Basi farmacologiche per l'impiego del solfuro d'idrogeno nella chemioterapia del carcinoma pancreatico

Riassunto analitico L’adenocarcinoma pancreatico duttale (PDAC) rappresenta la forma più comune di tumore del pancreas e figura tra le principali cause di morte per neoplasia nei paesi industrializzati; il ridotto tasso di sopravvivenza nei cinque anni successivi alla diagnosi (inferiore al 5 %) e l’elevato numero di morti per anno, sono poi indice di una prognosi decisamente infausta. Ad oggi, le uniche due possibilità di cura per pazienti affetti da carcinoma pancreatico sono rappresentate dall’intervento chirurgico (praticabile soltanto nel 10 % dei casi e caratterizzato da un alto rischio di complicanze piuttosto gravi) e dal trattamento farmacologico con Gemcitabina, analogo nucleosidico di uso consolidato in chemioterapia ma che fornisce scarsi risultati nel caso del tumore del pancreas; per questi motivi, è stata presa recentemente in considerazione la possibilità di co-somministrare Gemcitabina con altre molecole, nell’ottica di rendere le cellule più vulnerabili all’azione del chemioterapico di riferimento consentendo, allo stesso tempo, un regime posologico meno aggressivo e più efficace rispetto a quello attuale. In questo lavoro di tesi è stata focalizzata particolare attenzione sugli isotiocianati, composti di origine naturale ampiamente distribuiti nelle verdure edibili della famiglia delle Brassicaceae (tra cui broccoli, cavolo, cavolfiore, senape bianca e senape nera) che, in precedenti studi, si sono dimostrati in grado di esercitare un marcato effetto protettivo nei confronti di numerose neoplasie indotte da agenti chimici, comprese quelle del pancreas; a concentrazioni maggiori, inoltre, queste molecole sembrano espletare una spiccata azione anti-proliferativa e citotossica nei confronti di molteplici linee cellulari tumorali. In alcuni studi in vitro, diversi isotiocianati di origine naturale e sintetica sono stati poi riconosciuti come potenziali donatori di solfuro di idrogeno, mediatore endogeno di natura gassosa che, insieme a NO e CO, esplica svariate funzioni fisiologiche e fisiopatologiche; a concentrazioni più elevate, anche H2S possiede spiccate proprietà anti-proliferative e citotossiche, decisamente simili a quelle riportate in letteratura per molti isotiocianati. Alla luce di questa osservazione, risulta ragionevole ipotizzare che la loro azione su specifici sistemi biologici sia in gran parte attribuibile al probabile rilascio di solfuro di idrogeno a livello tissutale e, dunque, la valutazione di potenziali azioni antitumorali additive e non di derivati sintetici degli isotiocianati naturali ha rappresentato l’obiettivo principale del presente lavoro di tesi; in particolare, sono stati studiati gli effetti in vitro del composto di derivazione commerciale p-carbossifenilisotiocianato (p-COOHPhNCS) sulla linea cellulare di metastasi di adenocarcinoma pancreatico umano (AsPC-1). In una prima fase, sono state testate diverse concentrazioni di p-COOHPhNCS per studiarne l’effetto sulla vitalità (quantificata tramite saggio colorimetrico con WST-1) e per identificare, al contempo, una concentrazione d’elezione tale da provocare una significativa ma parziale riduzione della sopravvivenza cellulare; nel range di concentrazioni testate, il composto in esame ha determinato una significativa inibizione concentrazione-dipendente della proliferazione cellulare dopo 72 h di incubazione. Le doti di p-COOHPhNCS quale H2S donor, precedentemente registrate in vitro, sono state poi investigate su cellula per verificare il rilascio di solfuro d’idrogeno anche sul substrato biologico d’interesse (AsPC-1) attraverso una misurazione fluorimetrica con sonda WSP-1; questo nuovo metodo ha consentito di comprovare una reale generazione di H2S da parte di p-COOHPhNCS, avvalorando l’ipotesi secondo cui la liberazione di solfuro d’idrogeno possa contribuire ampiamente all’effetto antiproliferativo dell’isotiocianato. Successivamente, sono state testate concentrazioni crescenti di Gemcitabina (farmaco di riferimento nella terapia del tumore pancreatico) per identificarne una capace di abbattere in modo consistente la proliferazione cellulare senza causare un’eccessiva citotossicità; così come p-COOHPhNCS, anche Gemcitabina ha mostrato una significativa attività anti-proliferativa concentrazione-dipendente su cellule AsPC-1. In seguito, è stata valutata l’azione della co-somministrazione di concentrazioni scelte di Gemcitabina e p-COOHPhNCS sulla vitalità cellulare dopo incubazione di 72 h, al fine di evidenziare un potenziale effetto additivo/sinergico tra le due molecole; il trattamento in associazione ha permesso di osservare un effetto inibitorio sulla proliferazione di cellule AsPC-1 molto più marcato rispetto a quello raggiunto dopo incubazione dei singoli composti. Un’ultima fase dello studio è stata poi dedicata all’analisi di p-COOHPhNCS e Gemcitabina sul ciclo cellulare, sia separatamente che in associazione, in modo da identificare eventuali azioni anti-proliferative ciclo-specifiche (l’analisi dei campioni è stata effettuata con un microcitofluorimetro dopo 72 h di incubazione con il composto selezionato). I dati ottenuti hanno evidenziato uno spostamento significativo e quantitativamente rilevante del numero di cellule in fase G0/G1 dopo trattamento con p-COOHPhNCS ed un aumento altrettanto significativo del numero di cellule verso la fase S del ciclo dopo incubazione con il chemioterapico Gemcitabina; la co-somministrazione dei due composti, invece, si è rivelata in grado di indurre un effetto quali-quantitativamente analogo a quello mostrato dalla sola Gemcitabina ed ha permesso di avanzare l’ipotesi secondo cui p-COOHPhNCS possa sensibilizzare le cellule tumorali al trattamento con Gemcitabina facilitando così l’azione antiproliferativa del chemioterapico di riferimento

