In Vitro and In Vivo Characterization of Two C-11-Labeled PET Tracers for Vesicular Acetylcholine Transporter

Purpose

The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective ¹¹C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo.

Procedures

For both (?)-[¹¹C]2 and (?)-[¹¹C]6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (?)-[¹¹C]2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI.

Results

The resolved enantiomers (?)-2 and (?)-6 were very potent and selective for VAChT in vitro (K _i ?35-fold selectivity for VAChT vs. σ receptors); both radioligands, (?)-[¹¹C]2 and (?)-[¹¹C]6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (?)-[¹¹C]2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550?±?0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)-2, striatal uptake of (?)-[¹¹C]2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (?)-[¹¹C]2 displayed favorable in vivo stability in rat blood and brain. PET studies of (?)-[¹¹C]2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold.

Conclusions

The radioligand (?)-[¹¹C]2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects.     

Synthesis and Evaluation of 13N-Labelled Azo Compounds for β-Amyloid Imaging in Mice

Purpose

The aim of the present study was to develop short half-lived tools for in vitro and in vivo β-amyloid imaging in mice, for which no suitable PET tracers are available.

Procedures

Five ¹³N-labelled azo compounds (1–5) were synthesized using a three-step process using cyclotron-produced [¹³N]NO₃ ^?. Biodistribution studies were performed using positron emission tomography–computed tomography (PET–CT) on 20-month-old healthy, wild-type (WT) mice. In vivo and in vitro binding assays were performed using PET-CT and autoradiography, respectively, on 20-month-old healthy (WT) mice and transgenic (Tg2576) Alzheimer's disease model mice.

Results

¹³N-labelled azo compounds were prepared with decay corrected radiochemical yields in the range 27?±?4 % to 39?±?4 %. Biodistribution studies showed good blood–brain barrier penetration for compounds 1 and 3–5; good clearance data were also obtained for compounds 1–3 and 5. Compounds 2, 3 and 5 (but not 1) showed a significant uptake in β-amyloid-rich structures when assayed in in vitro autoradiographic studies. PET studies showed significant uptake of compounds 2 and 3 in the cortex of transgenic animals that exhibit β-amyloid deposits.

Conclusions

The results underscore the potential of compounds 2 and 3 as in vitro and in vivo markers for β-amyloid in animal models of Alzheimer's disease.     

Syntheses and Radiosyntheses of Two Carbon-11 Labeled Potent and Selective Radioligands for Imaging Vesicular Acetylcholine Transporter

Purpose

The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. The syntheses and C-11 labeling of two potent enantiopure VAChT inhibitors are reported here.

Procedures

Two VAChT inhibitors, (±)-2 and (±)-6, were successfully synthesized. A chiral HPLC column was used to resolve the enantiomers from each corresponding racemic mixture for in vitro characterization. The radiosyntheses of (?)-[¹¹C]2 and (?)-[¹¹C]6 from the corresponding desmethyl phenol precursor was accomplished using [¹¹C]methyl iodide or [¹¹C]methyl triflate, respectively.

Results

The synthesis of (?)-[¹¹C]2 was accomplished with 40–50 % radiochemical yield (decay-corrected), SA?>?480 GBq/μmol (EOB), and radiochemical purity >99 %. Synthesis of (?)-[¹¹C]6 was accomplished with 5–10 % yield, SA?>?140 GBq/μmol (EOB), and radiochemical purity >97 %. The radiosynthesis and dose formulation of each tracer was completed in 55–60 min.

Conclusions

Two potent enantiopure VAChT ligands were synthesized and ¹¹C-labeled with good radiochemical yield and specific activity.     

Comparison of Ga-68-Labeled Fusarinine C-Based Multivalent RGD Conjugates and [<Superscript>68</Superscript>Ga]NODAGA-RGD—<Emphasis Type="Italic">In Vivo</Emphasis> Imaging Studies in Human Xenograft Tumors

Purpose

Multimeric arginine-glycine-aspartic acid (RGD) peptides have advantages for imaging integrin α_vβ₃ expression. Here, we compared the in vitro and in vivo behavior of three different Ga-68-labeled multimeric Fusarinine C-RGD (FSC-RGD) conjugates, whereby RGD was coupled directly, via a succinic acid or PEG linker (FSC(RGDfE)₃, FSC(succ-RGD)₃, FSC(Mal-RGD)₃). The positron emission tomography/X-ray computed tomography (PET/CT) imaging properties were further compared using [⁶⁸Ga]FSC(succ-RGD)₃ with the monomeric [⁶⁸Ga]NODAGA-RGD in a murine tumor model.

