Targeting Pancreatic Ductal Adenocarcinoma (PDAC).

Pancreatic cancers are among the most ominous, and among the most studied. Their complexities have provided ample material for a huge investigative effort, which is briefly surveyed in this review. Eradication by surgery has proven extremely difficult, and a successful chemotherapeutic approach is desperately needed. Treatment with "traditional" anticancer drugs, such as benchmark gemcitabine or the current standard-of-care FOLFIRINOX quaternary combination increase the mean overall survival by only a few months and often leads to chemoresistance. Much work is therefore currently devoted to potentiating our pharmacological weapons by accurate targeting and, in particular, by acting on the dense tumoral stroma, a distinctive feature of PDAC accounting for much of the therapeutic difficulty. We give an overview of recent developments, touching on the major aspects of PDAC physiology and biochemistry, currently-used and experimental drugs, and targeting technologies under development. A few papers are discussed in some detail to help provide a sense of how the field is moving

Small-Molecule Modulators of Mitochondrial Channels as Chemotherapeutic Agents

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Quercetin can act either as an inhibitor or an inducer of the mitochondrial permeability transition pore: A demonstration of the ambivalent redox character of polyphenols

AbstractThe Ca2+- and oxidative stress-induced mitochondrial permeability transition (MPT) plays an important role in phenomena ranging from tissue damage upon infarction to muscle wasting in some forms of dystrophy. The process is due to the activation of a large pore in the inner mitochondrial membrane. Anti-oxidants are considered a preventive and remedial tool, and mitochondria-targeted redox-active compounds have been developed. Plant polyphenols are generally considered as anti-oxidants, and thus candidates to the role of mitochondria-protecting agents. In patch-clamp experiments, easily oxidizable polyphenols induced closure of the MPT channel. In swelling experiments with suspensions of mitochondria, high (20–50 μM) concentrations of quercetin, the most efficient inhibitor, promoted instead the onset of the MPT. Chelators of Fe2+/3+ and Cu+/2+ ions counteracted this effect. Fluorescent indicators of superoxide production confirmed that quercetin potentiates O2− generation by isolated mitochondria and cultured cells. Since this was not affected by chelating Fe and Cu ions, the MPT-inducing effect can be ascribed to a “secondary”, metal ion-catalyzed production of ROS. These results are a direct demonstration of the ambivalent redox character of polyphenols. Their mode of action in vivo cannot be taken for granted, but needs to be experimentally verified

New water-soluble carbamate ester derivatives of resveratrol

Low bioavailability severely hinders exploitation of the biomedical potential of resveratrol. Extensive phase-II metabolism and poor water solubility contribute to lowering the concentrations of resveratrol in the bloodstream after oral administration. Prodrugs may provide a solution—protection of the phenolic functions hinders conjugative metabolism and can be exploited to modulate the physicochemical properties of the compound. We report here the synthesis and characterization of carbamate ester derivatives of resveratrol bearing on each nitrogen atom a methyl group and either a methoxy-poly(ethylene glycol)-350 (mPEG-350) or a butyl-glucosyl promoiety conferring high water solubility. Ex vivo absorption studies revealed that the butyl-glucosyl conjugate, unlike the mPEG-350 one, is able to permeate the intestinal wall. In vivo pharmacokinetics confirmed absorption after oral administration and showed that no hydrolysis of the carbamate groups takes place. Thus, sugar groups can be attached to resveratrol to obtain soluble derivatives maintaining to some degree the ability to permeate biomembranes, perhaps by facilitated or active transport

