Automated production of [68Ga]Ga-DOTA-exendin-4 via fractionated radionuclide generator elution on a cassette based synthesis module.

BACKGROUND: PET/CT imaging of glucagon-like peptide receptor 1 has recently filled a gap in reliably diagnosing insulinoma through non-invasive means. 68Ga-labelled derivatives of exendin-4 show high sensitivity as well as sufficient serum stability to enable routine clinical application. Here, we provide data for automated production of [68Ga][Nle14,Lys40(Ahx-DOTA-Ga)NH2]exendin-4 ([68Ga]Ga-DOTA-exendin-4) on a cassette based synthesis module (Modular-Lab PharmTracer, Eckert & Ziegler) using commercially available cassettes in combination with an approved 68Ge/68Ga generator (GalliaPharm, Eckert & Ziegler). This setup ensured high reproducibility as well as low radiation burden for the production team. Quality control including determination of radiochemical purity was performed by RP-HPLC using a water/0.1 % TFA/acetonitrile gradient on a C18 column. A modified TLC system with ammonium acetate & methanol as mobile phase and a novel limit test for determination of polysorbate 80 content in the final formulation are also described in this study. MAIN FINDINGS: Reliable yields as well as high molar activity for patient use were only achieved using a fractionated elution approach. Batch data showed radiochemical purity of >93 % as determined by RP-HPLC and TLC as well as good stability over 2 h post production. Testing for polysorbate 80 confirmed a concentration <1 mg/mL in the final product solution. Specifications for routine production were established based on existing Pharmacopeia monographs for other radiopharmaceuticals and were validated with 5 master batches. CONCLUSION: The described synthesis method enables reproducible, automated in-house production of [68Ga]Ga-DOTA-exendin-4 for routine clinical application.

Effects of Side Chain and Peptide Bond Modifications on the Targeting Properties of Stabilized Minigastrin Analogs.

Different attempts have been made in the past two decades to develop radiolabeled peptide conjugates with enhanced pharmacokinetic properties in order to improve the application for tumor imaging and peptide receptor radionuclide therapy (PRRT), which targets the cholecystokinin-2 receptor (CCK2R). In this paper, the influence of different side chain and peptide bond modifications has been explored for the minigastrin analog DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 (DOTA-MGS5). Based on this lead structure, five new derivatives were synthesized for radiolabeling with trivalent radiometals. Different chemical and biological properties of the new derivatives were analyzed. Receptor interaction of the peptide derivatives and cell internalization of the radiolabeled peptides were studied in A431-CCK2R cells. The stability of the radiolabeled peptides in vivo was investigated using BALB/c mice. Tumor targeting of all 111In-labeled peptide conjugates, and of a selected compound radiolabeled with gallium-68 and lutetium-177, was evaluated in BALB/c nude mice xenografted with A431-CCK2R and A431-mock cells. All 111In-labeled conjugates, except [111In]In-DOTA-[Phe8]MGS5, showed a high resistance against enzymatic degradation. A high receptor affinity with IC50 values in the low nanomolar range was confirmed for most of the peptide derivatives. The specific cell internalization over time was 35.3-47.3% for all radiopeptides 4 h after incubation. Only [111In]In-DOTA-MGS5[NHCH3] exhibited a lower cell internalization of 6.6 ± 2.8%. An overall improved resistance against enzymatic degradation was confirmed in vivo. Of the radiopeptides studied, [111In]In-DOTA-[(N-Me)1Nal8]MGS5 showed the most promising targeting properties, with significantly increased accumulation of radioactivity in A431-CCK2R xenografts (48.1 ± 9.2% IA/g) and reduced accumulation of radioactivity in stomach (4.2 ± 0.5% IA/g). However, in comparison with DOTA-MGS5, a higher influence on the targeting properties was observed for the change of radiometal, resulting in a tumor uptake of 15.67 ± 2.21% IA/g for [68Ga]Ga-DOTA-[(N-Me)1Nal8]MGS5 and 35.13 ± 6.32% IA/g for [177Lu]Lu-DOTA-[(N-Me)1Nal8]MGS5.

Effect of N-Terminal Peptide Modifications on In Vitro and In Vivo Properties of 177Lu-Labeled Peptide Analogs Targeting CCK2R.

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.

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.

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting.

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.

Development of methylated cobalt-alkyne complexes with selective cytotoxicity against COX-positive cancer cell lines.

