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The present translational study aimed to verify whether serial 18F-fluoro-deoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT), predicts doxorubicin cardiotoxicity. Methods: Fifteen athymic mice were treated with intravenous administration of saline (n = 5), doxorubicin 5 mg/Kg (n = 5) or doxorubicin 7.5 mg/Kg (n = 5) and submitted to dynamic microPET scan to estimate left ventricular glucose consumption (LV-MRGlu) before and after chemotherapy. Thereafter, we retrospectively identified 69 patients successfully treated with Adriamycin, Bleomycin, Vinblastine, and Dacarbazine (ABVD) regimen for Hodgkin's Disease (HD) and submitted to four consecutive FDG-PET/CT scans. Volumes of interest were drawn on LV myocardium to quantify mean standardized uptake value (LV-SUV). All patients were subsequently interviewed by telephone (median follow-up: 30 months); 36 of them accepted to undergo electrocardiogram and transthoracic echocardiography. Results: In mice LV-MRGlu was 17.9±4.4 nMol x min-1 x g-1 at baseline. doxorubicin selectively and dose-dependently increased this value in the standard-dose (27.9±9 nMol x min-1 x g-1, p

Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, neurons, cardiomyocytes, and keratinocytes, is the precursor of various peptides that regulate the carbohydrate/lipid metabolism and the cardiovascular system. We have found that CgA, locally administered to injured mice, can accelerate keratinocyte proliferation and wound healing. This biological activity was abolished by the Asp(45)Glu mutation. CgA and its N-terminal fragments, but not the corresponding Asp(45)Glu mutants, could selectively recognize the alphavbeta6-integrin on keratinocytes (a cell-adhesion receptor that is up-regulated during wound healing) and regulate keratinocyte adhesion, proliferation, and migration. No binding was observed to other integrins such as alphavbeta3, alphavbeta5, alphavbeta8, alpha5beta1, alpha1beta1, alpha3beta1, alpha6beta4, alpha6beta7 and alpha9beta1. Structure-activity studies showed that the entire CgA(39-63) region is crucial for alphavbeta6 recognition (K (i)=7nM). This region contains an RGD site (residues CgA(43-45)) followed by an amphipathic alpha-helix (residues CgA(47-63)), both crucial for binding affinity and selectivity. These results suggest that the interaction of the RGD/alpha-helix motif of CgA with alphavbeta6 regulates keratinocyte physiology in wound healing.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.

Introduzione: le infezioni della ferita chirurgica (Surgical Site Infection=SSI) presentano nelle realtà occidentali una frequenza relativa del 15 - 20% e tassi di incidenza dal 2 - 3% al 12 - 15%, in relazione alla classe di intervento. Concorrono all’insorgenza di SSI le condizioni cliniche del paziente, la durata della degenza pre operatoria, il tipo di intervento chirurgico, le pratiche assistenziali. Lo studio di queste ultime consente di individuare e analizzare quali procedure siano adottate fra quelle definite in letteratura di provata efficacia nella prevenzione delle SSI e quali siano di uso comune pur non esistendo per esse evidenza di efficacia. Obiettivo: descrivere e analizzare le procedure di controllo delle SSI messe in atto nelle chirurgie generali degli ospedali del Piemonte sotto il profilo assistenziale e ambientale.Materiale e metodi: lo studio prevede una settimana di osservazione nelle sale operatorie di chirurgia generale da parte di personale formato. Saranno indagati con schede appositamente strutturate: 1) tutti gli interventi, per rilevarne le caratteristiche e le misure di controllo pre e intraoperatorie rivolte al paziente; 2) i comportamenti degli operatori in sala; 3) i dati relativi al controllo ambientale. Al fine di poter confrontare oggettivamente le diverse situazioni sarà assegnato ad ogni valutazione un punteggio, sulla base delle evidenze di efficacia disponibili.Risultati: i dati raccolti consentiranno di evidenziare situazioni di inosservanza delle misure di controllo efficaci, disomogeneità di comportamenti fra presidi ospedalieri o intra “team” chirurgici, di avviare interventi di correzione attraverso produzione di documenti regionali e attività di formazione mirata.

Neuroblastoma (NB) is the most common extracranial solid tumor encountered in childhood. Although there has been significant improvement in the outcomes of patients with high-risk disease, the prognosis for patients with metastatic relapse or refractory disease is poor. Hence, the clinical integration of genome sequencing into standard clinical practice is necessary in order to develop personalized therapy for children with relapsed or refractory disease. The PeRsonalizEdMEdicine (PREME) project focuses on the design of innovative therapeutic strategies for patients suffering from relapsed NB. We performed whole exome sequencing (WES) of patient-matched tumor-normal samples to identify genetic variants amenable to precision medicine. Specifically, two patients were studied (First case: a three-year-old male with early relapsed NB; Second case: a 20-year-old male who relapsed 10 years after the first diagnosis of NB). Results were reviewed by a multi-disciplinary molecular tumor board (MTB) and clinical reports were issued to the ordering physician. WES revealed the mutation c.G320C in the CUL4A gene in case 1 and the mutation c.A484G in the PSMC2 gene in case 2. Both patients were treated according to these actionable alterations, with promising results. The effective treatment of NB is one of the main challenges in pediatric oncology. In the era of precision medicine, the need to design new therapeutic strategies for NB is fundamental. Our results demonstrate the feasibility of incorporating clinical WES into pediatric oncology practice.

