Several exploratory studies have demonstrated the feasibility of cholecystokinin-2 receptor (CCK2R) targeting in patients with medullary thyroid carcinoma (MTC) and other neuroendocrine tumors (NETs). We report the results of a prospective phase I/IIA pilot study (clinicaltrials.gov NCT06155994) conducted at our center with the 68Ga-labeled peptide analog DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-Phe-NH2 (68Ga-DOTA-MGS5). Methods: Six patients with advanced MTC and 6 patients with gastroenteropancreatic and bronchopulmonary NETs confirmed by previous PET/CT imaging with other PET tracers received a single dose of 180 MBq of 68Ga-DOTA-MGS5. The first 6 patients enrolled in the study were included in the dosimetry evaluation, and safety was assessed in all 12 patients. PET/CT imaging was performed at different time points after injection to perform dosimetric calculations and to determine the optimal imaging time window. In addition, blood and urine samples were collected for pharmacokinetic assessments. Results: The administration of 68Ga-DOTA-MGS5 was well tolerated, with minor adverse drug reactions occurring only in 3 patients. 68Ga-DOTA-MGS5 was cleared rapidly from the blood, with less than 21% of the injected activity present in blood 215 ± 10 min after injection. Tracer elimination occurred mainly through the kidneys, with a cumulative urinary excretion greater than 40% 3 h after injection. A high percentage of intact radiopeptide was confirmed in plasma. The highest absorbed dose was found for the urinary bladder wall, the stomach wall, and the kidneys, with an effective dose of 0.023 ± 0.007 mSv/MBq. The time points of 1 and 2 h after injection proved to be optimal for PET/CT imaging. In the 6 patients included in the dosimetry evaluation, local metastasis was confirmed in 2 patients with advanced MTC, whereas only 1 of 4 patients with gastroenteropancreatic NETs was positive in 68Ga-DOTA-MGS5 PET/CT. Conclusion: Besides confirming the safety of administration, within the phase I part of the prospective clinical trial, an acceptable effective whole-body dose, an overall favorable biodistribution, and the feasibility of cholecystokinin-2 receptor imaging could be shown for 68Ga-DOTA-MGS5.

Introduction: Medullary thyroid cancer (MTC) is characterized by overexpression of cholecystokinin-2/gastrin receptors (CCK2R). There are limitations of calcitonin as a tumor marker in MTC diagnosis and prognosis. Procalcitonin is gaining a role as a complementary tumor marker. This study aimed to assess the feasibility of procalcitonin measurements on top of the calcitonin measurements after CCK2R agonist stimulation in patients with MTC. Material and methods: The assessment was part of the GRAN-T-MTC translational study conducted through a Phase I multicenter clinical trial in patients with locally advanced and/or disseminated MTC. Patients were administered intravenously the CCK2R agonist CP04 labelled with indium-111 ([In-111]In-CP04); the first four patients at a lower mass amount of 10 mu g, and afterwards the whole group at a higher mass amount of 50 mu g. Blood samples for calcitonin and procalcitonin measurements were obtained shortly before and 2, 5, 10, and 20 minutes after start of [In-111]In-CP04 administration. Results: Sixteen patients were included in the study. After injection of the higher mass amount of [In-111]In-CP04, the median maximum ratio for stimulated calcitonin was 2.97 (interquartile range [IQR] 2.35) pg/mL and procalcitonin 2.01 (IQR 2.07) pg/mL. The maximum stimulated/baseline calcitonin ratio was 5.2 +/- 4.0 and 4.1 +/- 3.8 in the low and high mass amount groups, respectively, and the maximum stimulated/baseline procalcitonin ratio was 4.6 +/- 5.1 and 2.9 +/- 3.1 in the low and high mass amount groups, respectively. There was a significant linear correlation between calcitonin and procalcitonin concentrations (p < 0.001) at each test time point and between the maximum procalcitonin and maximum calcitonin increment ratio (r = 0.94, p < 0.0001). Mild, short-lasting side effects (transient tachycardia, flushing) were observed in one patient during the injection of low and in two patients during the injection of high mass amount of [In-111] In-CP04. The side effects were not related to the baseline calcitonin or procalcitonin concentrations. Conclusion: Procalcitonin concentrations after CP04 stimulation were highly correlated with calcitonin concentrations. Unlabeled CP04, if available commercially, may be considered an alternative stimulating agent in MTC patients, even in lower mass amounts. Further studies, including healthy controls, are required to prove this concept and calculate the diagnostic thresholds.

