Lorenzo Lodola

Targeted radionuclide therapy (TRT) is gaining wide and rapid acceptance in clinical practice as it can deliver alpha or beta irradiation to a tumor-associated target which may be present in the tumor cell itself or in the microenvironment

Several studies investigated the use of ^99mTc-labelled Aprotinin as an amyloid seeker some years ago. In vitro tests showed high binding affinity for several types of amyloid fibrils accompanied by an excellent specificity. Initial human studies demonstrated good accuracy in detecting cardiac involvement. Scintigraphy results were confirmed in a group of 28 endomyocardial biopsies. Unfortunately, clinical studies were halted because of a temporary suspension of the vector protein (Trasylol) and public health concerns over prion contamination of the bovine origin compound. To obviate these limitations, efforts have been made to label a recombinant Aprotinin with 99mTc, which exhibits the same affinity for h-insulin fibrils. With the aim of developing a PET tracer, the same recombinant protein was labeled with Gallium. The introduction of a bifunctional chelator did not affect fibril affinity. Finally, a synthetic peptidic fragment, the cyclic 30-51 SS, was synthetized. After direct technetium labeling, an impressive increase in affinity was demonstrated. This peptide appears to be a potential candidate for Gallium labeling through a bifunctional chelator for PET imaging.

Background: Macroaggregated human serum albumin (MAA) properties are widely used in nuclear medicine, labelled with ^99mTc. The aim of this study is to improve the knowledge about the morphology, size, dimension and physical–chemical characteristics of MAA and their bond with ^99mTc and ⁶⁸Ga. Methods: Commercial kits of MAA (Pulmocis^®) were used. Characterisation through experiments based on SEM, DLS and Stokes’ Law were carried out. In vitro experiments for Langmuir isotherms and pH studies on radiolabelling were performed and the stability of the radiometal complex was verified through competition reactions. Results: The study settles the MAA dimension within the range 43–51 μm. The Langmuir isotherm reveals for [^99mTc]MAA: Bmax (46.32), h (2.36); for [⁶⁸Ga]MAA: Bmax (44.54), h (0.893). Dual labelling reveals that MAA does not discriminate different radioisotopes. Experiments on pH placed the optimal pH for labelling with ^99mTc at 6. Conclusion: Radiolabelling of MAA is possible with high efficiency. The nondiscriminatory MAA bonds make this drug suitable for radiolabelling with different radioisotopes or for dual labelling. This finding illustrates the need to continue investigating MAA chemical and physical characteristics to allow for secure labelling with different isotopes.

Nanoparticles of Human Serum Albumin (NC) labelled with ^99mTc are widely used in Nuclear Medicine and represent the gold-standard for the intraoperative detection of the sentinel lymph node in many kinds of cancer, mainly breast cancer and melanoma. A significant amount of radionuclides can be incorporated into the HSA particle, due to the multiple binding sites, and HSA-based nanocolloid catabolism is a fast and easy process that results in innocuous degradation products. NCs labelled with different isotopes represent an interesting radiopharmaceutical for extending diagnostic accuracy and surgical outcome, but the knowledge of the chemical bond between NCs and isotopes has not been fully elucidated, including information on its strength and specificity. The aim of this study is to investigate and compare the physicochemical characteristics of the bond between NCs and ^99mTc and ⁶⁸Ga isotopes. Commercial kits of HSA-based nanocolloid particles (NanoAlbumon^®) were used. For this purpose, we have primarily studied the kinetic orders of NC radiolabelling. Langmuir isotherms and pH effect on radiolabelling were tested and the stability of the radiometal complex was verified through competition reactions carried out in presence of different ligands. The future goal of our research is the development of inexpensive and instant kits, easily labelled with a wide spectrum of diagnostic and therapeutic isotopes, thus facilitating the availability of versatile and multipurpose radiopharmaceuticals.

