Development of the 99mTc-Labelled SST2 Antagonist TECANT-1 for a First-in-Man Multicentre Clinical Study.

Broad availability and cost-effectiveness of 99Mo/99mTc generators worldwide support the use, and thus the development, of novel 99mTc-labelled radiopharmaceuticals. In recent years, preclinical and clinical developments for neuroendocrine neoplasms patient management focused on somatostatin receptor subtype 2 (SST2) antagonists, mainly due to their superiority in SST2-tumour targeting and improved diagnostic sensitivity over agonists. The goal of this work was to provide a reliable method for facile preparation of a 99mTc-labelled SST2 antagonist, [99mTc]Tc-TECANT-1, in a hospital radiopharmacy setting, suitable for a multi-centre clinical trial. To ensure successful and reproducible on-site preparation of the radiopharmaceutical for human use shortly before administration, a freeze-dried three-vial kit was developed. The final composition of the kit was established based on the radiolabelling results obtained during the optimisation process, in which variables such as precursor content, pH and buffer, as well as kit formulations, were tested. Finally, the prepared GMP-grade batches met all predefined specification parameters together with long-term kit stability and stability of the product [99mTc]Tc-TECANT-1. Furthermore, the selected precursor content complies with micro-dosing, based on an extended single-dose toxicity study, where histopathology NOEL was established at 0.5 mg/kg BW, being more than 1000 times higher than the planned human dose of 20 µg. In conclusion, [99mTc]Tc-TECANT-1 is suitable to be advanced into a first-in-human clinical trial.

Radiolabelled CCK2 R Antagonists Containing PEG Linkers: Design, Synthesis and Evaluation.

The cholecystokinin-2/gastrin receptor (CCK2 R) is considered a suitable target for the development of radiolabelled antagonists, due to its overexpression in various tumours, but no such compounds are available in clinical use. Therefore, we designed novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated ligands based on CCK2 R antagonist Z360/nastorazepide. As a proof of concept that CCK2 R antagonistic activity can be retained by extending the Z360/nastorazepide structure using suitable linker, we present herein three compounds containing various PEG linkers synthesised on solid phase and in solution. The antagonistic properties were measured in a functional assay in the A431-CCK2 R cell line (in the presence of agonist G17), with IC50 values of 3.31, 4.11 and 10.4 nM for compounds containing PEG4 , PEG6 and PEG12 , respectively. All compounds were successfully radiolabelled with indium-111, lutetium-177 and gallium-68 (incorporation of radiometal >95 %). The gallium-68-labelled compounds were stable for up to 2 h (PBS, 37 °C). log D7.4 values were determined for indium-111- and gallium-68-labelled compounds, showing improved hydrophilicity compared to the reference compound.

Preclinical pharmacokinetics, biodistribution, radiation dosimetry and toxicity studies required for regulatory approval of a phase I clinical trial with (111)In-CP04 in medullary thyroid carcinoma patients.

INTRODUCTION: From a series of radiolabelled cholecystokinin (CCK) and gastrin analogues, (111)In-CP04 ((111)In-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) was selected for further translation as a diagnostic radiopharmaceutical towards a first-in-man study in patients with medullary thyroid carcinoma (MTC). A freeze-dried kit formulation for multicentre application has been developed. We herein report on biosafety, in vivo stability, biodistribution and dosimetry aspects of (111)In-CP04 in animal models, essential for the regulatory approval of the clinical trial. MATERIALS AND METHODS: Acute and extended single dose toxicity of CP04 was tested in rodents, while the in vivo stability of (111)In-CP04 was assessed by HPLC analysis of mouse blood samples. The biodistribution of (111)In-CP04 prepared from a freeze-dried kit was studied in SCID mice bearing double A431-CCK2R(±) xenografts at 1, 4 and 24h pi. Further 4-h animal groups were either additionally treated with the plasma expander gelofusine or injected with (111)In-CP04 prepared by wet-labelling. Pharmacokinetics in healthy mice included the 30min, 1, 4, 24, 48 and 72h time points pi. Dosimetric calculations were based on extrapolation of mice data to humans adopting two scaling models. RESULTS: CP04 was well-tolerated by both mice and rats, with an LD50>178.5µg/kg body weight for mice and a NOAEL (no-observed-adverse-effect-level) of 89µg/kg body weight for rats. After labelling, (111)In-CP04 remained >70% intact in peripheral mouse blood at 5min pi. The uptake of (111)In-CP04 prepared from the freeze-dried kit and by wet-labelling were comparable in the A431-CCK2R(+)-xenografts (9.24±1.35%ID/g and 8.49±0.39%ID/g, respectively; P>0.05). Gelofusine-treated mice exhibited significantly reduced kidneys values (1.69±0.15%ID/g vs. 5.55±0.94%ID/g in controls, P<0.001). Dosimetry data revealed very comparable effective tumour doses for the two scaling models applied, of 0.045 and 0.044mSv/MBq. CONCLUSION: The present study has provided convincing toxicology, biodistribution and dosimetry data for prompt implementation of the freeze-dried kit formulation without or with gelofusine administration in a multicentre clinical trial in MTC patients.

