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SINTESI E VALUTAZIONE BIOLOGICA DI NUOVI AGENTI POTENZIALMENTE UTILI NELLA TERAPIA DEL MELANOMA MALIGNO
Full text linkMalignant melanoma is an extremely aggressive tumour, which originates from the neoplastic transformation of melanocytes. The therapy of metastatic melanoma, whose incidence is dramatically increasing, continues to be a challenge since, regardless of the treatment approach used (chemotherapy, immunotherapy or immuno-chemotherapy), a long-term survival is quite uncommon.
In an attempt to improve the effectiveness of the anticancer drugs currently available and to decrease their systemic toxicity, in addition to exploit some biochemical characteristics rather specific of the melanoma cells, a useful approach might be also the use of prodrugs, targeted to the tumor cells, that would release the active drug directly into the tumor mass and/or their surrounding extracellular environment.
Aim of my Ph.D. work was the synthesis and the biological evaluation of three types of new compounds, designed as possible agents useful in an anti-melanoma therapy.
The first compound, which we synthesized and tested, was a peptide-paclitaxel conjugate containing three functional domains: a “targeting domain”, an “activation sequence” and the cytotoxic agent paclitaxel. The “targeting domain”, whose function was to direct the conjugate to the tumour mass, was represented by a cyclic peptide containing the RGD sequence that can bind selectively the αVβ3 integrin, a surface molecule overexpressed by both metastatic human melanoma cells and endothelial cells of tumour vessels. The “activation sequence”, which should allow a selective release of paclitaxel within the tumour mass, was represented by a short peptide, substrate of cathepsin B (a lysosomal and pericellular protease highly up-regulated in malignant tumours, including human melanomas). The third functional domain consisted of the anticancer drug paclitaxel.
The second type of prodrug, which we designed and tested, was a 4-mercaptophenol derivative, containing a butenone moiety, the 4-[(4-hydroxyphenyl)sulfanyl]-3-buten-2-one, obtained in its E and Z geometric isomers. This compound has been designed considering that: a) the 4-mercaptophenol moiety would be a good substrate for tyrosinase, an enzyme expressed at high levels in melanoma cells, where it is involved in the biosynthesis of melanin, and able to oxidize a variety of natural and synthetic phenols, giving rise to alkylating and cytotoxic o-quinones; b) an α,β unsaturated side chain, reactive towards GSH (present in relatively high concentrations in melanoma cells) would decrease cellular antioxidant defense against the oxygen free radicals (ROS) generated as by-products during melanin synthesis, in normal as well in tumor cells. Therefore, our prodrug should act as a bifunctional agent, capable of generating cytotoxic o-quinone species (following its oxidation by tyrosinase) and reducing GSH levels.
A third part of the present work has focused on the study of the naftoquinone alkannin and its two acetylated derivatives (1’-acetyl alkannin and 5,8,1’-triacetyl alkannin), as potential inhibitors of the human glutathione-S-transferase (GST) P1-1. This enzyme, which catalyzes the conjugation of GSH to a wide range of nucleophilic compounds, is expressed at high levels in many tumor cells, including melanoma cells, and is considered one of the factors responsible of tumor resistance towards anti-cancer agents. Therefore, a specific inhibitor of this enzyme could be useful in cancer therapy, making the tumor cells more sensitive to chemotherapeutic agents.Il melanoma maligno è un tumore molto aggressivo, la cui incidenza è in costante aumento, che origina dalla trasformazione di cellule chiamate melanociti. La terapia del melanoma metastatico continua ad essere una difficile sfida, in quanto, indipendentemente dall’approccio terapeutico utilizzato (chemioterapia, immunoterapia o immuno-chemioterapia), i soggetti affetti raramente presentano una sopravvivenza a lungo termine. Per migliorare l'efficacia dei farmaci antitumorali attualmente disponibili e per diminuirne la tossicità sistemica, oltre a sfruttare alcune caratteristiche biochimiche relativamente specifiche delle cellule tumorali, e del melanoma in particolare, un approccio utile potrebbe anche essere quello di indirizzare verso le cellule tumorali dei profarmaci, che rilascino il farmaco attivo direttamente all’interno della massa tumorale e/o nel loro intorno extracellulare.
I risultati riportati in questa tesi si riferiscono alla sintesi e alla valutazione biologica di tre nuovi tipi di composti progettati in questa ottica verso le cellule di melanoma.