DA7R: A 7-Letter Zip Code to Target PDAC

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and is among the most aggressive and still incurable cancers. Innovative and successful therapeutic strategies are extremely needed. Peptides represent a versatile and promising tool to achieve tumor targeting, thanks to their ability to recognize specific target proteins (over)expressed on the surface of cancer cells. A7R is one such peptide, binding neuropilin-1 (NRP-1) and VEGFR2. Since PDAC expresses these receptors, the aim of this study was to test if A7R-drug conjugates could represent a PDAC-targeting strategy. PAPTP, a promising mitochondria-targeted anticancer compound, was selected as the cargo for this proof-of-concept study. Derivatives were designed as prodrugs, using a bioreversible linker to connect PAPTP to the peptide. Both the retro-inverso (DA7R) and the head-to-tail cyclic (cA7R) protease-resistant analogs of A7R were tested, and a tetraethylene glycol chain was introduced to improve solubility. Uptake of a fluorescent DA7R conjugate, as well as of the PAPTP-DA7R derivative into PDAC cell lines was found to be related to the expression levels of NRP-1 and VEGFR2. Conjugation of DA7R to therapeutically active compounds or nanovehicles might allow PDAC-targeted drug delivery, improving the efficacy of the therapy and reducing off-target effects

Pterostilbene Improves Cognitive Performance in Aged Rats: An in Vivo Study

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Cytotoxicity of a mitochondriotropic quercetin derivative: Mechanisms

AbstractThe mitochondriotropic compound 7-O-(4-triphenylphosphoniumbutyl)quercetin iodide (Q-7BTPI) in the μM concentration range caused necrotic death of cultured cells by acting as a prooxidant, with generation of superoxide anion in the mitochondria. Externally added membrane-permeating superoxide dismutase or catalase largely prevented death. Rescue by permeant catalase indicates that the toxicant is H2O2, or reactive species derived from it. Rescue by permeant dismutase suggests the possibility of a chain mechanism of H2O2 production, in which dismutation of superoxide constitutes a termination step. Oxidative stress was due to the presence of free phenolic hydroxyls and to accumulation in mitochondria, since the analogous mitochondriotropic per-O-methylated compound -3,3′,4′,5-tetra-O-methyl,7-O-(4-triphenylphosphoniumbutyl) quercetin iodide (QTM-7BTPI)—or Quercetin itself induced no or little superoxide production and cell death. Q-7BTPI did not cause a significant perturbation of the mitochondrial transmembrane potential or of respiration in cells. On the other hand its presence led to inhibition of glutathione peroxidase, an effect expected to accentuate oxidative stress by interfering with the elimination of H2O2. An exogenous permeable glutathione precursor determined a strong increase of cellular glutathione levels but did not rescue the cells. Death induction was selective for fast-growing C-26 tumoral cells and mouse embryonic fibroblasts (MEFs) while sparing slow-growing MEFs. This suggests a possible use of Q-7BTPI as a chemotherapeutic agent

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (K(Ca)3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoK(Ca)3.1) of various cancer cell lines. The role mitoK(Ca)3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity K(Ca)3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the mu M range, depending on channel expression. In contrast, inhibition of the plasma membrane K(Ca)3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoK(Ca)3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoK(Ca)3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-kappa B (NF-kappa B) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-kappa B and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial K(Ca)3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo

Come sfruttare il potenziale farmacologico dei polifenoli vegetali: modificazioni reversibili per aumentarne la biodisponibilità/bioefficacia