Derivatives of the cytotoxic cyclooxygenase (COX) inhibitor [(prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS) with a methyl group in the 3, 4, 5, or 6 position of the acetylsalicylic acid (ASS) scaffold were synthesized with the aim to achieve enhanced selectivity for COX-2. From this modification, a higher specificity for COX-2-expressing tumors is expected, preventing COX-1-mediated side effects. The cobalt-alkyne complexes were tested for their COX-inhibitory and antiproliferative properties as well as their cellular uptake. Methylation reduced the effects at the isolated COX-1, whereas those at the isolated COX-2 remained nearly constant compared to Co-ASS. In cellular systems, the new compounds showed superior cytotoxicity toward the COX-positive HT-29 colon carcinoma cells than cisplatin. The reduced growth-inhibitory potency in T-24 cells, which express distinctly fewer COX enzymes (COX-1/COX-2 = 50/1) than HT-29 cells (COX-1/COX-2 = 50/50), and the only marginal activity in COX-negative MCF-7 breast cancer cells point to an interference in the arachidonic acid cascade through COX-2 inhibition as part of the mode of action, especially as the cellular uptake was even higher in MCF-7 cells than in T-24 cells. These findings clearly demonstrate that the methylated cobalt-alkyne complexes possess promising potential for further development as reasonable alternatives to the limited platinum-based antitumor agents.

Siderophore Scaffold as Carrier for Antifungal Peptides in Therapy of Aspergillus fumigatus Infections.

Antifungal resistance of human fungal pathogens represents an increasing challenge in modern medicine. Short antimicrobial peptides (AMP) display a promising class of antifungals with a different mode of action, but lack target specificity and metabolic stability. In this study the hexapeptide PAF26 (Ac-dArg-dLys-dLys-dTrp-dPhe-dTrp-NH2) and the three amino acid long peptide NLF (H2N-Asn-Leu-dPhe-COOH) were coupled to diacetylfusarinine C (DAFC), a derivative of the siderophore triacetylfusarinine C (TAFC) of Aspergillus fumigatus, to achieve targeted delivery for treatment of invasive aspergillosis. Conjugated compounds in various modifications were labelled with radioactive gallium-68 to perform in vitro and in vivo characterizations. LogD, serum stability, uptake- growth promotion- and minimal inhibitory concentration assays were performed, as well as in vivo stability tests and biodistribution in BALB/c mice. Uptake and growth assays revealed specific internalization of the siderophore conjugates by A. fumigatus. They showed a high stability in human serum and also in the blood of BALB/c mice but metabolites in urine, probably due to degradation in the kidneys. Only PAF26 showed growth inhibition at 8 µg/ml which was lost after conjugation to DAFC. Despite their lacking antifungal activity conjugates based on a siderophore scaffold have a potential to provide the basis for a new class of antifungals, which allow the combination of imaging by using PET/CT with targeted treatment, thereby opening a theranostic approach for personalized therapy.

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177.

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo.

Cholecystokinin-2 Receptor Targeting with Radiolabeled Peptides: Current Status and Future Directions.

A wide variety of radiolabeled peptide analogs for specific targeting of cholecystokinin- 2 receptors (CCK2R) has been developed in the last decades. Peptide probes based on the natural ligands Minigastrin (MG) and Cholecystokinin (CCK) have a high potential for molecular imaging and targeted radiotherapy of different human tumors, such as Medullary Thyroid Carcinoma (MTC) and Small Cell Lung Cancer (SCLC). MG analogs with high persistent uptake in CCK2R expressing tumors have been preferably used for the development of radiolabeled peptide analogs. The clinical translation of CCK2R targeting has been prevented due to high kidney uptake or low metabolic stability of the different radiopeptides developed. Great efforts in radiopharmaceutical development have been undertaken to overcome these limitations. Various modifications in the linear peptide sequence of MG have been introduced mainly with the aim to reduce kidney retention. Furthermore, improved tumor uptake could be obtained by in situ stabilization of the radiopeptide against enzymatic degradation through coinjection of peptidase inhibitors. Recent developments focusing on the stabilization of the Cterminal receptor binding sequence (Trp-Met-Asp-Phe-NH2) have led to new radiolabeled MG analogs with highly improved tumor uptake and tumor-to-kidney ratio. In this review, all the different aspects in the radiopharmaceutical development of CCK2R targeting peptide probes are covered, giving also an overview on the clinical investigations performed so far. The recent development of radiolabeled MG analogs, which are highly stabilized against enzymatic degradation in vivo, promises to have a high impact on the clinical management of patients with CCK2R expressing tumors in the near future.