Search

The drug mass balance, performed on the entire human body, allows building up mathematical models able to account for the simultaneous processes of in vivo drug release kinetics and ADME steps (Absorption, Distribution, Metabolism and Elimination) that determine the drug pharmacokinetics (PK). As this approach holds whatever the administration route and the delivery system considered, it could play a paramount role in the development of personalized medicine.

This chapter is aimed to illustrate and to discuss membrane applications in the delivery field focussing the attention on the mechanisms ruling mass transport. Accordingly, membranes physical characteristics such as intrinsic topological structure, viscoelastic nature and membrane-drug interaction are considered. In this frame the continuity equation is presented and particularised in relation to different scenarios such as polymeric matrices, osmotic systems and electrotransport. On the basis of this theoretical frame, some examples of mathematical models serving to get drug diffusion coefficient by means of data fitting are shown. In particular, it is stressed the necessity of experimentally match the assumptions on which interpretative models relay. Among the great variety of membrane applications in the delivery field, those related to the oral and transdermal administration route are presented and discussed. Consequently, coating processes, asymmetric membranes and membranes devoted to colon delivery are presented for what concerns oral delivery systems. Membranes for passive diffusion delivery systems and physically enhanced delivery systems constitute the topic of transdermal administration route. The final part of this chapter is devoted to some membranes applications in the nano-medicine field. In particular, the restenosis prevention and the bioavailability enhancement due to nano-crystals will be dealt with

The importance of hydrogels in the biomedical and biotechnological fields induced researchers to experimentally and theoretically study their properties. Among them, one of the most important is represented by the mesh size distribution of their polymeric network. Indeed, this characteristic heavily rules the mass exchange processes between the hydrogel and the surrounding. The aim of this chapter is to present and discuss some techniques devoted to the estimation of the polymeric network mesh size. In particular, attention will be focused on rheology, low-field NMR, crioporosimetry, and release tests

Although mathematics finds large application in physical sciences, its use in the medical and biological field is less usual. Nevertheless, mathematical models can help in understanding many medical and biological phenomena and they can be a powerful tool in the designing process of many technological devices connected to the above mentioned phenomena. Of course, fundamental pre-requisite for building up a reliable mathematical model is the deep cooperation of different competences such as those of medical doctors, biologists, pharmacists and engineers. This paper deals with four different examples of mathematical models developed by our group and dealing with restenosis, enzyme action on two dimensional DNA brushes, oral delivery of drug in amorphous or nano-crystalline form and tumour cell behaviour.

This work focusses on the characterisation, by means of rheology and low field NMR, of different hydrogels obtained by photo-crosslinking of an anphyphilic polymer (polyvinylpyrrolidone). This work allows to conclude that all the studied hydrogels have a homogeneous network characterised by rather wide meshes (40 – 80 nm).

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is a global health problem representing the sixth most common cancer and the third cause of cancer related death worldwide. The number of deaths per year in HCC is comparable to the incidence number, underlying the aggressive behavior of HCC and the modest efficacy of available curative treatments. Effective HCC treatment is problematic also due to the lack of early and specific diagnostic markers. In this regard, particular interest has been put on the tyrosine kinase with Ig and EGF homology domains 2 (TIE2), a receptor of angiopoietins, predominantly present on endothelial cells but also observed on monocytes (TIE-2-expressing monocytes – TEMs). Recently, a work by Matsubara et al showed that the amount of circulating TEMs is higher in HCV/HCC patients compared to HCV patients or healthy subjects. Additionally the authors showed that TEMs have a diagnostic potential for HCC. Whereas the molecular mechanisms responsible for this observation remain elusive and further studies are necessary to confirm this finding, the work of Matsubara et al may contribute to the identification of a novel HCC prognostic and diagnostic marker.

Prostate cancer diagnosis is increasing, being the second most frequently cancer in men worldwide. The treatment of castrate-resistant prostate cancer is often unsuccessfully and new therapeutic interventions are searching for. Nucleic acid aptamers targeting eEF1A proteins are emerging molecular tools for the control of cancer growth. We found that an aptamer named GT75 was able to bind to eEF1A proteins of human prostate cancer cell lines and to significantly and specifically reduce their growth with respect to the control oligomer CT75. The highest anti-proliferation effect was found in the androgen-independent PC-3 cells. Interestingly, GT75 was able to specifically inhibit the migration of PC-3 cells but not that of the nontumorigenic PZHPV-7 cells. The overall results suggest that the GT75 aptamer targeting eEF1A proteins is a promising molecular drug to develop for the control of the castrate-resistant prostate cancer.

