Development and Evaluation of User-Friendly Single Vial DOTA-Peptide Kit Formulations,Specifically Designed for Radiolabelling with <Superscript>68</Superscript>Ga from a Tin Dioxide <Superscript>68</Superscript>Ge/<Superscript>68</Superscript>Ga Generator

Purpose

This study was aimed to develop single vial 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide kits to be used with fractionated eluates from a SnO₂-based ⁶⁸Ge/⁶⁸Ga generator.

Procedures

Kits were formulated with 35 μg DOTA-Tyr³-Thre⁸-octreotide, DOTA-[Tyr³]-octreotide and DOTA-[NaI³]-octreotide (DOTATATE, DOTATOC and DOTANOC) and sodium acetate powder, vacuum-dried and stored at ?20 °C for up to 12 months. Labelling of the kits was carried out with 2 ml ⁶⁸Ga eluate. Comparative labelling was carried out using aqueous DOTA-peptide stock solutions kept frozen at ?20 °C for up to 12 months.

Results

The quality of the kits was found to be suitable over a 1-year storage period (pH, sterility, endotoxin content, radiolabelling efficiency and radiochemical yields of ⁶⁸Ga-labelled DOTA-peptides). Radiochemical yields ranged from 73 to 83 %, while those obtained from stock solutions from 64 to 79 %. No significant decline in kit labelling yields was observed over a 12-month storage period.

Conclusion

The single vial kit formulations met the quality release specifications for human administration and appear to be highly advantageous over using peptide stock solutions in terms of stability and user-friendliness.

     

Preliminary PET/CT Imaging with Somatostatin Analogs [<Superscript>68</Superscript>Ga]DOTAGA-TATE and [<Superscript>68</Superscript>Ga]DOTAGA-TOC

Purpose

Somatostatin receptor positron emission tomography/X-ray computed tomography (SSTR-PET/CT) is a well-established technique for staging and detection of neuroendocrine tumors (NETs). Ga-68-labeled DOTA-conjugated octreotide analogs are the privileged radiotracers for diagnosis and therapeutic monitoring of NETs. Hence, we were interested in assessing the influence of promising, newer variant DOTAGA on the hydrophilicity, pharmacokinetics, and lesion pick-up of somatostatin analogs. Herein, the potential of ([⁶⁸Ga]DOTAGA, Tyr³, Thr⁸) octreotide ([⁶⁸Ga]DOTAGA-TATE) and ([⁶⁸Ga]DOTAGA, Tyr³) octreotide ([⁶⁸Ga]DOTAGA-TOC) as NET imaging agents has been investigated.

Procedures

Amenability of [⁶⁸Ga]DOTAGA-(TATE/TOC) to kit-type formulation has been demonstrated. Biodistribution studies were carried out in normal rats at 1 h post-injection (p.i.). [⁶⁸Ga]DOTAGA-(TATE/TOC) PET/CT scans were carried out in patients (70–170 MBq, 1 h p.i.) with histologically confirmed well-differentiated NETs.

Results

[⁶⁸Ga]DOTAGA-TATE exhibited hydrophilicity similar to [⁶⁸Ga]DOTA-TATE (log P = ?3.51 vs ?3.69) whereas [⁶⁸Ga]DOTAGA-TOC was more hydrophilic than [⁶⁸Ga]DOTA-TOC (log P = ?3.27 vs ?2.93). [⁶⁸Ga]DOTAGA-TATE and [⁶⁸Ga]DOTA-TATE showed almost identical blood and kidney uptake in normal rats whereas significantly fast clearance (p < 0.05) of [⁶⁸Ga]DOTAGA-TATE was observed from other non-specific organs (liver, lungs, spleen, intestine). [⁶⁸Ga]DOTAGA-TOC also demonstrated rapid clearance from blood and kidneys (p < 0.05) in comparison to [⁶⁸Ga]DOTA-TOC. The metastatic lesions in NET patients were well identified by [⁶⁸Ga]DOTAGA-TATE and [⁶⁸Ga]DOTAGA-TOC.