Procedure

FSC-RGD conjugates were labeled with Ga-68, and stability properties were studied. For in vitro characterization, the partition coefficient, integrin α_vβ₃ binding affinity, and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET/CT were carried out using mice bearing either human M21/M21-L melanoma or human U87MG glioblastoma tumor xenografts.

Results

All FSC-RGD conjugates were quantitatively labeled with Ga-68 within 10 min at RT. The [⁶⁸Ga]FSC-RGD conjugates exhibited high stability and hydrophilic character, with only minor differences between the different conjugates. In vitro and in vivo studies showed enhanced integrin α_vβ₃ binding affinity, receptor-selective tumor uptake, and rapid renal excretion resulting in good imaging properties.

Conclusions

The type of linker between FSC and RGD had no pronounced effect on targeting properties of [⁶⁸Ga]FSC-RGD trimers. In particular, [⁶⁸Ga]FSC(succ-RGD)₃ exhibited improved properties compared to [⁶⁸Ga]NODAGA-RGD, making it an alternative for imaging integrin α_vβ₃ expression.

     

Radiolabeling and Preliminary Evaluation of Ga-68 Labeled NODAGA-Ubiquicidin Fragments for Prospective Infection Imaging

Purpose

The present work was aimed at the development of prospective positron emission tomography (PET) agents for infection imaging. Towards this aim, ubiquicidin (UBI) fragments conjugated with the macrocyclic NODAGA chelator were radiolabeled with Ga-68 and evaluated.

Procedures

Conformations of custom synthesized NODAGA-UBI (29–41) and NODAGA-UBI (31–38) conjugates were compared with UBI (29–41) by circular dichroism (CD) spectroscopy. Optimization of labeling of NODAGA conjugates of UBI peptides with Ga-68 was performed and quality control analysis was carried out by chromatography techniques. In vitro uptake of [⁶⁸Ga] NODAGA-UBI (29–41) and [⁶⁸Ga]NODAGA-UBI (31–38) was studied in Staphylococcus aureus cells. In vivo distribution of [⁶⁸Ga]GaCl₃ and [⁶⁸Ga]NODAGA-UBI complexes was performed in normal Swiss mice.

Results

Conformations of NODAGA-UBI (29–41) and NODAGA-UBI (31–38) conjugates were found to be similar to UBI (29–41). NODAGA-UBI conjugates could be consistently labeled with Ga-68 in high radiochemical yields (>95 %) with high radiochemical purity (>95 %). [⁶⁸Ga]NODAGA-UBI (29–41) and [⁶⁸Ga]NODAGA-UBI (31–38) complexes showed retention time of 14 and 14.5 min, respectively, by HPLC radiochromatogram. Specific uptake of [⁶⁸Ga]NODAGA-UBI fragments was observed in S.aureus cells. Greater than 64 % of the injected dose was cleared via the renal route at 1 h post injection, and no significant uptake in vital organs of mice was observed with both the agents.

Conclusion

This is the first report on Ga-68 labeled NODAGA-UBI fragments for infection imaging and the agents hold tremendous prospect in PET imaging.

     

Multimodal Imaging for Early Functional Response Assessment of 90Y-/177Lu-DOTATOC Peptide Receptor Targeted Radiotherapy with DW-MRI and 68Ga-DOTATOC-PET/CT

Purpose

The purpose of this study is to investigate the utility of contrast-enhanced magnetic resonance imaging (CE-MRI), diffusion-weighted MRI (DW-MRI), and ⁶⁸Ga-DOTATOC positron emission tomography/computer tomography (⁶⁸Ga-DOTATOC PET/CT) in the assessment of response to loco-regional peptide receptor radiotherapy (PRRT) with ⁹⁰Y-/¹⁷⁷Lu-DOTATOC in patients with hepatic metastases from gastro-entero-pancreatic neuroendocrine tumors (GEP-NET).

