Sara Rubagotti

Synthesis and Characterization of 68Ga-Labeled Curcumin and Curcuminoid Complexes as Potential Radiotracers for Imaging of Cancer and Alzheimer’s Disease

Curcumin (CUR) and curcuminoids complexes labeled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimers disease. Gallium-68 is a positron-emitting, generator-produced radionuclide, and its properties can be exploited in situ in medical facilities without a cyclotron. Moreover, CUR showed a higher uptake in tumor cells compared to normal cells, suggesting potential diagnostic applications in this field. In spite of this, no studies using labeled CUR have been performed in this direction, so far. Herein, (68)Ga-labeled complexes with CUR and two curcuminoids, namely diacetyl-curcumin (DAC) and bis(dehydroxy)curcumin (bDHC), were synthesized and characterized by means of experimental and theoretical approaches. Moreover, a first evaluation of their affinity to synthetic β-amyloid fibrils and uptake by A549 lung cancer cells was performed to show the potential application of these new labeled curcuminoids in these diagnostic fields. The radiotracers were prepared by reacting (68)Ga(3+) obtained from a (68)Ge/(68)Ga generator with 1 mg/mL curcuminoids solutions. Reaction parameters (precursor amount, reaction temperature, and pH) were optimized to obtain high and reproducible radiochemical yield and purity. Stoichiometry and formation of the curcuminoid complexes were investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR, ultraviolet-visible, and fluorescence spectroscopy on the equivalent (nat)Ga-curcuminoids (nat = natural) complexes, and their structure was computed by theoretical density functional theory calculations. The analyses evidenced that CUR, DAC, and bDHC were predominantly in the keto-enol form and attested to Ga(L)2(+) species formation. Identity of the (68)Ga(L)2(+) complexes was confirmed by coelution with the equivalent (nat)Ga(L)2(+) complexes in ultrahigh-performance liquid chromatography analyses.(68)Ga(CUR)2(+), (68)Ga(DAC)2(+), and (68)Ga(bDHC)2(+) were highly (87 ± 4, 90 ± 1%) and moderately (48 ± 2%), respectively, retained by synthetic β-amyloid fibrils in vitro. All the Ga-curcuminoid complexes showed an uptake in A549 lung cancer cells, at least equivalent to the respective free curcuminoids, confirming potential applications as cancer-detecting radiotracers.

Post-Synthesis Incorporation of 64Cu in CuS Nanocrystals to Radiolabel Photothermal Probes: A Feasible Approach for Clinics

We report a simple method for the incorporation of Cu(I) or (64)Cu(I) radionuclides in covellite nanocrystals (CuS NCs). After the in situ reduction of Cu(II) or (64)Cu(II) ions by ascorbic acid, their incorporation in PEG-coated CuS NCs takes place at room temperature. In all the reaction steps, the stability of the NCs under physiological conditions was ensured. The copper incorporation reaction could also take place on CuS NCs bearing biotin molecules at their surface, with no detrimental effects on the specific binding affinity of the NCs toward streptavidin after incorporation. At low loading of Cu ions, the strong near-infrared (NIR) absorption band of the starting CuS NCs was essentially preserved, which allowed for efficient plasmonic photothermal therapy. The combined presence in the NCs of (64)Cu ions, well suitable for positron emission tomography, and of free carriers responsible for the NIR absorption, should enable their theranostic use as radiotracers and as photothermal probes in tumor ablation treatments. Moreover, the simplicity of the preparation scheme, which involves the use of radioactive species only as a last step, makes the protocol easily transferable to the clinical practice.

Influence of different chelators on the radiochemical properties of a 68-Gallium labelled bombesin analogue