Conclusion

The phenomenal analogy was observed between [⁶⁸Ga]DOTAGA-TATE and [⁶⁸Ga]DOTA-TATE as well as between [⁶⁸Ga]DOTAGA-TOC and [⁶⁸Ga]DOTA-TOC in biodistribution studies in rats. The good lesion detection ability of the two radiotracers indicates their potential as NET imaging radiotracers.

     

Optimization of a Labeling and Kit Preparation Method for Ga-68 Labeled DOTATATE,Using Cation Exchange Resin Purified Ga-68 Eluates Obtained from a Tin Dioxide <Superscript>68</Superscript>Ge/<Superscript>68</Superscript>Ga Generator

Purpose

The aim of this study was to optimize a radiolabeling method using cationic processed Ga-68 eluates from a SnO₂-based ⁶⁸Ge/⁶⁸Ga generator, followed by the development of DOTA-Tyr³-Thre⁸-octreotide (DOTATATE) kits.

Procedures

Diluted generator eluates were adsorbed on a SCX resin and desorbed with acidified 5 M NaCl solution. Optimized labeling conditions were determined by variation of pH, using 35 μg DOTATATE and sodium acetate buffer. DOTATATE kits were developed based on optimized radiolabeling conditions, were labeled, and evaluated.

Results

Optimized labeling conditions resulted in a radiolabeling efficiency of around 99 % and radiochemical yield of almost 85 %. Different kit preparation methods did not significantly influence the radiolabeling results. Kits were found to be stable over 3 months.

Conclusion

A labeling method using SCX-processed Ga-68 eluates was optimized. DOTATATE kits specifically for these SCX-processed Ga-68 eluates were successfully formulated. A post-labeling Sep-Pak C18 purification should be optional.

     

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.

     

64Cu- and 68Ga-Labelled [Nle14,Lys40(Ahx-NODAGA)NH2]-Exendin-4 for Pancreatic Beta Cell Imaging in Rats

Purpose

Glucagon-like peptide-1 receptor (GLP-1R) is a molecular target for imaging of pancreatic beta cells. We compared the ability of [Nle¹⁴,Lys⁴⁰(Ahx-NODAGA-⁶⁴Cu)NH₂]-exendin-4 ([⁶⁴Cu]NODAGA-exendin-4) and [Nle¹⁴,Lys⁴⁰(Ahx-NODAGA-⁶⁸Ga)NH₂]-exendin-4 ([⁶⁸Ga]NODAGA-exendin-4) to detect native pancreatic islets in rodents.

Procedures

The stability, lipophilicity and affinity of the radiotracers to the GLP-1R were determined in vitro. The biodistribution of the tracers was assessed using autoradiography, ex vivo biodistribution and PET imaging. Estimates for human radiation dosimetry were calculated.

Results

We found GLP-1R-specific labelling of pancreatic islets. However, the pancreas could not be visualised in PET images. The highest uptake of the tracers was observed in the kidneys. Effective dose estimates for [⁶⁴Cu]NODAGA-exendin-4 and [⁶⁸Ga]NODAGA-exendin-4 were 0.144 and 0.012 mSv/MBq, respectively.

Conclusion

[⁶⁴Cu]NODAGA-exendin-4 might be more effective for labelling islets than [⁶⁸Ga]NODAGA-exendin-4. This is probably due to the lower specific radioactivity of [⁶⁸Ga]NODAGA-exendin-4 compared to [⁶⁴Cu]NODAGA-exendin-4. The radiation dose in the kidneys may limit the use of [⁶⁴Cu]NODAGA-exendin-4 as a clinical tracer.     

