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Ricercatore Universitario presso: Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103

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- “Nuovi modulatori allosterici positivi delrecettore AMPA” [Brevetto]
U. Battisti ; G. Cannazza ; G. Puja; Carozzo M.; Braghiroli D.; Parenti C.; Troisi L.; Jozwiak K


2018 - A Metabolomic Approach Applied to a LiquidChromatography Coupled to High-ResolutionTandem Mass Spectrometry Method (HPLC-ESI-HRMS/MS): Towards the ComprehensiveEvaluation of the Chemical Composition ofCannabis Medicinal Extracts [Articolo su rivista]
Citti, Cinzia; Battisti, UMBERTO MARIA; Braghiroli, Daniela; Ciccarella, Giuseppe; Schmid, Martin; Vandelli, Maria Angela; Cannazza, Giuseppe

Introduction – Cannabis sativa L. is a powerful medicinal plant and its use has recently increased for the treatment of several pa-thologies. Nonetheless, side effects, like dizziness and hallucinations, and long-term effects concerning memory and cognition,can occur. Most alarming is the lack of a standardised procedure to extract medicinal cannabis. Indeed, each galenical prepara-tion has an unknown chemical composition in terms of cannabinoids and other active principles that depends on the extractionprocedure.Objective – This study aims to highlight the main differences in the chemical composition of Bediol® extracts when the extractionis carried out with either ethyl alcohol or olive oil for various times (0, 60, 120 and 180 min for ethyl alcohol, and 0, 60, 90 and120 min for olive oil).Methodology.Cannabis medicinal extracts (CMEs) were analysed by liquid chromatography coupled to high-resolution tandem mass spec-trometry (LC–MS/MS) using an untargeted metabolomics approach. The data sets were processed by unsupervised multivariateanalysis.Results – Our results suggested that the main difference lies in the ratio of acid to decarboxylated cannabinoids, which dramat-ically influences the pharmacological activity of CMEs. Minor cannabinoids, alkaloids, and amino acids contributing to this differ-ence are also discussed. The main cannabinoids were quantified in each extract applying a recently validated LC–MS and LC-UVmethod.Conclusions – Notwithstanding the use of a standardised starting plant material, great changes are caused by different extractionprocedures. The metabolomics approach is a useful tool for the evaluation of the chemical composition of cannabis extracts.

2018 - Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA) [Articolo su rivista]
Citti, Cinzia; Pacchetti, Barbara; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe

Hemp seed oil from Cannabis sativa L. is a very rich natural source of important nutrients, not only polyunsaturated fatty acids and proteins, but also terpenes and cannabinoids, which contribute to the overall beneficial effects of the oil. Hence, it is important to have an analytical method for the determination of these components in commercial samples. At the same time, it is also important to assess the safety of the product in terms of amount of any psychoactive cannabinoid present therein. This work presents the development and validation of a highly sensitive, selective and rapid HPLC-UV method for the qualitative and quantitative determination of the main cannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG) and cannabidivarin (CBDV), present in 13 commercial hemp seed oils. Moreover, since decomposition of cannabinoid acids generally occurs with light, air and heat, decarboxylation studies of the most abundant acid (CBDA) were carried out in both open and closed reactor and the kinetics parameters were evaluated at different temperatures in order to evaluate the stability of hemp seed oil in different storage conditions.

2018 - Cell-penetrating CaCO3nanocrystals for improved transport of NVP-BEZ235 across membrane barrier in T-cell lymphoma [Articolo su rivista]
Vergaro, Viviana; Civallero, Monica; Citti, Cinzia; Cosenza, Maria; Baldassarre, Francesca; Cannazza, Giuseppe; Pozzi, Samantha; Sacchi, Stefano; Fanizzi, Francesco Paolo; Ciccarella, Giuseppe

Owing to their nano-sized porous structure, CaCO3nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

2018 - Deletion of Maged1 in mice abolishes locomotor and reinforcing effects of cocaine [Articolo su rivista]
De Backer, Jean-François; Monlezun, Stéphanie; Detraux, Bérangère; Gazan, Adeline; Vanopdenbosch, Laura; Cheron, Julian; Cannazza, Giuseppe; Valverde, Sébastien; Cantacorps, Lídia; Nassar, Mérie; Venance, Laurent; Valverde, Olga; Faure, Philippe; Zoli, Michele; De Backer, Olivier; Gall, David; Schiffmann, Serge N; de Kerchove d'Exaerde, Alban

Melanoma antigen genes (Mage) were first described as tumour markers. However, some of Mage are also expressed in healthy cells where their functions remain poorly understood. Here, we describe an unexpected role for one of these genes, Maged1, in the control of behaviours related to drug addiction. Mice lacking Maged1 are insensitive to the behavioural effects of cocaine as assessed by locomotor sensitization, conditioned place preference (CPP) and drug self-administration. Electrophysiological experiments in brain slices and conditional knockout mice demonstrate that Maged1 is critical for cortico-accumbal neurotransmission. Further, expression of Maged1 in the prefrontal cortex (PFC) and the amygdala, but not in dopaminergic or striatal and other GABAergic neurons, is necessary for cocaine-mediated behavioural sensitization, and its expression in the PFC is also required for cocaine-induced extracellular dopamine (DA) release in the nucleus accumbens (NAc). This work identifies Maged1 as a critical molecule involved in cellular processes and behaviours related to addiction.

2018 - Development of a simple and sensitive liquid chromatography triple quadrupole mass spectrometry (LC–MS/MS) method for the determination of cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and its metabolites in rat whole blood after oral administration of a single high dose of CBD [Articolo su rivista]
Palazzoli, Federica; Citti, Cinzia; Licata, Manuela; Vilella, Antonietta; Manca, Letizia; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe

The investigation of the possible conversion of cannabidiol (CBD) into Δ 9 -tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50 mg/kg) was administered orally to rats and their blood was collected after 3 and 6 h. A highly sensitive and selective LC–MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6 h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice.

2018 - Inihibition of glycolysis by using a micro/nano-lipid bromopyruvic chitosan carrier as a promising tool to improve treatment of hepatocellular carcinoma [Articolo su rivista]
Hanafy, Nemany A.; Dini, Luciana; Citti, Cinzia; Cannazza, Giuseppe; Leporatti, Stefano

Glucose consumption in many types of cancer cells, in particular hepatocellular carcinoma (HCC), was followed completely by over-expression of type II hexokinase (HKII). This evidence has been used in modern pharmacotherapy to discover therapeutic target against glycolysis in cancer cells. Bromopyruvate (BrPA) exhibits antagonist property against HKII and can be used to inhibit glycolysis. However, the clinical application of BrPA is mostly combined with inhibition effect for healthy cells particularly erythrocytes. Our strategy is to encapsulate BrPA in a selected vehicle, without any leakage of BrPA out of vehicle in blood stream. This structure has been constructed from chitosan embedded into oleic acid layer and then coated by dual combination of folic acid (FA) and bovine serum albumin (BSA). With FA as specific ligand for cancer folate receptor and BSA that can be an easy binding for hepatocytes, they can raise the potential selection of carrier system.

