Although increasing used in the preclinical testing of new anti-fibrotic drugs, a thorough validation of micro-computed tomography (CT) in pulmonary fibrosis models has not been performed. Moreover, ...no attempts have been made so far to define density thresholds to discriminate between aeration levels in lung parenchyma. In the present study, a histogram-based analysis was performed in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis by micro-CT, evaluating longitudinal density changes from 7 to 21 days after BLM challenge, a period representing the progression of fibrosis. Two discriminative densitometric indices (i.e. 40th and 70th percentiles) were extracted from Hounsfield Unit density distributions and selected for lung fibrosis staging. The strong correlation with histological findings (r
= 0.76, p < 0.01) confirmed that variations in 70th percentile could reflect a pathological lung condition and estimate the effect of antifibrotic treatments. This index was therefore used to define lung aeration levels in mice distinguishing in hyper-inflated, normo-, hypo- and non-aerated pulmonary compartments. A retrospective analysis performed on a large cohort of mice confirmed the correlation between the proposed preclinical density thresholds and the histological outcomes (r
= 0.6, p < 0.01), strengthening their suitability for tracking disease progression and evaluating antifibrotic drug candidates.
Background: Nowadays, in Nuclear Medicine, clinically applied radiopharmaceuticals must meet quality release criteria such as high radiochemical purity and radiochemical yield. Many ...radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within European Pharmacopeia (Ph. Eur.); therefore, general monographs on quality controls (QCs) have to be applied for clinical application. These criteria require standardization and validation in labeling and preparation, including quality controls measurements, according to well defined standard operation procedures. However, QC measurements are often based on detection techniques that are specific to a certain chromatographic system. Several radiosyntheses of 68GaGa-radiopharmaceuticals are more efficient and robust when they are performed with 2-4-(2-hydroxyethyl)piperazin-1-yl ethanesulfonic acid (HEPES) buffer, which is considered as an impurity to be assessed in the QC procedure, prior to clinical use. Thus, Ph. Eur. has introduced a thin-layer chromatography (TLC) method to quantify the HEPES amount that is present in 68GaGa-radiopharmaceuticals. However, this is only qualitative and has proven to be unreliable. Here we develop and validate a new high-performance liquid chromatography (UV-Radio-HPLC) method to quantify the residual amount of HEPES in 68Ga-based radiopharmaceuticals. Method: To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document that was adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. A range of concentrations of HEPES (100, 80, 60, 40, 20, 10, 5, 3 μg/mL) were analyzed. Moreover, to test the validity and pertinence of our new HPLC method, we analyzed samples of 68GaGa-DOTATOC; 68GaGa-PSMA; 68GaGa-DOTATATE; 68GaGa-Pentixafor; and 68GaGa-NODAGA-Exendin-4 from different batches that were prepared for clinical use. Results: In the assessed samples, HEPES could not be detected by the TLC method that was described in Ph. Eur. within 4 min incubation in an iodine-saturated chamber. Our developed HPLC method showed excellent linearity between 3 and 100 μg/mL for HEPES, with a correlation coefficient (R2) for calibration curves that was equal to 0.999, coefficients of variation (CV%) < 2%, and percent deviation value of bias from 100% to 5%, in accordance with acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed and the limit-of-quantification (LOQ) was 3 μg/mL, confirming the high sensitivity of the method. The amount of HEPES that was detected with our developed HPLC method in the tested 68GaGa-radiopharmaceuticals resulted well below the Ph. Eur. limit, especially for 68GaGa-NODAGA-Exendin-4. Conclusions: The TLC method that is described in Ph. Eur. to assess residual HEPES in 68Ga-based radiopharmaceuticals may not be sufficiently sensitive and thus unsuitable for QC release. Our new HPLC method was sensitive, quantitative, reproducible, and rapid for QCs, allowing us to exactly determine the residual HEPES amount in 68GaGa-radiopharmaceuticals for safe patient administration.
The association between amyloid deposition and cognitive, behavioral and physical performance in mild cognitive impairment (MCI) due to Alzheimer's disease (AD) has been poorly investigated, ...especially in older persons.
We studied the in vivo correlation between the amyloid deposition at Positron Emission Tomography (amyloid-PET) and the presence of memory loss, reduced executive function, neuropsychiatric symptoms and physical performance in older persons with MCI. Amyloid-PET was performed with 18F-flutemetamol and quantitatively analyzed.