Imaging Characteristics and First Experience of [<Superscript>68</Superscript>Ga]THP-PSMA,a Novel Probe for Rapid Kit-Based Ga-68 Labeling and PET Imaging: Comparative Analysis with [<Superscript>68</Superscript>Ga]PSMA I&amp;T

Purpose

[⁶⁸Ga]Trishydroxypyridinone (THP)–prostate-specific membrane antigen (PSMA) is a novel tracer that can be labeled in one step by cold reconstitution of a kit with unprocessed generator eluate, targeting PSMA via the lysine-urea-glutamate (KuE) motif. The aim of this study was to evaluate the human imaging characteristics of [⁶⁸Ga]THP-PSMA.

Procedures

[⁶⁸Ga]THP-PSMA positron emission tomography (PET)/x-ray computed tomography (CT) was performed in 25 patients with biochemical recurrence after radical prostatectomy for prostate cancer. Urinary and biliary excretion and tumor lesion uptake were quantified using standardized uptake values (SUVs). Imaging characteristics were assessed in terms of non-target organ uptake, background activity, target-to-background ratios (TBRs) of tumor lesions, and frequency of bladder halo artifacts. Findings were compared to a matched cohort of 25 patients undergoing PET/CT with the established agent [⁶⁸Ga]PSMA I&T.

Results

Physiologic uptake of [⁶⁸Ga]THP-PSMA was significantly lower in salivary glands (P?<?0.0001), liver (P?<?0.0001), spleen (P?<?0.0001), and kidneys (P?<?0.0001) than with [⁶⁸Ga]PSMA I&T. While biliary tracer excretion of [⁶⁸Ga]THP-PSMA was negligible, urinary tracer excretion of [⁶⁸Ga]THP-PSMA was fast, and significantly higher than for [⁶⁸Ga]PSMA I&T, contributing to a higher frequency of bladder artifacts. Malignant lesion uptake of [⁶⁸Ga]THP-PSMA assessed as either SUV or TBR was significantly lower than with [⁶⁸Ga]PSMA I&T.

Conclusion

[⁶⁸Ga]THP-PSMA yields suitable in vivo uptake characteristics. The simplified synthesis method for [⁶⁸Ga]THP-PSMA may facilitate wider application and higher patient throughput with PSMA imaging. However, direct intraindividual comparison studies are needed to assess the relative performance of [⁶⁸Ga]THP-PSMA vs other PSMA ligands in terms of clinical detection rate and image quality.

     

Comparison of Somatostatin Receptor 2-Targeting PET Tracers in the Detection of Mouse Atherosclerotic Plaques

Purpose

Rupture-prone atherosclerotic plaques are characterized by accumulation of macrophages, which have shown to express somatostatin type 2 receptors. We aimed to investigate whether somatostatin receptor-targeting positron emission tomography (PET) tracers, [⁶⁸Ga]DOTANOC, [¹⁸F]FDR-NOC, and [⁶⁸Ga]DOTATATE, can detect inflamed atherosclerotic plaques.

Procedures

Atherosclerotic IGF-II/LDLR^?/?ApoB^100/100 mice were studied in vivo and ex vivo for tracer uptake into atherosclerotic plaques. Furthermore, [⁶⁸Ga]DOTANOC and [⁶⁸Ga]DOTATATE were compared in a head-to-head setting for in vivo PET/X-ray computed tomography (CT) imaging characteristics.

Results

Ex vivo uptake of [⁶⁸Ga]DOTANOC and [⁶⁸Ga]DOTATATE in the aorta was higher in atherosclerotic mice compared to control C57Bl/6N mice, while the aortic uptake of [¹⁸F]FDR-NOC showed no genotype difference. Unlike [¹⁸F]FDR-NOC, [⁶⁸Ga]DOTANOC and [⁶⁸Ga]DOTATATE showed preferential binding to atherosclerotic plaques with plaque-to-wall ratio of 1.7?±?0.3 and 2.1?±?0.5, respectively. However, the aortic uptake and aorta-to-blood ratio of [⁶⁸Ga]DOTANOC were higher compared to [⁶⁸Ga]DOTATATE in in vivo PET/CT imaging.