UNLABELLED The radiolabelled bombesin analogue AMBA shows high potential for diagnosis and treatment of prostate and breast cancer, but the influence of different chelators, which differ in terms of radiochemical reactivity and stability, have not been explored so far. In order to find the best suitable chelator for labelling of AMBA, we synthesized AMBA analogues linked to the most commonly used chelators DOTA, NOTA and NODAGA and compared their reactivity and stability after labelling with 68-Gallium. METHODS For the synthesis of DO3A-, NO2A- and NODAGA-AMBA, a solid-phase synthesis approach was used. The influence of concentration, pH and temperature on the radiolabelling was analysed. The in vitro stability of all complexes in saline, human serum, human whole blood and against transchelation and transmetallation was analysed. RESULTS The peptides were synthesised in high yield and purity. Purity and identity of products and impurities were confirmed using UHPLC coupled to ESI-MS. Radiolabelling of these peptides was optimal at elevated temperature, although room temperature labelling was reported previously for NOTA and NODAGA chelators. The highest reactivity was observed for NODAGA-AMBA. On preparation of NO2A-AMBA, the formation of a by-product was detected with HPLC. More detailed analysis revealed the formation of an isomer with the same mass to charge ratio which led to the conclusion that a coordination isomer was formed. All complexes showed high stability in saline, human serum or when challenged with DTPA, transferrin and varying metals (Fe(3+), Cu(2+), Zn(2+)). Conversely, the stability in human blood was low, and varying metabolites were detected and identified by ESI-MS. CONCLUSION All three precursors are available in high yields suitable for routine production. NODAGA-AMBA showed the most favoured features when labelled with 68-gallium, but a further comparison in vivo should be performed in order to confirm the superior features found in vitro.

Development of a simple kit-based method for preparation of pharmaceutical-grade 68Ga-DOTATOC

IntroductionThe use of germanium-68 (68Ge)/gallium-68 (68Ga) generators is still limited when compared with the 99Mo/99mTc counterpart, mainly because of the absence of commercial kits and a kit-based method for preparing 68Ga-labelled radiopharmaceuticals in a reliable way. The present study aimed to develop and optimize a reliable direct labelling of DOTATOC with 68Ga through a kit-based approach. Materials and methodsA fraction of the eluate of two different 68Ge/68Ga generators was directly injected into a vial prefilled with precursor, buffer and scavenger (the prototype of a potential lyophilized kit). The vial was directly warmed to 100°C and then buffered with a 1.5 mol/l sodium ascorbate solution. The parameters influencing the reaction were studied and optimized. The efficacy of the method in terms of incorporation yield and quality of the final radiotracer was compared with preparations performed with two commercial automatic synthesizers by applying the tests prescribed in the European Pharmacopeia monograph for 68Ga-DOTATOC. ResultsUnder optimal conditions, the overall radiochemical yields of the kit-based process were 73±4 and 69±3% not decay-corrected for the IGG100 and itG generator, respectively. The radiochemical purity was 95±3% and the preparations were compliant with all specifications given in the pharmacopoeia monograph. ConclusionThe feasibility of a kit-based approach for the preparation of 68Ga-DOTATOC was proved and a first home-made version of a putative lyophilized kit was proposed.

Somatostatin receptor positron emission tomography/computed tomography imaging in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an uncommon aggressive primary cutaneous carcinoma with neuroendocrine differentiation. However, literature data about the use of somatostatin receptor positron emission tomography/computed tomography (PET/CT) imaging in MCC are limited and its role is not clearly stated.

Affinity of nat/68Ga-Labelled Curcumin and Curcuminoid Complexes for β-Amyloid Plaques: Towards the Development of New Metal-Curcumin Based Radiotracers

Curcumin derivatives labelled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer’s disease. Nevertheless, no study by exploiting the labelling with gallium-68 has been performed so far, in spite of its suitable properties (positron emitter, generator produced radionuclide). Herein, an evaluation of the affinity for synthetic β-amyloid fibrils and for amyloid plaques of three nat/68Ga-labelled curcumin analogues, namely curcumin curcumin (CUR), bis-dehydroxy-curcumin (bDHC) and diacetyl-curcumin (DAC), was performed. Affinity and specificity were tested in vitro on amyloid synthetic fibrils by using gallium-68 labelled compounds. Post-mortem brain cryosections from Tg2576 mice were used for the ex vivo visualization of amyloid plaques. The affinity of 68Ga(CUR)2+, 68Ga(DAC)2+, and 68Ga(bDHC)2+ for synthetic β-amyloid fibrils was moderate and their uptake could be observed in vitro. On the other hand, amyloid plaques could not be visualized on brain sections of Tg2576 mice after injection, probably due to the low stability of the complexes in vivo and of a hampered passage through the blood–brain barrier. Like curcumin, all nat/68Ga-curcuminoid complexes maintain a high affinity for β-amyloid plaques. However, structural modifications are still needed to improve their applicability as radiotracers in vivo.