The unbalanced production of pro- and antiangiogenic factors in tumors can lead to aberrant vasculature morphology, angiogenesis, and disease progression. In this study, we report that disease progression in various murine models of solid tumors is associated with increased cleavage of full-length chromogranin A (CgA), a circulating vasoregulatory neurosecretory protein. Cleavage of CgA led to the exposure of the highly conserved PGPQLR site, which corresponds to residues 368–373 of human CgA1-373, a fragment that has proangiogenic activity. Antibodies against this site, unable to bind full-length CgA, inhibited angiogenesis and reduced tumor perfusion and growth. The PGPQLR sequence of the fragment, but not of the precursor, bound the VEGF-binding site of neuropilin-1; the C-terminal arginine (R373) of the sequence was crucial for binding. The proangiogenic activity of the CgA1-373 was blocked by anti-neuropilin-1 antibodies as well as by nicotinic acetylcholine receptor antagonists, suggesting that these receptors, in addition to neuropilin-1, play a role in the proangiogenic activity of CgA1-373. The R373 residue was enzymatically removed in plasma, causing loss of neuropilin-1 binding and gain of antiangiogenic activity. These results suggest that cleavage of the R373R374 site of circulating human CgA in tumors and the subsequent removal of R373 in the blood represent an important “on/off” switch for the spatiotemporal regulation of tumor angiogenesis and may serve as a novel therapeutic target. Significance: This work reveals that the interaction between fragmented chromogranin A and neuropilin-1 is required for tumor growth and represents a novel potential therapeutic target.

NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.

The therapeutic index of anti-cancer drugs is increased when encapsulating them in tumor-targeted liposomes. Liposome-entrapped doxorubicin (DXR), targeting the tumor vasculature marker, aminopeptidase N (APN), displayed enhanced anti-tumor effects and prolonged survival in human neuroblastoma (NB)-bearing mice. Here we exploited a peptide ligand of aminopeptidase A (APA), discovered by phage display technology for delivery of liposomal DXR to perivascular tumor cells. Immunohistochemistry, performed in NB-bearing mice, showed APA expression in the vascular wall of NB primary and metastatic lesions. APA-targeted peptides displayed specific binding to APA-transfected cells in vitro, and also accumulation in the tumor of NB-bearing mice. Consequently, novel, APA-targeted, DXR-liposomes were developed and in vivo proof-of-principle was established, alone and in combination with APN-targeted DXR-loaded liposomes, in NB-bearing mice. Mice receiving APA-targeted liposomal DXR exhibited an increased life span in comparison to control mice, but to a lesser extent relative to that in mice treated with APN-targeted formulation, moreover the greatest increase in TUNEL-positive tumor cells was observed in animals treated with APN-targeted formulations. Mice treated with a combination of APA- and APN-targeted, liposomal DXR had a significant increase in life span compared to each treatment administered separately. There was a significant increase in the level of apoptosis in the tumors of mice on the combination therapy, and a pronounced destruction of the tumor vasculature with nearly total ablation of endothelial cells and pericytes. The availability of novel ligands binding to additional tumor vasculature-associated antigens will allow the design of sophisticated combinations of ligand-targeted liposomal anti-cancer drugs. (C) 2010 Elsevier B.V. All rights reserved.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycinearginine (isoDGR), a ligand of alpha v beta 3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alpha v beta 3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alpha v beta 3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (alpha v beta 5, alpha v beta 6, alpha v beta 8, and alpha 5 beta 1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.

none 18 no Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies. Zuccari, G.; Milelli, A.; Pastorino, F.; Loi, M.; Petretto, A.; Parise, A.; Marchetti, C.; Minarini, A.; Cilli, M.; Emionite, L.; Di Paolo, D.; Brignole, C.; Piaggio, F.; Perri, P.; Tumiatti, V.; Pistoia, V.; Pagnan, G.; Ponzoni, M. Zuccari, G.; Milelli, A.; Pastorino, F.; Loi, M.; Petretto, A.; Parise, A.; Marchetti, C.; Minarini, A.; Cilli, M.; Emionite, L.; Di Paolo, D.; Brignole, C.; Piaggio, F.; Perri, P.; Tumiatti, V.; Pistoia, V.; Pagnan, G.; Ponzoni, M.