From preclinical development to clinical application: Kit formulation for radiolabelling the minigastrin analogue CP04 with In-111 for a first-in-human clinical trial.

INTRODUCTION: A variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04) after labelling with (111)In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with (111)In-CP04 in MTC patients. MATERIALS AND METHODS: The kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50 µg) were prepared and radiolabelled with (111)In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis. RESULTS: Use of ascorbic acid buffer (pH4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met(11)-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10 µg formulation. The final formulations contained 10 or 50 µg CP04, 25mg ascorbic acid, 0.5mg gentisic acid and 5mg L-methionine. The radiolabelling performed by incubation of 200-250 MBq (111)InCl3 at 90 °C for 15 min resulted in reproducible radiochemical purity (RCP) >94%. Kit-stability was proven for >6 months at +5 °C and at +25 °C. The radiolabelled product was stable for >4h at +25 °C. CONCLUSION: A kit formulation to prepare (111)In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product.

A novel, self-shielded modular radiosynthesis system for fully automated preparation of PET and therapeutic radiopharmaceuticals.

OBJECTIVE: With the vast development of theranostics and, recently, (68)Ga-radiolabeled molecules, there is also a need for novel, smaller, flexible, safe, and efficient modular automated synthesis systems in different clinical settings. The aim of our study was to determine the shielding properties of the modular self-shielded automated radiosynthesis box and determine its suitability for routine preparation of different radiopharmaceuticals to be used for diagnosis and therapy. METHODS: To evaluate shielding properties, shielding factors were determined using two different radiation sources: (137)Cs and (68)Ga. The dose rates were measured at critical points at the surface and 1 m distance from the surface. Three different methods were used to concentrate and purify (68)Ga generator eluate. Performance of the system was tested by evaluating several radiolabeling applications using (68)Ga, (177)Lu, and (90)Y. RESULTS: Dose rates measured at the surface did not exceed 9 µSv/h for (68)Ga and 20 µSv/h when using (137)Cs. On average, dose rates at the surface were reduced for factors of 1665 and 906, respectively. Different DOTA peptides were labeled successfully with (68)Ga with radiochemical purities more than 94% using three different radiolabeling methods. (177)Lu-DOTATATE and (90)Y-DOTATATE were synthesized reproducibly with a radiochemical purity of more than 99% and more than 97%, respectively. CONCLUSION: A self-shielded radiosynthesis box is a unique solution for nuclear medicine departments that lack space for installation of standard automated synthesis systems set in large and heavy dedicated PET synthesis boxes. Shielding properties are sufficient for safe clinical use for both PET and ß(-) radioisotopes. Because of its modular design and the simple adaptability of system parameters, the system can be used for the preparation of different clinically used radiopharmaceuticals and is also useful for research purposes.

Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2/gastrin receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Influence of radiation source geometry on determination of (111)In and (90)Y activity of radiopharmaceuticals.

OBJECTIVE: Yttrium-90 (Y)-labelled peptides such as DOTATOC and antibodies such as Zevalin are widely used in radionuclide therapy. Indium-111 (In) is used as a Y surrogate for imaging and dosimetry purposes. We aimed to investigate accuracy, geometry (vials and syringes) and volume dependencies for both radionuclides in several different radionuclide calibrators. METHODS: YCl3 and InCl3 solutions were gravimetrically dispensed into the most frequently used containers. In each container several dilutions of the parent solutions were performed. Mass, activity and time were recorded for each calibrator and measurement. Aliquots of both parent solutions were calibrated at the National Metrology Laboratory, Vienna, Austria (BEV). From our measurements and results from BEV, correction factors were determined and further partitioned into calibration, geometry and volume correction factors. RESULTS: Using the nominal calibration factors provided by the manufacturer, measured activity in P6 vials was overestimated by up to 25% for In, depending on the calibrator. Y activity was either underestimated (by up to 20%) or overestimated (by up to 25%) using different radionuclide calibrators. This is the result of the difference in containers used to set the manufacturer's calibration factor values and the containers used in nuclear medicine departments and in this study. There was little geometry dependence for glass vials but strong geometry dependence for syringes for both radionuclides in all calibrators. CONCLUSION: The results should constitute a warning for all personnel responsible for preparation of radiopharmaceuticals. Every nuclear medicine department should incorporate a proper quality-control regimen for radionuclide calibrators and a quality-assurance system.