Procedures

CE-MRI, DW-MRI, and ⁶⁸Ga-DOTATOC-PET/CT images were acquired before and 3 months after one to two cycles of intra-arterial ⁹⁰Y-/¹⁷⁷Lu-DOTATOC therapy in 14 patients (nine female, five male; mean age, 54?±?9 years; range, 41–69 years) with hepatic metastases from GEP-NET. A total of 38 liver metastases were defined as target lesions for which the longest diameter, mean apparent diffusion coefficient (ADC_mean) and maximum standardized uptake value (SUV_max) were assessed. Based on changes in size on follow-up imaging, target lesions were classified as responding (RL) or nonresponding (NRL). Relative changes in tumor size, ADC_mean, and SUV_max were compared between the two subgroups.

Results

A total of 27 responding and 11 nonresponding lesions were successfully evaluated. Mean ADC_mean increased significantly in RL (p?=?0.011) as well as NRL (p?=?0.025). A significant correlation was found between baseline ADC_mean and both the percent ADC_mean change (p?=?0.033) and decrease in lesion size after therapy (diameter p?=?0.006; volume p?=?0.002). SUV_max of RL declined significantly by 24.1 % (p?=?0.014) and remained nearly unchanged in NRL. The change of SUV_max correlated significantly with the pretreatment SUV_max (p?<?0.001) and the change in lesion diameter (p?=?0.009). NRL with an ADC_mean change >0.31?×?10^?3 mm²/s on first follow-up imaging showed a decrease in size in the long-term course.

Conclusion

These results suggest that both DW-MRI and DOTATOC-PET imaging provide potential biomarkers for early assessment of treatment and stratification of therapy response, but that DW-MRI should be interpreted only in combination with SSTR expression and morphologic changes.     

TEG® and RapidTEG® are unreliable for detecting warfarin-coagulopathy: a prospective cohort study

Background

Thromboelastography^® (TEG) utilizes kaolin, an intrinsic pathway activator, to assess clotting function. Recent published studies suggest that TEG results are commonly normal in patients receiving warfarin, despite an increased International Normalized Ratio (INR). Because RapidTEG? includes tissue factor, an extrinsic pathway activator, as well as kaolin, we hypothesized that RapidTEG would be more sensitive in detecting a warfarin-effect.

Methods

Included in this prospective study were 22 consecutive patients undergoing elective cardioversion and receiving warfarin. Prior to cardioversion, blood was collected to assess INR, Prothrombin Time, TEG, and RapidTEG.

Results

INR Results: 2.8?±?0.5 (1.6 to 4.2). Prothrombin Time Results: 19.1?±?2.2 (13.9. to 24.3). TEG Results (Reference Range): R-Time: 8.3?±?2.7 (2–8); K-Time: 2.1?±?1.4 (1–3); Angle: 62.5?±?10.3 (55–78); MA: 63.2?±?10.3 (51–69); G: 9.4?±?3.5 (4.6-10.9); R-Time within normal range: 10 (45.5%) with INR 2.9?±?0.3; Correlation coefficients for INR and each of the 5 TEG variables were insignificant (P?>?0.05). RapidTEG Results (Reference Range): ACT: 132?±?58 (86–118); K-Time: 1.2?±?0.5 (1–2); Angle: 75.4?±?5.2 (64–80); MA: 63.4?±?5.1 (52–71); G: 8.9?±?2.0 (5.0-11.6); ACT within normal range: 9 (40.9%) with INR 2.7?±?0.5; Correlation coefficients for INR and each of the 5 RapidTEG variables were insignificant (P?>?0.05).

Conclusions

TEG, using kaolin activation, and RapidTEG, with kaolin and tissue factor activation, were normal in a substantial percent of warfarin patients, despite an increased INR. The false-negative rate for detecting warfarin coagulopathy with either test is unacceptable. The lack of correlation between INR and all TEG and RapidTEG components further indicates that these methodologies are insensitive to warfarin effects. Findings suggest that intrinsic pathway activation may mitigate detection of an extrinsic pathway coagulopathy.     

Quantification of Iron-Labeled Cells with Positive Contrast in Mouse Brains

Purpose

To quantify small amounts of iron-labeled cells in mouse brains with magnetic resonance imaging (MRI).

