Improving Routine 89Zr-Immuno-PET Applications: Mild Iron Removal Can Favor the Use of Fe-DFO-N-suc-TFP Ester Over p-NCS-Bz-DFO.

A key aspect for the applicability of 89Zr-radioimmunoconjugates is inert modification and radiolabeling. The two commercially available bifunctional variants of the siderophore desferrioxamine (DFO), Fe-DFO-N-suc-TFP-ester and p-NCS-Bz-DFO, are most often used for clinical 89Zr-immuno-PET. The use of Fe-DFO-N-suc-TFP-ester is advantageous with regard to higher radiolysis stability and more facile assessment of radiochemical purity as well as chelator-to-mAb ratio. However, not all mAbs withstand the Fe-removal step at relatively low pH (4-4.5) using EDTA, which is needed after conjugation to allow 89Zr labeling. In this study, it was investigated whether hydroxybenzyl ethylenediamine (HBED) or the clinically approved deferiprone (DFP) can serve as an alternative for EDTA to establish a pH-independent mild method for Fe-removal and thereby broaden the applicability of Fe-DFO-N-suc-TFP-ester. Carrier-added [59Fe]Fe-DFO-N-suc-TFP-ester was used for mAb modification to enable direct tracking of the Fe-removal efficiency under various conditions. Whereas incomplete Fe-removal with HBED was observed at pH 5 or higher, Fe-removal with DFP was possible at a broad pH range (4-9). This provides a mild, pH-independent method for Fe-removal, improving the applicability and attractiveness of Fe-DFO-N-suc-TFP-ester for 89Zr-mAb preparation.

Synthesis of 18F-labeled Aryl Trifluoromethyl Sulfones, -Sulfoxides, and -Sulfides for Positron Emission Tomography.

Positron emission tomography (PET) is becoming increasingly important in nuclear medicine and drug discovery. To date, the development of many potential PET tracers is hampered by the lack of suitable synthetic pathways for their preparation. This is particularly true for the highly desired radiolabeling of compounds bearing [18F]CF3-groups. For instance, S(O)nCF3-groups (n=0, 1, 2) serve as structural motif in a range of biologically active compounds, but their radiosynthesis remains largely unprecedented (for n=1, 2). Herein, we describe general methods for the radiosynthesis of 18F-labeled aryl trifluoromethyl sulfones, -sulfoxides, and -sulfides. All three methods are operationally straightforward, start from widely available precursors, i.e., sulfonyl fluorides and thiophenols, and make use of the recently established [18F]Ruppert-Prakash reagent. Further, the syntheses display good functional group tolerance as demonstrated by the 18F-labeling of more than 40 compounds. The applicability of the new method is demonstrated by the radiolabeling of three bioactive molecules, optionally to be used as PET tracers. In a broader context, this work presents a substantial expansion of the chemical space of radiofluorinated structural motifs to be used for the development of new PET tracers.

89Zr-immuno-PET using the anti-LAG-3 tracer [89Zr]Zr-BI 754111: demonstrating target specific binding in NSCLC and HNSCC.

PURPOSE: Although lymphocyte activation gene-3 (LAG-3) directed therapies demonstrate promising clinical anti-cancer activity, only a subset of patients seems to benefit and predictive biomarkers are lacking. Here, we explored the potential use of the anti-LAG-3 antibody tracer [89Zr]Zr-BI 754111 as a predictive imaging biomarker and investigated its target specific uptake as well as the correlation of its tumor uptake and the tumor immune infiltration. METHODS: Patients with head and neck (N = 2) or lung cancer (N = 4) were included in an imaging substudy of a phase 1 trial with BI 754091 (anti-PD-1) and BI 754111 (anti-LAG-3). After baseline tumor biopsy and [18F]FDG-PET, patients were given 240 mg of BI 754091, followed 8 days later by administration of [89Zr]Zr-BI 754111 (37 MBq, 4 mg). PET scans were performed 2 h, 96 h, and 144 h post-injection. To investigate target specificity, a second tracer administration was given two weeks later, this time with pre-administration of 40 (N = 3) or 600 mg (N = 3) unlabeled BI 754111, followed by PET scans at 96 h and 144 h post-injection. Tumor immune cell infiltration was assessed by immunohistochemistry and RNA sequencing. RESULTS: Tracer uptake in tumors was clearly visible at the 4-mg mass dose (tumor-to-plasma ratio 1.63 [IQR 0.37-2.89]) and could be saturated by increasing mass doses (44 mg: 0.67 [IQR 0.50-0.85]; 604 mg: 0.56 [IQR 0.42-0.75]), demonstrating target specificity. Tumor uptake correlated to immune cell-derived RNA signatures. CONCLUSIONS: [89Zr]Zr-BI-754111 PET imaging shows favorable technical and biological characteristics for developing a potential predictive imaging biomarker for LAG-3-directed therapies. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03780725. Registered 19 December 2018.