The research project I have been engaged in during my graduate studies is meant to open the way to the pharmacological exploitation of plant polyphenols, a vast family of natural compounds present in many foods and drinks. A large amount of data shows that many of them have noteworthy biological properties, but their efficacy is severely hindered by the low bioavailability of these compounds. As a result of a low level of absorption and of a rapid metabolism/degradation in the intestinal and hepatic compartments only small amounts of polyphenols are found in the bloodstream, and then mostly as metabolites. The first part of my doctorate work was aimed at circumventing the obstacle posed by low bioavailability through the development of “pro-drugs” of polyphenols, resistant to metabolism during absorption and capable of regenerating the natural compound thanks to the action of ubiquitous enzymes. Quercetin (3,3’,4’,5,7-pentahydroxyflavone) and resveratrol (3,4’,5-trihydroxystilbene), both much studied molecules with interesting properties, were chosen as model polyphenols for our proof-of-principle project. Since the most relevant metabolic modifications involve the hydroxyls, and since enzymes with esterase activity are ubiquitous in the body, it seemed logical to begin work by developing carboxyester derivatives. I have thus synthesised a series of quercetin precursors, most of which comprised an aminoacidic functionality. I have studied the transport/diffusion of these derivatives across supported monolayers of epithelial cells. In this experimental system the ester precursors turned out to be more resistant to metabolic conjugation than quercetin as such. The studies on absorption/metabolism with monolayers of colonic Caco-2 cells revealed a remarkable heterogeneity in the expression of Phase II metabolism enzymes (sulfo- and glucuronosyl-transferases) within the same cell line. It has been possible to regenerate a uniform activity in the different populations by cultivating the cells with low concentrations of xenobiotic compound (in our case quercetin). To assess the bioavailability of the precursors in vivo it was first necessary to set up a protocol for the quantitative extraction and analysis of quercetin and resveratrol from blood. It soon became evident that it is more appropriate to analyse samples of whole blood, rather than blood plasma as generally done, to avoid underestimates due to the association of polyphenols with the cellular fraction. Since polyphenols generally have a low solubility in water, and solubility is a key factor contributing to the bioavailability of a compound, we obtained a first resveratrol derivative functionalising the hydroxyls with glucose moieties, linked via a succinic acid group. Pharmacokinetic studies with this compound are under way. We have furthermore synthesised two other carboxyester derivatives (carrying methyl or polyethyleneglycol groups), and two less hydrolysis-prone ones (the per-methylether and the per-mesylate). The results of stability studies in blood, of absorption experiments using ex vivo intestine segments and of pharmacokinetic determinations have pointed out an excessive instability of the carboxyester linkage in these biological contexts. A second part of the project originated from the consideration that the action of polyphenols can be enhanced not only by increasing systemic levels, but also by causing them to accumulate specifically at desired sites of action. Polyphenols are redox-active molecules, and reactive oxygen species are involved in several pathologies. Mitochondria are the major site of oxygen radical production and of key events for cell death. In the genesis of this part of the project an important role was played by the observation that quercetin can either inhibit or induce the mitochondrial permeability transition, depending on whether its anti- or pro-oxidant activity prevails. We have thus synthesised derivatives of quercetin and resveratrol capable of accumulating into mitochondria thanks to functionalisation with the triphenylphosphonium (TPP+) group, a cation capable of diffusing through biological membranes and to concentrate in regions at negative electrical potential, such as the mitochondrial matrix. A first investigation into the biological effects of these new compounds has been performed with some of the quercetin derivatives and isolated mitochondria; these compounds turned out to be potential co-inducers of the mitochondrial permeability transition, as well as inhibitors of the respiratory chain and of mitochondrial ATP synthase. We have thus carried out a preliminary study to test the possible cytostatic/cytotoxic activity of the mitochondriotropic compounds and of a few constructs produced during the project work, on cultured tumour and non-tumoural cells. These experiments have shown that some of the derivatives exhibit the typical effects of chemotherapeutic agents.Il progetto di ricerca a cui ho lavorato durante il corso di dottorato