Procedures

Iron-labeled cells (from 500 to 7,500) were stereotaxically transplanted into the brain of living mice that were subsequently imaged with MRI at 4.7 T. We compared four quantitative methods: (1) T2 relaxometry, (2) T2* relaxometry, (3) the volume of the cloverleaf hypointense artifact generated on T2*-weighted images, and (4) the volume of the cloverleaf hyperintense artifact generated on positive contrast images.

Results

The methods based on relaxometry, whether T2 or T2*, did not correlate with the number of injected cells. By contrast, those based on measurement of cloverleaf artifact volume, whether using negative or positive enhancement, showed a significant linear relationship for the given range of cells (R [0.92?C0.95], p?<?0.05).

Conclusions

T2* artifact volume imaging (negative or positive) appears promising for the quantification of magnetically labeled cells following focal injection in the brain.     

Fully Automated Radiosynthesis of 2-[18F]Fludarabine for PET Imaging of Low-Grade Lymphoma

Purpose

An efficient and fully automated radiosynthesis of 2-[¹⁸F]fluoro-9-β-d-arabinofuranosyl-adenine (2-[¹⁸F]fludarabine, [¹⁸F]-5) based on a GE TRACERlab? FX-FN module has been developed.

Procedures

A 2-nitro purine derivative 3 was developed as precursor for labeling with fluorine-18. The radiosynthesis of [¹⁸F]-5 was performed in two steps in a single reactor with an intermediary purification on Sep-Pak® silica which involved the addition of a three-way valve on the original module. After hydrolysis, [¹⁸F]-5 was purified by semi-preparative high-pressure liquid chromatography (HPLC) and a quality control was established.

Results

The labeling precursor 3 was obtained in 45 % overall yield. Nucleophilic substitution with K¹⁸F/K_2.2.2 afforded protected 2-[¹⁸F]fludarabine ([¹⁸F]-4) in 73?±?4 % , radiochemical yield (decay corrected to the end of bombardment (EOB)) and based on the initial [¹⁸F]F^? activity. An aqueous ammonia/methanol solution was used for the deprotection reaction and gave the desired [¹⁸F]-5 in 67?±?3 % yield after 20 min at 70 °C based on HPLC profile.

Conclusions

The process afforded pure 2-[¹⁸F]fludarabine in 48?±?3 % yield (decay corrected to the EOB) in 85 min, with a specific activity of 310?±?72 GBq/μmol at the end of synthesis (EOS) and a radiochemical purity up to 99 %.     

Capacity building of nurses providing neonatal care in Rio de Janeiro, Brazil: methods for the POINTS of care project to enhance nursing education and reduce adverse neonatal outcomes

Background

Increased survival of preterm infants in developing countries has often been accompanied by increased morbidity. A previous study found rates of severe retinopathy of prematurity varied widely between different neonatal units in Rio de Janeiro. Nurses have a key role in the care of high-risk infants but often do not have access to ongoing education programmes. We set out to design a quality improvement project that would provide nurses with the training and tools to decrease neonatal mortality and morbidity. The purpose of this report is to describe the methods and make the teaching package (POINTS of care--six modules addressing Pain control; optimal Oxygenation; Infection control; Nutrition interventions; Temperature control; Supportive care) available to others.

Methods/Design

Six neonatal units, caring for 40% of preterm infants in Rio de Janeiro were invited to participate. In Phase 1 of the study multidisciplinary workshops were held in each neonatal unit to identify the neonatal morbidities of interest and to plan for data collection. In Phase 2 the teaching package was developed and tested. Phase 3 consisted of 12 months data collection utilizing a simple tick-sheet for recording. In Phase 4 (the Intervention) all nurses were asked to complete all six modules of the POINTS of care package, which was supplemented by practical demonstrations. Phase 5 consisted of a further 12 months data collection. In Phase 1 it was agreed to include inborn infants with birthweight ?? 1500 g or gestational age of ?? 34 weeks. The primary outcome was death before discharge and secondary outcomes included retinopathy of prematurity and bronchopulmonary dysplasia. Assuming 400-450 infants in both pre- and post-intervention periods the study had 80% power at p = < 0.05 to detect an increase in survival from 68% to 80%; a reduction in need for supplementary oxygen at 36 weeks post menstrual age from 11% to 5.5% and a reduction in retinopathy of prematurity requiring treatment from 7% to 2.5%.