Conclusion

Our results demonstrate superior applicability for [⁶⁸Ga]DOTANOC and [⁶⁸Ga]DOTATATE in the detection of atherosclerotic plaques compared to [¹⁸F]FDR-NOC.

     

The effects of trace metal impurities on Ga-68-radiolabelling with a tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelator

GMP-grade ⁶⁸Ge/⁶⁸Ga generators provide access to positron-emitting ⁶⁸Ga, enabling preparation of Positron Emission Tomography (PET) tracers and PET imaging at sites that do not have access to cyclotron-produced radionuclides. Radiotracers based on tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelators enable simple one-step preparations of ⁶⁸Ga PET radiopharmaceuticals from pre-fabricated kits without pre-processing of generator eluate or post-purification. However, trace metal impurities eluted along with ⁶⁸Ga could compete for THP and reduce radiochemical yields (RCY). We have quantified trace metal impurities in ⁶⁸Ga eluate from an Eckert & Ziegler (E&Z) generator using ICP-MS. The metals Al, Fe, ^natGa, Pb, Ti and ^natZn were present in generator eluate in significantly higher concentrations than in the starting eluent solution. Concentrations of Fe and ^natGa in eluate were in the range of 0.01–0.1 μM, Al, Zn and Pb in the range of 0.1–1 μM, and Ti in the range of 0.9–1.5 μM. To assess the ability of THP to chelate ⁶⁸Ga in the presence of such metal ions, radiolabelling reactions were undertaken in which selected metal ions were added to make them equimolar with THP, or higher. Al³⁺, Fe³⁺, ^natGa³⁺ and Ti⁴⁺ reduced RCY at concentrations equimolar with THP and higher, but at lower concentrations they did not affect RCY. Pb²⁺, Zn²⁺, Ni²⁺ and Cr³⁺ had no effect on RCY (even under conditions in which each metal ion was present in 100-fold molar excess over THP). The multi-sample ICP-MS analysis reported here is (to date) the most comprehensive and robust quantification of metal impurities in the widely used E&Z ⁶⁸Ga generator. ⁶⁸Ga from an E&Z generator enables near-quantitative radiolabelling of THP at chelator concentrations as low as 5 μM (lower than other common gallium chelators) without pre-processing. The combination of Al³⁺, Fe³⁺, ^natGa³⁺ and Ti⁴⁺ in unprocessed ⁶⁸Ga eluate is likely to decrease RCY of ⁶⁸Ga radiolabelling if a lower amount of THP chelator is used, and future kit design should take this into account. To increase specific activities by using even lower THP concentrations, purification of ⁶⁸Ga from trace metal ions will likely be required.

We have quantified trace metal impurities present in ⁶⁸Ga generator eluant from the widely used Eckert & Ziegler ⁶⁸Ga generator, and measured the effect of these metal impurities on ⁶⁸Ga radiolabelling of a THP chelator.     

Diagnostic performance of somatostatin receptor PET/CT using 68Ga-DOTANOC in gastrinoma patients with negative or equivocal CT findings

Purpose

Contrast-enhanced CT (CECT) is a standard investigative procedure in the localization of gastrinomas. Small tumors are often missed and metastatic lesions may remain occult on CT. The purpose of present study was to prospectively evaluate the diagnostic performance of ⁶⁸Ga-labeled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-NaI³-Octreotide (⁶⁸Ga-DOTANOC) positron emission tomography/computed tomography (PET/CT) in gastrinoma patients with negative or equivocal CT findings.

Methods

Twenty-five patients (age 46.6 ± 13.3 years; male 60%) with clinical/biochemical diagnosis of gastrinoma and negative or equivocal findings on CECT were prospectively evaluated. All of them underwent ⁶⁸Ga-DOTANOC PET/CT which was evaluated by two nuclear medicine physicians in consensus. Combination of histopathology, serum gastrin, endoscopy, and follow-up imaging were taken as reference standard.