The problems connected with the use of traditional strategies (stent and coated stent) for the treatment of coronary restenosis (lumen re-occlusion due to smooth muscle cells iper-proliferation following balloon angioplasty and stent implantation) impose the development of novel solutions. Among the possible alternatives, the use of a stent embodied in a gel-polymeric film containing nucleic acid based drugs (NABDs), with antiproliferative action, seems to be promising. As gel macroscopic and structural properties are of paramount importance for the success of this approach (erosion resistance, desired release kinetics of the NABDs agent and small pressure drop), this paper is aimed to study the effect of some gel formulative variables (polymer concentration, crosslinking procedure) in order to select the best promising gel

Introduzione Nella suddivisione dei carcinomi mammari in sottotipi Luminal A e B, la quantificazione della proliferazione tumorale tramite colorazione immunoistochimica con anticorpo Ki-67 (MIB-1), sembra avere un ruolo determinante . Tuttavia la quantificazione precisa è gravata da numerose variabili , tra le quali un’importante grado di soggettività. Vi sono inoltre incertezze nel determinare un preciso cut-off di positività di Ki-67 utile per inserire il singolo caso nel sottotipo corretto. Lo studio valuta il rischio di un’erronea classificazione quando deve essere valutata la fascia di positività con maggior ricaduta clinica (8%-25%). A tale scopo è stata comparata la lettura tradizionale con quella ottenuta da un’analisi digitale semi-automatizzata. Materiale e Metodi: Sono stati selezionati 66 casi di carcinoma mammario duttale infiltrante (anno 2012) che presentavano una percentuale di Ki-67, valutata dal patologo, tra l’8 e il 25%. I preparati immunoistochimici sono stati acquisiti tramite scanner (D-Sight Instrument) ottenendo vetrini digitali, sui quali è stata eseguita un’analisi quantitativa semi-automatica (software AVISIA imaging S.r.l). L’analisi digitale è stata eseguita ed analizzata ad ingrandimento 20X per ogni caso, selezionando un’ area di 20 mm2 a maggior positività. Sono state comparate le letture (ottica e digitale) rispetto ai due cut-off di riferimento (S1=14% ed S2=20%) suggeriti dagli studi più recenti [4,5] ed è stato poi valutato il grado di concordanza con la metodica statistica kappa (k), attribuendo alla lettura digitale il gold standard. Risultati: Nel nostro studio la concordanza tra le due letture (ottica e digitale) per il cut-off 14% (S1) è risultata statisticamente moderata (17 casi discordanti); modificando il cut-off al 20% (S2), questa non è variata (k=0.45 p-value

The role of breastfeeding as protective factor for breast cancer incidence is controversial among scientific literature. The Interdisciplinary Senology Unit of Trieste has analyzed the relation between breast cancer incidence and lactation habits among a total of 4307 Trieste’s women. In this study, the statistical analysis revealed that over having children, the breastfeeding is a further protective factor for breast cancer. In particular, the decrease of breast cancer risk by breastfeeding resulted to be significant for lactation more than 12 months. It is worth noting that breastfeeding resulted protective only for women in childbearing age and it didn’t result to decrease breast cancer risk among post-menopausal women.