2018 - Pharmaceutical and biomedical analysis of cannabinoids: A critical review [Articolo su rivista]
Cittia, Cinzia; Braghiroli, Daniela; Vandelli, Maria Angela; Cannazza, Giuseppe

Cannabis products have recently regained much attention due to the high pharmacological potential of their cannabinoid content. In this review, the most widely used sample preparation strategies for the extraction of cannabinoids are described for the specific application to either plant materials or biological matrices. Several analytical techniques are described pointing out their respective advantages and drawbacks. In particular, chromatographic methods, such as TLC, GC and HPLC, are discussed and compared in terms of selectivity and sensitivity. Various detection methods are also presented based on the specific aim of the cannabinoids analysis. Lastly, critical considerations are mentioned with the aim to deliver useful suggestions for the selection of the optimal and most suitable method of analysis of cannabinoids in either biomedical or cannabis derived samples.

2018 - Polymeric nano-micelles as novel cargo-carriers for LY2157299 liver cancer cells delivery [Articolo su rivista]
Hanafy, Nemany Abdelhamid Nemany; Quarta, Alessandra; Ferraro, Marzia Maria; Dini, Luciana; Nobile, Concetta; De Giorgi, Maria Luisa; Carallo, Sonia; Citti, Cinzia; Gaballo, Antonio; Cannazza, Giuseppe; Rinaldi, Rosaria; Giannelli, Gianluigi; Leporatti, Stefano

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFβ. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

2018 - Quality Traits of "Cannabidiol Oils": Cannabinoids Content, Terpene Fingerprint and Oxidation Stability of European Commercially Available Preparations [Articolo su rivista]
Pavlovic, Radmila; Nenna, Giorgio; Calvi, Lorenzo; Panseri, Sara; Borgonovo, Gigliola; Giupponi, Luca; Cannazza, Giuseppe; Giorgi, Annamaria

Cannabidiol (CBD)-based oil preparations are becoming extremely popular, as CBD has been shown to have beneficial effects on human health. CBD-based oil preparations are not unambiguously regulated under the European legislation, as CBD is not considered as a controlled substance. This means that companies can produce and distribute CBD products derived from non-psychoactive hemp varieties, providing an easy access to this extremely advantageous cannabinoid. This leaves consumers with no legal quality guarantees. The objective of this project was to assess the quality of 14 CBD oils commercially available in European countries. An in-depth chemical profiling of cannabinoids, terpenes and oxidation products was conducted by means of GC-MS and HPLC-Q-Exactive-Orbitrap-MS in order to improve knowledge regarding the characteristics of CBD oils. Nine out of the 14 samples studied had concentrations that differed notably from the declared amount, while the remaining five preserved CBD within optimal limits. Our results highlighted a wide variability in cannabinoids profile that justifies the need for strict and standardized regulations. In addition, the terpenes fingerprint may serve as an indicator of the quality of hemp varieties, while the lipid oxidation products profile could contribute in evaluation of the stability of the oil used as milieu for CBD rich extracts.

2018 - Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol [Articolo su rivista]
Citti, Cinzia; Palazzoli, Federica; Licata, Manuela; Vilella, Antonietta; Leo, Giuseppina; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Pacchetti, Barbara; Cannazza, Giuseppe

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds’ concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds’ concentrations were correlated with some of the pharmacological properties of this cannabinoid.

2017 - Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To InhibitTrypanosoma bruceiPteridine Reductase in Support of Early-Stage Drug Discovery [Articolo su rivista]
Linciano, Pasquale; Dawson, Alice; Pöhner, Ina; Costa, David M; Sá, Monica S; Cordeiro-da-Silva, Anabela; Luciani, Rosaria; Gul, Sheraz; Witt, Gesa; Ellinger, Bernhard; Kuzikov, Maria; Gribbon, Philip; Reinshagen, Jeanette; Wolf, Markus; Behrens, Birte; Hannaert, Véronique; Michels, Paul A M; Nerini, Erika; Pozzi, Cecilia; di Pisa, Flavio; Landi, Giacomo; Santarem, Nuno; Ferrari, Stefania; Saxena, Puneet; Lazzari, Sandra; Cannazza, Giuseppe; Freitas-Junior, Lucio H; Moraes, Carolina B; Pascoalino, Bruno S; Alcântara, Laura M; Bertolacini, Claudia P; Fontana, Vanessa; Wittig, Ulrike; Müller, Wolfgang; Wade, Rebecca C; Hunter, William N; Mangani, Stefano; Costantino, Luca; Costi, Maria P

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme-and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 mu M, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

2017 - Probing an allosteric pocket of CDK2 with small-molecules [Articolo su rivista]
Christodoulou, Michael S; Caporuscio, Fabiana; Restelli, Valentina; Carlino, Luca; Cannazza, Giuseppe; Costanzi, Elisa; Citti, Cinzia; Lo Presti, Leonardo; Pisani, Pasquale; Battistutta, Roberto; Broggini, Massimo; Passarella, Daniele; Rastelli, Giulio

The availability of well characterized allosteric modulators is of crucial importance for investigating allosteric regulation of protein function. In a recently identified inactive conformation of CDK2 an open allosteric pocket has been detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Here, we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from the one previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by X-ray. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. Then, the activity of the obtained compounds was evaluated with different biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of A549 and SKOV3 cancer cell lines. Therefore, this paper presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.

2017 - Rescue of IL-1β-induced reduction of human neurogenesis by omega-3 fatty acids and antidepressants [Articolo su rivista]
Borsini, Alessandra; Alboni, Silvia; Horowitz, Mark A.; Tojo, Luis M.; Cannazza, Giuseppe; Su, Kuan-Pin; Pariante, Carmine M.; Zunszain, Patricia A.