Results

⁶⁸Ga-DOTANOC PET/CT was positive in 17 patients and negative in 8 patients, yielding an overall detection rate of 68%. It was positive 13/20 patients who underwent baseline evaluation and in 4/5 post-treatment patients. Of the 11 patients who had a negative CT result, ⁶⁸Ga-DOTANOC PET/CT was positive in four cases (detection rate 36.4%), while it was abnormal in 13/14 patients who had equivocal CT findings (detection rate 92.8%). Diagnostic performance of ⁶⁸Ga-DOTANOC PET/CT was superior in patients with equivocal CECT findings than that in patients with negative CECT (P = 0.010).

Conclusion

⁶⁸Ga-DOTANOC PET/CT appears to be useful in patients with gastrinoma with negative or equivocal results on CECT, especially the latter group.     

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.

     

Adventures in radiosynthesis of clinical grade [68Ga]Ga-DOTA-Siglec-9

We finally managed to establish a protocol for generating Good Manufacturing Practice (GMP)-grade gallium-68-labelled 1,4,7,0-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([⁶⁸Ga]Ga-DOTA-Siglec-9), the first radiopharmaceutical for positron emission tomography imaging of vascular adhesion protein 1.

[⁶⁸Ga]Ga-DOTA-Siglec-9 is the first vascular adhesion protein-1 targeting radiopharmaceutical for positron emission tomography imaging of inflammation, and here we present its long-awaited clinical grade radiosynthesis.     

Effects of Chelator Modifications on 68Ga-Labeled [Tyr3]Octreotide Conjugates

Purpose

Somatostatin receptors (SSTR) have been reported as promising targets for imaging agents for cancer. Recently, ⁶⁸Ga-DOTATOC-based PET imaging has been used successfully for diagnosis and management of SSTR-expressing tumors. The purpose of this study was to evaluate the influence of chelator modifications and charge on ⁶⁸Ga-labeled peptide conjugates.

Procedures

We have synthesized a series of [Tyr³]octreotide conjugates that consisted of different NOTA-based chelators with two to five carboxylate moieties, and compared our results with ⁶⁸Ga-DOTATOC in both in vitro and in vivo studies.

Results

With the exception of ⁶⁸Ga-1 (three carboxylates), the increased number of carboxylates on the NOTA-based chelators resulted in a reduced binding affinity and internalization. Additionally, the tumor uptake for ⁶⁸Ga-2 (four carboxylates) and ⁶⁸Ga-3 (five carboxylates) was reduced compared to that of ⁶⁸Ga-DOTATOC (three carboxylates) and ⁶⁸Ga-NO2ATOC (two carboxylates) and ⁶⁸Ga-1 (three carboxylates) at 2 h p.i. suggesting the presence of an optimal charge for this compound.

Conclusions

Chelator modifications can lead to the altered pharmacokinetics. These results may impact further design considerations for peptide-based imaging agents.     

Repeatability of [<Superscript>68</Superscript>Ga]DKFZ11-PSMA PET Scans for Detecting Prostate-specific Membrane Antigen-positive Prostate Cancer

Purpose

We studied the effect of varying specific activity of [⁶⁸Ga]DKFZ-PSMA11 ([⁶⁸Ga]DP11) on repeated imaging of prostate-specific membrane antigen-positive (PSMA+) xenograft tumors.

Procedures

Athymic nude mice bearing PC3-PIP (PSMA+) and PC3 (PSMA?) bilateral flank tumors were assessed to study intra- and inter-day repeatability of [⁶⁸Ga]DP11 imaging in mice administered [⁶⁸Ga]DP11 or [⁶⁷Ga]DP11 (as a dilution tracer) using imaging and biodistribution studies.

Results

Region of interest (ROI) analysis of the [⁶⁸Ga]DP11 imaging study indicated that the uptake was constant on the same day or consecutive days. Prior imaging with [⁶⁸Ga]DP11 did not significantly influence the subsequent uptake of [⁶⁸Ga]DP11. Uptake of [⁶⁸Ga]DP11 (60 min) and [⁶⁷Ga]DP11 (24 h) in PC3-PIP tumors was 12.37 ± 4.19 %ID/g and 12.49 ± 6.88 %ID/g, respectively; [⁶⁸Ga]DP11 was 13.83 ± 3.77 and 17.76 ± 1.84 on same-day and 15.98 ± 5.82 %ID/g on second-day imaging.