Il primo composto studiato è stato un coniugato peptidico del paclitaxel contenente tre domini funzionali: un dominio di "indirizzamento", una sequenza di "attivazione" e il paclitaxel, appunto, quale agente citotossico. Il dominio di “indirizzamento", la cui funzione era quella di dirigere il coniugato verso la massa tumorale, era rappresentato da un peptide ciclico contenente la sequenza RGD, in grado di legarsi selettivamente e con alta affinità all’integrina αVβ3, una molecola di superficie sovra-espressa sia dalle cellule di melanoma che dalle cellule endoteliali dei nuovi vasi tumorali. La sequenza di “attivazione ", che avrebbe dovuto consentire un rilascio selettivo del paclitaxel all’interno della massa tumorale, era rappresentata da una sequenza peptidica substrato della catepsina B, una proteasi a localizzazione lisosomiale e associata a membrana, altamente sovraespressa in molti tumori, compreso il melanoma. Il terzo dominio funzionale era costituito dal farmaco antitumorale vero e proprio, cioè dal paclitaxel, il cui legame con il peptide - nelle aspettative - avrebbe dovuto causarne la momentanea inattivazione fino al momento della sua effettiva liberazione in sede tumorale.
Il secondo profarmaco testato è stato un derivato del 4-mercaptofenolo contenente una catena laterale butenonica, il 4-[(4-idrossifenil)sulfanil]-3-buten-2-one, ottenuto nei suoi due isomeri geometrici E e Z. Questo composto è stato progettato considerando che: a) la parte derivata dal 4-mercaptofenolo avrebbe dovuto essere un buon substrato per l’enzima tirosinasi (TYRase), un enzima espresso ad alti livelli nelle cellule di melanoma, perchè coinvolto nel processo di biosintesi della melanina, e in grado di ossidare una varietà di fenoli naturali e di sintesi, dando luogo a forme alchilanti e citotossiche come gli o-chinoni; b) la catena laterale butenonica (α,β insatura), essendo dotata di elevata reattività nei confronti del GSH, presente in concentrazioni relativamente elevate nelle cellule di melanoma, avrebbe dovuto sottrarre GSH alla cellula stessa, indebolendone di fatto le difese antiossidanti nei confronti dei radicali liberi dell’ossigeno (ROS) che si generano, come sottoprodotti, durante la sintesi di melanina sia in cellule normali che tumorali. Nelle aspettative, il profarmaco così progettato si sarebbe dovuto comportare come un agente bifunzionale in grado di formare o-chinoni citotossici in seguito alla sua ossidazione da parte della tirosinasi e in grado di ridurre al tempo stesso i livelli di GSH.
Infine, un terzo approccio ha riguardato lo studio dell’alcannina (colorante naturale a struttura naftochinonica) e di due suoi derivati acetilati, quali possibili inibitori dell’enzima umano glutatione-S-transferasi (GST) di tipo P1-1, enzima espresso ad alti livelli in molti tumori tra cui appunto il melanoma. GSTP1-1 è un enzima coinvolto nel metabolismo di fase II degli xenobiotici, in quanto catalizza la coniugazione del GSH ad una vasta gamma di composti nucleofili, ma esso è anche ritenuto uno dei fattori promuoventi la farmaco-resistenza del tumore. Un inibitore specifico di questo enzima potrebbe essere quindi estremamente utile in una terapia antitumorale, rendendo le cellule tumorali più sensibili agli agenti chemioterapici
Enzyme induction is independent on liver functional status
No full textBackground and objectives: Drug-drug interactions have became an important issue in health care. Since metabolism is the major pathway of elimination of drugs, and cytochromes P450 (CYPs) are the most common enzymes involved, most drug-drug interactions arise from either inhibition or induction of CYP enzymes. Although the effect of liver disease on the magnitude of drug interactions consequent upon enzyme induction has been extensively investigated, highly discrepant results have been obtained.Human studies, because of insurmountable ethic problems (the impossibility to administer repeated doses of a non-therapeutic drug to patients with serious liver dysfunction), had to use hepatopathic patients taking inducers for therapeutic purposes and, consequently, often lack rigorous methodology. Therefore, animal studies are necessary to clarify the influence of liver disease on CYP induction. This study was designed to compare enzyme induction in animals with normal and impaired liver function,rigorously stratified according to the degree of liver dysfunction.