Recent developments in sentinel lymph node (SLN) and radio occult lesion localization (ROLL) highlight the need for a multimodal contrast agent, providing better presurgical PET imaging and improved intraoperative mapping thanks to fluorescence detection. For this reason, we have studied a trimodal SLN/ROLL targeting agent (^99mTc-⁶⁸Ga-ICG) with commercially available kits of macroaggregated or nanocolloidal albumin (MA/NC-HSA). ⁶⁸Ga PET imaging does provide better spatial resolution and makes it possible to predict signal intensity during surgery. The presence of ^99mTc assesses the efficacy of these compounds in vitro and also during the surgery procedure. The aim of this study was to optimise the labelling and tagging of these two radiopharmaceuticals and assess their yields and stability. Kits of MA/NC-HSA particles (Pulmocis® and NanoAlbumon®) were used for sequential radiolabelling with ^99mTc and ⁶⁸Ga. Fluorescent tagging was performed using indocyanine green, a tricarbocyanine dye. The ITLC radiochemical purity of the trilabelled MA/NC-HSA was >95%. Fluorescent purity was measured by scanning the strips with a PhotoDynamicEye probe. Finally, in vitro stability tests, performed with DTPA and human serum solutions, assessed the efficacy of fluorescent tagging and radiolabelling.

Background: Neurotensin receptors are overexpressed in several cancer types including pancreatic ductal adenocarcinoma. Three NTR subtypes have been cloned: NTR-1, NTR-2 and NTR-3. The most expressed NTR-1 is not present in normal pancreatic tissue and has a low expression in chronic pancreatitis. Objective: Objective of this study was to test in vitro affinity of the new 68Ga labelled neurotensin analogue DOTA-NT-20.3 (fragment 6-13, Ac-Lys(DOTA)-Pro-Arg(N-CH3)-Arg-Pro-Tyr-Tle-Leu) on the human pancreatic ductal adenocarcinoma cell line AsPC-1. Method: For the preparation of 68Ga-DOTA-NT-20.3, 68GaCl3 solution (eluted from 68Ge/68Ga generator) and 50 μg of precursor (Iason, Graz, Austria) water dissolved were used in an automatic synthesis module. The labeled compound was added to cell culture flask and incubated at 37°C. At various time points after tracer addition up to 80min, cells were recovered, rinsed and counted for radioactivity. Results were expressed as percent binding normalized to 200000 cells and affinity parameters were calculated. Results: Labeling yield was ≥98 %. The molar ratio between labelled and total peptide was about 1/400. AsPC-1 cell line showed rapid uptake of the tracer including surface and internalized binding, tending to a plateau phase 80 min after tracer addition (11%/200.000 cells). The Kd (7.335 pmol) and Bmax (90.52 kBq) value indicated high tracer affinity for AsPC-1cell line especially if compared with the literature data regarding other malignancies (e.g. colonic cancer cell line). Binding sites were 1.09x106 sites per cell. Conclusion: New tracer 68Ga-DOTA-NT-20.3 can be a suitable candidate for the clinical use in patients with pancreatic ductal adenocarcinoma.

Background: Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas often recur. Differentiation between post-treatment changes and recurrence is difficult. ¹⁸F-methyl-choline (¹⁸F-FCH) is frequently used in staging and detection of recurrent prostate cancer disease as well as some brain tumours; however accumulation in inflammatory tissue limits its specificity. The ¹⁸F-ethyl-tyrosine (¹⁸F-FET) shows a specific uptake in malignant cells, resulting from increased expression of amino acid transporters or diffusing through the disrupted blood-brain barrier. ¹⁸F-FET exhibits lower uptake in machrophages and other inflammatory cells. Aim of this study was to evaluate ¹⁸F-FCH and ¹⁸F-FET uptake by human glioblastoma T98G cells. Material and methods: Human glioblastoma T98G or human dermal fibroblasts cells, seeded at a density to obtain 2 × 10⁵ cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO₂ in complete medium. Equimolar amounts of radiopharmaceuticals were added to cells for different incubation times (20 to 120 minutes) for ¹⁸F-FCH and ¹⁸F-FET respectively. The cellular radiotracer uptake was determined with a gamma counter. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 × 10⁵ cells. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p18F-FCH was seen in T98G cells at 60, 90 and 120 minutes. The percentage uptake of ¹⁸F-FET in comparison to ¹⁸F-FCH was lower by a factor of more than 3, with different kinetic curves.¹⁸F-FET showed a more rapid initial uptake up to 40 minutes and ¹⁸F-FCH showed a progressive rise reaching a maximum after 90 minutes. Conclusions: ¹⁸F-FCH and ¹⁸F-FET are candidates for neuro-oncological PET imaging. ¹⁸F-FET could be the most useful oncological PET marker in the presence of reparative changes after therapy, where the higher affinity of ¹⁸F-FCH to inflammatory cells makes it more difficult to discriminate between tumour persistence and non-neoplastic changes. Additional studies on the influence of inflammatory tissue and radionecrotic cellular components on radiopharmaceutical uptake are necessary.