Labelling of Y- and Lu-DOTA-Bioconjugates for Targeted Radionuclide Therapy: A Comparison among Manual, Semiautomated, and Fully Automated Synthesis

In spite of the hazard due to the radiation exposure, preparation of 90Y- and 177Lu-labelled radiopharmaceuticals is still mainly performed using manual procedures. In the present study the performance of a commercial automatic synthesizer based on disposable cassettes for the labelling of 177Lu- and 90Y-DOTA-conjugated biomolecules (namely, DOTATOC and PSMA-617) was evaluated and compared to a manual and a semiautomated approach. The dose exposure of the operators was evaluated as well. More than 300 clinical preparations of both 90Y- and 177Lu-labelled radiopharmaceuticals have been performed using the three different methods. The mean radiochemical yields for 90Y-DOTATOC were 96.2 ± 4.9%, 90.3 ± 5.6%, and 82.0 ± 8.4%, while for 177Lu-DOTATOC they were 98.3%  ± 0.6, 90.8%  ± 8.3, and 83.1 ± 5.7% when manual, semiautomated, and automated approaches were used, respectively. The mean doses on the whole hands for yttrium-90 preparations were 0.15 ± 0.4 mSv/GBq, 0.04 ± 0.1 mSv/GBq, and 0.11 ± 0.3 mSv/GBq for manual, semiautomated, and automated synthesis, respectively, and for lutetium-177 preparations, they were 0.02 ± 0.008 mSv/GBq, 0.01 ± 0.03 mSv/GBq, and 0.01 ± 0.02 mSv/GBq, respectively. In conclusion, the automated approach guaranteed reliable and reproducible preparations of pharmaceutical grade therapeutic radiopharmaceuticals in a decent RCY. The radiation exposure of the operators remained comparable to the manual approach mainly due to the fact that a dedicated shielding was still not available for the system.

Radiopharmaceuticals for Treatment of NETs

Neuroendocrine tumours (NETs) are a group of unusual cancers which develop from cells of the diffuse endocrine system. They are found most commonly in lungs or gastrointestinal system, but they can also originate in other tissues such as pancreas, ovary and testes. A common feature of NETs is that they almost all overexpress somatostatin receptors. For this reason somatostatin receptors have been considered as a target for radiolabelled radiopharmaceuticals. These molecules are constituted by a peptide chain (i.e. a somatostatin-like structure), a partially or totally electron emitter radionuclide and a suitable bifunctional chelator able both to firmly complex the radionuclide as well as to be connected to the peptide chain by means of proper molecular spacers. Nowadays, the most used radiopharmaceuticals for treatments of NETs are [DOTA]0-Tyr3-octreotide (DOTATOC) and [DOTA0]-Tyr3-octreotate (DOTATATE) labelled with yttrium-90 and lutetium-177.

Uptake of Ga-curcumin derivatives in different cancer cell lines: Toward the development of new potential 68Ga-labelled curcuminoids-based radiotracers for tumour imaging

Thanks to the ability to suppress the proliferation and to kill tumour cells, several studies have shown the anti-cancer effects of curcumin (CUR) and its derivatives, i.e. diacetylcurcumin (DAC) and bis-dehydroxycurcumin (bDHC). This study is focused onto the development of curcuminoid complexes with gallium-68 employed as potential new radio-labelled probes to detect neoplastic tissues through imaging techniques such as positron emission tomography. To this purpose, the uptake of three Ga-curcuminoid complexes, namely Ga(CUR)2+, Ga(DAC)2+, Ga(bDHC)2+, by various tumour cell lines was compared with the uptake of the same compounds by normal human lymphocytes by flow cytometry using the intrinsic fluorescence of the curcuminoids. Ga(CUR)2+, and particularly Ga(DAC)2+, showed a higher uptake by colorectal carcinoma (HT29) and lymphoma (K562) cell lines than lymphocytes, while the uptake of Ga(bDHC)2+ was higher in lymphocytes than in all the other cell lines. Based on the fluorescence data, Gallium-68 labelled complexes were then tested in HT29 cell line. 68Ga(DAC)2+ showed the highest uptake by HT29 cells (higher internalization with a lower externalization) and the highest affinity. The obtained results are promising and the findings foster further investigation on the development of curcumin-metal-based radiopharmaceuticals.