[99mTc]Tc-GSA, an albumin-based glycoprotein, is routinely used in Japan to measure the asialoglycoprotein receptor (ASGR) density via single photon emission tomography. Here we describe the development of 68Ga-labeled peptide-based alternatives. Peptides were assembled on a solid support using a fragment coupling strategy. Glycosylation was carried out via a click chemistry approach resulting in a set of three peptides with increasing amounts of d-galactose (n = 3, 6, and 9) as well as one glycopeptide bearing nine N-acetylgalactosamine residues. 68Ga-labeling of all compounds could be achieved in high radiochemical yields (>95%). Radiotracers exhibited high hydrophilicity, good metabolic stability in human serum and protein binding between 12 and 22%. The IC50 values improved in the series tri-, hexa-, and nonamer with an IC50 of 50 ± 30 pM for the latter one. In analogy, the in vivo biodistribution studies revealed increased liver uptake in the series of [68Ga]Ga-NODAGA-TriLysan (9.4 ± 2.0% ID/g, 30 min p.i.), [68Ga]Ga-NODAGA-HexaLysan (55.5 ± 7.4% ID/g, 30 min p.i.), and [68Ga]Ga-NODAGA-NonaLysan (79.6 ± 8.0% ID/g, 30 min p.i.). [68Ga]Ga-NODAGA-GalNAc-NonaLysan reached comparable liver uptake to [68Ga]Ga-NODAGA-NonaLysan, but showed higher accumulation in nontarget organs. The impressive imaging properties of [68Ga]Ga-NODAGA-NonaLysan were also confirmed by the PET/MR imaging studies in mice. Hence, [68Ga]Ga-NODAGA-NonaLysan represents a new PET radiopharmaceutical with even better imaging properties than [99mTc]Tc-GSA.

Background/Objectives: PET imaging of bacterial infection could potentially provide added benefits for patient care through non-invasive means. [68Ga]Ga-desferrioxamine B—a radiolabelled siderophore—shows specific uptake by human-pathogenic bacteria like Staphylococcus aureus or Pseudomonas aeruginosa and sufficient serum stability for clinical application. In this report, we present data for automated production of [68Ga]Ga-desferrioxamine B on two different cassette-based synthesis modules (Modular-Lab PharmTracer and GRP 3V) utilising commercially obtainable cassettes together with a licensed 68Ge/68Ga radionuclide generator. Methods: Quality control, including the determination of radiochemical purity, as well as a system suitability test, was set up via RP-HPLC on a C18 column. The two described production processes use an acetic acid/acetate buffer system with ascorbic acid as a radical scavenger for radiolabelling, yielding ready-to-use formulations with sufficient activity yield. Results: Batch data analysis demonstrated radiochemical purity of >95% by RP-HPLC combined with ITLC and excellent stability up to 2 h after synthesis. Specifications for routine production were set up and validated with four masterbatches for each synthesis module. Conclusions: Based on this study, an academic clinical trial for imaging of bacterial infection was initiated. Both described synthesis methods enable automated production of [68Ga]Ga-desferrioxamine B in-house with high reproducibility for clinical application.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