Both increased inflammation and reduced neurogenesis have been associated with the pathophysiology of major depression. We have previously described how interleukin-1 (IL-1) β, a pro-inflammatory cytokine increased in depressed patients, decreases neurogenesis in human hippocampal progenitor cells. Here, using the same human in vitro model, we show how omega-3 (ω-3) polyunsaturated fatty acids and conventional antidepressants reverse this reduction in neurogenesis, while differentially affecting the kynurenine pathway. We allowed neural cells to proliferate for 3 days and further differentiate for 7 days in the presence of IL-1β (10 ng/ml) and either the selective serotonin reuptake inhibitor sertraline (1 µM), the serotonin and norepinephrine reuptake inhibitor venlafaxine (1 µM), or the ω-3 fatty acids eicosapentaenoic acid (EPA, 10 µM) or docosahexaenoic acid (DHA, 10 µM). Co-incubation with each of these compounds reversed the IL-1β-induced reduction in neurogenesis (DCX- and MAP2-positive neurons), indicative of a protective effect. Moreover, EPA and DHA also reversed the IL-1β-induced increase in kynurenine, as well as mRNA levels of indolamine-2,3-dioxygenase (IDO); while DHA and sertraline reverted the IL-1β-induced increase in quinolinic acid and mRNA levels of kynurenine 3-monooxygenase (KMO). Our results show common effects of monoaminergic antidepressants and ω-3 fatty acids on the reduction of neurogenesis caused by IL-1β, but acting through both common and different kynurenine pathway-related mechanisms. Further characterization of their individual properties will be of benefit towards improving a future personalized medicine approach.

2017 - Stimulatory effect on pea of Typha Angustifolia L. extracts and their chemical composition [Articolo su rivista]
Ghezal, Nadia; Rinez, Asma; Zribi, Ines; Farooq, Muhammad; Troisi, Luigino; Cannazza, Giuseppe; Granito, Catia; Haouala, Rabiaa

In this study, the influence of aqueous and organic extracts of different plant parts (flowers, leaves, and stems) of Typha angustifolia on the germination and early seedling growth of field pea (Pisum sativum L.) was evaluated. Chemical composition of extracts of different plant parts of Typha was also determined. Aqueous (20, 40, 60, 80, and 100 g L−1) and organic extracts (at 0.5, 1, and 2 mg mL−1) were applied to the seeds of two pea cultivars, Douce de Provence and Lincoln, placed in Petri dishes. Application of extracts had a beneficial effect on germination and early seedling growth of both pea cultivars. However, aqueous extract of leaves showed the most beneficial effect at 60 and 40 g L−1for the cultivars Douce de Provence and Lincoln, respectively. The effect could be attributed to the allelochemicals present in the aqueous extracts. Petroleum ether and chloroform extracts of leaves had the most stimulating effect on the germination and early seedling growth of pea. Analysis of Typha extracts indicated the presence of vitamin E in leaves, which could be responsible forthis stimulation. Moreover, Typha leaves also had substantial amount of flavonoids. In conclusion, the allelopathic activity of of Typha was dependent on the plant part, the solvent nature, the concentration of the extracts tested, and on the pea cultivar. Application of leaf extract was the most effective in improving the germination rate and early seedling growth of pea.

2016 - "Heart-cut" bidimensional achiral-chiral liquid chromatography applied to the evaluation of stereoselective metabolism, in vivo biological activity and brain response to chiral drug candidates targeting the central nervous system [Articolo su rivista]
Battisti, Umberto M.; Citti, Cinzia; Larini, Martina; Ciccarella, Giuseppe; Stasiak, Natalia; Troisi, Luigino; Braghiroli, Daniela; Parenti, Carlo; Zoli, Michele; Cannazza, Giuseppe

A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC-LC-MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the "heart-cut" two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d4eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs.Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal.

2016 - 7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-Dioxide as Positive Allosteric Modulator of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor. The End of the Unsaturated-Inactive Paradigm? [Articolo su rivista]
Citti, Cinzia; Battisti, UMBERTO MARIA; Cannazza, Giuseppe; Jozwiak, Krzysztof; Stasiak, Natalia; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino; Zoli, Michele

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1.

2016 - Alterations in alpha5* nicotinic acetylcholine receptors result in midbrain- and hippocampus-dependent behavioural and neural impairments [Articolo su rivista]
Besson, Morgane; Guiducci, Stefania; Granon, Sylvie; Guilloux, Jean Philippe; Guiard, Bruno; Repérant, Christelle; Faure, Philippe; Pons, Stéphanie; Cannazza, Giuseppe; Zoli, Michele; Gardier, Alain M.; Maskos, Uwe

Rationale: Evidence links alterations in α5-containing nicotinic receptors (α5*-nAChRs) to nicotine addiction. Notably, the rs16969968 polymorphism in the α5 gene (α5SNP) increases the risk for heavy smoking and impairs nicotine-rewarding properties in mice. Additional work is needed to understand how native and polymorphic α5*-nAChRs contribute to processes associated with the risk for nicotine addiction. Objectives: We aimed at understanding the contribution of α5*-nAChRs to endophenotypes like increased responses to novelty and anxiety, known to promote vulnerability to addiction, and to the response of the dopamine and serotonin systems to nicotine. Methods: Behavioural phenotypes were investigated in mice lacking the α5 gene (α5−/−). Nicotine injections were performed to test the consequences of nicotine exposure on the phenotypes identified. Dopamine and serotonin signalling were assessed using in vivo microdialysis and electrophysiology. We used lentiviral vectors to compare the consequences of re-expressing either the α5 wild-type allele or the α5SNP in specific brain areas of α5−/− mice. Results: α5−/− mice did not exhibit high responses to novelty but showed decreased novelty-induced rearing behaviour together with high anxiety. Exposure to high doses of nicotine rescued these phenotypes. We identified altered spontaneous and nicotine-elicited serotonin and dopamine activity in α5−/− mice. Re-expression of α5 in the ventral tegmental area and hippocampus rescued rearing and anxiety levels in α5−/− mice, respectively. When expressing the α5SNP instead, this resulted in a knockout-like phenotype for both behaviours. Conclusions: We propose that altered α5*-nAChR cholinergic signalling contributes to emotional/behavioural impairments that may be alleviated by nicotine consumption.

2016 - An unexpected reversal in the pharmacological stereoselectivity of benzothiadiazine AMPA positive allosteric modulators [Articolo su rivista]
Battisti, UMBERTO MARIA; Citti, Cinzia; Rastelli, Giulio; Pinzi, Luca; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Cannazza, Giuseppe

Benzothiadiazine type compounds (BTDs) have gained great attention for their potential therapeutic activity as nootropic and neuroprotective agents. BTDs, acting as AMPA positive allosteric modulators, potentiate the glutamatergic neurotransmission without the side effects typically associated with direct agonists. Studies regarding the binding mode of racemic BTDs into the receptor binding pocket demonstrated that one enantiomer establishes a more favourable interaction and possesses a higher biological activity with respect to the other one. The S enantiomer was proved to be the eutomer for both IDRA21 and S18986, two of the most studied BTD AMPA positive allosteric modulators. However, recent data highlighted an opposite stereoselectivity for some substituted BTDs (7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide and 7-chloro-2,3,4-trimethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide) showing unexpected structure-activity relationships. In this work, the synthesis and configuration assignment of the stereoisomers of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, one of the most active BTDs, are reported. Electrophysiological tests demonstrated that the R form is the eutomer. Docking and molecular dynamics simulations on the AMPA GluA2 binding site revealed new insights into the stereodiscrimination process. Lastly, metabolic studies disclosed a stereoselective hepatic metabolization of this chiral BTD.

