The radiometal makes a difference. Synthesis and preliminary characterisation of DOTA-minigastrin analogue complexes with Ga, Lu and Y

BACKGROUND: The minigastrin analogue — CP04: DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 has been developed for CCK2R targeting. This analogue can be radiolabelled with 111In or 68Ga for imaging, or with 90Y and 177Lu for therapy. However, affinity of the chelator-peptide conjugates to the cell membrane receptors may vary depending on the metal incorporated into the complex. So far, there are no such studies for the ligands of gastrin/cholecystokinin receptor CCK2R. It is supposed that the reason for the differentiation of receptor affinity to the respective receptors is in the changes of structure of chelating system and their influence on the bioactive conformations of the metal conjugated peptides. Herein, we report on the radiolabeling of CP04 with 90Y, 177Lu and 68Ga and synthesis of cold CP04 complexes with respective stable metals for further structural and physico-chemical and biological studies. MATERIALS AND METHODS: From 200 to 600 MBq of 90Y, 177Lu or 68Ga were used for radiolabelling of 20 μg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5). Non-radioactive complexes with Lu and Ga were synthesized in milligram amounts starting from 0.5 mg up to 5 mg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5) when using 2-molar excess of the metal ions. Complex formation needed 5 min in microwave oven or 12 min in thermo-block at 95°C. RP-HPLC isocratic method (Kinetex 150/4.6 mm; 25% AcN/0.1% TFA, 1 mL/min) with UV/Vis and radiometric detection was developed for investigation of the radiolabelled and “cold” complexes. For LC-MS investigations, HPLC method was modified replacing TFA by formic acid. RESULTS AND DISCUSSION: Yields of CP04 radiolabelling were greater than 90% for all three radionuclides. The HPLC method enabled identification of these radio-complexes based on comparison to their non-radioactive equivalents. In all cases, chromatograms revealed peaks that could be attributed to the metal-CP04 complexes and to impurities (including methionine oxidation). LC-MS analysis of Ga and Lu complexes revealed conformity of the observed molecular ions to the predicted formulas (m/z 2116 and 2220 Da for Ga and Lu, respectively). Different chromatographic behaviour observed for Ga-CP04 complex comparing to Lu- and Y- labelled peptide (relative retention to CP04: 1.08, 0.86 and 0.85, respectively) suggest different coordination of the metal ions. Therefore, further studies are planned using the non-radioactive complexes in order to assess their structural conformations

Comparison of chromatographic methods for quality control of DMSA complexes with 99mTc and 188Re at (III) and (V) oxidation states