We evaluated 48 subjects, 21 men and 27 women. We performed in each patient a comprehensive geriatric assessment (CGA) including Mini Mental State Examination (MMSE), Clock Drawing Test (CDT), Activity Daily Living (ADL), Instrumental Activity of Daily Living (IADL), Neuropsychiatric inventory (NPI) questionnaire, 15 Geriatric Depression Scale (GDS), Short Physical Performance Battery (SPPB) and Hand Grip strength. Then, each patient underwent amyloid-PET. Mean age of the enrolled subjects was 74.6 ± 7.8 years. All of these subjects showed preserved cognitive function at MMSE > 24, while 29 of 48 subjects (61.0%) had altered CDT. Mean NPI score was 6.9 ± 5.9. The mean value of SPPB score was 9.0 ± 2.6, while the average muscle strength of the upper extremities measured by hand grip was 25.6 ± 7.7 Kg. CT/MRI images showed cortical atrophic changes in 26 of the 48 examined subjects (54.0%), while cerebrovascular modifications were present in 31 subjects (64.5%). Pathological burden of amyloid deposits was detected in 25 of 48 (52.0%) patients with a mean value of global z-score of 2.8 (subjects defined as MCI due to AD). After stratifying subjects in subclasses of clinical alterations, more probability of pathological amyloid deposition was found in subjects with impaired CDT and higher NPI score (O.R. = 3.45 1.01-11.2, p = 0.04), with both impaired CDT and low physical performance (O.R. = 5.80 1.04-32.2, p = 0.04), with altered CDT and high NPI score (O.R. = 7.98 1.38-46.0, p = 0.02), and finally in those subjects with altered CDT, high NPI and low physical performance (O.R. = 5.80 1.05-32.2, p = 0.04).
Our findings support the recent hypothesis that amyloid deposition could be associated with multiple cerebral dysfunction, mainly affecting executive, behavioral and motor abilities.
Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in β-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several ...GLP-1R-avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic β-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of
GaGa-NODAGA-exendin-4, to be used in the clinical routine.
Ga-68 was obtained from a
Ge/
Ga Generator (GalliaPharma
) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP
). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 μg/mL) and
GaGa-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of
GaGa-NODAGA-exendin-4 were tested.
Excellent linearity was found between 5-0.75 μg/mL for both the analytes (NODAGA-exendin-4 and
Ga-NODAGA-exendin-4), with a correlation coefficient (R
) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of
GaGa-NODAGA-exendin-4 was linear with a R
of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 μg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of
GaGa-NODAGA-exendin-4. The radiochemical purity of
GaGa-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches.
The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of
GaGa-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.
Background
Recently it has been identified a short peptide that showed allosteric antagonism against C–C motif chemokine receptor 2 (CCR2) expressed on inflammatory monocyte and macrophages. A 7-
d
...-amino acid peptidic CCR2 inhibitor called extracellular loop 1 inverso (ECL1i), d(LGTFLKC) has been identified and labeled to obtain a new probe for positron emission tomography in pulmonary fibrosis, heart injury, abdominal aortic aneurysm inflammation, atherosclerosis, head and neck cancer. Our goal was to develop, optimize and validate an automated synthesis method for 68Ga68Ga-DOTA-ECL1i to make it available for a broader community. The synthesis of 68Ga68Ga-DOTA-ECL1i was done using the Scintomics GRP
®
module with the already estabilished synthesis template for 68Ga68Ga-DOTATOC/68Ga68Ga-PSMA. The radiopharmaceutical production was optimized scaling down the amount of DOTA-ECL1i (from 50 to 10 μg), evaluating synthesis efficiency and relevant quality control parameters in accordance with the European Pharmacopeia.
Results
Best results were yielded with 20 μg DOTA-ECL1i and then the process validation was carried out by producing three different batches on three different days obtaining an optimal radiochemical yield (66.69%) as well as radiochemical purity (100%) and molar activity (45.41 GBq/µmol).
Conclusions
68Ga68Ga-DOTA-ECL1i was successfully synthesized and it is, thus, available for multi-dose application in clinical settings.
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive degenerative lung disease leading to respiratory failure and death. Although anti-fibrotic drugs are now available for treating IPF, their ...clinical efficacy is limited and lung transplantation remains the only modality to prolong survival of IPF patients. Despite its limitations, the bleomycin (BLM) animal model remains the best characterized experimental tool for studying disease pathogenesis and assessing efficacy of novel potential drugs. In the present study, the effects of oropharyngeal (OA) and intratracheal (IT) administration of BLM were compared in C57BL/6 mice. The development of lung fibrosis was followed
in vivo
for 28 days after BLM administration by micro-computed tomography and
ex vivo
by histological analyses (bronchoalveolar lavage, histology in the left lung to stage fibrosis severity and hydroxyproline determination in the right lung). In a separate study, the antifibrotic effect of Nintedanib was investigated after oral administration (60 mg/kg for two weeks) in the OA BLM model. Lung fibrosis severity and duration after BLM OA and IT administration was comparable. However, a more homogeneous distribution of fibrotic lesions among lung lobes was apparent after OA administration. Quantification of fibrosis by micro-CT based on % of poorly aerated tissue revealed that this readout correlated significantly with the standard histological methods in the OA model. These findings were further confirmed in a second study in the OA model, evaluating Nintedanib anti-fibrotic effects. Indeed, compared to the BLM group, Nintedanib inhibited significantly the increase in % of poorly aerated areas (26%) and reduced
ex vivo
histological lesions and hydroxyproline levels by 49 and 41%, respectively. This study indicated that micro-computed tomography is a valuable
in vivo
technology for lung fibrosis quantification, which will be very helpful in the future to better evaluate new anti-fibrotic drug candidates.