Validation of simplified uptake measures against dynamic Patlak Ki for quantification of lesional 89Zr-Immuno-PET antibody uptake.

PURPOSE: Positron emission tomography imaging of zirconium-89-labelled monoclonal antibodies (89Zr-Immuno-PET) allows for visualisation and quantification of antibody uptake in tumours in vivo. Patlak linearization provides distribution volume (VT) and nett influx rate (Ki) values, representing reversible and irreversible uptake, respectively. Standardised uptake value (SUV) and tumour-to-plasma/tumour-to-blood ratio (TPR/TBR) are often used, but their validity depends on the comparability of plasma kinetics and clearances. This study assesses the validity of SUV, TPR and TBR against Patlak Ki for quantifying irreversible 89Zr-Immuno-PET uptake in tumours. METHODS: Ten patients received 37 MBq 10 mg 89Zr-anti-EGFR with 500 mg/m2 unlabelled mAbs. Five patients received two doses of 37 MBq 89Zr-anti-HER3: 8-24 mg for the first administration and 24 mg-30 mg/kg for the second. Seven tumours from four patients showed 89Zr-anti-EGFR uptake, and 18 tumours from five patients showed 89Zr-anti-HER3 uptake. SUVpeak, TPRpeak and TBRpeak values were obtained from one to six days p.i. Patlak linearization was applied to tumour time activity curves and plasma samples to obtain Ki. RESULTS: For 89Zr-anti-EGFR, there was a small variability along the linear regression line between SUV (- 0.51-0.57), TPR (- 0.06‒0.11) and TBR (- 0.13‒0.16) on day 6 versus Ki. Similar doses of 89Zr-anti-HER3 showed similar variability for SUV (- 1.3‒1.0), TPR (- 1.1‒0.53) and TBR (- 1.5‒0.72) on day 5 versus Ki. However, for the second administration of 89Zr-anti-HER3 with a large variability in administered mass doses, SUV showed a larger variability (- 1.4‒2.3) along the regression line with Ki, which improved when using TPR (- 0.38-0.32) or TBR (- 0.56‒0.46). CONCLUSION: SUV, TPR and TBR at late time points were valid for quantifying irreversible lesional 89Zr-Immuno-PET uptake when constant mass doses were administered. However, for variable mass doses, only TPR and TBR provided reliable values for irreversible uptake, but not SUV, because SUV does not take patient and mass dose-specific plasma clearance into account.

[11C]CO2 BOP fixation with amines to access 11C-labeled ureas for PET imaging.

Carbon-11 (11 C) is a widely used radionuclide for positron emission tomography (PET) owing to the omnipresence of carbon atoms in organic molecules. While its half-life of 20.4 min is ideal for imaging and dosimetry, it also limits the synthetic possibilities. As such, the development of fast and easy, high-yielding synthesis methods is crucial for the application of 11 C-labeled tracers in humans. In this study, we present a novel and efficient method for the reaction of [11 C]CO2 with amine precursors using benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) to access 11 C-labeled ureas. Our method is extremely fast as it only requires transfer of [11 C]CO2 into a solution with precursor and BOP at room temperature, where it reacts momentary into the desired 11 C-labeled urea. This simple procedure makes it possible to radiolabel urea directly from [11 C]CO2 without the need for advanced equipment, making the method applicable for all laboratories where [11 C]CO2 is available. We synthesized a small series of aliphatic symmetrical and non-symmetrical 11 C-labeled ureas using this method, and achieved good to excellent yields. The novelty of our study lies in the fact that peptide coupling reagent BOP is used for the first time in radiochemistry to activate [11 C]CO2 , facilitating its reaction with amines to obtain 11 C-labeled ureas.

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Pharmacodynamic evaluation and safety assessment of treatment with antibodies to serum amyloid P component in patients with cardiac amyloidosis: an open-label Phase 2 study and an adjunctive immuno-PET imaging study.

