Mattia Asti

Validation of 68Ge/68Ga generator processing by chemical purification for routine clinical application of 68Ga-DOTATOC

INTRODUCTION Imaging of somatostatin receptor expressing tumours has been greatly enhanced by the use of (68)Ga-DOTATOC and PET/CT. METHODS In this work, a purification method for the (68)Ge/(68)Ga generator eluate and a method to produce (68)Ga-DOTATOC suitable for clinical use were evaluated. The generator eluate was purified and concentrated on a cation-exchange cartridge in HCl/acetone media. The efficacy of this procedure in eliminating metal impurities from the (68)Ga solution was investigated by ICP-MS. The radiotracer quality was evaluated by radio-TLC, GC and gamma-ray spectrometry. RESULTS (68)Ga-DOTATOC preparations (n=33) were carried out with a mean synthesis yield of 59.3+/-2.8% (not corrected for decay) and a batch activity ranging from 555 to 296 MBq. The radiochemical and radionuclidic purity were >98% and 99.9999%, respectively. With this purification process, >95% of the Fe(III), Zn(II) and Mn(II) were eliminated from the solution. CONCLUSIONS (68)Ga-DOTATOC produced with this method can be efficiently used in nuclear medicine departments for PET evaluations.

Ga-68 DOTATOC PET, endoscopic ultrasonography, and multidetector CT in the diagnosis of duodenopancreatic neuroendocrine tumors: a single-centre retrospective study.

Purpose: In this report, we compared endoscopic ultrasonography (EUS), multidetector CT (MDCT), and Ga-68 DOTATOC PET/CT in patients with neuroendocrine tumors (NETs). We report our experience with use of these methods in patients suspected to have duodenopancreatic primitive NET. Methods: Nineteen consecutive patients (mean age, 56; 21–80), who underwent both Ga-68 DOTATOC PET/CT and EUS between March 2007 and November 2008 were retrospectively included in the study (16 underwent MDCT). Suspicion of NET was confirmed by EUS-FNA and/or surgery. Operative characteristics of PET, EUS, and MDCT were compared. Results: Twenty-three neuroendocrine lesions were diagnosed in 13/19 patients. EUS, PET, and MDCT correctly identified as affected 13/13 (100%), 12/13 (92%), and 10/11 (91%) patients, respectively. On a lesion basis, EUS, PET, and MDCT identified correctly as NETs 22/23 (96%), 20/23 (87%), and 13/18 (72%) lesions (P = 0.08 EUS vs. CT). Both on a patient and on a lesion basis, specificity was 67%, 83%, and 80% for EUS, PET, and MDCT, respectively. Conclusions: EUS, Ga-68 DOTATOC PET, and MDCT seem to have comparable accuracy in diagnosis of duodenopancreatic NET and their combination may allow an optimal preoperative diagnosis.

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.

Differentiated Thyroid Cancer: A New Perspective with Radiolabeled Somatostatin Analogues for Imaging and Treatment of Patients