Development of In Vitro Corneal Models: Opportunity for Pharmacological Testing

The human eye is a specialized organ with a complex anatomy and physiology, because it is characterized by different cell types with specific physiological functions. Given the complexity of the eye, ocular tissues are finely organized and orchestrated. In the last few years, many in vitro models have been developed in order to meet the 3Rs principle (Replacement, Reduction and Refinement) for eye toxicity testing. This procedure is highly necessary to ensure that the risks associated with ophthalmic products meet appropriate safety criteria. In vitro preclinical testing is now a well-established practice of significant importance for evaluating the efficacy and safety of cosmetic, pharmaceutical, and nutraceutical products. Along with in vitro testing, also computational procedures, herein described, for evaluating the pharmacological profile of potential ocular drug candidates including their toxicity, are in rapid expansion. In this review, the ocular cell types and functionality are described, providing an overview about the scientific challenge for the development of three-dimensional (3D) in vitro models

Searching for novel hydrogen sulfide donors: The vascular effects of two thiourea derivatives

The gasotransmitter hydrogen sulfide (H2S) is involved in the regulation of the vascular tone and an impairment of its endogenous production may play a role in hypertension. Thus, the administration of exogenous H2S may be a possible novel and effective strategy to control blood pressure. Some natural and synthetic sulfur compounds are suitable H2S-donors, exhibiting long-lasting H2S release; however, novel H2S-releasing agents are needed to improve the pharmacological armamentarium for the treatment of cardiovascular diseases. For this purpose, N-phenylthiourea (PTU) and N,N'-diphenylthiourea (DPTU) compounds have been investigated as potential H2S-donors. The thioureas showed long-lasting H2S donation in cell free environment and in human aortic smooth muscle cells (HASMCs). In HASMCs, DPTU caused membrane hyperpolarization, mediated by activation of KATP and Kv7 potassium channels. The thiourea derivatives promoted vasodilation in rat aortic rings, which was abolished by KATP and Kv7 blockers. The vasorelaxing effects were also observed in angiotensin II-constricted coronary vessels. In conclusion, thiourea represents an original H2S-donor functional group, which releases H2S with slow and long lasting kinetic, and promotes typical H2S-mediated vascular effects. Such a moiety will be extremely useful for developing original cardiovascular drugs and new chemical tools for investigating the pharmacological roles of H2S

Potential Effects of Natural H2S-Donors in Hypertension Management

After the discovery of hydrogen sulfide (H2S) in the central nervous system by Abe and Kimura in 1996, the physiopathological role of H2S has been widely investigated in several systems such as the cardiovascular. In particular, H2S plays a pivotal role in the control of vascular tone, exhibiting mechanisms of action able to induce vasodilation: for instance, activation of potassium channels (KATP and Kv7) and inhibition of 5-phosphodiesterase (5-PDE). These findings paved the way for the research of natural and synthetic exogenous H2S-donors (i.e., molecules able to release H2S) in order to have new tools for the management of hypertension. In this scenario, some natural molecules derived from Alliaceae (i.e., garlic) and Brassicaceae (i.e., rocket or broccoli) botanical families show the profile of slow H2S-donors able to mimic the endogenous production of this gasotransmitter and therefore can be viewed as interesting potential tools for management of hypertension or pre-hypertension. In this article, the preclinical and clinical impacts of these natural H2S-donors on hypertension and vascular integrity have been reviewed in order to give a complete panorama of their potential use for the management of hypertension and related vascular diseases

