Automated radiosynthesis and preclinical evaluation of two new PSMA-617 derivatives radiolabelled via [18F]AlF2+ method.

BACKGROUND: In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation. RESULTS: The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands. CONCLUSION: PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

Evaluating [18F]FDG and [18F]FLT Radiotracers as Biomarkers of Response for Combined Therapy Outcome in Triple-Negative and Estrogen-Receptor-Positive Breast Cancer Models.

Breast cancer (BC) is the most frequent cancer and the second leading cause of death in women. A typical feature of BC cells is the metabolic shift toward increased glycolysis, which has become an interesting therapeutic target for metabolic drugs such as metformin (MET). Recently, the administration of the antihypertensive syrosingopine (SYRO) in combination with MET has shown a synergistic effect toward a variety of cancers. However, a fundamental need remains, which is the development of in vivo biomarkers that are able to detect early clinical response. In this study, we exploited a triple-negative murine BC cell line (4T1) and a metastatic ER+ murine BC cell line (TS/A) in order to investigate, in vivo, the early response to treatment, based on MET and/or SYRO administration, evaluating [18F]FDG and [18F]FLT as potential biomarkers via PET/CT. The study provides evidence that SYRO plus MET has a synergistic effect on tumor growth inhibition in both 4T1 and TS/A experimental models and has showed the highest efficacy on the TNBC xenograft mice (4T1) via the expression reduction in the lactate transporter MCT4 and in the epithelial-mesenchymal transition biomarker Snail, promoting its potential application in therapy settings. In addition, the selective reduction in the [18F]FLT tumor uptake (at 7 dd), observed in the SYRO plus MET treated mice in comparison with the vehicle group, suggests that this radiotracer could be potentially used as a biomarker for the early detection of therapy response, in both evaluated xenografts models.

A stapled chromogranin A-derived peptide homes in on tumors that express αvß6 or αvß8 integrins.

Rationale: The αvß6- and αvß8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFß complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called "5a"), which selectively recognizes the LAP/TGFß complex-binding site of αvß6 and αvß8. Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvß6/αvß8 integrins and various αvß6/αvß8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvß8-positive prostate tumors. Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFß activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvß6/αvß8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvß6/αvß8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvß8-positive prostate tumors. Conclusions: The results indicate that 5a can home to αvß6- and/or αvß8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvß6/αvß8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFß activators.

Imaging Metformin Efficacy as Add-On Therapy in Cells and Mouse Models of Human EGFR Glioblastoma.

Glioblastoma (GBM) is a highly aggressive tumor of the brain. Despite the efforts, response to current therapies is poor and 2-years survival rate ranging from 6-12%. Here, we evaluated the preclinical efficacy of Metformin (MET) as add-on therapy to Temozolomide (TMZ) and the ability of [18F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [18F]VC701 (translocator protein, TSPO) Positron Emission Tomography (PET) radiotracers to predict tumor response to therapy. Indeed, TSPO is expressed on the outer mitochondrial membrane of activated microglia/macrophages, tumor cells, astrocytes and endothelial cells. TMZ-sensitive (Gli36ΔEGFR-1 and L0627) or -resistant (Gli36ΔEGFR-2) GBM cell lines representative of classical molecular subtype were tested in vitro and in vivo in orthotopic mouse models. Our results indicate that in vitro, MET increased the efficacy of TMZ on TMZ-sensitive and on TMZ-resistant cells by deregulating the balance between pro-survival (bcl2) and pro-apoptotic (bax/bad) Bcl-family members and promoting early apoptosis in both Gli36ΔEGFR-1 and Gli36ΔEGFR-2 cells. In vivo, MET add-on significantly extended the median survival of tumor-bearing mice compared to TMZ-treated ones and reduced the rate of recurrence in the TMZ-sensitive models. PET studies with the cell proliferation radiopharmaceutical [18F]FLT performed at early time during treatment were able to distinguish responder from non-responder to TMZ but not to predict the duration of the effect. On the contrary, [18F]VC701 uptake was reduced only in mice treated with MET plus TMZ and levels of uptake negatively correlated with animals' survival. Overall, our data showed that MET addition improved TMZ efficacy in GBM preclinical models representative of classical molecular subtype increasing survival time and reducing tumor relapsing rate. Finally, results from PET imaging suggest that the reduction of cell proliferation represents a common mechanism of TMZ and combined treatment, whereas only the last was able to reduce TSPO. This reduction was associated with the duration of treatment response. TSPO-ligand may be used as a complementary molecular imaging marker to predict tumor microenvironment related treatment effects.

99mTc-Radiolabeled Silica Nanocarriers for Targeted Detection and Treatment of HER2-Positive Breast Cancer.