Introduzione: Il riconoscimento intra-operatorio delle metastasi nel linfonodo sentinella, in pazienti affetti da cancro della mammella, è utile al fine di procedere ad una linfoadenectomia ascellare immediata nel corso del medesimo intervento. Ciò è fondamentale per evitare una terapia adiuvante tardiva ed un incremento della morbilità e dei costi sanitari. Scopo di tale lavoro è dimostrare che, la metodica citologica “touch-imprint” (TIC), utilizzata presso l’ Azienda Ospedaliero-Universitaria di Trieste, risponde a tale necessità e nel contempo valutarne l’affidabilità. Materiali e metodi: Nel periodo 2009-2012, presso la nostra struttura sono stati operati 690 carcinomi mammari, di cui 479 (69.4%) sono stati selezionati per la ricerca del linfonodo sentinella con radiocolloide. In 347 carcinomi di dimensioni inferiori o uguali a T2 è stata eseguita la metodica citologica TIC; il linfonodo sentinella è stato successivamente sottoposto all’ analisi istologica standard (sezioni seriate ogni 100 micron per ematossilina-eosina e immunoistochimica) che è stato assunta come “gold standard”. Risultati: Su 347 TIC 298 casi erano concordanti con la diagnosi istologica (38 veri positivi e 260 veri negativi). Dei 49 casi risultati falsi negativi, l’analisi definitiva ha dimostrato 28 casi di micrometastasi e 21 casi di macrometastasi, mentre dei 38 casi positivi al TIC, 35 risultavano macrometastasi, 2 micrometastasi e 1 caso di cellule tumorali isolate. La sensibilità e specificità del TIC per macrometastasi sono rispettivamente 64.4% e 100%. Solo 29 casi su 347, pari all’ 8.3%, sono stati sottoposti a radicalizzazione ascellare differita, mentre nel periodo 2005-2008, prima dell’introduzione del TIC,utilizzando l’analisi istologica standard del linfonodo sentinella, tale percentuale era del 18.2%. Discussione e conclusioni: Grazie alla metodica TIC il tasso di dissezioni ascellari in differita è diminuito notevolmente (dal 18.2% all’8.3%). Se gli studi, in corso di validazione definitiva, dimostreranno che la linfoadenectomia ascellare nel trattamento delle micrometastasi non è necessaria, si può ipotizzare che il numero di reinterventi sull’ascella possa ulteriormente ridursi. Nella nostra esperienza, grazie alla discreta sensibilità del TIC per le macrometastasi legata all’ ottima confidenza del patologo con i preparati citologici, è stato possibile migliorare in maniera rilevante l’approccio chirurgico sull’ascella ed il timing oncologico, senza impegno aggiuntivo di risorse economiche.

Nucleic-acid-based drugs1 are a novel class of drugs with great therapeutic potential. Among these are DNAzymes, which contain short stretches of single-stranded DNA molecules synthesised in vitro. A DNAzyme can recognize through Watson-Crick basepairing a specific messenger RNA (mRNA) sequence and induce cleavage between a purine and a pyrimidine residue. Upon cleavage, the target mRNA undergoes accelerated degradation, which leads to a reduction in the concentration of the encoded protein and its related biological functions. Thus, DNAzymes can potentially be used to target specific mRNA in diseases dependent on individual genes. Nevertheless, although the great therapeutic potential of these drugs has been noted in preclinical studies, data about their use in human beings are missing. In The Lancet, Eun-Ae Cho and colleagues report a first-in-human study of the DNAzyme Dz13 in patients with nodular basal-cell carcinoma. The therapeutic promise of Dz13 is strongly supported by previous animal studies. The drug targets the mRNA of the JUN proto-oncogene: this target is particularly well suited for the purpose of the study, as c-Jun protein is minimally expressed in normal skin tissue but concentrations are notably increased in skin cancer tumours. Additionally, the model tumour pathology chosen is well suited for clinical investigation because basal-cell carcinomas are easily accessible, monitoring is straightforward, the risk of systemic tumour spread is low, and the therapeutic drug can be injected locally.

