Early investigation of a novel SI306 theranostic prodrug for glioblastoma treatment.

Glioblastoma multiforme (GBM) is one of the most aggressive malignancies with a high recurrence rate and poor prognosis. Theranostic, combining therapeutic and diagnostic approaches, arises as a successful strategy to improve patient outcomes through personalized medicine. Src is a non-receptor tyrosine kinase (nRTK) whose involvement in GBM has been extensively demonstrated. Our previous research highlighted the effectiveness of the pyrazolo[3,4-d]pyrimidine SI306 and its more soluble prodrug CMP1 as Src inhibitors both in in vitro and in vivo GBM models. In this scenario, we decided to develop a theranostic prodrug of SI306, ProSI-DOTA(68 Ga) 1, which was designed to target GBM cells after hydrolysis and follow-up on the disease's progression and improve the therapy's outcome. First, the corresponding nonradioactive prodrug 2 was tested to evaluate its ADME profile and biological activity. It showed good metabolic stability, no inhibition of CYP3A4, suboptimal aqueous solubility, and slight gastrointestinal and blood-brain barrier passive permeability. Compound 2 exhibited a drastic reduction of cell vitality after 72 h on two different GBM cell lines (GL261 and U87MG). Then, 2 was subjected to complexation with the radionuclide Gallium-68 to give ProSI-DOTA(68 Ga) 1. The cellular uptake of 1 was evaluated on GBM cells, highlighting a slight but significant time-dependent uptake. The data obtained from our preliminary studies reflect the physiochemical properties of 1. The use of an alternative route of administration, such as the intranasal route, could overcome the physiochemical limitations and enhance the pharmacokinetic properties of 1, paving the way for its future development.

Synthesis and Characterization of Multifunctional Nanovesicles Composed of POPC Lipid Molecules for Nuclear Imaging.

The integration of nuclear imaging analysis with nanomedicine has tremendously grown and represents a valid and powerful tool for the development and clinical translation of drug delivery systems. Among the various types of nanostructures used as drug carriers, nanovesicles represent intriguing platforms due to their capability to entrap both lipophilic and hydrophilic agents, and their well-known biocompatibility and biodegradability. In this respect, here we present the development of a labelling procedure of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)-based liposomes incorporating an ad hoc designed lipophilic NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) analogue, derivatized with an oleic acid residue, able to bind the positron emitter gallium-68(III). Based on POPC features, the optimal conditions for liposome labelling were studied with the aim of optimizing the Ga(III) incorporation and obtaining a significant radiochemical yield. The data presented in this work demonstrate the feasibility of the labelling procedure on POPC liposomes co-formulated with the ad hoc designed NOTA analogue. We thus provided a critical insight into the practical aspects of the development of vesicles for theranostic approaches, which in principle can be extended to other nanosystems exploiting a variety of bioconjugation protocols.

Assessment of tissue response in vivo: PET-CT imaging of titanium and biodegradable magnesium implants.

To study in vivo the bioactivity of biodegradable magnesium implants and other possible biomaterials, we are proposing a previously unexplored application of PET-CT imaging, using available tracers to follow soft tissue and bone remodelling and immune response in the presence of orthopaedic implants. Female Wistar rats received either implants (Ti6Al7Nb titanium or WE43 magnesium) or corresponding transcortical sham defects into the diaphyseal area of the femurs. Inflammatory response was followed with [18F]FDG and osteogenesis with [18F]NaF, over the period of 1.5 months after surgery. An additional pilot study with [68Ga]NODAGA-RGD tracer specific to αvß3 integrin expression was performed to follow the angiogenesis for one month. [18F]FDG tracer uptake peaked on day 3 before declining in all groups, with Mg and Ti groups exhibiting overall higher uptake compared to sham. This suggests increased cellular activity and tissue response in the presence of Mg during the initial weeks, with Ti showing a subsequent increase in tracer uptake on day 45, indicating a foreign body reaction. [18F]NaF uptake demonstrated the superior osteogenic potential of Mg compared to Ti, with peak uptake on day 7 for all groups. [68Ga]NODAGA-RGD pilot study revealed differences in tracer uptake trends between groups, particularly the prolonged expression of αvß3 integrin in the presence of implants. Based on the observed differences in the uptake trends of radiotracers depending on implant material, we suggest that PET-CT is a suitable modality for long-term in vivo assessment of orthopaedic biomaterial biocompatibility and underlying tissue reactions. STATEMENT OF SIGNIFICANCE: The study explores the novel use of positron emission tomography for the assessment of the influence that biomaterials have on the surrounding tissues. Previous related studies have mostly focused on material-related effects such as implant-associated infections or to follow the osseointegration in prosthetics, but the use of PET to evaluate the materials has not been reported before. The approach tests the feasibility of using repeated PET-CT imaging to follow the tissue response over time, potentially improving the methodology for adopting new biomaterials for clinical use.