Discussion

The results of the POINTS of Care intervention will be presented in a separate publication.

Trial registration

Current Controlled Trials: ISRCTN83110114     

68Ga-Triacetylfusarinine C and 68Ga-Ferrioxamine E for Aspergillus Infection Imaging: Uptake Specificity in Various Microorganisms

Purpose

⁶⁸Ga-triacetylfusarinine C (⁶⁸Ga-TAFC) and ⁶⁸Ga-ferrioxamine E (⁶⁸Ga-FOXE) showed excellent targeting properties in Aspergillus fumigatus rat infection model. Here, we report on the comparison of specificity towards different microorganisms and human lung cancer cells (H1299).

Procedures

The in vitro uptake of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE was studied in various fungal, bacterial and yeast cultures as well as in H1299 cells. The in vivo imaging was studied in fungal and bacterial rat infection and inflammation models.

Results

⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed rapid uptake in A. fumigatus cultures, significantly lower in other fungal species and almost no uptake in other microorganisms and H1299 cells, except for ⁶⁸Ga-FOXE in Staphylococcus aureus. ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE revealed rapid uptake in the lungs of A. fumigatus-infected rats, low accumulation in sterile inflammation and no uptake in bacterial abscess.

Conclusions

We have shown that ⁶⁸Ga-FOXE and ⁶⁸Ga-TAFC have high uptake in A. fumigatus both in vitro and in vivo. ⁶⁸Ga-TAFC showed higher specificity, while ⁶⁸Ga-FOXE showed higher sensitivity.     

Development of Single Vial Kits for Preparation of 68Ga-Labelled Peptides for PET Imaging of Neuroendocrine Tumours

Purpose

The present work was aimed at the formulation and evaluation of freeze-dried kits of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptides for the preparation of ⁶⁸Ga-labelled peptides for PET imaging of neuroendocrine tumours. The ⁶⁸GaCl₃ was obtained from the locally produced nanoceria-PAN, composite-sorbent-based ⁶⁸Ge/⁶⁸Ga generator.

Procedures

Single vial kits of somatostatin analogues DOTA-[Tyr³]-octreotide (DOTA-TOC), DOTA-[NaI³]-octreotide (DOTA-NOC) and DOTA-Tyr³-Thre⁸-octreotide (DOTA-TATE) were formulated. Optimization of radiolabelling with ⁶⁸Ga from the in-house generator, characterization, long term evaluation of stability of kits and bioevaluation studies in animals was carried out.

Results

DOTA-TOC, DOTA-NOC and DOTA-TATE kits could be successfully formulated. Consistently high radiochemical yields (>95 %) were obtained on radiolabelling with ⁶⁸Ga. The radiolabelled peptides exhibited excellent in vitro stability. Biodistribution studies in normal non-tumour bearing Swiss mice revealed fast clearance of activity via renal route as reported for the respective peptides.

Conclusion

Availability of ready to use DOTA-peptide kits in conjunction with ⁶⁸Ge/⁶⁸Ga generators would pave way for the establishment of ⁶⁸Ga radiopharmacy, a long-felt need of the nuclear medicine community.     

Evaluation of Radioiodinated 1,4-Naphthoquinones as Necrosis Avid Agents for Rapid Myocardium Necrosis Imaging

Purpose

Identifying necrotic myocardium in ischemic regions is of great importance for risk stratification and clinical decision-making. However, rapid noninvasive imaging of necrotic myocardium is still challenging. This study sought to evaluate the potential of 1,4-naphthoquinones to rapidly visualize necrotic myocardium and the possible mechanisms of necrosis avidity.

Procedures

Six 1,4-naphthoquinones were radiolabeled with iodine-131 and the necrosis avidity was estimated in mouse models with muscular necrosis by gamma counting and autoradiography. The necrotic myocardium imaging property and biodistribution of [¹³¹I]naphthazarin (6) were determined in rat models with re-perfused myocardial infarction. A possible mechanism of necrosis avidity was explored by in vitro DNA-binding and in vivo blocking experiments.