BACKGROUND: The reliable method for determination ofidentity and radiochemical purity (RCP) is of great importancein radiopharmaceutical development. This is especially relevantwhen more than one form of radiometal/ligand complex can beformed during radiolabelling, such as complexes of 99mTc or 188Rewith meso-2,3-dimercaptosuccinic acid (DMSA), where dependingon the pH, metal can occur either at +3 or +5 oxidation state.The aim of our study was to evaluate possibilities for optimizationof chromatographic systems leading to specific and reliableanalytical method for determination of the identity and RCP ofDMSA complexes with 99mTc or 188Re.MATERIAL AND METHODS: The commercial DMSA kits(POLATOM) were used for preparation of technetium-99m (III) and (V) complexes with DMSA. 99mTc(V)-DMSA complexeswere prepared by addition of NaHCO3 to the kit vial prior to99mTc-eluate to obtain pH ~8. 188Re(V)-DMSA was prepared eitherdirectly or using intermediate 188Re(III)-EDTA complex addedto DMSA. RCP was evaluated by TLC using: ITLC-SG developedin methylethylketon, SG60 coated plates developed in:n-BuOH/H2O/CH3COOH and n-PrOH/H2O/CH3COOH systems,and in H2O. Comparative biodistribution studies were performedin normal Wistar rats.RESULTS: Using silica gel plates and n-PrOH, H2O and aceticacid in the developing solution, we observed that 99mTc/188Re(III)-DMSA and 99mTc/188Re(V)-DMSA complexes could be wellseparated from each other and from the impurities in the formof free pertechnetate/perrhenate. In vivo studies showed quitedifferent biodistribution of 99mTc(III)- and 99mTc(V)-DMSA. Thetrivalent complex accumulated mainly in kidneys (>40%ID),while 99mTc(V)-DMSA revealed high excretion with urine andrelatively high concentration in osseous tissue (ca. 2 %ID/g).Accumulation of this complex in kidneys was very low (ca.2.5 %ID). Biodistribution pattern of 188Re(V)-DMSA prepareddirectly was almost identical to that of 99mTc(V)-DMSA. Biodistributionresults of the 188Re preparation obtained using 188Re(III)-EDTA intermediate indicated that the preparation contained themixture of penta- and trivalent 188Re complexes. The quite highaccumulation of radioactivity in kidneys (23 %ID) gave evidenceof the presence of 188Re(III)-DMSA in this preparation, what wasalso confirmed by the results of TLC analysis performed usingsilica gel plate and n-propanol/water/acetic acid as developingsystem. CONCLUSIONS: Based on our study, we have made recommendationon the suitable methods for investigations of RCP ofDMSA complexes, i.e.: SG60 plates developed in the mixtureof n-propanol/water/acetic acid, which enable determination of the tri- and pentavalent DMSA complexes, as well as, thepertechnetate/perrhenate impurity, and developed in water fordetermination of the colloidal residue.BACKGROUND: The reliable method for determination ofidentity and radiochemical purity (RCP) is of great importancein radiopharmaceutical development. This is especially relevantwhen more than one form of radiometal/ligand complex can beformed during radiolabelling, such as complexes of 99mTc or 188Rewith meso-2,3-dimercaptosuccinic acid (DMSA), where dependingon the pH, metal can occur either at +3 or +5 oxidation state.The aim of our study was to evaluate possibilities for optimizationof chromatographic systems leading to specific and reliableanalytical method for determination of the identity and RCP ofDMSA complexes with 99mTc or 188Re.MATERIAL AND METHODS: The commercial DMSA kits(POLATOM) were used for preparation of technetium-99m (III) and (V) complexes with DMSA. 99mTc(V)-DMSA complexeswere prepared by addition of NaHCO3 to the kit vial prior to99mTc-eluate to obtain pH ~8. 188Re(V)-DMSA was prepared eitherdirectly or using intermediate 188Re(III)-EDTA complex addedto DMSA. RCP was evaluated by TLC using: ITLC-SG developedin methylethylketon, SG60 coated plates developed in:n-BuOH/H2O/CH3COOH and n-PrOH/H2O/CH3COOH systems,and in H2O. Comparative biodistribution studies were performedin normal Wistar rats.RESULTS: Using silica gel plates and n-PrOH, H2O and aceticacid in the developing solution, we observed that 99mTc/188Re(III)-DMSA and 99mTc/188Re(V)-DMSA complexes could be wellseparated from each other and from the impurities in the formof free pertechnetate/perrhenate. In vivo studies showed quitedifferent biodistribution of 99mTc(III)- and 99mTc(V)-DMSA. Thetrivalent complex accumulated mainly in kidneys (>40%ID),while 99mTc(V)-DMSA revealed high excretion with urine andrelatively high concentration in osseous tissue (ca. 2 %ID/g).Accumulation of this complex in kidneys was very low (ca.2.5 %ID). Biodistribution pattern of 188Re(V)-DMSA prepareddirectly was almost identical to that of 99mTc(V)-DMSA. Biodistributionresults of the 188Re preparation obtained using 188Re(III)-EDTA intermediate indicated that the preparation contained themixture of penta- and trivalent 188Re complexes. The quite highaccumulation of radioactivity in kidneys (23 %ID) gave evidenceof the presence of 188Re(III)-DMSA in this preparation, what wasalso confirmed by the results of TLC analysis performed usingsilica gel plate and n-propanol/water/acetic acid as developingsystem.CONCLUSIONS: Based on our study, we have made recommendationon the suitable methods for investigations of RCP ofDMSA complexes, i.e.: SG60 plates developed in the mixtureof n-propanol/water/acetic acid, which enable determination of the tri- and pentavalent DMSA complexes, as well as, thepertechnetate/perrhenate impurity, and developed in water fordetermination of the colloidal residue