Targeting the Translocator Protein (18 kDa) in Cardiac Diseases: State of the Art and Future Opportunities.

Mitochondria dysfunctions are typical hallmarks of cardiac disorders (CDs). The multiple tasks of this energy-producing organelle are well documented, but its pathophysiologic involvement in several manifestations of heart diseases, such as altered electromechanical coupling, excitability, and arrhythmias, is still under investigation. The human 18 kDa translocator protein (TSPO) is a protein located on the outer mitochondrial membrane whose expression is altered in different pathological conditions, including CDs, making it an attractive therapeutic and diagnostic target. Currently, only a few TSPO ligands are employed in CDs and cardiac imaging. In this Perspective, we report an overview of the emerging role of TSPO at the heart level, focusing on the recent literature concerning the development of TSPO ligands used for fighting and imaging heart-related disease conditions. Accordingly, targeting TSPO might represent a successful strategy to achieve novel therapeutic and diagnostic strategies to unravel the fundamental mechanisms and to provide solutions to still unanswered questions in CDs.

MicroPET/CT imaging of αvß3 integrin via a novel 68Ga-NOTA-RGD peptidomimetic conjugate in rat myocardial infarction.

PURPOSE: The αvß3 integrin is expressed in angiogenic vessels and is a potential target for molecular imaging of evolving pathological processes. Its expression is upregulated in cancer lesions and metastases as well as in acute myocardial infarction (MI) as part of the infarct healing process. The purpose of our study was to determine the feasibility of a new imaging approach with a novel (68)Ga-2,2',2″-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA)-arginine-glycine-aspartic acid (RGD) construct to assess integrin expression in the evolving MI. METHODS: A straightforward labelling chemistry to attach the radionuclide (68)Ga to a NOTA-based chelating agent conjugated with a cyclic RGD peptidomimetic is described. Affinity for αvß3 integrin was assessed by in vitro receptor binding assay. The proof-of-concept in vivo studies combined the (68)Ga-NOTA-RGD with the flow tracer (13)N-NH3 imaging in order to obtain positron emission tomography (PET)/CT imaging of both integrin expression and perfusion defect at 4 weeks after infarction. Hearts were then processed for immunostaining of integrin ß3. RESULTS: NOTA-RGD conjugate displayed a binding affinity for αvß3 integrin of 27.9 ± 6.8 nM. (68)Ga-NOTA-RGD showed stability without detectable degradation or formation of by-products in urine up to 2 h following injection in the rat. MI hearts exhibited (68)Ga-NOTA-RGD uptake in correspondence to infarcted and border zone regions. The tracer signal drew a parallel with vascular remodelling due to ischaemia-induced angiogenesis as assessed by immunohistochemistry. CONCLUSION: As compared to similar imaging approaches using the (18)F-galacto-derivative, we documented for the first time with microPET/CT imaging the (68)Ga-NOTA-RGD derivative that appears eligible for PET imaging in animal models of vascular remodelling during evolving MI. The simple chemistry employed to synthesize the (68)Ga-based radiotracer may greatly facilitate its translation to a clinical setting.

Extracellular Vesicles and Intercellular Communication: Challenges for In Vivo Molecular Imaging and Tracking.