INTRODUCTION: The overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is usually associated with aggressive and infiltrating breast cancer (BC) phenotype, and metastases. Functionalized silica-based nanocarriers (SiNPs) can be labeled for in vivo imaging applications and loaded with chemotherapy drugs, making possible the simultaneous noninvasive diagnosis and treatment (theranostic) for HER2-positive BC. METHODS: Firstly, FITC-filled SiNPs, were engineered with two different amounts of Hc-TZ (trastuzumab half-chain) per single nanoparticle (1:2 and 1:8, SiNPs to Hc-TZ ratio), which was 99mTc-radiolabeled at histidine residues for ex vivo and in vivo biodistribution evaluations. Secondly, nanoparticles were loaded with DOX and their in vitro and ex vivo/in vivo delivery was assessed, in comparison with liposomal Doxorubicin (Caelyx). Finally, the treatment efficacy of DOX-SiNPs-TZ (1:8 Hc-TZ) was evaluated in vivo by PET and supported by MS-based proteomics profiling of tumors. RESULTS: SiNPs-TZ (1:8 Hc-TZ) tumor uptake was significantly greater than that of SiNPs-TZ (1:2 Hc-TZ) at 6 hours post-injection (p.i.) in ex vivo biodistribution experiment. At 24 h p.i., radioactivity values remained steady. Fluorescence microscopy, confirmed the presence of radiolabeled SiNPs-TZ (1:8 Hc-TZ) within tumor even at later times. SiNPs-TZ (1:8 Hc-TZ) nanoparticles loaded with Doxorubicin (DOX-SiNPs-TZ) showed a similar DOX delivery capability than Caelyx (at 6 h p.i.), in in vitro and ex vivo assays. Nevertheless, at the end of treatment, tumor volume was significantly reduced by DOX-SiNPs-TZ (1:8 Hc-TZ), compared to Caelyx and DOX-SiNPs treatment. Proteomics study identified 88 high stringent differentially expressed proteins comparing the three treatment groups with controls. CONCLUSION: These findings demonstrated a promising detection specificity and treatment efficacy for our system (SiNPs-TZ, 1:8 Hc-TZ), encouraging its potential use as a new theranostic agent for HER2-positive BC lesions. In addition, proteomic profile confirmed that a set of proteins, related to tumor aggressiveness, were positively affected by targeted nanoparticles.

Molecular and Cellular Complexity of Glioma. Focus on Tumour Microenvironment and the Use of Molecular and Imaging Biomarkers to Overcome Treatment Resistance.

This review highlights the importance and the complexity of tumour biology and microenvironment in the progression and therapy resistance of glioma. Specific gene mutations, the possible functions of several non-coding microRNAs and the intra-tumour and inter-tumour heterogeneity of cell types contribute to limit the efficacy of the actual therapeutic options. In this scenario, identification of molecular biomarkers of response and the use of multimodal in vivo imaging and in particular the Positron Emission Tomography (PET) based molecular approach, can help identifying glioma features and the modifications occurring during therapy at a regional level. Indeed, a better understanding of tumor heterogeneity and the development of diagnostic procedures can favor the identification of a cluster of patients for personalized medicine in order to improve the survival and their quality of life.

Radiosynthesis and Preclinical Evaluation of 11C-VA426, a Cyclooxygenase-2 Selective Ligand.

Cyclooxygenase-2 (COX-2) is involved in the inflammatory response, and its recurrent overexpression in cancers as well as in neurodegenerative disorders has made it an important target for therapy. For this reason, noninvasive imaging of COX-2 expression may represent an important diagnostic tool. In this work, a COX-2 inhibitor analogue, VA426 [1-(4-fluorophenyl)-3-(2-methoxyethyl)-2-methyl-5-(4-(methylsulfonil)phenyl)-1H-pyrrole], was synthesized and radiolabelled with the 11C radioisotope. The ex vivo biodistribution profile of 11C-VA426 was evaluated in the brain and periphery of healthy rats and mice and in brain and periphery of inflammation models, based on the administration of LPS. 11C-VA426 synthesis with the tBuOK base showed optimal radiochemical yield (15 ± 2%) based on triflate activity, molar activity (range 37-148 GBq/µmol), and radiochemical purity (>95%). Ex vivo biodistribution studies showed a fast uptake of radioactivity but a rapid washout, except in regions expressing COX-2 (lungs, liver, and kidney) both in rats and in mice, with maximum values at 30 and 10 minutes p.i., respectively. LPS administration did not show significant effect on radioactivity accumulation. Celecoxib competition experiments performed in rats and mice treated with LPS produced a general target unrelated reduction of radioactivity concentration in all peripheral tissues and brain areas examined. Finally, in agreement with the negative results obtained from biodistribution experiments, radiometabolites analysis revealed that 11C-VA426 is highly unstable in vivo. This study indicates that the compound 11C-VA426 is not currently suitable to be used as radiopharmaceutical for PET imaging. This family of compounds needs further implementation in order to improve in vivo stability.