BACKGROUND: In a Phase I study treatment with the serum amyloid P component (SAP) depleter miridesap followed by monoclonal antibody to SAP (dezamizumab) showed removal of amyloid from liver, spleen and kidney in patients with systemic amyloidosis. We report results from a Phase 2 study and concurrent immuno-positron emission tomography (PET) study assessing efficacy, pharmacodynamics, pharmacokinetics, safety and cardiac uptake (of dezamizumab) following the same intervention in patients with cardiac amyloidosis. METHODS: Both were uncontrolled open-label studies. After SAP depletion with miridesap, patients received ≤ 6 monthly doses of dezamizumab in the Phase 2 trial (n = 7), ≤ 2 doses of non-radiolabelled dezamizumab plus [89Zr]Zr-dezamizumab (total mass dose of 80 mg at session 1 and 500 mg at session 2) in the immuno-PET study (n = 2). Primary endpoints of the Phase 2 study were changed from baseline to follow-up (at 8 weeks) in left ventricular mass (LVM) by cardiac magnetic resonance imaging and safety. Primary endpoint of the immuno-PET study was [89Zr]Zr-dezamizumab cardiac uptake assessed via PET. RESULTS: Dezamizumab produced no appreciable or consistent reduction in LVM nor improvement in cardiac function in the Phase 2 study. In the immuno-PET study, measurable cardiac uptake of [89Zr]Zr-dezamizumab, although seen in both patients, was moderate to low. Uptake was notably lower in the patient with higher LVM. Treatment-associated rash with cutaneous small-vessel vasculitis was observed in both studies. Abdominal large-vessel vasculitis after initial dezamizumab dosing (300 mg) occurred in the first patient with immunoglobulin light chain amyloidosis enrolled in the Phase 2 study. Symptom resolution was nearly complete within 24 h of intravenous methylprednisolone and dezamizumab discontinuation; abdominal computed tomography imaging showed vasculitis resolution by 8 weeks. CONCLUSIONS: Unlike previous observations of visceral amyloid reduction, there was no appreciable evidence of amyloid removal in patients with cardiac amyloidosis in this Phase 2 trial, potentially related to limited cardiac uptake of dezamizumab as demonstrated in the immuno-PET study. The benefit-risk assessment for dezamizumab in cardiac amyloidosis was considered unfavourable after the incidence of large-vessel vasculitis and development for this indication was terminated. Trial registration NCT03044353 (2 February 2017) and NCT03417830 (25 January 2018).

Towards PET imaging of the dynamic phenotypes of microglia.

There is increasing evidence showing the heterogeneity of microglia activation in neuroinflammatory and neurodegenerative diseases. It has been hypothesized that pro-inflammatory microglia are detrimental and contribute to disease progression, while anti-inflammatory microglia play a role in damage repair and remission. The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype relies heavily upon a clearer understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. To that end, molecular imaging techniques are required to assess microglia dynamics and study their role in disease progression as well as to evaluate the outcome of therapeutic interventions. Positron emission tomography (PET) is such a molecular imaging technique, and provides unique capabilities for non-invasive quantification of neuroinflammation and has the potential to discriminate between microglia phenotypes and define their role in the disease process. However, several obstacles limit the possibility for selective in vivo imaging of microglia phenotypes mainly related to the poor characterization of specific targets that distinguish the two ends of the microglia activation spectrum and lack of suitable tracers. PET tracers targeting translocator protein 18 kDa (TSPO) have been extensively explored, but despite the success in evaluating neuroinflammation they failed to discriminate between microglia activation statuses. In this review, we highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We also discuss the current and emerging PET imaging targets, the tracers and their potential in discriminating between the pro- and anti-inflammatory microglia activation states.

State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET.

Inert and stable radiolabeling of monoclonal antibodies (mAb), antibody fragments, or antibody mimetics with radiometals is a prerequisite for immuno-PET. While radiolabeling is preferably fast, mild, efficient, and reproducible, especially when applied for human use in a current Good Manufacturing Practice compliant way, it is crucial that the obtained radioimmunoconjugate is stable and shows preserved immunoreactivity and in vivo behavior. Radiometals and chelators have extensively been evaluated to come to the most ideal radiometal-chelator pair for each type of antibody derivative. Although PET imaging of antibodies is a relatively recent tool, applications with 89Zr, 64Cu, and 68Ga have greatly increased in recent years, especially in the clinical setting, while other less common radionuclides such as 52Mn, 86Y, 66Ga, and 44Sc, but also 18F as in [18F]AlF are emerging promising candidates for the radiolabeling of antibodies. This review presents a state of the art overview of the practical aspects of radiolabeling of antibodies, ranging from fast kinetic affibodies and nanobodies to slow kinetic intact mAbs. Herein, we focus on the most common approach which consists of first modification of the antibody with a chelator, and after eventual storage of the premodified molecule, radiolabeling as a second step. Other approaches are possible but have been excluded from this review. The review includes recent and representative examples from the literature highlighting which radiometal-chelator-antibody combinations are the most successful for in vivo application.

Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET.

PURPOSE: Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. METHODS: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. RESULTS: [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. CONCLUSION: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

Evaluating N-difluoromethyltriazolium triflate as a precursor for the synthesis of high molar activity [18 F]fluoroform.

The trifluoromethyl group is a prominent motif in biologically active compounds and therefore of great interest for the labeling with the positron emitter fluorine-18 for positron emission tomography (PET) imaging. Multiple labeling strategies have been explored in the past; however, most of them suffer from low molar activity due to precursor degradation. In this study, the potential of 1-(difluoromethyl)-3-methyl-4-phenyl-1H-1,2,3-triazol-3-ium triflate as precursor for the synthesis of the [18 F]trifluoromethylation building block [18 F]fluoroform with high molar activity was investigated. The triazolium precursor was reacted under various conditions with [18 F]fluoride, providing [18 F]fluoroform with radiochemical yields (RCY) and molar activities (Am ) comparable and even superior with already existing methods. Highest molar activities (Am = 153 ± 14 GBq/µmol, dc, EOS) were observed for the automated procedure on the Neptis® perform module. Due to its easy handling and good RCY and Am in the [18 F]fluoroform synthesis, the triazolium precursor is a valuable alternative to already known precursors.

PET imaging of P2X7R in the experimental autoimmune encephalomyelitis model of multiple sclerosis using [11C]SMW139.

BACKGROUND: Non-invasive imaging of the activation status of microglia and the ability to identify a pro- or anti-inflammatory environment can provide valuable insights not only into pathogenesis of neuro-inflammatory and neurodegenerative diseases but also the monitoring of the efficacy of immunomodulatory therapies. P2X7R is highly expressed on pro-inflammatory microglia and [11C]SMW139, a specific P2X7R tracer for positron emission tomography imaging, showed good pharmacokinetics, stability, and brain permeability in vivo. Our objective was to evaluate the potential of [11C]SMW139 for PET imaging of neuroinflammation in vivo in the experimental autoimmune encephalomyelitis (EAE) model. METHODS: We induced EAE in Lewis rats by immunization with MBP 69-88 in complete Freund's adjuvant (CFA). We determined the affinity of [11C]SMW139 to human and rat P2X7R using saturation binding assay. Using this tracer, PET imaging was performed at the peak of disease and in the recovery phase. In vivo blocking experiments were conducted to validate the specific brain uptake of the tracer. Immunohistochemistry staining and autoradiography were performed to evaluate the level of neuroinflammation and validate the specific binding of [11C]SMW139. RESULTS: [11C]SMW139 showed good affinity for the rat P2X7R with a Kd of 20.6 ± 1.7 nM. The uptake of [11C]SMW139 was significantly higher in EAE animals at the peak of disease compared to the recovery phase but not in CFA control animals. The amplitude of increase of [11C]SMW139 uptake showed significant positive correlation with clinical scores mainly in the spinal cord (Pearson = 0.75, Spearman = 0.76; p < 0.0001). Treating EAE animals with P2X7R antagonist JNJ-47965567 blocked the uptake of [11C]SMW139 in the spinal cord, cerebellum, and brain stem, demonstrating specific accumulation of the tracer. P-glycoprotein blocking with tariquidar (30 mg/kg) did not affect tracer penetration in the brain showing that [11C]SMW139 is not a Pgp substrate. CONCLUSION: Our data shows that [11C]SMW139 is a promising PET tracer for imaging neuroinflammation and evaluating the dynamics of pro-inflammatory microglia in the brain. This can provide crucial insights into the role of microglia in disease progression and enables the development of novel treatment strategies aimed at modulating the immune response in order to promote neuroprotection.

Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers.

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

The P2X7 receptor tracer [11C]SMW139 as an in vivo marker of neuroinflammation in multiple sclerosis: a first-in man study.