Results

The radiochemical purities of the six radiotracers were greater than 95 %. The uptakes in necrotic muscles of all six radiotracers were higher than those in viable muscles, and [¹³¹I]naphthazarin (6) showed the highest necrotic-to-viable ratio and necrosis-to-blood ratio at all tested time points. The necrotic myocardium could be clearly visualized by single-photon emission computed tomography/x-ray computed tomography (SPECT/CT) using [¹³¹I]naphthazarin (6) as early as 3 h post-injection. Post-mortem biodistribution showed the uptake of [¹³¹I]naphthazarin (6) in necrotic myocardium was 11.67-fold higher than that in viable myocardium. Absorption spectra and emission spectra suggested naphthazarin (6) could bind to DNA through intercalation. The uptake of [¹³¹I]naphthazarin (6) in necrotic muscle could be significantly blocked by excessive ethidium bromide (a typical DNA intercalator) and cold naphthazarin (6) with 63.49 and 71.96 % decline at 3 h post-injection in vivo, respectively.

Conclusions

1,4-Naphthoquinones retained necrosis avidity and [¹³¹I]naphthazarin (6) rapidly visualized necrotic myocardium. The necrosis avidity mechanism of [¹³¹I]naphthazarin (6) may be attributed to its binding with exposed DNA in necrotic tissues.

     

PET Imaging of Stroke-Induced Neuroinflammation in Mice Using [18F]PBR06

Purpose

The purpose of this study is to evaluate the 18 kDa translocator protein (TSPO) radioligand [¹⁸F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([¹⁸F]PBR06) as a positron emission tomography (PET) imaging biomarker of stroke-induced neuroinflammation in a rodent model.

Procedures

Stroke was induced by transient middle cerebral artery occlusion in Balb/c mice. Dynamic PET/CT imaging with displacement and preblocking using PK111195 was performed 3 days later. PET data were correlated with immunohistochemistry (IHC) for the activated microglial markers TSPO and CD68 and with autoradiography.

Results

[¹⁸F]PBR06 accumulation peaked within the first 5 min postinjection, then decreased gradually, remaining significantly higher in infarct compared to noninfarct regions. Displacement or preblocking with PK11195 eliminated the difference in [¹⁸F]PBR06 uptake between infarct and noninfarct regions. Autoradiography and IHC correlated well spatially with uptake on PET.

Conclusions

[¹⁸F]PBR06 PET specifically images TSPO in microglial neuroinflammation in a mouse model of stroke and shows promise for imaging and monitoring microglial activation/neuroinflammation in other disease models.     

MR Signal Amplification for Imaging of the Mutant EGF Receptor in Orthotopic Human Glioma Model

Purpose

To investigate the potential of targeted MR signal amplification strategy for imaging of EGF receptor variant III (EGFRvIII) overexpression associated with the infiltrating margin of aggressive orthotopic brain tumors.

Procedures

F(ab′)₂ fragments of humanized anti-EGFRvIII monoclonal antibody (EMD72000) were linked to deglycosylated horseradish peroxidase (HRP) and glucose oxidase (GOX). Detection of the F(ab′)₂ conjugate pair colocalization in vivo was enabled by a subsequent IV injection of a low molecular weight paramagnetic substrate of HRP, diTyr-GdDTPA.

Results

The delivery of the targeted fragments to the tumor was validated using SPECT/CT imaging of radiolabeled anti-EGFRvIII F(ab′)₂ conjugates. Further, by using 3 T MRI, we observed time-dependent differences in tumor signal intensity and signal retention at the endpoint depending on whether or not the animals were pre-injected with the anti-EGFRvIII F(ab′)₂ conjugates.

Conclusions

Imaging of EGFRvIII expression in vivo was enabled by consecutive administration of targeted F(ab′)₂ conjugates and a paramagnetic substrate resulting in a tumor-specific receptor detection with high specificity and resolution.     

Incongruity of Imaging Using Fluorescent 2-DG Conjugates Compared to 18F-FDG in Preclinical Cancer Models

Purpose

We compared the use of near-infrared conjugates of 2-deoxyglucose (NIR 2-DG) to 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG) for the purposes of imaging tumors, as well as response to therapy.