Micro-computed tomography (CT) imaging provides densitometric and functional assessment of lung diseases in animal models, playing a key role either in understanding disease progression or in drug ...discovery studies. The generation of reliable and reproducible experimental data is strictly dependent on a system's stability. Quality controls (QC) are essential to monitor micro-CT performance but, although QC procedures are standardized and routinely employed in clinical practice, detailed guidelines for preclinical imaging are lacking. In this work, we propose a routine QC protocol for in vivo micro-CT, based on three commercial phantoms. To investigate the impact of a detected scanner drift on image post-processing, a retrospective analysis using twenty-two healthy mice was performed and lung density histograms used to compare the area under curve (AUC), the skewness and the kurtosis before and after the drift. As expected, statistically significant differences were found for all the selected parameters AUC 532 ± 31 vs. 420 ± 38 (p < 0.001); skewness 2.3 ± 0.1 vs. 2.5 ± 0.1 (p < 0.001) and kurtosis 4.2 ± 0.3 vs. 5.1 ± 0.5 (p < 0.001), confirming the importance of the designed QC procedure to obtain a reliable longitudinal quantification of disease progression and drug efficacy evaluation.
Recently a new iterative reconstruction algorithm named Sinogram Affirmed Iterative Reconstruction (SAFIRE) has been released by Siemens. This algorithm works in the raw data domain with noise ...reduction as main purpose, providing five different strengths. In this study, the effect of SAFIRE on image quality has been investigated using selected phantoms and a comparison with standard filtered back projection (FBP) has been carried out. The following quantitative parameters have been evaluated: image noise, impact of different reconstruction kernels on noise reduction, noise power spectrum (NPS), contrast‐to‐noise ratio (CNR), spatial resolution, and linearity and accuracy of CT numbers. The influence of strengths on image quality parameters has also been examined. Results show that image noise reduction is independent of reconstruction kernel and strongly related to the strength of SAFIRE applied. The peak of NPS curve for SAFIRE reconstructions is shifted towards low frequencies; this effect is more marked at higher levels of strength. Contrast‐to‐noise ratio is always improved in SAFIRE reconstruction and increases with higher strength. At different dose levels SAFIRE preserves CT number accuracy, linearity, and spatial resolution, both in transversal and coronal planes. These results confirm that SAFIRE allows for image noise reduction with preserved image quality. First clinical data to validate this phantom analysis and confirm that commercially available iterative algorithms can play an effective role in dose containment.
PACS number: 87.57.Q
We report on a 66-year-old man who presented with a right axillary lymphadenopathy approximately 10 days after receiving the third dose of the BNT162b2 vaccine. The lymphadenopathy gradually ...enlarged, and physical examination and ultrasound (US) revealed one right axillary 6.99 cm and one right supraclavicular 2.36 cm lymphadenopathy. Histologic examination of the right axillary nodule revealed anaplastic large-cell lymphoma that was ALK negative and CD30 positive. A total body computerized tomography (CT) scan, positron emission tomography (PET) and bone-marrow biopsy showed a stage-II non-Hodgkin lymphoma (NHL). The patient was treated with chemotherapy and a scheme of Brentuximab Vedotin, Cyclophosphamide, Doxorubicin and Prednisone (BV-CHP) for six cycles and is now well and in complete remission. The revision of the literature revealed eight additional cases of NHL developed shortly after COVID-vaccination. There were four cases of diffuse large-B-cell lymphoma (DLBCL) (one in a patient who was a heart transplant recipient and developed an Epstein-Bar-virus-positive DLBCL), one case of extranodal NK/T-cell lymphoma, one patient with subcutaneous panniculitis-like T-cell lymphoma, one case of marginal zone B-cell lymphoma and one primary cutaneous anaplastic large-cell lymphoma (PC-ALCL). In five cases, the lymphoma developed after BNT162b2 mRNA vaccination, including one case after ChAdOx1 nCOV-19, one case after the adenovirus type 26 (Ad26) vaccine and one after mRNA-1273/Spikevax (ModernaTX). We are aware that the link between COVID-19 vaccination and lymphoma most likely is a chance phenomenon, and that COVID-19 vaccines represent very efficient products for many people around the world. However, we believe that clinical events, even if only temporally associated with novel treatments or novel vaccines, should be reported for the benefit of the patients and the scientific community.
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