Extracellular vesicles (EVs) are a heterogeneous class of cell-derived membrane vesicles released by various cell types that serve as mediators of intercellular signaling. When released into circulation, EVs may convey their cargo and serve as intermediaries for intracellular communication, reaching nearby cells and possibly also distant organs. In cardiovascular biology, EVs released by activated or apoptotic endothelial cells (EC-EVs) disseminate biological information at short and long distances, contributing to the development and progression of cardiovascular disease and related disorders. The significance of EC-EVs as mediators of cell-cell communication has advanced, but a thorough knowledge of the role that intercommunication plays in healthy and vascular disease is still lacking. Most data on EVs derive from in vitro studies, but there are still little reliable data available on biodistribution and specific homing EVs in vivo tissues. Molecular imaging techniques for EVs are crucial to monitoring in vivo biodistribution and the homing of EVs and their communication networks both in basal and pathological circumstances. This narrative review provides an overview of EC-EVs, trying to highlight their role as messengers of cell-cell interaction in vascular homeostasis and disease, and describes emerging applications of various imaging modalities for EVs visualization in vivo.

Spatial Inhomogeneity of Cardiac Norepinephrine Transport Protein and Meta-[123I]Iodobenzylguanidine Uptake in Swine Myocardial Tissue.

PURPOSE: The aim of the present study was to evaluate the expression of the cardiac norepinephrine transporter (NET) in the left ventricle (LV) of healthy pigs and its relationship with regional meta-[123I]iodobenzylguanidine ([123I]MIBG) myocardial uptake. PROCEDURES: Experiments were performed on animals injected with [123I]MIBG and acquired 2 h later using an ultrafast CZT gamma camera to assess the regional myocardial uptake. After image acquisition, animals were euthanized; the heart was quickly excised and underwent to an ex vivo single photon emission tomography (SPECT) imaging. Four small samples of tissue were then harvested from mid-walls and apex of the left ventricle; NET densities were evaluated and further normalized for protein loading per cardiac region. RESULTS: Three variants of NET protein with different molecular weights were detected. The expression of NET was not homogenous in the LV, with the highest density in the inferior wall and the lowest one in the apical area. The regional in vivo [123I]MIBG uptake revealed an analogous trend, showing a good linear relationship with NET expression. Parallel results were obtained from the ex vivo study. CONCLUSION: This study elucidates the expression of three different variants of NET proteins into the left ventricular myocardium of a healthy pig. NET expression into the LV was not homogeneous and paralleled by differences in regional [123I]MIBG uptake. Moreover, the correlation and the agreement between measurements of regional expression of NET variants and [123I]MIBG uptake represent a relevant finding for inferences about NET expression in the context of clinical imaging.

Spectroscopic and photoacoustic characterization of encapsulated iron oxide super-paramagnetic nanoparticles as a new multiplatform contrast agent.

Recently, a number of photoacoustic (PA) agents with increased tissue penetration and fine spatial resolution have been developed for molecular imaging and mapping of pathophysiological features at the molecular level. Here, we present bio-conjugated near-infrared light-absorbing magnetic nanoparticles as a new agent for PA imaging. These nanoparticles exhibit suitable absorption in the near-infrared region, with good photoacoustic signal generation efficiency and high photo-stability. Furthermore, these encapsulated iron oxide nanoparticles exhibit strong super-paramagnetic behavior and nuclear relaxivities that make them useful as magnetic resonance imaging (MRI) contrast media as well. Their simple bio-conjugation strategy, optical and chemical stability, and straightforward manipulation could enable the development of a PA probe with magnetic and spectroscopic properties suitable for in vitro and in vivo real-time imaging of relevant biological targets.

In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature.

Although the angiogenic effect of vascular endothelial growth factor (VEGF) is widely recognized, a central question concerns whether the vessels formed on its overexpression effectively increase tissue perfusion in vivo. To explore this issue, here we exploit AAV vectors to obtain the prolonged expression of VEGF and angiopoietin-1 (Ang1) in rat skeletal muscle. Over a period of 6 months, muscle blood flow (MBF) and vascular permeability were measured by positron emission tomography and single-photon emission computed tomography, respectively. All measurements were performed under resting conditions and after electrically induced muscle exercise. Despite the potent angiogenic effect of VEGF, documented by vessel counting and intravascular volume assessment, the expression of this factor did not improve resting MBF, and it even decreased perfusion after exercise. This deleterious effect was related to the formation of leaky vascular lacunae, which accounted for the occurrence of arteriovenous shunts that excluded the downstream microcirculation. These effects were significantly counteracted by the coinjection of VEGF and Ang1, which determined a marked increase in resting MBF and, most notably, a significant improvement after exercise that persisted over time. Taken together, these results challenge the effectiveness of VEGF as a sole factor to induce angiogenesis and suggest the use of factor combinations to achieve competent vessel formation.