2016 - Analytical and preparative enantioseparation and main chiroptical properties of Iridium(III) bis(4,6-difluorophenylpyridinato)picolinato [Articolo su rivista]
Citti, Cinzia; Battisti, Umberto M.; Ciccarella, Giuseppe; Maiorano, Vincenzo; Gigli, Giuseppe; Abbate, Sergio; Mazzeo, Giuseppe; Castiglioni, Ettore; Longhi, Giovanna; Cannazza, Giuseppe

Almost all Iridium(III) complexes employed both as dopants in PhOLEDs and as pharmaceuticals and fluorescence bioprobes are racemic mixtures. In this study the single enantiomers of the most stable diastereomeric form fac-trans-N–N, bis[2-(4,6-difluorophenyl)pyridinato-C2,N](picolinato)iridium(III) (FIrpic) were separated and analysed. The data obtained showed that the complex can be separated into stable optically active Λ and Δ isomers employing cellulose based chiral stationary phase both in normal and polar phase mode. Their chirality was confirmed and their absolute configuration assigned employing several methods (DFT and TDDFT calculations, CD and VCD). The CPL spectroscopy of the isolated enantiomers of FIrpic was also recorded due to its possible value in the OLEDs field. The chromatographic method was applied for a semipreparative purpose demonstrating that polar organic solvent chromatography (POSC) could be used to avoid the low-solubility issues associated with these Iridium(III) complexes. Finally, the chemical and stereochemical stability of the single isomers was evaluated under thermal stress by liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF) on both chiral and achiral columns. No racemization and/or isomerization was observed; however, the dissociation of the ancillary ligand was demonstrated employing LC-QTOF.

2016 - Biocatalytic Synthesis of Phospholipids and Their Application as Coating Agents for CaCO3Nano-crystals: Characterization and Intracellular Localization Analysis [Articolo su rivista]
Baldassarre, Francesca; Allegretti, Chiara; Tessaro, Davide; Carata, Elisabetta; Citti, Cinzia; Vergaro, Viviana; Nobile, Concetta; Cannazza, Giuseppe; D'Arrigo, Paola; Mele, Andrea; Dini, Luciana; Ciccarella, Giuseppe

Inorganic nanoparticles are widely investigated as drug delivery systems. In particular micro and nanoparticles of CaCO3 offer smart features for different biomaterials applications. In this work we exploit the phospholipids coating of nano-CaCO3. We prepare modified phospholipids through a chemo-enzymatic approach using Phospholipase D to efficiently transform the most abundant natural phospholipids in two products, snglycero-3-phosphocholine (GPC) and sn-glycero-3-phosphoserine (GPS). We have investigated and modified the Spray Drier process to obtain nano-crystals with specific phase, surface zeta-potential and morphology. The intracellular localization of coated nano-crystals was achieved by ultrastructural microscopy analysis. The synthetized phospholipids, GPC and GPS, improve nano-CaCO3 proprieties and promote a specific targeting as opposed to a widespread and nonspecific localization in the cell.

2016 - Medicinal cannabis: Principal cannabinoids concentration and their stability evaluated by a high performance liquid chromatography coupled to diode array and quadrupole time of flight mass spectrometry method [Articolo su rivista]
Citti, Cinzia; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Vandelli, Maria Angela; Cannazza, Giuseppe

In the last few years, there has been a boost in the use of cannabis-based extracts for medicinal purposes, although their preparation procedure has not been standardized but rather decided by the individual pharmacists. The present work describes the development of a simple and rapid high performance liquid chromatography method with UV detection (HPLC-UV) for the qualitative and quantitative determination of the principal cannabinoids (CBD-A, CBD, CBN, THC and THC-A) that could be applied to all cannabis-based medicinal extracts (CMEs) and easily performed by a pharmacist. In order to evaluate the identity and purity of the analytes, a high-resolution mass spectrometry (HPLC-ESI-QTOF) analysis was also carried out. Full method validation has been performed in terms of specificity, selectivity, linearity, recovery, dilution integrity and thermal stability. Moreover, the influence of the solvent (ethyl alcohol and olive oil) was evaluated on cannabinoids degradation rate. An alternative extraction method has then been proposed in order to preserve cannabis monoterpene component in final CMEs.

2016 - Synthesis of β-enamino acid and heteroaryl acetic acid derivatives by Pd-catalyzed carbonylation of α-chloroimines and 2-chloromethyl aza-heterocycles [Articolo su rivista]
Perrone, Serena; Capua, Martina; Cannazza, Giuseppe; Salomone, Antonio; Troisi, Luigino

β-Enamino esters or amides can be synthesized in a single step by a carbonylative coupling of α-chloroimines with alcohols or amines under Pd-catalysis. The methodology has been also applied to the preparation of heteroaryl acetic acid derivatives starting from chloromethyl heteroaromatic rings containing a C-N double bond. The in situ generation of a β-imino acylpalladium species has been proposed as a key step for the process.

2015 - A direct synthesis of 3-acyl-4-hydroxy-2-pyranone derivatives via palladium-catalyzed carbonylation of α-chloroketones. A cascade reaction involving acylketenes [Articolo su rivista]
Perrone, Serena; Caroli, Antonio; Cannazza, Giuseppe; Granito, Catia; Salomone, Antonio; Troisi, Luigino

A simple and direct method to obtain 3-acyl-4-hydroxy-2-pyranone derivatives by the palladium-catalyzed carbonylation of α-chloroketones has been described. The methodology can be applied to a variety of aromatic and aliphatic ketones to afford valuable products from both a synthetic and a biological point of view. A mechanistic hypothesis involving an acylketene intermediate has also been proposed.