Conclusions

This study demonstrates that [⁶⁸Ga]DP11, in a given PSMA+ lesion, is constant under several same-day or serial-day imaging conditions.

     

[68Ga]-Albumin-PET in the Monitoring of Left Ventricular Function in Murine Models of Ischemic and Dilated Cardiomyopathy: Comparison with Cardiac MRI

Purpose

The purpose of this study is to evaluate left ventricular functional parameters in healthy mice and in different murine models of cardiomyopathy with the novel blood pool (BP) positron emission tomography (PET) tracer [⁶⁸Ga]-albumin.

Procedures

ECG-gated microPET examinations were obtained in healthy mice, and mice with dilative (DCM) and ischemic cardiomyopathy (ICM) using the novel BP tracer [⁶⁸Ga]-albumin (Alb_BP), as well as [¹⁸F]-FDG microPET. Cine-magnetic resonance imaging (MRI) examination performed on a clinical 1.5-T MRI provided the reference standard measurements.

Results

When considering the combined group of healthy controls, DCM and ICM Alb_BP-PET significantly overestimated the magnitudes of EDV (Alb_BP, 181?±?86 μl; cine-MRI, 125?±?80 μl; P?<?0.001) and ESV (Alb_BP, 136?±?92 μl; cine-MRI, 96?±?77 μl; P?<?0.001), whereas the EF (Alb_BP, 31?±?16 %; cine-MRI, 33?±?21 %; P?=?0.910) matched closely to cine-MRI results, as did findings with [¹⁸F]-FDG. High correlations were found between the measured cardiac parameters (EDV: R?=?0.978, ESV: R?=?0.989, and LVEF: R?=?0.992).

Conclusions

Measuring left ventricular function in mice with [⁶⁸Ga]-albumin BP PET is feasible and showed a high correlation compared to cine-MRI, which was used as a reference standard.     

Assessing Glomerular Filtration in Small Animals Using [<Superscript>68</Superscript>Ga]DTPA and [<Superscript>68</Superscript>Ga]EDTA with PET Imaging

Purpose

Determining the glomerular filtration rate (GFR) is essential for clinical medicine but also for pre-clinical animal studies. Functional imaging using positron emission tomography (PET) allows repetitive almost non-invasive measurements. The aim of the study was the development and evaluation of easily synthesizable PET tracers for GFR measurements in small animals.

Procedures

Diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) were labeled with Ga-68. The binding to blood cells and plasma proteins was tested in vitro. The distribution of the tracers in rats was analyzed by PET imaging and ex vivo measurements. From the time-activity-curve of the blood compartment (heart) and the total tracer mass excreted by the kidney, the GFR was calculated. These values were compared directly with the inulin clearance in the same animals.

Results

Both tracers did not bind to blood cells. [⁶⁸Ga]DPTA but not [⁶⁸Ga]EDTA showed strong binding to plasma proteins. For this reason, [⁶⁸Ga]DPTA stayed much longer in the blood and only 30 % of the injected dose was eliminated by the kidney within 60 min whereas the excretion of [⁶⁸Ga]EDTA was 89 ± 1 %. The calculated GFR using [⁶⁸Ga]EDTA was comparable to the measured inulin clearance in the same animal. Using [⁶⁸Ga]-DPTA, the measurements led to values which were 80 % below the normal GFR. The results also revealed that definition of the volume of interest for the blood compartment affects the calculation and may lead to a slight overestimation of the GFR.

Conclusions

[⁶⁸Ga]EDTA is a suitable tracer for GFR calculation from PET imaging in small animals. It is easy to be labeled, and the results are in good accordance with the inulin clearance. [⁶⁸Ga]DTPA led to a marked underestimation of GFR due to its strong binding to plasma proteins and is therefore not an appropriate tracer for GFR measurements.