Procedures

Uptake of both ¹⁸F-FDG and NIR 2-DG within gastrointestinal stromal tumor xenografts were imaged before and after nilotinib treatment. Confocal microscopy was performed to determine NIR 2-DG distribution in tumors.

Results

Treatment with nilotinib resulted in a rapid reduction in ¹⁸F-FDG uptake and reduced tumor cell viability which was predictive of long-term antitumor efficacy. In contrast, optical imaging with NIR 2-DG probes was unable to differentiate control from niltonib-treated animals, and microscopic analysis revealed no change in probe distribution as a result of treatment.

Conclusions

These results suggest that conjugation of large bulky fluorophores to 2-DG disrupts the facilitated transport and retention of these probes in cells. Therefore, optical imaging of NIR 2-DG probes cannot substitute for ¹⁸F-FDG positron emission tomography imaging as a biomarker of tumor cell viability and metabolism.     

In Vivo Imaging of Sentinel Nodes Using Fluorescent Silica Nanoparticles in Living Mice

Purpose

We examine the feasibility of fluorescent imaging system for sentinel lymph node detection by using functionalized silica nanoparticles.

Materials and Methods

We developed a functionalized RITC-SiO₂ nanoparticles containing fluorescent dye, C₂₈H₃₁N₂O₃Cl (rhodamine B isothiocyanate) inside, and subsequently synthesized ⁶⁸Ga-NOTA-RITC-SiO₂ nanoparticles.

Results

At 5 min after RITC-doped silica nanoparticles injection, fluorescent signals were shown in both right axillary lymph node (ALN) and injection site of living mice. Fluorescent signals were also observed at these locations in a biodistribution study. In addition, fluorescence was detected in frozen ALN sections microscopically. The percentages of doses injected per gram of tissue of axillary and brachial lymph nodes near footpad treated with ⁶⁸Ga-NOTA-RITC-SiO₂ nanoparticles were 308.3?±?3.4 and 41.5?±?6.1, respectively. Little ⁶⁸Ga radioactivity was found in other organs.

Conclusion

Our data provide strong evidence that functionalized silica nanoparticles has a promising potential as organic lymphatic tracer in biomedical imaging such as pre- and intraoperative surgical guidance.     

In Vitro and In Vivo Structure–Property Relationship of 68Ga-Labeled Schiff Base Derivatives for Functional Myocardial PET Imaging

Purpose

SPECT (e.g., with ^99mTc-sestamibi) is routinely used for imaging myocardial damage, even though PET could offer a higher spatial resolution. Using the generator-gained isotope ⁶⁸Ga would allow a rapid supply of the tracer in the diagnostic unit. For this reason, the aim of the study was to develop ⁶⁸Ga-labeled PET tracers based on different Schiff base amines and to evaluate the cardiomyocyte uptake in vitro as well as the biodistribution of the tracers in vivo.

Procedures

Fifteen different Schiff bases (basing on 3 different backbones) were synthesized and labeled with ⁶⁸Ga. Lipophilicity varied between 0.87?±?0.24 and 2.72?±?0.14 (logD value). All tracers were positively charged and stable in plasma and apo-transferrin solution. In vitro uptake into cardiomyocytes was assessed in HL-1 cells in the absence and presence of the ionophor valinomycin. In vivo accumulation in the heart and in various organs was assessed by small animal PET imaging as well as by ex vivo biodistribution. The results were compared with ^99mTc-sestamibi and ¹⁸F-flurpiridaz.

Results

All cationic Schiff bases were taken up into cardiomyocytes but the amount varied by a factor of 10. When destroying the membrane potential, the cellular uptake was markedly reduced in most of the tracers, indicating the applicability of these tracers for identifying ischemic myocardium. PET imaging revealed that the in vivo myocardial uptake reached a constant value approximately 10 min after injection but the intracardial amount of the tracer varied profoundly (SUV 0.46 to 3.35). The most suitable tracers showed a myocardial uptake which was comparable to that of ^99mTc-sestamibi.

Conclusions

⁶⁸Ga-based Schiff bases appear suitable for myocardial PET images with uptake comparable to ^99mTc-sestamibi but offering higher spatial resolution. By systematical variation of the backbone and the side chains, tracers with optimal properties can be identified for further clinical evaluation.     