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 111In 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 111In-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 111In. 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 (pH 4.5) 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 Met11-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, 25 mg ascorbic acid, 0.5 mg gentisic acid and 5 mg l-methionine. The radiolabelling performed by incubation of 200-250 MBq 111InCl3 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 > 4 h at + 25°C. Conclusion A kit formulation to prepare 111In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product

IAEA Contribution to Nanosized Targeted Radiopharmaceuticals for Drug Delivery

The rapidly growing interest in the application of nanoscience in the future design of radiopharmaceuticals and the development of nanosized radiopharmaceuticals in the late 2000 ' s, resulted in the creation of a Coordinated Research Project (CRP) by the International Atomic Energy Agency (IAEA) in 2014. This CRP entitled 'Nanosized delivery systems for radiopharmaceuticals' involved a team of expert scientist from various member states. This team of scientists worked on a number of cutting-edge areas of nanoscience with a focus on developing well-defined, highly effective and site-specific delivery systems of radiopharmaceuticals. Specifically, focus areas of various teams of scientists comprised of the development of nanoparticles (NPs) based on metals, polymers, and gels, and their conjugation/encapsulation or decoration with various tumor avid ligands such as peptides, folates, and small molecule phytochemicals. The research and development efforts also comprised of developing optimum radiolabeling methods of various nano vectors using diagnostic and therapeutic radionuclides including Tc-99m, Ga-68, Lu-177 and Au-198. Concerted efforts of teams of scientists within this CRP has resulted in the development of various protocols and guidelines on delivery systems of nanoradiopharmaceuticals, training of numerous graduate students/post-doctoral fellows and publications in peer reviewed journals while establishing numerous productive scientific networks in various participating member states. Some of the innovative nanoconstructs were chosen for further preclinical applications-all aimed at ultimate clinical translation for treating human cancer patients. This review article summarizes outcomes of this major international scientific endeavor

H.E.S.S. observations of gamma-ray bursts in 2003-2007

Very-high-energy (VHE; >~100 GeV) gamma-rays are expected from gamma-ray bursts (GRBs) in some scenarios. Exploring this photon energy regime is necessary for understanding the energetics and properties of GRBs. GRBs have been one of the prime targets for the H.E.S.S. experiment, which makes use of four Imaging Atmospheric Cherenkov Telescopes (IACTs) to detect VHE gamma-rays. Dedicated observations of 32 GRB positions were made in the years 2003-2007 and a search for VHE gamma-ray counterparts of these GRBs was made. Depending on the visibility and observing conditions, the observations mostly start minutes to hours after the burst and typically last two hours. Results from observations of 22 GRB positions are presented and evidence of a VHE signal was found neither in observations of any individual GRBs, nor from stacking data from subsets of GRBs with higher expected VHE flux according to a model-independent ranking scheme. Upper limits for the VHE gamma-ray flux from the GRB positions were derived. For those GRBs with measured redshifts, differential upper limits at the energy threshold after correcting for absorption due to extra-galactic background light are also presented.Comment: 9 pages, 4 tables, 3 figure

Detection of extended TeV emission around the Geminga pulsar with H.E.S.S.

Highly extended gamma-ray emission around the Geminga pulsar was discovered by Milagro and verified by HAWC. Despite many observations with Imaging Atmospheric Cherenkov Telescopes (IACTs), detection of gamma-ray emission on angular scales exceeding the IACT field-of-view has proven challenging. Recent developments in analysis techniques have enabled the detection of significant emission around Geminga in archival data with H.E.S.S.. In 2019, further data on the Geminga region were obtained with an adapted observation strategy. Following the announcement of the detection of significant TeV emission around Geminga in archival data, in this contribution we present the detection in an independent dataset. New analysis results will be presented, and emphasis given to the technical challenges involved in observations of highly extended gamma-ray emission with IACTs