2015 - Calcium-Carbonate Nanocapsules Improve the Efficacy of BEZ235 in Lymphoma a Cell Line: A Promising New Technology of Drug Delivery [Abstract in Rivista]
Civallero, Monica; Vergaro, Viviana; Citti, Cinzia; Cosenza, Maria; Cannazza, Giuseppe; Parenti, Carlo; Bari, Alessia; Ciccarella, Giuseppe; Sacchi, Stefano; Pozzi, Samantha

Nanotechnology is a promising branch of the medical field, directed to improve diagnostic and therapeutics strategies, applying nanovectors as drug delivery systems. Efficient encapsulation of anticancer drugs in nanocolloids and microcapsules was recently developed by G. Ciccarella research group (1). Based on our collaboration with the Nantional Nanotechnology Laboratory of the University of Salento and our previous experience with target therapies, we encapsulated BEZ235, a phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR). BEZ235 efficiently blocks the dysfunctional activation of the PI3K/mTOR pathway in cellular and in vivo settings, thus inhibiting the growth and proliferation of various cancer cells, and phase I/II clinical trials were open in solid cancer. However the scarse solubility limited further development of this promising compound. In order to overcome the solubility issue BEZ235-loaded nanocapsules were generated by the stepwise adsorption of oppositely charged polyelectrolytes into biocompatible CaCO3 cores. First nanocapsules were tested for biocompatibility. The exposition of lymphoma cell lines to empty nanocapsules up to 48 hours, did not induce any cititoxicity, confirming their biocompatibility. Second, encapsulated BEZ235 was compared with free-drug to test the cytotoxicity in a T lymphoma cell line (HUT78) by MTT assay. The results suggested that nanoencapsulated-BEZ235 was extremely efficient compared with free-BEZ235, reaching IC50 just after 5 hours of exposure compared with an IC65% at 48 hours with the free drug. A validated LC-MS/MS method was developed in order to quantify intracellular concentration of BEZ235 over time. Intracellular concentration of BEZ235 in the lymphoma cell line was consistent with biological results since the internalization kinetic and efficiency was increased by the coating. In order to confirm that the encapsuled-BEZ235 was still effective on cell apoptosis, we tested free BEZ and encapsulated BEZ235 at a concentration of 1µM in T cell lymphoma cell lines. Encapsulated-BEZ235 induced apoptosis evidenced by the cleavage of caspase 8, 9 and 3 at an earlier time point compared with free BEZ235 and at significantly lower concentration. We also confirmed that the encapsulated-BEZ235 maintained its effect on the target mTOR/AKT pathway: p-AKT was dephosphorylated at 5h while the free BEZ235 operates at least after 24 hours at concentrations 100 times higher, as previously demonstrated. Keeping in mind a future clinical application of these polymeric particles/capsules, our data can be regarded as a promising new nanotechnology-based strategy to improve the efficacy and bioavailability of old and new drugs. Functional biological studies of BEZ235-encapsulated carrier and its mechanism of internalization are already under way, and animal in vivo studies to evaluated toxicity and distribution of the nanocapsuled compound are ongoing.

2015 - Different physiological and behavioural effects of e-cigarette vapour and cigarette smoke in mice [Articolo su rivista]
Ponzoni, L; Moretti, M; Sala, M; Fasoli, F; Mucchietto, V; Lucini, V; Cannazza, G; Gallesi, G; Castellana, Cn; Clementi, F; Zoli, M; Gotti, C; Braida, D

Nicotine is the primary addictive substance in tobacco smoke and electronic cigarette (e-cig) vapour. Methodological limitations have made it difficult to compare the role of the nicotine and non-nicotine constituents of tobacco smoke. The aim of this study was to compare the effects of traditional cigarette smoke and e-cig vapour containing the same amount of nicotine in male BALB/c mice exposed to the smoke of 21 cigarettes or e-cig vapour containing 16.8mg of nicotine delivered by means of a mechanical ventilator for three 30-min sessions/day for seven weeks. One hour after the last session, half of the animals were sacrificed for neurochemical analysis, and the others underwent mecamylamine-precipitated or spontaneous withdrawal for the purposes of behavioural analysis. Chronic intermittent non-contingent, second-hand exposure to cigarette smoke or e-cig vapour led to similar brain cotinine and nicotine levels, similar urine cotinine levels and the similar up-regulation of α4β2 nicotinic acetylcholine receptors in different brain areas, but had different effects on body weight, food intake, and the signs of mecamylamine-precipitated and spontaneous withdrawal episodic memory and emotional responses. The findings of this study demonstrate for the first time that e-cig vapour induces addiction-related neurochemical, physiological and behavioural alterations. The fact that inhaled cigarette smoke and e-cig vapour have partially different dependence-related effects indicates that compounds other than nicotine contribute to tobacco dependence.

2015 - Interaction between human serum albumin and different anatase TiO2 nanoparticles: A nano-bio interface study [Articolo su rivista]
Vergaro, Viviana; Carlucci, Claudia; Cascione, Mariafrancesca; Lorusso, Caterina; Conciauro, Francesca; Scremin, Barbara Federica; Congedo, Paolo Maria; Cannazza, Giuseppe; Citti, Cinzia; Ciccarella, Giuseppe

In this investigation, differently shaped and surface functionalized TiO2 anatase nanoparticles and human serum albumin (HSA) were selected to study proteinnanoparticles interaction both in a solution and on flat surfaces, thereby mimicking a medical device. Anatase nanocrystals were characterized by transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET) surface analysis and dynamic light scattering (DLS). The proteinnanoparticles' interactions and their eventual reversibility were studied by pH dependent ζ- potential measurements in different media: ultra-pure water, a phosphate buffer simulating physiological conditions and in a culture medium supplemented with foetal bovine serum. The protein corona masking effect was evidenced and the interaction HSA-nanocrystals resulted irreversible. The interaction HSA-silicon supported TiO2 nanocrystals films was studied by atomic force microscopy (AFM), and resulted driven by the substrate hydrophilicity degree plus was different for the diverse range of nanocrystals tested. Surface roughness measurements showed that on some of the nanocrystals, HSA were arranged in a more globular manner. A lower protein affinity was found for nanocrystals that had a smaller primary particle size, which may correspond to their higher biocompatibility. This nano-bio interface research aimed to study the HSA protein-TiO2 anatase nanocrystals under conditions similar to those for in vitro and in vivo toxicity analyses.

2015 - Nanoencapsulation of an hTS inhibitor octapeptide against ovarian cancer in solid lipid matrix [Abstract in Atti di Convegno]
Francesca, Sacchetti; Chiara, Marraccini; Giuseppe, Cannazza; Valentina, Iannuccelli; Miriam, Hanuskova; Eleonora, Maretti; Maria Paola, Costi; Eliana, Leo

New octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), cannot cross the cell membrane alone and require an appropriate delivery system. In the aim to transport hTS inhibiting LR octapeptide (LR-op) into the cells, Solid Lipid Nanoparticles (SLNs) were developed and evaluated in vitro. The optimized SLNs were formulated in the absence and presence of squalene (7S and 7Sq) both in the LR-op loaded and unloaded form. All the SLNs produced had dimensions below 150 nm, negative Zpotential and a good stability both in suspension and after freeze-drying. Only the sample obtained in the absence of squalene showed to stably incorporate the LR-op promoting its cell internalization, as demonstrated by in vitro studies on C13* ovarian carcinoma cell line.