     

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.     

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.     

[<Superscript>68</Superscript>Ga]PSMA-HBED-CC Uptake in Osteolytic,Osteoblastic, and Bone Marrow Metastases of Prostate Cancer Patients

Purpose

The aim of this study was to evaluate potential differences in “Glu-NH-CO-NH-Lys” radio-labeled with [⁶⁸Ga]gallium N,N-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N-diacetic acid ([⁶⁸Ga]PSMA-HBED-CC) uptake in osteolytic, osteoblastic, mixed, and bone marrow metastases in prostate cancer (PC) patients.

Procedures

This retrospective study was approved by the local ethics committee. Patients who received [⁶⁸Ga]PSMA-HBED-CC positron emission tomography/computed tomography ([⁶⁸Ga]PSMA-PET/CT) with at least one positive bone metastasis were included in this study. Only patients who have not received systemic therapy for their PC were included. Bone metastases had to be confirmed by at least one other imaging modality or follow-up investigation. The maximum standardized uptake value (SUV_max) and mean Hounsfield units (HU_mean) of each metastasis were measured. Based on CT, each metastasis was classified as osteolytic (OL), osteoblastic (OB), bone marrow (BM), or mixed (M).

Results

One hundred fifty-four bone metastases in 30 patients were evaluated. Eighty out of 154 (51.9%) metastases were classified as OB, 21/154 (13.6%) as OL, 23/154 (14.9%) as M, and 30/154 (19.5%) as BM. The SUV_max for the different types of metastases were 10.6 ± 7.07 (OB), 24.0 ± 19.3 (OL), 16.0 ± 21.0 (M), and 14.7 ± 9.9 (BM). The SUV_max of OB vs. OL and OB vs. BM metastases differed significantly (p ≤ 0.025). A significant negative correlation between HU_mean and SUV_max (r = ?0.23, p < 0.05) was measured.

Conclusions

[⁶⁸Ga]PSMA-HBED-CC uptake is higher in osteolytic and bone marrow metastases compared to osteoblastic metastases. Information derived from [⁶⁸Ga]PSMA-PET and CT complement each other for the reliable diagnosis of the different types of bone metastases in PC patients.

     

<Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Comparison of Selected Ga-68 and Zr-89 Labelled Siderophores

Purpose

Some [⁶⁸Ga]siderophores show promise in specific and sensitive imaging of infection. Here, we compare the in vitro and in vivo behaviour of selected Ga-68 and Zr-89 labelled siderophores.

Procedures

Radiolabelling was performed in HEPES or sodium acetate buffer systems. Radiochemical purity of labelled siderophores was determined using chromatography. Partition coefficients, in vitro stability and protein binding affinities were determined. Ex vivo biodistribution and animal imaging was studied in mice.

Results

Certain differences among studied siderophores were observed in labelling efficiency. Protein binding and stability tests showed highest stabilities and lowest protein binding affinities for Ga-68 and [⁸⁹Zr]triacetylfusarinine C (TAFC). All studied Ga-68 and [⁸⁹Zr]siderophores exhibited a similar biodistribution and pharmacokinetics in mice with the exception of [⁸⁹Zr]ferrioxamine E (FOXE).

Conclusions

Zr-89 and [⁶⁸Ga]siderophores showed analogous in vitro and in vivo behaviour. Tested [⁸⁹Zr]siderophores could be applied for longitudinal positron emission tomography (PET) studies of fungal infections and especially TAFC for the development of novel bioconjugates.

     

Tumor Targeting <Emphasis Type="Italic">via</Emphasis> Sialic Acid: [<Superscript>68</Superscript>Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET

Purpose

The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia).

Procedures

The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [⁶⁸Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [⁶⁸Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent.

Results

The affinity of [⁶⁸Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36?±?2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [⁶⁸Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection.

Conclusions

Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.