Astronomy outreach in Namibia : H.E.S.S. and beyond

Astronomy plays a major role in the scientific landscape of Namibia. Because of its excellent sky conditions, Namibia is home to ground-based observatories like the High Energy Spectroscopic System (H.E.S.S.), in operation since 2002. Located near the Gamsberg mountain, H.E.S.S. performs groundbreaking science by detecting very-high-energy gamma rays from astronomical objects. The fascinating stories behind many of them are featured regularly in the "Source of the Month", a blog-like format intended for the general public with more than 170 features to date. In addition to other online communication via social media, H.E.S.S. outreach activities have been covered locally, e.g. through 'open days' and guided tours on the site itself. An overview of the H.E.S.S. outreach activities are presented in this contribution, along with discussions relating to the current landscape of astronomy outreach and education in Namibia. There has also been significant activity in the country in recent months, whereby astronomy is being used to further sustainable development via human capacity-building. Finally, as we take into account the future prospects of radio astronomy in the country, momentum for a wider range of astrophysics research is clearly building — this presents a great opportunity for the astronomy community to come together to capitalise on this movement and support astronomy outreach, with the overarching aim to advance sustainable development in Namibia

Detection of new Extreme BL Lac objects with H.E.S.S. and Swift XRT

Extreme high synchrotron peaked blazars (EHBLs) are amongst the most powerful accelerators found in nature. Usually the synchrotron peak frequency of an EHBL is above 10$^{17}$ Hz, i.e., lies in the range of medium to hard X-rays making them ideal sources to study particle acceleration and radiative processes. EHBL objects are commonly observed at energies beyond several TeV, making them powerful probes of gamma-ray absorption in the intergalactic medium. During the last decade, several attempts have been made to increase the number of EHBL detected at TeV energies and probe their spectral characteristics. Here we report new detections of EHBLs in the TeV energy regime, each at a redshift of less than 0.2, by the High Energy Stereoscopic System (H.E.S.S.). Also, we report on X-ray observations of these EHBLs candidates with Swift-XRT. In conjunction with the very high energy observations, this allows us to probe the radiation mechanisms and the underlying particle acceleration processes

Evidence of 100 TeV γ-ray emission from HESS J1702-420 : a new PeVatron candidate

The identification of active PeVatrons, hadronic particle accelerators reaching the knee of the cosmic-ray spectrum (at the energy of few PeV), is crucial to understand the origin of cosmic rays in the Galaxy. In this context, we report on new H.E.S.S. observations of the PeVatron candidate HESS J1702-420, which bring evidence for the presence of γ-rays up to 100 TeV. This is the first time in the history of H.E.S.S. that photons with such high energy are observed. Remarkably, the new deep observations allowed the discovery of a new γ-ray source component, called HESS J1702-420A, that was previously hidden under the bulk emission traditionally associated with HESS J1702-420. This new object has a power-law spectral slope < 2 and a γ-ray spectrum that, extending with no sign of curvature up to 100 TeV, makes it an excellent candidate site for the presence of PeV-energy cosmic rays. This discovery brings new information to the ongoing debate on the nature of the unidentified source HESSJ1702-420, and on the origin of Galactic cosmic rays

Search for dark matter annihilation signals from unidentified Fermi-LAT objects with H.E.S.S.

Cosmological N-body simulations show that Milky-Way-sized galaxies harbor a population of unmerged dark matter subhalos. These subhalos could shine in gamma rays and be eventually detected in gamma-ray surveys as unidentified sources. We search for very-high-energy (VHE, E $\geq$ 100~GeV) gamma-ray emission using H.E.S.S. observations carried out from a thorough selection of unidentified Fermi-LAT Objects (UFOs) as dark matter subhalo candidates. Provided that the dark matter mass is higher than a few hundred GeV, the emission of the UFOs can be well described by dark matter annihilation models. No significant VHE gamma-ray emission is detected in any UFO dataset nor in their combination. We, therefore, derive constraints on the product of the velocity-weighted annihilation cross-section \left by the J-factor on dark matter models describing the UFO emissions. Upper limits at 95% confidence level are derived on \left J in W$^{+}$W$^{-}$ and τ$^{+}$τ$^{-}$ annihilation channels for the TeV dark matter particles. Focusing on thermal WIMPs, strong constraints on the J-factors are obtained from H.E.S.S. observations. Adopting model-dependent predictions from cosmological N-body simulations on the J-factor distribution function for Milky Way (MW)-sized galaxies, only $\lesssim$ 0.3 ~TeV mass dark matter models marginally allow to explain observed UFO emission