This paper is an report of the investigation of the in vitro binding of ^99mTc-DPD for synthetic amyloid fibrils used for the diagnosis of cardiac amyloidosis (CA), as compared with the use of ^99mTc-HMDP and 99mTc-PPI. It also includes an inquiry into the role played by Ca²⁺ ions and serum proteins on binding to amyloid like materials, as well as the saturability and specificity of DPD for fibrils versus amorphous precipitates (AP). In the work, synthetic insulin fibrils (SIF) and AP were characterized by Congo red staining and TEM imaging. An equal amount of three radiopharmaceuticals were then added to fibrils in Ca²⁺ (0-4.2 mmol/L) or human serum (HS) adjoined samples and radiopharmaceutical uptake was assessed. To test the saturability of amyloid binding sites, a displacement assays with cold DPD was performed, while adding 50-1500 nmol of ^99mTc-DPD to SIF or AP, saturation binding tests were subsequently carried out for evaluating its specificity for amyloid. Herein, synthetic fibrils and AP showed conformational differences at TEM and polarized microscopy analysis. In our study, ^99mTc-DPD fibrils uptake was seen to be the highest and increased with calcium ions concentration. What is more, serum proteins reduced the bound fraction to the amyloid deposits of about 15%, and the Kd values of 90 nM and 114 nM relative to SIF and AP, respectively, did not significantly differ. We saw that ^99mTc-DPD is the best seeker for amyloid fibrils in cardiac amyloidosis, and that Ca²⁺ concentration positively influenced DPD fibrils binding. Furthermore, the radioactivity bound to the serum protein clear up the idea of nuclide exchanging dynamic balance between amyloid and circulating proteins. Moreover, non-labeled DPD did not exert a competition for ^99mTc-DPD binding sites, and, finally, DPD cannot be defined a radiopharmaceutical specific for amyloid deposits.

Tumor and chemo/radiotherapy-damaged brain tissues are hardly distinguishable by conventional morphological imaging. (18)F-FCH was compared against (18)F-FDG in the T98G glioblastoma cell line with regard to their radiopharmaceutical uptake, in order to test its diagnostic power on brain tumor lesions. Equimolar amounts of (18)F-FCH and (18)F-FDG were added to human glioblastoma T98G cells and human dermal fibroblasts for 20, 40, 60, 90 and 120 min of incubation. Radiopharmaceutical uptake was expressed as a percentage of the administered dose. Cold choline was used for binding competition experiments. In T98G cells (18)F-FCH was taken-up in higher amounts than 18F-FDG after 60 min. In fibroblasts, uptake was lower than 1% for both radiopharmaceuticals. Cold choline reduced the uptake of FCH to 1% similarly to fibroblasts. Our results prove the efficacy of (18)F-FCH as a promising tracer, better than (18)F-FDG in establishing the tumor-to-background ratio in brain tumors.

Several parameters affect the biodistribution of administered nanocolloids (NC) for Sentinel Lymph Node (SLN) detection: particle size distribution, number of Tc atoms per particle and specific activity (SA). Relatively few data are available with frequently conflicting results. ^99mTc‐NC‐human serum albumin (HSA) Nanocoll®, Nanoalbumon® and Nanotop® were analysed for particles' dimensional and radioactivity distribution, and a mathematical model was elaborated to estimate the number of particles involved. Commercially available kits were reconstituted at maximal SA of 11 MBq/µg HSA. Particles size distribution was evaluated by Dynamic Light Scattering. These data were related to the radioactivity distribution analysis passing labelled NC through three polycarbonate filters (15‐30‐50‐nm pore size) under vacuum. Highest radioactivity was carried by 30–50 nm particles. The smallest ones, even though most numerous, carried only the 10% of ^99mTc atoms. Nanocoll and Nanotop are not significantly different, while Nanoalbumon is characterized by largest particles (>30 nm) that carried the most of radioactivity (80%). Smallest particles could saturate the clearing capacity of macrophages; therefore, if the tracer is used for SLN detection, more node tiers could be visualized, reducing accuracy of SLN mapping. Manufacturers could implement technical leaflets with particle size distribution and could improve the labelling protocol to provide clinicians useful information.