PURPOSE: The novel PET tracer [11C]SMW139 binds with high affinity to the P2X7 receptor, which is expressed on pro-inflammatory microglia. The purposes of this first in-man study were to characterise pharmacokinetics of [11C]SMW139 in patients with active relapsing remitting multiple sclerosis (RRMS) and healthy controls (HC) and to evaluate its potential to identify in vivo neuroinflammation in RRMS. METHODS: Five RRMS patients and 5 age-matched HC underwent 90-min dynamic [11C]SMW139 PET scans, with online continuous and manual arterial sampling to generate a metabolite-corrected arterial plasma input function. Tissue time activity curves were fitted to single- and two-tissue compartment models, and the model that provided the best fits was determined using the Akaike information criterion. RESULTS: The optimal model for describing [11C]SMW139 kinetics in both RRMS and HC was a reversible two-tissue compartment model with blood volume parameter and with the dissociation rate k4 fixed to the whole-brain value. Exploratory group level comparisons demonstrated an increased volume of distribution (VT) and binding potential (BPND) in RRMS compared with HC in normal appearing brain regions. BPND in MS lesions was decreased compared with non-lesional white matter, and a further decrease was observed in gadolinium-enhancing lesions. In contrast, increased VT was observed in enhancing lesions, possibly resulting from disruption of the blood-brain barrier in active MS lesions. In addition, there was a high correlation between parameters obtained from 60- to 90-min datasets, although analyses using 60-min data led to a slight underestimation in regional VT and BPND values. CONCLUSIONS: This first in-man study demonstrated that uptake of [11C]SMW139 can be quantified with PET using BPND as a measure for specific binding in healthy controls and RRMS patients. Additional studies are warranted for further clinical evaluation of this novel neuroinflammation tracer.

F8-IL10: A New Potential Antirheumatic Drug Evaluated by a PET-Guided Translational Approach.

Antibody fragment F8-mediated interleukin 10 (IL10) delivery is a novel treatment for rheumatoid arthritis (RA). F8 binds to the extra-domain-A of fibronectin (ED-A). In this study, in vivo biodistribution and arthritis targeting of radiolabeled F8-IL10 were investigated in RA patients, followed by further animal studies. Therefore, three RA patients (DAS28 > 3.2) received 0.4 mg of 30-74 megabecquerel [124I]I-F8-IL10 for PET-CT and blood sampling. In visually identified PET-positive joints, target-to-background was calculated. Healthy mice, rats, and arthritic rats were injected with iodinated F8-IL10 or KSF-IL10 control antibody. Various organs were excised, weighed, and counted for radioactivity. Tissue sections were stained for fibronectin ED-A. In RA patients, [124I]I-F8-IL10 was cleared rapidly from the circulation with less than 1% present in blood after 5 min. PET-CT showed targeting in 38 joints (11-15 per patient) and high uptake in the liver and spleen. Mean target-to-background ratios of PET-positive joints were 2.5 ± 1.2, 1.5 times higher for clinically active than clinically silent joints. Biodistribution of radioiodinated F8-IL10 in healthy mice showed no effect of the radioiodination method. [124I]I-F8-IL10 joint uptake was also demonstrated in arthritic rats, ∼14-fold higher than that of the control antibody [124I]I-KSF-IL10 ( p < 0.001). Interestingly, liver and spleen uptake were twice as high in arthritic than in healthy rats and were related to increased (∼7×) fibronectin ED-A expression in these tissues. In conclusion, [124I]I-F8-IL10 uptake was observed in arthritic joints in RA patients holding promise for visualization of inflamed joints by PET-CT imaging and therapeutic targeting. Patient observations and, subsequently, arthritic animal studies pointed to awareness of increased [124I]I-F8-IL10 uptake in the liver and spleen associated with moderate systemic inflammation. This translational study demonstrated the value of in vivo biodistribution and PET-CT-guided imaging in development of new and potential antirheumatic drugs'.

Fluorine-18 labelled building blocks for PET tracer synthesis.

Positron emission tomography (PET) is an important driver for present day healthcare. Fluorine-18 is the most widely used radioisotope for PET imaging and a thorough overview of the available radiochemistry methodology is a prerequisite for selection of a synthetic approach for new fluorine-18 labelled PET tracers. These PET tracers can be synthesised either by late-stage radiofluorination, introducing fluorine-18 in the last step of the synthesis, or by a building block approach (also called modular build-up approach), introducing fluorine-18 in a fast and efficient manner in a building block, which is reacted further in one or multiple reaction steps to form the PET tracer. This review presents a comprehensive overview of the synthesis and application of fluorine-18 labelled building blocks since 2010.

PET Imaging of Microglial Activation-Beyond Targeting TSPO.