Antioxidant and Antisenescence Effects of Bergamot Juice

Aging is one of the main risk factor for the onset of cardiovascular diseases; one of the possible explanations could be linked to the age-associated overproduction of free radicals. This increase of oxidative stress can be overcome with a high intake of food antioxidants. In this context, a number of studies have been addressed to assess the antiaging potential of natural antioxidant compounds. Recently, it has been shown that the juice of bergamot (Citrus bergamia Risso et Poiteau), a fruit mostly produced in the Ionian coastal areas of Southern Italy (Calabria), is a valuable source of health-promoting constituents with, among other, antioxidant properties. In order to investigate the potential antiaging effects of this Mediterranean natural antioxidant source, bergamot juices of three different cultivars ("fantastico," "femminello," and "castagnaro") were herein characterized by the mean of high-performance liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry. Then, juices were investigated for the evaluation of total polyphenolic and flavonoid contents, cell-free model antioxidant activities, and in vitro antiaging properties on two different cellular models of induced myocardial senescence. The best performing juice was also assessed in vivo. The phytochemical profiles confirmed that juices were rich in flavonoids, both flavone and flavanone glycosides. In addition, two limonoid glycosides were also identified in all cultivars. Each cultivar showed different phenolic and flavonoid contents. In tube results showed the juice robust antioxidant activities that correlate with their phenolic and flavonoid contents. Moreover, for the first time, the ability of juice to counteract the chemical-induced senescence was here demonstrated in both cellular models. Lastly, the in vivo data obtained from mouse hearts evidenced an increase in transcription of genes involved in antiaging and antioxidant responses. The overall results suggest that bergamot juice exerts antioxidant and antisenescence effects, making it useful for nutraceutical purposes

Design, Synthesis and Evaluation of Novel Molecular Hybrids between Antiglaucoma Drugs and H2S Donors

Glaucoma is a group of eye diseases consisting of optic nerve damage with corresponding loss of field vision and blindness. Hydrogen sulfide (H2S) is a gaseous neurotransmitter implicated in various pathophysiological processes. It is involved in the pathological mechanism of glaucomatous neuropathy and exerts promising effects in the treatment of this disease. In this work, we designed and synthetized new molecular hybrids between antiglaucoma drugs and H2S donors to combine the pharmacological effect of both moieties, providing a heightened therapy. Brinzolamide, betaxolol and brimonidine were linked to different H2S donors. The H2S-releasing properties of the new compounds were evaluated in a phosphate buffer solution by the amperometric approach, and evaluated in human primary corneal epithelial cells (HCEs) by spectrofluorometric measurements. Experimental data showed that compounds 1c, 1d and 3d were the hybrids with the best properties, characterized by a significant and long-lasting production of the gasotransmitter both in the aqueous solution (in the presence of L-cysteine) and in the intracellular environment. Because, to date, the donation of H2S by antiglaucoma H2S donor hybrids using non-immortalized corneal cells has never been reported, these results pave the way to further investigation of the potential efficacy of the newly synthesized compounds

Anti-Inflammatory Effect of the Natural H2S-Donor Erucin in Vascular Endothelium

Vascular inflammation (VI) represents a pathological condition that progressively affects the integrity and functionality of the vascular wall, thus leading to endothelial dysfunction and the onset of several cardiovascular diseases. Therefore, the research of novel compounds able to prevent VI represents a compelling need. In this study, we tested erucin, the natural isothiocyanate H2S-donor derived from Eruca sativa Mill. (Brassicaceae), in an in vivo mouse model of lipopolysaccharide (LPS)-induced peritonitis, where it significantly reduced the amount of emigrated CD11b positive neutrophils. We then evaluated the anti-inflammatory effects of erucin in LPS-challenged human umbilical vein endothelial cells (HUVECs). The pre-incubation of erucin, before LPS treatment (1, 6, 24 h), significantly preserved cell viability and prevented the increase of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-alpha) levels. Moreover, erucin downregulated endothelial hyperpermeability and reduced the loss of vascular endothelial (VE)-Cadherin levels. In addition, erucin decreased vascular cell adhesion molecule 1 (VCAM-1), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E-synthase 1 (mPGES-1) expression. Of note, erucin induced eNOS phosphorylation and counteracted LPS-mediated NF-kappa B nuclear translocation, an effect that was partially abolished in the presence of the eNOS inhibitor L-NAME. Therefore, erucin can control endothelial function through biochemical and genomic positive effects against VI