BACKGROUND The expression of somatostatin receptors (SSTR) in thyroid cells may offer the possibility to identify metastatic lesions and to select patients for peptide receptor radionuclide therapy (PRRT). We investigated (68)Ga-DOTATOC positron emission tomography/computed tomography (PET/CT) to select patients with progressive differentiated thyroid cancer (DTC) for PRRT as well as treatment response and toxicity in treated patients. METHODS We enrolled 41 patients with progressive radioiodine-negative DTC (24 women and 17 men; mean age=54.3 years, median=59 years, range=19-78 years). In all patients, [(18)F]FDG-PET/CT was performed to determine recurrent disease with enhanced glucose metabolism, and (68)Ga-DOTATOC PET/CT was used to identify SSTR expression. Dosimetric evaluation was performed with (111)In-DOTATOC scintigraphy. Eleven patients were treated with PRRT receiving a fractionated injection of 1.5-3.7 GBq (90)Y-DOTATOC/administration. Serial (68)Ga-DOTATOC PET/CT scans were performed in all treated patients to evaluate treatment response. Parameters provided by (68)Ga-DOTATOC PET/CT were analyzed as potential therapeutic predictors to differentiate responding from nonresponding. In all treated patients, adverse events and toxicity were recorded. RESULTS (68)Ga-DOTATOC PET/CT were positive in 24/41 of radioiodine-negative DTC patients. Based on the high expression of SSTR detected by (68)Ga-DOTATOC PET/CT, 13 patients were suitable for PRRT. Two out of 13 patients were not treated due to the lack of fulfillment of other study inclusion criteria. PRRT induced disease control in 7/11 patients (two partial response and five stabilization) with a duration of response of 3.5-11.5 months. Objective response was associated with symptoms relief. Functional volume (FV) over time obtained by PET/CT was the only parameter demonstrating a significant difference between lesions responding and nonresponding to PRRT (p=0.001). Main PRRT adverse events were nausea, asthenia, and transient hematologic toxicity. One patient experienced permanent renal toxicity. CONCLUSIONS In our series, SSTR imaging provided positive results in more than half of the cases with radioiodine-negative DTC, and about one third of patients were eligible for PRRT. (68)Ga-DOTATOC PET/CT seems a reliable tool both for patient selection and evaluation of treatment response. In our experience, FV determination over time seems to represent a reliable parameter to determine tumor response to PRRT, although further investigations are needed to better define its role.

Radiolabeled Somatostatin Analogues Therapy in Advanced Neuroendocrine Tumors: A Single Centre Experience

The aim of this study was to assess the efficacy of PRRT in patients with advanced neuroendocrine tumors (NETs). Patients and Methods. From January 2007 to August 2011, we enrolled 65 patients (m/f 38/27; mean age 65 years, range 33–83) with advanced NETs having enhanced SSTR expression, treated with PRRT. The enhanced expression of SSTR was assessed using 68Ga-DOTATOC/DOTATATE PET/CT. Among all the enrolled patients, 6 of them were excluded from the present analysis since they voluntarily interrupted treatment. Mean activity/cycle of 2.6 GBq (90Y-DOTATOC/DOTATATE) or 6.0 GBq (177Lu-DOTATOC/DOTATATE) was administrated intravenously (max 9 cycles). Results. Complete response (CR) was found in 1/59 (2%) patients, partial remission (PR) in 24/59 (40.5%) patients, stable disease (SD) in 24/59 (40.5%), and progression (PD) in 10/59 (17%) patients. The overall tumor response rate (CR + PR) was 42.5%. In 40.5% of patients, the disease could be stabilized. Overall, 49 out of 59 patients had no tumor progression (83%). Twelve patients out of 59 (20%) had grade 2-3 hematological side effects including anemia, thrombocytopenia, and leukopenia. Long-term nephrotoxicity was observed in 3 patients (2 moderate, 1 severe). Conclusions. PRRT is a promising perspective for patients with advanced NETs.

Curcumin derivatives as metal-chelating agents with potential multifunctional activity for pharmaceutical applications.

Curcuminoids represent new perspectives for the development of novel therapeutics for Alzheimers disease (AD), one probable mechanism of action is related to their metal complexing ability. In this work we examined the metal complexing ability of substituted curcuminoids to propose new chelating molecules with biological properties comparable with curcumin but with improved stability as new potential AD therapeutic agents. The K2T derivatives originate from the insertion of a -CH2COOC(CH3)3 group on the central atom of the diketonic moiety of curcumin. They retain the diketo-ketoenol tautomerism which is solvent dependent. In aqueous solution the prevalent form is the diketo one but the addition of metal ion (Ga(3+), Cu(2+)) causes the dissociation of the enolic proton creating chelate complexes and shifting the tautomeric equilibrium towards the keto-enol form. The formation of metal complexes is followed by both NMR and UV-vis spectroscopy. The density functional theory (DFT) calculations on K2T21 complexes with Ga(3+) and Cu(2+) are performed and compared with those on curcumin complexes. [Ga(K2T21)2(H2O)2](+) was found more stable than curcumin one. Good agreement is detected between calculated and experimental (1)H and (13)C NMR data. The calculated OH bond dissociation energy (BDE) and the OH proton dissociation enthalpy (PDE), allowed to predict the radical scavenging ability of the metal ion complexed with K2T21, while the calculated electronic affinity (EA) and ionization potential (IP) represent yardsticks of antioxidant properties. Eventually theoretical calculations suggest that the proton-transfer-associated superoxide-scavenging activity is enhanced after binding metal ions, and that Ga(3+) complexes display possible superoxide dismutase (SOD)-like activity.