BACKGROUND:Determination of the functional liver mass is important in a variety of clinical settings including liver surgery and transplantation. [99mTc]Tc-diethylenetriamine-pentaacetic acid galactosyl human serum albumin (99mTc-GSA) is a radiotracer targeting the asialoglycoprotein receptor (ASGR) and is routinely used in Japan for this purpose. Here we describe the development and evaluation of [68Ga]Ga-NODAGA-TriGalactan a low molecular weight PET-tracer targeting this structure.RESULTS:For synthesis TRIS as branching unit and NODAGA as chelator for labelling with [68Ga]Ga are included. Three galactose moieties are conjugated via a click chemistry approach resulting in the desired labelling precursor.68Ga-labelling could be accomplished in high radiochemical yield and purity. [68Ga]Ga-NODAGA-TriGalactan is very hydrophilic and revealed high plasma stability and low plasma protein binding. Fluorescence imaging showed binding on ASGR-positive organoids and the IC50-value was in the nanomolar range. Most importantly, both biodistribution as well as animal imaging studies using normal mice demonstrated high liver uptake with rapid elimination from all other organs leading to even higher liver-to-background ratios as found for 99mTc-GSA.CONCLUSION:[68Ga]Ga-NODAGA-TriGalactan shows high in vitro stability and selectively binds to the ASGR allowing imaging of the functional liver mass with high contrast. Thus, our first generation compound resulted already in an alternative to 99mTc-GSA for imaging the functional liver reserve and might allow the broader use of this imaging technique.

The exclusive asialoglycoprotein receptor (ASGR) expression on hepatocytes makes it an attractive target for imaging of the functional liver reserve. Here, we present a set of TRAP-based glycoside trimers and evaluate their imaging properties compared to the gold standard [99mTc]Tc-GSA. The click-chemistry-based synthesis approach provided easy access to trimeric low-molecular-weight compounds. Labeling with 68Ga was carried out in high radiochemical yields (>99%). Complexes showed high stability and hydrophilicity. Protein binding ranged between 10 and 25%. Highest binding affinity (IC50) and best liver accumulation were found for [68Ga]Ga-T3N3, followed by [68Ga]Ga-T3G3 and [68Ga]Ga-T0G3. Rapid elimination from the rest of the body resulted in excellent target-to-background ratios. Our studies confirmed that high ASGR uptake depends on the correct spacer design and that N-acetylgalactosamine improves targeting properties in vivo. Thus, [68Ga]Ga-T3N3 represents a new low-molecular-weight radiopharmaceutical with pharmacokinetics similar to those of [99mTc]Tc-GSA.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The therapeutic potential of minigastrin (MG) analogs for the treatment of cholecystokinin-2 receptor (CCK2R)-expressing cancers is limited by poor in vivo stability or unfavorable accumulation in non-target tissues. Increased stability against metabolic degradation was achieved by modifying the C-terminal receptor-specific region. This modification led to significantly improved tumor targeting properties. In this study, further N-terminal peptide modifications were investigated. Two novel MG analogs were designed starting from the amino acid sequence of DOTA-MGS5 (DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2). Introduction of a penta-DGlu moiety and replacement of the four N-terminal amino acids by a non-charged hydrophilic linker was investigated. Retained receptor binding was confirmed using two CCK2R-expressing cell lines. The effect on metabolic degradation of the new 177Lu-labeled peptides was studied in human serum in vitro, as well as in BALB/c mice in vivo. The tumor targeting properties of the radiolabeled peptides were assessed using BALB/c nude mice bearing receptor-positive and receptor-negative tumor xenografts. Both novel MG analogs were found to have strong receptor binding, enhanced stability, and high tumor uptake. Replacement of the four N-terminal amino acids by a non-charged hydrophilic linker lowered the absorption in the dose-limiting organs, whereas introduction of the penta-DGlu moiety increased uptake in renal tissue.