2014 - Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide [Articolo su rivista]
Carrozzo, Marina Maria; Battisti, Umberto Maria; Cannazza, Giuseppe; Puia, Giulia; Ravazzini, Federica; Falchicchio, Aurelia; Perrone, Serena; Citti, Cinzia; Jozwiak, Krzysztof; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino

Chiral 5-arylbenzothiadiazine derivatives have recently attracted particular attention because they exhibit an interesting pharmacological activity as AMPA receptor (AMPAr) positive modulators. However, investigations on their configurational stability suggest a rapid enantiomerization in physiological conditions. In order to enhance configurational stability, preserving AMPAr activity, we have designed the novel compound (R,S)-7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzothiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute configuration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-flow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound.

2014 - Development of an in vitro liquid chromatography-mass spectrometry method to evaluate stereo and chemical stability of new drug candidates employing immobilized artificial membrane column [Articolo su rivista]
Cannazza, Giuseppe; Battisti, Umberto M.; Carrozzo, Marina M.; Cazzato, Addolorata S.; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino

A stopped-flow HPLC method was developed to evaluate configurational and chemical stability of pharmaceutical compounds employing immobilized artificial membranes (IAM) column to simulate conditions that pharmaceutical compounds will meet in vivo. The method was applied to recent developed chiral 5-arylbenzothiadiazine derivatives possessing high positive allosteric modulatory (PAM) activity on AMPA receptor. In particular the stopped-flow HPLC method developed used a chiral column to separate single enantiomer of the compounds that are forced into an IAM column where configurational and chemical stability was evaluated in simulated gastrointestinal fluids (pH 1.2 and 6.8 at 37.5. °C) to simulate in vivo conditions. The results were compared to those obtained by dynamic and off-column methods to evaluate the effects of stationary phases on kinetic constant of enantiomerization and hydrolysis. The results suggested that the phospholipids environment of IAM stationary phases, which mimes biological membrane, greatly influence the hydrolysis process increasing the chemical stability of tested compounds while no influence on enantiomerization kinetic was observed. Therefore it is possible to suppose that 5-arylbenzothiadiazine derivatives should not hydrolysed in vivo while they should rapidly racemized in aqueous solvents. The method could represents a rapid and value tool to predict chemical and configurational stability of new chemical entities to decrease the number of animal studies.

2014 - Development of lipid nanocarriers as delivery systems for a small peptide with anti-ovarian activity [Abstract in Atti di Convegno]
F. Sacchetti; A.S. Cazzato; C. Marraccini; G. Cannazza; V. Iannuccelli; E. Maretti; M.P. Costi; E. Leo

The encapsulation of a small peptide in SLN was achieved modifying the hot high shear homogenization method. The data obtained by the comparison of SLN to standard Liposomes suggested that even if Liposomes are more efficient carrier for hydrophilic peptides, it is possible to embed this kind of molecules in a solid lipid matrix achieving carriers with higher in vitro stability and lower cytotoxicity. Moreover, the ability of the loaded carriers to reduce the cell viability more efficiently than the unloaded vectors, indicates that the peptide was released inside the cell environment being able to exert its action.

Cazzato, ADDOLORATA STEFANIA; Cannazza, Giuseppe; Ponterini, Glauco; Marraccini, Chiara; Pirondi, Silvia; Genovese, Filippo; Costi, Maria Paola

In the present work the degradation profile of an anticancer peptide in different biological matrixes like DMEM (Dulbecco’s Modified Eagle Medium) and cell lysates by LC Chip Q-TOF was shown. Subsequently, an LC-MS/MS method for the quantitative analysis of LR in cell lysates was developed and fully validated.

2014 - Internalization and stability of a thymidylate synthase peptide inhibitor in ovarian cancer cells [Articolo su rivista]
Cannazza, Giuseppe; Cazzato, ADDOLORATA STEFANIA; Marraccini, Chiara; Pavesi, Giorgia; Pirondi, Silvia; R., Guerrini; M., Pelà; Frassineti, Chiara; Ferrari, Stefania; Marverti, Gaetano; Ponterini, Glauco; Costi, Maria Paola

Information on the cellular internalization and stability of the ovarian cancer cell growth inhibitor peptide, LSCQLYQR (LR), is vital for lead optimization. Ad-hoc-synthesized LR/fluorescent-probe conjugates were used to monitor the internalization of the peptide. Mass spectrometry was used to identify adducts resulting from the thiol reactivity of the cysteine residue in LR. A mechanistic model is proposed to explain the observed change in intracellular peptide amount over time. Structural modifications can be foreseen to improve the peptide stability.

2014 - One-pot synthesis of azobenzene derivatives by oxidation of 2,3-dihydrobenzothiadiazines [Articolo su rivista]
Cannazza, Giuseppe; Perrone, Serena; Rosato, Francesca; D'Accolti, Lucia; Parenti, Carlo; Troisi, Luigino

An oxidative route to N-substituted sulfonamidic azobenzene derivatives is reported. A mechanism, based on a rationalization of previous findings, is proposed. This simple one-pot method could be adapted to the synthesis of a range of substituted sulfonylazobenzenes with potential applications in the pharmaceutical and industrial fields

2014 - Ring opening of heterocycles containing a C–N double bond: a simple synthesis of imides promoted by acyl palladium species [Articolo su rivista]
Perrone, Serena; Cannazza, Giuseppe; Caroli, Antonio; Salomone, Antonio; Troisi, Luigino

A detailed study on the reactivity of various heterocycles, containing a C-N double bond, with acyl palladium species, generated in situ from allyl or benzyl halides and CO, has been performed. While the cyclic imine 2-methyl-1-pyrroline reacted with acyl-palladium intermediates to give a bicyclic β-lactam, other heterocycles containing a C-N double bond conjugated with a heteroatom (O or N), showed a ring-opening reaction leading to functionalized imides with high structural diversity. Such methodology represents a simple and direct way to prepare structurally complex imides. Moreover, a reaction mechanism, involving cationic intermediates, was also proposed.