Metal binding ability of curcumin derivatives: a theoretical vs. experimental approach

Theoretical calculations employing DFT at the B3LYP/6-311G++** level are used to investigate the tautomeric equilibrium in curcumin derivatives. The solvent effect is evaluated using the CPCM continuum solvation method. The results are compared with experimental data obtained from the X-ray crystal structure of K2A23 and UV-vis data. The KE tautomer is more stable in a vacuum and in the solid state, while in water the DK tautomer reaches a population of 90%. In agreement with spectroscopic data, theoretical calculations predict a slight prevalence of the DK form in non-aqueous solvent systems. The ability to chelate metal ions [Fe(3+), Ga(3+) and Cu(2+)] is then explored by means of (1)H, (13)C NMR and UV-Vis spectroscopy. From the calculation of the overall stability constants of metal complexes and (1)H NMR titrations with Ga(3+), it is clear that the more stable species has a 1 : 2 M/L molar ratio. The curcuminoid coordinates the metal ion through the keto-enol function in the dissociated form; in addition 2D (1)H (13)C NMR experiments suggest the involvement of carboxylic oxygen in metal coordination it was found in the solid state for the complex [Ga(K2A33)2]PF6. The rate of the complexation reaction is strongly influenced by the type of substituent on the aromatic ring of the curcuminoid (K2A33 ≈ K2A23 ≫ K2A21). In addition DPPH assay evidences how antioxidant ability of curcumin derivatives is mainly due to the presence of a phenolic group and metal coordination by a keto-enolic moiety does not affect it, especially for K2A21.

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.

Brief Report on the Use of Radiolabeled Somatostatin Analogs for the Diagnosis and Treatment of Metastatic Small-Cell Lung Cancer Patients

Introduction: The demonstration of type 2 somatostatin receptors (SSTRs) in small-cell lung cancer (SCLC) represents the rationale for the use of positron emission tomography/computed tomography (PET/CT) to determine SSTR expression, and select patients suitable for peptide radioreceptor radionuclide therapy (PRRT) in extensive-disease stage (ED) SCLC. Methods: We evaluated 24 ED-SCLC patients with radiolabeled SST-analog PET/CT. Lesions at PET/CT scan were semiquantitatively scored (from 0 to 3+) and compared with contrast-enhanced CT findings. Patients scored as 3+ were admitted to PRRT after dosimetric evaluation. Average injected activity/cycle was 2.6 GBq (90yttrium-PRRT) or 6.0 GBq (177lutetium-PRRT). PRRT efficacy was clinically and radiologically assessed. Results: PET/CT was negative in four of 24 patients, whereas in the remaining 20 cases uptake was scored as 1+ in seven of 20, 2+ in one of 20, and 3+ in 12 of 20. Primary tumor lesions showed uptake in 16 of 24 patients. Uptake in metastatic lesions was observed in four of four adrenals, two of five brain, 12 of 16 bone, three of eight liver, and 17 of 20 lymph node lesions. Of the 12 patients eligible for PRRT, 11 were eventually treated and four of 11 patients received multiple PRRT administrations. Dosimetry resulted in a BED for kidney of 7.5 Gy (range, 4–21); bone marrow provisional dosage was 0.43 Gy (range, 0.1–1.7). Hematological PRRT toxicity occurred in three of 11 patients. No clinical or objective responses were observed with disease progression occurring approximately 48 days (range, 9–32) after PRRT. Conclusion: Radiolabeled SST-analog PET/CT demonstrated enhanced SSTR expression in 50% of cases. Nevertheless, PRRT in ED-SCLC was ineffective, suggesting the need to anticipate or combine PRRT in a multimodality approach.