Methods: To reach this goal, we used 3 groups of animals: normal rats and two groups of rats with experimentally-induced cirrhosis, obtained by treatment with carbon tetrachloride (CCl4) administered by inhalation; one group with compensated (corresponding to human Child grade A or B) liver cirrhosis, the other group with decompensated (human Child grade C) liver cirrhosis. Each group was divided in 2 subgroups of 6 rats, one treated with vehicle (olive oil), the other with the inducing agent benzo[α]pyrene (bap). The inducing effect was assessed by measuring the activity of the prototypical enzymes induced by bap, CYP1A1 and CYP1A2, using liver microsomes obtained from normal and CCl4-treated rats. 7-ethoxyresorufin and 7-methoxyresorufin (both metabolized to resorufin) were used as validated probes for CYP1A1 and CYP1A2, respectively.The severity of liver cirrhosis was defined on the basis of histological examination (Ishak score), the presence or absence of ascites and laboratory data (PT, albumin, AST and ALT).
Results and conclusion: Total CYP content per gram of liver tissue decreased in proportion to the decline of liver function (14.04 ± 3.20 nmol/g in normal rats vs 11.00 ± 1.99 nmol/g and 9.16 ± 1.08 nmol/g in rats with compensated and decompensated cirrhosis, respectively). In vehicle-treated rats, Vmax for ethoxyresorufin-7-dealkylase (CYP1A1 marker reaction) decreased from 570.2 ± 118.5 nmol/min/mg protein in normal animals to 229.8 ± 64.3 nmol/min/mg protein and 199.6 ± 29.6 nmol/min/mg protein in rats with compensated and decompensated cirrhosis, respectively. Intrinsic metabolic clearance (CLint) decreased in a similar fashion. Treatment with bapincreased Vmax to 6282 ± 1231 nmol/min/mg protein in normal rats,and even higher induction was observed in rats with compensated and decompensated cirrhosis. In vehicle-treated rats, Vmax for methoxyresorufin-7-dealkylase (CYP1A2 marker reaction) decreased from 78.8 ± 23.9 nmol/min/mg protein in normal animals to 39.3 ± 9.2 nmol/min/mg protein and 29.5 ± 6.1 nmol/min/mg protein in rats with compensated and decompensated cirrhosis, respectively, and a similar decrease was observed for CLint. In the bap-treated rats, Vmax of normal animals was 8 times higher (635 ± 168 nmol/min/mg protein) than in the vehicle-treated groups, and a greater inducing effect was again observed in cirrhotic rats.
In conclusion, our study clearly show that, at variance with the results of some previous human studies, enzyme induction does not decrease with the decline of liver function. Determination of CYP1A1, CYP1A2 and AhR mRNA and of their protein expression (by Western blot) is in progress in our laboratory to confirm these findings
Flavonoids diosmetin and luteolin inhibit midazolam metabolism by human liver microsomes and recombinant CYP 3A4 and CYP3A5 enzymes
No full textWe evaluated the effects of increasing concentrations of the flavonoids salvigenin, diosmetin and luteolin on the in vitro metabolism of midazolam (MDZ), a probe substrate for cytochrome P450 (CYP) 3A enzymes, which is converted into 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) by human liver microsomes. Salvigenin had only a modest effect on MDZ metabolism, whereas diosmetin and luteolin inhibited in a concentration-dependent manner the formation of both 1'-OH-MDZ and 4-OH-MDZ, with apparent K(i) values in the 30-50mumol range. Both diosmetin and luteolin decreased 1'-OH-MDZ formation by human recombinant CYP3A4, but not CYP3A5, whereas they decreased 4-OH-MDZ formation by both recombinant enzymes. To assess whether any relationship exists between the physico-chemical characteristics of flavones and their effects on MDZ metabolism, we tested the effects of three other flavones (flavone, tangeretin, chrysin) on MDZ metabolism by human liver microsomes. Whereas flavones possessing more than two hydroxyl groups (luteolin, diosmetin) inhibited MDZ biotransformation, flavones lacking hydroxyl groups in their A and B rings (flavone, tangeretin) stimulated MDZ metabolism. We also found close relationships between the maximum stimulatory or inhibitory effects of flavones on 1'-OH-MDZ and 4-OH-MDZ formation rates and their log of octanol/water partition coefficients (logP) or their total number of hydroxyl groups. The results of the study may be of clinical relevance since they suggest that luteolin and diosmetin may cause pharmacokinetic interactions with co-administered drugs metabolized via CYP3A
Relative contribution of cytochrome P450 3A to midazolam oxidation in cattle liver microsomes.