The limited efficacy of current therapeutic approaches for a number of socially relevant human diseases requires the exploration of alternative and more effective therapeutic strategies. In this regard, the researchers have pursued on one hand the identification of novel and more effective therapeutic molecules and on the other the optimization of drug delivery systems. So far, many therapeutic molecules, especially those used as anticancer drugs, are plagued by a low therapeutic index being the efficacious dose very close to the lethal one; moreover, they often lack any specificity of action. This aspect can be improved by the use of drug delivery systems composed of different drug carriers including lipids and polymers. The carriers, often in the shape of nanoparticles , can be loaded by the therapeutic molecule and directed against the target cells via the presence of targeting moieties allocated on the nanoparticle surface. The specificity of the complex carrier/drug can be further improved by the use of therapeutic molecules preferentially/exclusively active on the target diseased cells. Molecules active against diseased-associated target (oncogenes etc) may hit the diseased cells leaving healthy cells substantially unaffected. In this regard, in the last three decades, nucleic acid based drugs (NABDs) have emerged as an attractive and novel alternative with great therapeutic potential. NABDs, which include antisense oligonucleotides, decoys, aptamers, triple helix forming oligonucleotides, DNAzymes, Ribozymes and small interfering RNAs, have been shown to be able to efficiently and specifically counteract pathological gene expression in many different experimental systems. Notably as they can be engineered to hit virtually any cell target, their potential applicability is very broad. Despite NABD broad potential applicability, their use in the clinic is limited by the lack of optimal delivery systems. Due to their hydrophilic nature, NABDs cannot efficiently cross cellular membrane for which appropriate carriers are needed. Moreover, their instability in serum requires a proper protection to prevent a fast degradation which would invariably lead to the abrogation of any significant therapeutic effect. The present special issue will be focused on the critical description of some aspects related to the optimization of drug delivery with a particular emphasis on NABD; despite this, a discussion about the possibility to use/adapt NABD developed delivery systems for more conventional drugs, is also present. The papers of Chan et al., of Marrache et al, of Schaffert et al., Jung et al. and Grassi et al. describe different delivery approaches for NABD and other commonly used therapeutic molecules for several pathological conditions. In the paper of Chan et al. attention is given to liposome and polymeric based delivery systems with regard to DNA enzymes; the described studies offer perspectives on future methodologies for improved DNAzyme delivery and utility as novel drugs. Marrache et al. describe the use of nanoparticles (made by polymer, liposome and other delivery agents), as delivery devices which can be engineered to load multiple drugs with varied physicochemical properties, contrast agents, and cellular or intracellular component targeting moieties. Schaffert et al draw their attention on the description of delivery systems based on the polycation linear polyethylenimine, where peptide based ligands are attached to the polycation via heterobifunctional polyethylene glycol linker molecules. Conjugate synthesis, in vitro testing and in vivo cancer models in rodents are discussed. Jung et al describe the employment of the thermo sensitive pluronic-based core/shell nanoparticles, formed using various strategies such as self-assembly and temperature induced-phase transition. Particular emphasis is given to the use of the nanoparticles for tumor targeting, stimulated release of proteins, and cancer imaging capabilities. Grassi et al, beside discussing the above mentioned delivery systems, for most of the different types of NABDs, draw their attention on the complex situation of NABD delivery to the arteries describing the advantages and dis-advantages of three different administration routes i.e. systemic, perivascular and intravascular. The papers of Lico et al, Pagliari et al and Castronovo et al report the use of “living delivery systems” and describe the influences of nano-systems on NABD. Lico et al. focus their attention on the use of a different approach for NABD delivery based on plant viruses which have a size particularly suitable for nanoscale applications and can offer several advantages being structurally uniform, robust, biodegradable and easy to produce. Pagliari et al. continue the description of “living vector” reporting the possible and very innovative use of stem cells as delivery devices for therapeutic molecules to the injured myocardium. Finally, in the paper of Castronovo et al., a completely innovative point of view about NABD complexation in nano-carriers is provided. The author show that the functionality of NABD in nano-systems is highly dependent upon the local density, molecular flexibility and network of weak interactions between adjacent molecules. The understanding of these properties can enable the development of powerful molecular tools for nano-medicine. In conclusion, whereas the developmental process of many delivery systems is still at the beginning, other delivery strategies are closer to possible applications. Regardless of the fact that the delivery systems are used for NABD or clinically available drugs, we believe the target tissue will mainly determine the nature of the optimal strategy. Despite the delivery issue can and should be further optimized, the encouraging results displayed so far in different experimental models using NABD or clinically used drugs, fully justify further economic and scientific efforts.

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon.

Oncogene proteins are proteins coded by oncogenes. They include proteins resulting from the fusion of an oncogene and another gene (oncogene proteins, fusion). An oncogene is a modified gene, or a set of nucleotides that codes for a protein and is believed to cause cancer. This book brings together the latest research in this field from around the world.

Chronic oxidative stress that characterizes uremia has potentially devastating effects on the vasculature and has been advocated in the pathogenesis of accelerated atherosclerosis in this disease. Recent advances have been made in our understanding of the molecular mechanisms that regulate expression and activity of key enzymes of vascular oxidative stress (eg, nicotinamide adenine dinucleotide phosphate [NAD{P}H] oxidase) and that dissect their interactions with signalling pathways of inflammation. The finding that NAD(P)H oxidase is upregulated in experimental uremia has important consequences from a physiologic and a therapeutic standpoint. In addition, identification of novel proteins involved in systemic oxidative stress has shed some new light on the pathogenesis of vascular disease. p66(shc) is a cytoplasmic protein that is expressed in a wide range of cell types. Initially believed to be involved in signalling pathways that regulate cell growth and oxidative stress, it has now been shown to play a pivotal role in promoting endothelial dysfunction and atherosclerosis. Although a specific role in uremia-related vascular disease has not yet been shown, available data in humans suggest involvement of p66(shc) in clinical conditions associated with increased oxidative stress.