Minigastrin (MG) analogs for therapy of CCK2R-expressing malignancies are limited by low stability in vivo or excessive accumulation in non-target organs. By modifying the C-terminal receptor-binding sequence, metabolization could be prevented and tumor targeting significantly improved. In this work, N-terminal changes of the peptide length were evaluated. Based on the amino acid sequence of DOTA-MGS5 (DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2), two new MG analogs were synthesized, by either introduction of a penta-DGlu moiety or depletion of the four N-terminal amino acids and introduction of a non-charged hydrophilic linker. Two CCK2R-expressing cell lines were used to demonstrate receptor interaction. Stability of the 177Lu-labeled peptide analogs was evaluated in human serum up to 24 h after incubation and in BALB/c mice up to 30 min after injection. The biodistribution profile and tumor targeting potential was evaluated in xenografted BALB/c nude mice. For both new MG analogs, the combination of strong receptor-specific cell interaction, high stability and enhanced tumor targeting could be demonstrated. Shortening of the peptide sequence lowered the absorption in the dose-limiting organs, whereas elongation increased uptake in renal tissue.

The new minigastrin analog DOTA-MGS8 targeting the cholecystokinin-2 receptor (CCK2R) used in this study displays the combination of two site-specific modifications within the C-terminal receptor binding sequence together with an additional N-terminal amino acid substitution preventing fast metabolic degradation. Within this study, the preparation of 68Ga-labeled DOTA-MGS8 was validated using an automated synthesis module, describing the specifications and analytical methods for quality control for possible clinical use. In addition, preclinical studies were carried out to characterize the targeting potential. [68Ga]Ga-DOTA-MGS8 showed a high receptor-specific cell internalization into AR42J rat pancreatic cells (~40%) with physiological expression of rat CCK2R as well as A431-CCK2R cells transfected to stably express human CCK2R (~47%). A favorable biodistribution profile was observed in BALB/c nude mice xenografted with A431-CCK2R cells and mock-transfected A431 cells as control. The high tumor uptake of ~27% IA/g together with low background activity and limited uptake in non-target tissue confirms the potential for high-sensitivity positron emission tomography of stabilized MG analogs in patients with MTC and other CCK2R-related malignancies.

INTRODUCTION:Medullary thyroid cancer (MTC) is a rare malignant tumour of the parafollicular C-cells with an unpredictable clinical course and currently suboptimal diagnostic and therapeutic options, in particular in advanced disease. Overexpression of cholecystokinin-2 receptors (CCK2R) represents a promising avenue to diagnostic imaging and targeted therapy, ideally through a theranostic approach. MATERIALS AND METHODS:A translational study (GRAN-T-MTC) conducted through a Phase I multicentre clinical trial of the indium-111 labelled CP04 ([111In]In-CP04), a CCK2R-seeking ligand was initiated with the goal of developing a theranostic compound. Patients with proven advanced/metastatic MTC or short calcitonin doubling time were enrolled. A two-step concept was developed through the use of low- and high-peptide mass (10 and 50 μg, respectively) for safety assessment, with the higher peptide mass considered appropriate for therapeutic application. Gelofusine was co-infused in a randomized fashion in the second step for the evaluation of potential reduction of the absorbed dose to the kidneys. Imaging for the purpose of biodistribution, dosimetry evaluation, and diagnostic assessment were performed as well as pre-, peri-, and postprocedural clinical and biochemical assessment. RESULTS:Sixteen patients were enrolled. No serious adverse events after application of the compound at both peptide amounts were witnessed; transient tachycardia and flushing were observed in two patients. No changes in biochemistry and clinical status were observed on follow-up. Preliminary dosimetry assessment revealed the highest dose to urinary bladder, followed by the kidneys and stomach wall. The effective dose for 200 MBq of [111In]In-CP04 was estimated at 7±3 mSv and 7±1 mSv for 10 μg and 50 μg CP04, respectively. Administration of Gelofusine reduced the dose to the kidneys by 53%, resulting in the organ absorbed dose of 0.044±0.019 mSv/MBq. Projected absorbed dose to the kidneys with the use of [177Lu]Lu-CP04 was estimated at 0.9±0.4 Gy/7.4 GBq. [111In]In-CP04 scintigraphy was positive in 13 patients (detection rate of 81%) with superior diagnostic performance over conventional imaging. CONCLUSION:In the present study, [111In]In-CP04 was shown to be a safe and effective radiopharmaceutical with promising theranostic characteristics for patients with advanced MTC.