No full textINTRODUCTION
In humans and rodents, members of the cytochrome P450 3A subfamily (CYP3A) are major contributors to midazolam (MDZ)
biotransformation into 1-hydroxy-MDZ (1-OHMDZ) and 4-hydroxy-MDZ (4-OHMDZ), and 1-OHMDZ activity is commonly
used as a surrogate marker for CYP3A in humans. In veterinary species, it is still crucial to identify isoform- and speciesspecific
CYP substrates, to better characterize drug biotransformation and potential for drug–drug-interactions. The aim of
this study was to characterize MDZ oxidation in cattle liver microsomes.
MATERIALS AND METHODS
Pooled microsomes were prepared from the liver of male Piedmontese beef cattle, and the formation of 1-OHMDZ and 4-
OHMDZ was evaluated using a slightly modified HPLC-UV method; all the incubations were carried out under linear conditions
of metabolite formation, with respect to incubation time and microsomal protein concentration. A confirmatory immunoinhibition
study was performed by pre-incubating the pooled liver microsomes with increasing amounts of a polyclonal antibody
raised against rat CYP3A1. Finally, MDZ hydroxylation was evaluated in 300 single-donor Piedmontese cattle liver microsomes,
and analyzed for correlation with 6b-hydroxylation of testosterone (TST).
RESULTS AND CONCLUSIONS
Under the adopted chromatographic conditions, 4-OHMDZ, 1-OHMDZ and MDZ were eluted and well separated; the retention
times were 13.9, 15.3 and 20.1 min, respectively. Formation of both metabolites conformed to single-enzyme Michaelis-Menten
kinetics; Vmax and Km values were 665 pmol/min/mg protein
and 6.16 micromolar for 4-OHMDZ, and 64 pmol/min/mg protein and 10.08 micromolar for 1-OHMDZ. The anti-rat CYP3A1
polyclonal antibody inhibited 4-OHMDZ formation up to 94%; however, only a 50% inhibition was noticed for 1-OHMDZ.
The rates of formation of 4-OHMDZ and 6b-OHTST in single donor liver microsomes were poorly correlated. In conclusion,
cattle liver microsomes are capable of metabolizing MDZ to 1-OHMDZ and 4-OHMDZ. Furthermore, the immunoinhibition
results indicate a major contribution of cattle CYP3A to 4-OHMDZ formation, while other CYPs might be involved in
drug oxidation to 1-OHMDZ. Finally, the observed poor relationship between 6b-OHTST and 4-OHMDZ deserve further
investigation to clarify the specific role played either by individual cattle CYP3A isoforms or other CYPs in MDZ and TST
hydroxylation.
ACKNOWLEDGEMENTS
Project supported by MIUR (2009ZE5HJP)
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Regulation of SIRT1 in Vascular Smooth Muscle Cells from Streptozotocin-Diabetic Rats
Full text linkSirtuins enzymes are a conserved family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases and ADP-ribosyltransferases that mediate responses to oxidative stress, fasting and dietary restriction in mammals. Vascular smooth muscle cells (VSMCs) are involved in many mechanisms that regulate vascular biology in vivo but the role of SIRT1 has not been explored in much detail. Therefore, we investigated the regulation of SIRT1 in cultured VSMCs under various stress conditions including diabetes. Sprague-Dawley rats were made diabetic by injecting a single dose of streptozotocin (65 mg/Kg), and aortic VSMCs were isolated after 4 weeks. Immunocytochemistry showed that SIRT1 was localized predominantly in the nucleus, with lower staining in VSMCs from STZ-diabetic as compared with normoglycemic rats. Previous diabetes induction in vivo and high glucose concentrations in vitro significantly downregulated SIRT1 amounts as detected in Western blot assays, whereas TNF-α (30 ng/ml) stimulation failed to induce significant changes. Because estrogen signaling affects several pathways of oxidative stress control, we also investigated SIRT1 modulation by 17β-estradiol. Treatment with the hormone (10 nM) or a selective estrogen receptor-α agonist decreased SIRT1 levels in VSMCs from normoglycemic but not in those from STZ-diabetic animals. 17β-estradiol treatment also enhanced activation of AMP-dependent kinase, which partners with SIRT1 in a signaling axis. SIRT1 downregulation by 17β-estradiol could be observed as well in human peripheral blood mononuclear cells, a cell type in which SIRT1 downregulation is associated with insulin resistance and subclinical atherosclerosis. These data suggest that SIRT1 protein levels are regulated by diverse cellular stressors to a variable extent in VSMCs from diabetic and normoglycemic rats, warranting further investigation on SIRT1 as a modulator of VSMC activity in settings of vascular inflammation
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