Study of the Tissue Distribution of TLQP-21 in Mice Using [18F]JMV5763, a Radiolabeled Analog Prepared via [18F]Aluminum Fluoride Chelation Chemistry.

TLQP-21 is a neuropeptide that is involved in the control of several physiological functions, including energy homeostasis. Since TLQP-21 could oppose the early phase of diet-induced obesity, it has raised a huge interest, but very little is known about its mechanisms of action on peripheral tissues. Our aim was to investigate TLQP-21 distribution in brain and peripheral tissues after systemic administration using positron emission tomography. We report here the radiolabeling of NODA-methyl phenylacetic acid (MPAA) functionalized JMV5763, a short analog of TLQP-21, with [18F]aluminum fluoride. Labeling of JMV5763 was initially performed manually, on a small scale, and then optimized and implemented on a fully automated radiosynthesis system. In the first experiment, mice were injected in the tail vein with [18F]JMV5763, and central and peripheral tissues were collected 13, 30, and 60 min after injection. Significant uptake of [18F]JMV5763 was found in stomach, intestine, kidney, liver, and adrenal gland. In the CNS, very low uptake values were measured in all tested areas, suggesting that the tracer does not efficiently cross the blood-brain barrier. Pretreatment with non-radioactive JMV5763 caused a significant reduction of tracer uptake only in stomach and intestine. In the second experiment, PET analysis was performed in vivo 10-120 min after i.v. [18F]JMV5763 administration. Results were consistent with those of the ex vivo determinations. PET images showed a progressive increase of [18F]JMV5763 uptake in intestine and stomach reaching a peak at 30 min, and decreasing at 120 min. Our results demonstrate that 18F-labeling of TLQP-21 analogs is a suitable method to study its distribution in the body.

Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity.

Live-cell microscopy has highlighted that transcription factors bind transiently to chromatin but it is not clear if the duration of these binding interactions can be modulated in response to an activation stimulus, and if such modulation can be controlled by post-translational modifications of the transcription factor. We address this question for the tumor suppressor p53 by combining live-cell single-molecule microscopy and single cell in situ measurements of transcription and we show that p53-binding kinetics are modulated following genotoxic stress. The modulation of p53 residence times on chromatin requires C-terminal acetylation-a classical mark for transcriptionally active p53-and correlates with the induction of transcription of target genes such as CDKN1a. We propose a model in which the modification state of the transcription factor determines the coupling between transcription factor abundance and transcriptional activity by tuning the transcription factor residence time on target sites.Both transcription binding kinetics and post-translational modifications of transcription factors are thought to play a role in the modulation of transcription. Here the authors use single-molecule tracking to directly demonstrate that p53 acetylation modulates promoter residence time and transcriptional activity.

Development of 99mTc-radiolabeled nanosilica for targeted detection of HER2-positive breast cancer.

The human epidermal growth factor receptor 2 (HER2) is normally associated with a highly aggressive and infiltrating phenotype in breast cancer lesions with propensity to spread into metastases. In clinic, the detection of HER2 in primary tumors and in their metastases is currently based on invasive methods. Recently, nuclear molecular imaging techniques, including positron emission tomography and single photon emission computed tomography (SPECT), allowed the detection of HER2 lesions in vivo. We have developed a 99mTc-radiolabeled nanosilica system, functionalized with a trastuzumab half-chain, able to act as drug carrier and SPECT radiotracer for the identification of HER2-positive breast cancer cells. To this aim, nanoparticles functionalized or not with trastuzumab half-chain, were radiolabeled using the 99mTc-tricarbonyl approach and evaluated in HER2 positive and negative breast cancer models. Cell uptake experiments, combined with flow cytometry and fluorescence imaging, suggested that active targeting provides higher efficiency and selectivity in tumor detection compared to passive diffusion, indicating that our radiolabeling strategy did not affect the nanoconjugate binding efficiency. Ex vivo biodistribution of 99mTc-nanosilica in a SK-BR-3 (HER2+) tumor xenograft at 4 h postinjection was higher in targeted compared to nontargeted nanosilica, confirming the in vitro data. In addition, viability and toxicity tests provided evidence on nanoparticle safety in cell cultures. Our results encourage further assessment of silica 99mTc-nanoconjugates to validate a safe and versatile nanoreporter system for both diagnosis and treatment of aggressive breast cancer.