Neuroinflammation, which involves microglial activation, is thought to play a key role in the development and progression of neurodegenerative diseases and other brain pathologies. Positron emission tomography is an ideal imaging technique for studying biochemical processes in vivo, and particularly for studying the living brain. Neuroinflammation has been traditionally studied using radiotracers targeting the translocator protein 18 kDa, but this comes with certain limitations. The current review describes alternative biological targets that have gained interest for the imaging of microglial activation over recent years, such as the cannabinoid receptor type 2, cyclooxygenase-2, the P2X7 receptor and reactive oxygen species, and some promising radiotracers for these targets. Although many advances have been made in the field of neuroinflammation imaging, current radiotracers all target the pro-inflammatory (M1) phenotype of activated microglia, since the number of known biological targets specific for the anti-inflammatory (M2) phenotype that are also suited as a target for radiotracer development is still limited. Next to proceeding the currently available tracers for M1 microglia into the clinic, the development of a suitable radiotracer for M2 microglia would mean a great advance in the field, as this would allow for imaging of the dynamics of microglial activation in different diseases.

Imaging of neuroinflammation in Alzheimer's disease, multiple sclerosis and stroke: Recent developments in positron emission tomography.

Neuroinflammation is thought to play a pivotal role in many diseases affecting the brain, including Alzheimer's disease, multiple sclerosis and stroke. Neuroinflammation is characterised predominantly by microglial activation, which can be visualised using positron emission tomography (PET). Traditionally, translocator protein 18kDa (TSPO) is the target for imaging of neuroinflammation using PET. In this review, recent preclinical and clinical research using PET in Alzheimer's disease, multiple sclerosis and stroke is summarised. In addition, new molecular targets for imaging of neuroinflammation, such as monoamine oxidases, adenosine receptors and cannabinoid receptor type 2, are discussed. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Comparison of the octadentate bifunctional chelator DFO*-pPhe-NCS and the clinically used hexadentate bifunctional chelator DFO-pPhe-NCS for 89Zr-immuno-PET.

PURPOSE: All clinical 89Zr-immuno-PET studies are currently performed with the chelator desferrioxamine (DFO). This chelator provides hexadentate coordination to zirconium, leaving two coordination sites available for coordination with, e.g., water molecules, which are relatively labile ligands. The unsaturated coordination of DFO to zirconium has been suggested to result in impaired stability of the complex in vivo and consequently in unwanted bone uptake of 89Zr. Aiming at clinical improvements, we report here on a bifunctional isothiocyanate variant of the octadentate chelator DFO* and the in vitro and in vivo comparison of its 89Zr-DFO*-mAb complex with 89Zr-DFO-mAb. METHODS: The bifunctional chelator DFO*-pPhe-NCS was prepared from previously reported DFO* and p-phenylenediisothiocyanate. Subsequently, trastuzumab was conjugated with either DFO*-pPhe-NCS or commercial DFO-pPhe-NCS and radiolabeled with Zr-89 according to published procedures. In vitro stability experiments were carried out in saline, a histidine/sucrose buffer, and blood serum. The in vivo performance of the chelators was compared in N87 tumor-bearing mice by biodistribution studies and PET imaging. RESULTS: In 0.9 % NaCl 89Zr-DFO*-trastuzumab was more stable than 89Zr-DFO-trastuzumab; after 72 h incubation at 2-8 °C 95 % and 58 % intact tracer were left, respectively, while in a histidine-sucrose buffer no difference was observed, both products were ≥ 92 % intact. In vivo uptake at 144 h post injection (p.i.) in tumors, blood, and most normal organs was similar for both conjugates, except for skin, liver, spleen, ileum, and bone. Tumor uptake was 32.59 ± 11.95 and 29.06 ± 8.66 % ID/g for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, respectively. The bone uptake was significantly lower for 89Zr-DFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. At 144 h p.i. for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, the uptake in sternum was 0.92 ± 0.16 and 3.33 ± 0.32 % ID/g, in femur 0.78 ± 0.11 and 3.85, ± 0.80 and in knee 1.38 ± 0.23 and 8.20 ± 2.94 % ID/g, respectively. The uptake in bone decreased from 24 h to 144 h p.i. about two fold for the DFO* conjugate, while it increased about two fold for the DFO conjugate. CONCLUSIONS: Zr-DFO*-trastuzumab showed superior in vitro stability and in vivo performance when compared to 89Zr-DFO-trastuzumab. This makes the new octadentate DFO* chelator a candidate successor of DFO for future clinical 89Zr-immuno-PET.