intende aprire la strada alla sfruttamento farmacologico dei polifenoli vegetali, una vasta famiglia di composti naturali presenti in molti cibi e bevande. Una grande quantità di dati dimostra che molti di essi hanno proprietà biologiche degne di nota, che trovano però un grosso ostacolo nella loro esplicazione a causa della scarsa biodisponibilità di questi composti. Come risultato del loro scarso assorbimento e della metabolizzazione/degradazione a livello intestinale ed epatico, solo piccole quantità di composto entrano nel circolo sanguigno, e per lo più sotto forma di metaboliti. La prima parte del mio lavoro di dottorato ha avuto come scopo quello di aggirare la barriera della bassa biodisponibilità attraverso lo sviluppo di “precursori” di polifenoli, più resistenti alla metabolizzazione durante l’assorbimento e in grado di rigenerare il composto naturale grazie all’azione di enzimi ubiquitari. Sono stati scelti come polifenoli modello la quercetina (3,3’,4’,5,7-pentaidrossiflavone) e il resveratrolo (3,4’,5-triidrossi-t-stilbene), entrambi ampiamente studiati e dotati di interessanti proprietà. Poichè le principali modificazioni metaboliche avvengono sugli ossidrili, e poiché enzimi con attività esterasica sono ubiquitari, è sembrato logico iniziare questo lavoro sviluppando dei derivati esterei. Ho quindi sintetizzato una serie di precursori della quercetina, la maggior parte dei quali comprendeva una funzionalità amminoacidica. Ho studiato il trasporto/diffusione di questi derivati attraverso monostrati supportati di cellule epiteliali. In questo sistema sperimentale i precursori esterei hanno dimostrato una maggiore resistenza al metabolismo rispetto alla quercetina tal quale. Gli studi di assorbimento/metabolismo con monostrati di cellule intestinali Caco-2 hanno messo in luce l’esistenza di eterogeneità nell’espressione degli enzimi metabolici di Fase II (solfo- e glucuronosil-trasferasi) all’interno della stessa linea cellulare. E’ stato possibile uniformare nuovamente popolazioni con diversa attività metabolica mediante coltivazione con concentrazioni minime di xenobiotico (nel nostro caso quercetina). Per studiare la biodisponibilità dei precursori sintetizzati è stato necessario mettere a punto innanzitutto un protocollo efficace per l’estrazione di quercetina e resveratrolo dal sangue. Questo lavoro ha messo in evidenza l’opportunità di eseguire l’analisi con campioni di sangue intero anzichè sul plasma sanguigno come viene generalmente fatto, per evitare sottostime dovute all’associazione dei polifenoli con la frazione cellulare. Poiché i polifenoli sono tutti scarsamente solubili in acqua, e la solubilità è un fattore determinante per la biodisponibilità di un composto, un primo precursore del resveratrolo è stato ottenuto funzionalizzando gli ossidrili con gruppi glucosio, attraverso un linker succinico (RGS); studi di farmacocinetica su questo composto sono in corso. Sono stati inoltre sintetizzati altri due derivati esterei (con gruppi metile o polietilenglicole), e due meno suscettibili all’idrolisi (metil-etere e mesilato). I risultati ottenuti dagli studi di stabilità in sangue, di assorbimento con intestino ex vivo e di farmacocinetica hanno evidenziato l’eccessiva instabilità dei legami esterei in questi contesti biologici. Una seconda parte del progetto è nata dalla considerazione che l’azione dei polifenoli può essere potenziata non solo cercando di innalzare il loro livello sistemico, ma anche accumulandoli specificamente in opportuni siti di azione. I polifenoli sono infatti molecole redox-attive, e i radicali sono coinvolti in molte patologie. Sito principale di produzione dei radicali sono i mitocondri, che sono inoltre la sede di eventi chiave nella morte cellulare. Importante per la genesi di questa parte del progetto è stata l’osservazione che la quercetina è in grado di inibire o indurre l’apertura del poro di transizione di permeabilità mitocondriale, a seconda che prevalga la sua attività anti- o pro-ossidante. Sono stati quindi sintetizzati dei derivati di quercetina e resveratrolo in grado di accumularsi nei mitocondri grazie alla funzionalizzazione con un gruppo trifenilfosfonio (TPP+), un catione in grado di diffondere attraverso le membrane biologiche e di accumularsi in regioni a potenziale negativo, quali la matrice mitocondriale. Una prima indagine su quale sia l’effetto biologico di questi nuovi composti è stata eseguita con dei derivati della quercetina e mitocondri isolati; questi composti si sono rivelati potenziali co-induttori della transizione di permeabilità mitocondriale, nonché inibitori della respirazione e dell’ATP sintasi mitocondriale. E’ stato quindi condotto uno studio preliminare per saggiare la possibile azione citostatica/citotossica dei derivati mitocondriotropici e di alcuni altri costrutti prodotti nel corso del progetto, su colture cellulari tumorali e non. Da questi esperimenti è emerso come alcuni dei derivati abbiano il comportamento tipico degli agenti chemioterapici