1,721,005 research outputs found
Come sfruttare il potenziale farmacologico dei polifenoli vegetali: modificazioni reversibili per aumentarne la biodisponibilità/bioefficacia
Full text linkThe 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
Resveratrol and health: the starting point.
No full textCascade of youth? Resveratrol, the celebrated phytoalexin of red wine, was known to activate AMPK indirectly, but how this happened was unclear. In a paper recently published in Cell, S.-J. Park, J. H. Chung and co-workers identify the signalling cascade, which begins with the inhibition of phosphodiesterases, in particular PDE4. But questions remain, even while new perspectives open up
Synthesis of resveratrol sulfates: turning a nightmare into a dream
No full textAbstract The growing interest in the bioactivity of natural polyphenols and of their metabolites requires pure metabolites to be used in bioassays and as standards in analytical protocols. We report here the synthesis of all five resveratrol sulfates achieved using a known approach of selective protection via silyl ether and sulfation but new reaction conditions and isolation procedures, which result in simplified protocols and greatly improved yields. A rationale for the problems so far encountered in handling and isolating resveratrol sulfates is provided as well as a solution based on avoidance of low pressure conditions during purification/isolation. These conclusions are probably of more general scope and might be usefully taken into consideration for obtaining other phenolic sulfates in high yield
Nanocostrutti comprendenti nanoparticelle lipidiche solide rivestite da un guscio idrofilo
No full textSynthesis and Testing of Novel Isomeric Mitochondriotropic Derivatives of Resveratrol and Quercetin
No full textPeptides as Pharmacological Carriers to the Brain: Promises, Shortcomings and Challenges
Full text linkCentral nervous system (CNS) diseases are among the most difficult to treat, mainly because the vast majority of the drugs fail to cross the blood-brain barrier (BBB) or to reach the brain at concentrations adequate to exert a pharmacological activity. The obstacle posed by the BBB has led to the in-depth study of strategies allowing the brain delivery of CNS-active drugs. Among the most promising strategies is the use of peptides addressed to the BBB. Peptides are versatile molecules that can be used to decorate nanoparticles or can be conjugated to drugs, with either a stable link or as pro-drugs. They have been used to deliver to the brain both small molecules and proteins, with applications in diverse therapeutic areas such as brain cancers, neurodegenerative diseases and imaging. Peptides can be generally classified as receptor-targeted, recognizing membrane proteins expressed by the BBB microvessels (e.g., Angiopep2, CDX, and iRGD), "cell-penetrating peptides" (CPPs; e.g. TAT(47-57), SynB1/3, and Penetratin), undergoing transcytosis through unspecific mechanisms, or those exploiting a mixed approach. The advantages of peptides have been extensively pointed out, but so far few studies have focused on the potential negative aspects. Indeed, despite having a generally good safety profile, some peptide conjugates may display toxicological characteristics distinct from those of the peptide itself, causing for instance antigenicity, cardiovascular alterations or hemolysis. Other shortcomings are the often brief lifetime in vivo, caused by the presence of peptidases, the vulnerability to endosomal/lysosomal degradation, and the frequently still insufficient attainable increase of brain drug levels, which remain below the therapeutically useful concentrations. The aim of this review is to analyze not only the successful and promising aspects of the use of peptides in brain targeting but also the problems posed by this strategy for drug delivery
Synthesis and Evaluation as Prodrugs of Hydrophilic Carbamate Ester Analogues of Resveratrol
No full textResveratrol (3,5,4'-trihydroxy-trans-stilbene) is an unfulfilled promise for health care: its exploitation is hindered by rapid conjugative metabolism in enterocytes and hepatocytes; low water solubility is a serious practical problem. To advantageously modify the physicochemical properties of the compound we have developed prodrugs in which all or part of the hydroxyl groups are linked via an N-monosubstituted carbamate ester bond to promoieties derived from glycerol or galactose, conferring higher water solubility. Kinetic studies of hydrolysis in aqueous solutions and in blood indicated that regeneration of resveratrol takes place in an appropriate time frame for delivery via oral administration. Despite their hydrophilicity some of the synthesized compounds were absorbed in the gastrointestinal tract of rats. In these cases the species found in blood after administration of a bolus consisted mainly of partially deprotected resveratrol derivatives and of the products of their glucuronidation, thus providing proof-of-principle evidence of behavior as prodrugs. The soluble compounds largely reached the lower intestinal tract. Upon administration of resveratrol, the major species found in this region was dihydroresveratrol, produced by enzymes of the intestinal flora. In experiments with a fully protected (trisubstituted) deoxygalactose containing prodrug, the major species were the prodrug itself and partially deprotected derivatives, along with small amounts of dihydroresveratrol. We conclude that the N-monosubstituted carbamate moiety is suitable for use in prodrugs of polyphenols
Acetal Derivatives as Prodrugs of Resveratrol.
No full textThe pharmacological exploitation of resveratrol is hindered by rapid phase-II conjugative metabolism in enterocytes and hepatocytes. One approach to the solution of this problem relies on prodrugs. We report the synthesis and characterization as well as the assessment of in vivo absorption and metabolism of a set of prodrugs of resveratrol in which the OH groups are engaged in the formal (−OCH2OR) or the more labile acetal (−OCH(CH3)OR) linkages. As carrier group (R) of the prodrug, we have used short ethyleneglycol oligomers (OEG) capped by a terminal methoxy group: −O–(CH2CH2O)n–CH3 (n = 0, 1, 2, 3, 4, 6). These moieties are expected to exhibit, to a degree, the favorable properties of longer polyethyleneglycol (PEG) chains, while their relatively small size makes for a more favorable drug loading capacity. After administration of formal-based prodrugs to rats by oral gavage, significant concentrations of derivatives were measured in blood samples over several hours, in all cases except for n = 0. Absorption was maximal for n = 4. Complete deprotection to give resveratrol and its metabolites was however too slow to be of practical use. Administration of the acetal prodrug carrying tetrameric OEG chains resulted instead in the protracted presence of resveratrol metabolites in blood, consistent with a progressive regeneration of the parent molecule from the prodrug after its absorption. The results suggest that prodrugs of polyphenols based on the acetal bond and short ethyleneglycol oligomers of homogeneous size may be a convenient tool for the systemic delivery of the unconjugated parent compound
Mitochondria-targeted resveratrol derivatives act as cytotoxic pro-oxidants.
No full textResveratrol derivatives bearing an O-linked mitochondria-targeting 4-triphenylphosphoniumbutyl group at either position 3 or position 4' are prooxidant and cytotoxic for cultured cells, selectively killing fast-growing cells when supplied in the low mu M range. Resveratrol is essentially without effect under these experimental conditions, while the cytotoxicity of the mitochondriotropic derivatives increases if they are methylated on the remaining hydroxyls. Experiments with Bax(-/-)/Bak(-/-) cells and a pan-caspase inhibitor show that cell death is mostly of the necrotic type. Cytotoxicity is due to ROS produced upon accumulation of the compounds into mitochondria, and specifically to H2O2, since externally added membrane-permeant catalase largely prevents cell death while superoxide dismutase potentiates toxicity. The mitochondriotropic compounds cause ROS-independent depolarization of in situ mitochondria. Effectiveness is increased if resveratrol hydroxyls are acetylated or methylated; this excludes the involvement of autooxidation of the polyphenolic nucleus and a protonophoric cycle as the causes of ROS generation and of depolarization, respectively. Resveratrol-triphenylphosphonium conjugates may thus represent a new class of chemotherapeutic agents, redox-active "mitocans", whose mechanisms of action and in vivo activity are worthy of further investigation
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