Synthesis and In Vivo Imaging of N-(3-[11C]Methoxybenzyl)-2-(3-Methoxyphenyl)ethylaniline as a Potential Targeting Agent for P-glycoprotein.

PURPOSE: The plasma membrane P-glycoprotein (Pgp) is an efflux transporter involved in multidrug resistance and in the onset of neurodegenerative disease. Its function and most mechanisms of action are still under investigation. We developed a C-11-labeled 2-arylethylphenylamine-([11C]AEPH) derivative for positron emission tomography (PET), as a novel probe to better understand the activity and the function of Pgp in vivo. PROCEDURES: The synthetic procedure and the quality control of the selected lead compound, [11C]AEPH-1, were set up and optimized. The biodistribution and the dynamic extraction in target organs of [11C]AEPH-1 were studied in vivo by PET in healthy rats at baseline and after pre-treatment with a Pgp inhibitor (tariquidar). RESULTS: In vivo dynamic imaging was consistent with the results of ex vivo extraction on explanted organs. An adequate stability for in vivo studies, as well as a high activity of [11C]AEPH-1 in intestine and barrier tissues, has been demonstrated. Results of the blockade study showed a decrease of uptake after the pre-treatment, indicating a behavior attributable to a Pgp ligand. CONCLUSIONS: The suitable pharmacokinetics and the specificity tested in the pre-treated animals have indicated the potentiality of this AEPH derivative to act as Pgp ligand, providing new opportunities for further studies on expression and function of this important efflux transporter in the fields of neurology and oncology.

Toward PET imaging of A2B adenosine receptors: a carbon-11 labeled triazinobenzimidazole tracer: Synthesis and imaging of a new A2B PET tracer.

INTRODUCTION: A2B adenosine receptors (ARs) are commonly defined as "danger" sensors because they are triggered during cell injury when the endogenous molecule, adenosine, increases rapidly. These receptors, together with the other receptor subtypes (A1, A2A and A3), exert a wide variety of immunomodulating and (cyto)protective effects, thus representing a pivotal therapeutic target for different pathologies including diabetes, tumors, cardiovascular diseases, pulmonary fibrosis and others. The limited availability of potent and selective ligands for A2B ARs has prevented this receptor to emerge both as therapeutic and diagnostic target. METHODS: Recently, a new class of potent A2B ARs antagonists was developed featuring the triazinobenzimidazole scaffold. Starting from this chemotype, we synthesized a new radiotracer, [(11)C]-4 (1-[(11)C]methyl-3-phenyl triazino[4,3-a]benzimidazol-4(1H)-one), and investigated the pharmacokinetics of this compound in vivo to define its potential use in the imaging of A2B AR with positron emission tomography. RESULTS: [(11)C]-4 showed a very high chemical and blood stability. Results of in vivo and ex vivo experiments underlined the ability of this molecule to bind the A2B AR and correlated with the A2B AR protein and gene expression data. CONCLUSIONS: Although further studies are necessary, these data suggest that [(11)C]-4 may represent a good lead compound for the development of novel selective and potent A2B AR radiotracers, and a new option for the clinical investigation of several pathophysiological processes and chronic diseases.

[¹¹C]diclofenac sodium: synthesis and PET assessment of transdermal penetration.

The aim of this work was to study the feasibility of using Positron Emission Tomography (PET) imaging as a new tool to detect transdermal penetration of topical drugs in human subjects. The compound used in the study is sodium 2-[(2,6-dichlorophenyl)amino]phenyl]acetate, better known as diclofenac sodium. This molecule belongs to the family of non-steroidal anti-inflammatory drugs and is considered one of the first choices among non-steroidal anti-inflammatory drugs for the treatment of inflammatory diseases; it is widely used and commercially present in a large number of pharmaceutical forms and formulations. (11)C-labeled diclofenac has been synthesized and coformulated, as an internal indicator, with a proprietary preparation based on the use of a sprayer. The radiolabeled preparation was topically administered to healthy volunteers, and PET imaging was used to evaluate transdermal penetration. Results obtained have demonstrated the efficacy of PET and radiolabeled tracers for the evaluation of transdermal penetration of active pharmaceutical ingredients as topical formulations.