The Citrus Flavonoid Naringenin Protects the Myocardium from Ageing-Dependent Dysfunction: Potential Role of SIRT1

Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. In particular, it is implicated in ageing-related diseases, such as cardiac hypertrophy, myocardial infarct, and endothelial dysfunction; moreover, its expression decreases with age. Therefore, an effective strategy to extend the lifespan and improve cardiovascular function is the enhancement of the expression/activity of SIRT1 with exogenous agents. The Citrus flavonoid naringenin (NAR) presents structural similarity with the natural SIRT1 activator resveratrol. In this study, we demonstrate through in vitro assays that NAR significantly activates SIRT1 enzyme and shows antisenescence effects. The binding mode of NAR into SIRT1 was detailed investigated through in silico studies. Moreover, chronic administration (for six months) of NAR (100 mg/kg/day) to 6-month-old mice leads to an enhancement of SIRT1 expression and a marked reduction of reactive oxygen species production in myocardial tissue. Furthermore, at the end of the treatment, the plasma levels of two well-known markers of cardiovascular inflammation, TNF-α and IL6, are significantly reduced in 12-month-old mice treated with NAR, as well as the cardiovascular risk (total cholesterol/HDL ratio) compared to control mice. Finally, the age-associated fibrotic remodeling, which is well detected through a Mallory trichrome staining in the vehicle-treated 12-month-old mice, is significantly reduced by the chronic treatment with NAR. Moreover, an improvement of myocardium functionality is highlighted by the enhancement of citrate synthase activity and stabilization of the mitochondrial membrane potential after NAR treatment. Taken together, these results suggest that a nutraceutical approach with NAR may have positive impacts on many critical hallmarks of myocardial senescence, contributing to improve the cardiac performance in aged subjects

Pharmacological modulation of intracellular signalling pathways involved in oxi-inflammageing and age-related cardiovascular pathologies by naturally occurring molecules

In the last decades, the average lifespan has progressively increased. Ageing dramatically predisposes to the onset of cardiovascular disorders (i.e. hypertension, ischemic stroke and heart failure), which still represent the leading cause of mortality worldwide. Therefore, the identification of novel pharmacological strategies for the prevention of cardiovascular ageing is a timely challenge. In this context, SIRT1 enzyme plays a crucial role, since it elegantly regulates downstream pathways involved in cell senescence, oxidative stress and inflammation. Interestingly, many naturally occurring compounds, including polyphenols, are SIRT1 activators. In the first part of this study, the anti-ageing effects of the Citrus flavonoid naringenin have been demonstrated in the myocardium of mice; moreover, the beneficial effects of a Citrus bergamia juice in the aged heart have been also investigated. Recently, it has been also proposed that the gasotransmitter hydrogen sulfide (H2S) is an endogenous activator of SIRT1 in the cardiovascular system. Hence, in the second part of this thesis the preventive effects of the H2S-donor Erucin (an isothiocyanate from Eruca sativa Mill.) against vascular inflammation, hypertension and myocardial ischemia/reperfusion injury have been demonstrated both in vitro and in vivo. Moreover, the beneficial effects of dietary intake of an extract of Eruca sativa Mill. (titled in the glucosinolate glucoerucin) have been investigated in a murine model of metabolic syndrome. Results showed that a pharmacological/nutraceutical approach with Citrus polyphenols and isothiocyanates from Brassicaceae may be useful in both the prevention and treatment of age-related cardiovascular diseases

The Renal Outer Medullary Potassium channel (ROMK): an intriguing pharmacological target for an innovative class of diuretic drugs

In the last four decades, the several classes of diuretics, currently available for clinical use, have been the first line option for the therapy of widespread cardiovascular and non-cardiovascular diseases. Diuretic drugs generally exhibit an overall favourable risk/benefit balance. However, they are not devoid of side effects. In particular, all the classes of diuretics cause alteration of potassium homeostasis. In recent years, understanding of the physiological role of the renal outer medullary potassium (ROMK) channels, has shown an intriguing pharmacological target for developing an innovative class of diuretic agents: the ROMK inhibitors. This novel class is expected to promote diuretic activity comparable to (or even higher than) that provided by the most effective drugs used in clinics (such as furosemide), with limited effects on potassium homeostasis. In this review, the physio-pharmacological roles of ROMK channels in the renal function are reported, along with the most representative molecules which have been currently developed as ROMK inhibitors