Synthesis and Ex Vivo Autoradiographic Evaluation of Ethyl-��-d-galactopyranosyl-(1,4��)-2��-deoxy-2��-[18F]fluoro-��-d-glucopyranoside��A Novel Radioligand for Lactose-Binding Protein: Implications for Early Detection of Pancreatic Carcinomas with PET

Introduction

Previous studies demonstrated that the lactose-binding protein (hepatocellular carcinoma?Cintestine?Cpancreas and pancreatitis-associated proteins (HIP/PAP)) is upregulated >130 times in peritumoral pancreatic tissue as compared to normal pancreatic tissue. Therefore, we developed a new radiolabeled ligand of HIP/PAP, the ethyl-??-d-galactopyranosyl-(1,4??)-2??-deoxy-2??-[¹⁸F]fluoro-??-d-glucopyranoside (Et-[¹⁸F]FDL) for noninvasive imaging of pancreatic carcinoma using positron emission tomography and computerized tomography (PET/CT).

Methods

The novel precursor and radiolabeling methods for synthesis of Et-[¹⁸F]FDL produced no isomers; the average decay-corrected radiochemical yield was 68%, radiochemical purity >99%, and specific activity >74 GBq/µmol. The radioligand properties of Et-[¹⁸F]FDL were evaluated using an ex vivo autoradiography and immunohistochemistry in pancreatic tissue sections obtained from mice-bearing orthotopic pancreatic tumor xenografts.

Results and Discussion

Et-[¹⁸F]FDL binding to peritumoral pancreatic tissue sections strongly correlated with HIP/PAP expression (r?=?0.81) and could be completely blocked by treatment with 1 mM lactose.

Conclusion

These results suggest that Et-[¹⁸F]FDL is a promising agent which should be evaluated for detection of early pancreatic carcinomas by PET/CT imaging.     

Evaluation of Cu-64 and Ga-68 Radiolabeled Glucagon-Like Peptide-1 Receptor Agonists as PET Tracers for Pancreatic β cell Imaging

Purpose

Copper-64 (Cu-64) and Galium-68 (Ga-68) radiolabeled DO3A and NODA conjugates of exendin-4 were used for preclinical imaging of pancreatic β cells via targeting of glucagon-like peptide-1 receptor (GLP-1R).

Procedures

DO3A-VS- and NODA-VS-tagged Cys⁴⁰exendin-4 (DO3A-VS-Cys⁴⁰-exendin-4 and NODA-VS-Cys⁴⁰-exendin-4, respectively) were labeled with Cu-64 and Ga-68 using standard techniques. Biodistribution and dynamic positron emission tomography (PET) were carried out in normal Sprague–Dawley (SD) rats. Ex vivo autoradiography imaging was conducted with freshly frozen pancreatic thin sections.

Results

DO3A-VS- and NODA-VS-Cys⁴⁰-exendin-4 analogues were labeled with Cu-64 and Ga-68 to a specific activity of 518.7?±?3.7 Ci/mmol (19.19?±?0.14 TBq/mmol) and radiochemical yield above 98 %. Biodistribution data demonstrated pancreatic uptake of 0.11?±?0.02 %ID/g for [⁶⁴Cu]DO3A-VS-, 0.14?±?0.02 %ID/g for [⁶⁴Cu]NODA-VS-, 0.11?±?0.03 for [⁶⁸Ga]DO3A-VS-, and 0.26?±?0.03 for [⁶⁸Ga]NODA-VS-Cys⁴⁰-exendin-4. Excess exendin-4 and exendin-(9–39)-amide displaced all four Cu-64 and Ga-68 labeled exendin-4 derivatives in blocking studies.

Conclusions

[⁶⁴Cu]/[⁶⁸Ga]DO3A-VS-Cys⁴⁰- and [⁶⁴Cu]/[⁶⁸Ga]NODA-VS-Cys⁴⁰-exendin-4 can be used as PET imaging agents specific for GLP-1R expressed on β cells. Here, we report the first evidence of pancreatic uptake visualized with exendin-4 derivative in a rat animal model via in vivo dynamic PET imaging.