2014 - Stereoselective synthesis of α-alkylidene β-oxo amides by palladium-catalyzed carbonylation [Articolo su rivista]
Perrone, Serena; Salomone, Antonio; Caroli, Antonio; Falcicchio, Aurelia; Citti, Cinzia; Cannazza, Giuseppe; Troisi, Luigino

A direct method to obtain α-alkylidene β-oxo amides by the palladium-catalyzed carbonylation of α-chloro ketones in the presence of aromatic imines has been described. The methodology can be applied to a variety of C-aryl imines bearing N-aryl or N-alkyl substituents. The entire process is highly stereoselective and affords the α-alkylidene β-oxo amides only as (Z) isomers. A mechanistic hypothesis involving an acyl-β-lactam intermediate has also been proposed.

2014 - The cytisine derivatives, CC4 and CC26, reduce nicotine-induced conditioned place preference in zebrafish by acting on heteromeric neuronal nicotinic acetylcholine receptors [Articolo su rivista]
Ponzoni, Luisa; Braida, Daniela; Pucci, Luca; Andrea, Donzelli; Fasoli, Francesca; Manfredi, Irene; Papke, Roger L.; Stokes, Clare; Cannazza, Giuseppe; Clementi, Francesco; Gotti, Cecilia; Sala, Mariaelvina

Rationale: Cigarette smoking is one of the most serious health problems worldwide and people trying to stop smoking have high rates of relapse. Zebrafish (Danio rerio), by combining pharmacological and behavioral assays, is a promising animal model for rapidly screening new compounds to induce smoking cessation. Objectives: This study aims to identify possible acetylcholine nicotinic receptors (nAChRs) involved in mediating nicotine (NIC)-induced conditioned place preference (CPP) in zebrafish and investigate the effect of the CC4 and CC26 cytisine derivatives in reducing NIC-induced CPP. Methods: CPP was evaluated using a two-compartment chamber, and the zebrafish were given CC4 (0.001-5 mg/kg), CC26 (0.001-1 mg/kg), cytisine (0.1-2.5 mg/kg), and varenicline (1-10 mg/kg) alone or with NIC (0.001 mg/kg). Swimming activity was evaluated using a square observational chamber. The affinity of the nicotinic ligands for native zebrafish brain nAChRs was evaluated by binding studies using [3H]-Epibatidine (Epi) and [125I]-αBungarotoxin (αBgtx) radioligands, and their subtype specificity was determined by means of electrophysiological assay of oocyte-expressed α4β2 and α7 subtypes. Results: CC4 and CC26 induced CPP with an inverted U-shaped dose-response curve similar to that of NIC. However, when co-administered with NIC, they blocked its reinforcing or slightly aversive effect. Binding and electrophysiological studies showed that this effect was due to binding to high-affinity heteromeric but not α7-containing receptors. Conclusions: We have further characterized CC4 and identified a new compound (CC26) that may be active in inducing smoking cessation. Zebrafish is a very useful model for screening new compounds that can affect the rewarding properties of NIC.

2013 - Interferon alpha exposure increases the expression of the enzymes belonging to the kynurenine pathway in an in vitro model of human neurons: SH-SY5Y cells [Abstract in Rivista]
Silvia, Alboni; Cristina, Benatti; Claudia, Montanari; Benatti, Stefania; Fabio, Tascedda; Giuseppe, Cannazza; Pariante Carmine, M; Nicoletta, Brunello .

The past two decades have witnessed a burgeoning area of pre-clinical and clinical research linking psychiatric illnesses – particularly major depression (MD) – to activation of the inflammatory immune system. One of the stronger evidence supporting a causal role for inflammation in leading MD comes from reports indicating that depressive symptoms frequently develop in patients undergoing immunotherapy with cytokines, such as interferon (IFN)-α, for the treatment of malignancies or chronic viral infection. Although INF-alpha- induced effects on the brain made of IFN-α a model to study the influence of pro-inflammatory cytokines in the CNS and behavior the molecular mechanisms underlying these effects are far from being fully understood. It has been proposed that IFN-α may contribute to the etiology of MD by inducing indolamine 2,3-dioxygenase (IDO) expression and thus unbalancing in the tryptophan/kynurenine metabolism toward the production of neurotoxic metabolites and\or reducing serotonin (5-HT) availability. IDO catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway (KP). Kynurenine, the initial product of tryptophan degradation, is further catalysed into neurotoxic end-products through steps catalyzed by kynurenine 3-monooxygenase (KMO) and kynureninase (Kynu). However, Kynurenine can also be catabolised by kynurenine aminotransferase (KAT), into kynurenic acid, a potentially neuroptotective agent. A role for a disturbance in the equilibrium between neurotoxic/ neuropoptective end KP endproducts producing an alteration in the neuroprotective–neurodegenerative balance in the brain of patients with MD, has been proposed in the neurodegeneration hypothesis of depression. Given that we previously demonstrated that IFN-α induces toxic effects in an in vitro model of human neurons (human SH-SY5Y neuroblastoma cells) we were aim to investigate the effects of IFN-α on KP in these cells. Our studies show that IFN-α exposure increased the expression of all the kynurenergic enzymes investigated (IDO, KMO, Kynu and KAT). More particularly strongly induced the expression of IDO mRNA (more than 900 –fold) in SH-SY5Y cells. Similar effects on kynurenergic enzyme expression were also observed when SH-SY5Y cells where differentiated with all-trans retinoic acid (in presence of neurotrophic support and in serum deprived conditions). We also demonstrated that INF-α decreased 5-HT levels whereas increased the kynurenine levels in the medium of both differentiated as well not differentiated SH-SY5Y cells.

2013 - On the oxidation of different iminic bonds by excess of 3-chloroperbenzoic acid [Articolo su rivista]
Troisi, L.; Carrozzo, ; M., M.; Citti, Ca; Falcicchio, A; Mansueto, R; Rosato, F; Cannazza, Giuseppe

In the present work the behavior of different substituted iminic bonds toward the oxidative action of 3-chloroperbenzoic acid is reported. The C=N bond was or was not oxidized to oxaziridines, amides, oximes, nitroso-, nitro-, and azodioxy compounds depending on the substituents at the iminic group and on the imine/MCPBA stoichiometric ratio.

2012 - 5-Arylbenzothiadiazine Type Compounds as Positive Allosteric Modulators of AMPA/Kainate Receptors [Articolo su rivista]
Umberto M., Battisti; Krzysztof, Jozwiak; Cannazza, Giuseppe; Puja, Giulia; Gabriella, Stocca; Braghiroli, Daniela; Parenti, Carlo; Brasili, Livio; Marina M., Carrozzo; Cinzia, Citti; Luigino, Troisi

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPAr) has led to the search for new AMPAr positive modulators. On the basis of crystallographic data of the benzothiadiazines binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-dihydrobenzothiadiazine type compounds has been synthesized and tested. Electrophysiological results suggested that 5-heteroaryl substituents on the benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the activity as positive modulators of AMPAr with respect to IDRA21 and cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-5-(3-furyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide crosses the blood–brain barrier after intraperitoneal injection. Biological results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-positive modulator candidates.

