribution of [18F]atorvastatin may help to better understand the origin of off-target effects and potentially allow to distinguish between statin-resistant and non-resistant patients.