2012 - An improved LC-S/MS method for the quantitation of adenosine concentration in mice brain microdialysates. [Articolo su rivista]
Carrozzo, Mm; Troisi, L; Cannazza, Giuseppe; Cazzato, As; Braghiroli, Daniela; Parenti, Carlo; Guiducci, Stefania; Zoli, Michele

A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the determination of adenosine concentrations in mouse brain microdialysis samples was developed. High method sensitivity (LLOQ of 1.25 fmol) was achieved by on-line switching column. A C18 was employed as enrichment column and a cyano based (CN-SB) as analytical column. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. It was successfully applied to measure quantitatively adenosine in brain of freely moving mice after different stimuli.

2012 - Dottorato regionale Spinner-Nuove molecole per il controllo e la differenziazione delle cellule staminali-NovaMolStam- Università di Bologna, Ferrara, Modena e Reggio Emilia, Parma-Anno 2012-2015- [Altro]
Costi, Maria Paola; Costantino, Luca; Ponterini, Glauco; Cannazza, Giuseppe

L’uso di cellule staminali rappresenta un approccio promettente nel trattamento di malattie degenerative e di altre patologie che richiedono la rigenerazione tissutale. Tuttavia, nell’applicazione di questa strategia si incontrano numerosi problemi, quali il controllo della differenziazione, le reazioni immunitarie che si scatenano nell’applicazione, la specificità della differenziazione e della produzione stessa delle cellule. Per affrontare questi problemi è necessario conoscere la biologia cellulare, e i cammini (“pathway”) metabolici più importanti che governano i processi di differenziazione. Alcuni prodotti naturali e piccole molecole di origine sintetica si sono rivelati utili nel controllo e nella differenziazione delle cellule staminali. Tra questi, ad es., l’acido retinoico, il forskolin, il desametasone, l’azacitidina, che tuttavia mancano di specificità d’azione ed in generale hanno caratteristiche non ottimali. Sulla base delle conoscenze esistenti e dei composti attualmente in uso, appare quindi necessario e possibile ricercare nuove molecole dotate di profili biologici specifici. Il presente progetto ha l’obiettivo di identificare nuovi composti dotati di specificità d’azione verso cammini metabolici importanti per il controllo e la differenziazione delle cellule staminali. Tali composti saranno molecole di origine naturale e/o sintetica e potranno essere utilizzati per ottimizzare la produzione delle staminali da usare per scopi clinici. Non sono da intendere come farmaci, ma come sostanze che supportano e promuovono il miglioramento della tecnologia di produzione delle cellule per uso clinico. Per raggiungere questo obiettivo, il progetto si articolerà in fasi successive che prevedono: a) l’identificazione dei cammini metabolici più adatti per controllare le cellule staminali e la selezione dei bersagli molecolari più adeguati all’interno dei pathway; b) la progettazione di molecole potenzialmente attive su questi bersagli mediante strategie quali il “virtual screening”, l’analogia strutturale rispetto a composti attivi esistenti e lo screening biologico diretto; c) la sintesi dei composti o il loro isolamento da fonti naturali; d) la valutazione biologica su panel di enzimi e proteine e su cellule; e) l’ottimizzazione dei prodotti migliori selezionati dalle fasi b-d mediante criteri di “drug-likeness” e successivi cicli di sintesi e valutazione biologica. Per raggiungere i suddetti obiettivi verranno applicate strategie proprie della ricerca farmaceutica. Il punto a) prevede l’utilizzo di strumenti bioinformatici per la ricerca e l’analisi di database, lo studio di proprietà strutturali e funzionali delle proteine coinvolte per la prioritizzazione dei bersagli più adeguati. Il punto b) prevede l’utilizzo di protocolli per l’identificazione di composti nuovi da banche dati di composti noti (“virtual screening”), la progettazione di composti basati sulla conoscenza delle strutture 3D dei bersagli selezionati, lo screening sperimentale di librerie molecolari disponibili nei laboratori di ricerca dei partecipanti al progetto e/o la loro valutazione diretta sulle linee cellulari staminali presso i laboratori di collaboratori interessati al progetto (Centro di Medicina Rigenerativa di Modena). Il punto c) prevede l’utilizzo di tecniche di sintesi chimica per ottenere i composti progettati, l’estrazione di prodotti naturali da piante studiate nei laboratori coinvolti nel progetto, la caratterizzazione, separazione e sintesi dei prodotti naturali identificati come interessanti. Il punto d) prevede la valutazione biologica dei nuovi composti sintetizzati verso le biomolecole bersaglio scelte. Successivamente i composti migliori saranno valutati sulle linee cellulari staminali presso i laboratori del Centro di Medicina Rigenerativa di Modena, similmente al punto b), ma con metodiche più raffinate per val

2012 - Efficient synthesis of 5,6-dihydro-8H-[1,2,4]thiadiazino[6,5,4-de] phenanthridine 4,4-dioxide and 5,6-dihydro-8H-[1,2,4]-thiadiazino[6,5,4-ij] thieno[2,3-c]quinoline 4,4-dioxide [Articolo su rivista]
Battisti, UMBERTO MARIA; Carrozzo, Marina Maria; Cannazza, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Brasili, Livio; Cinzia, Citti; Luigino, Troisi

A new efficient and versatile synthesis to obtain different substituted 5,6-dihydro-8H-[1,2,4]thiadiazino[6,5,4-de]phenanthridine 4,4-dioxide and 5,6-dihydro-8H-[1,2,4]-thiadiazino[6,5,4-ij]thieno[2,3-c]quinolone 4,4-dioxide was developed. The four cyclic systems are achieved by a three-step synthesis proceeding under mild conditions in high yields.

2012 - Regioselective cyclization of chloroacylaminobenzenesulfonamide derivatives [Articolo su rivista]
Carrozzo, Marina Maria; Battisti, UMBERTO MARIA; Cannazza, Giuseppe; C., Citti; Parenti, Carlo; L., Troisi

Chloroacylaminobenzensulfonamides regioselectively thermally cyclize under solvent free conditions to 1,2,4-benzothiadiazines with five- and six-membered rings fused on face b.

2012 - Simultaneous measurement of adenosine, dopamine, acetylcholine and 5-hydroxytryptamine in cerebral mice microdialysis samples by LC–ESI-MS/MS [Articolo su rivista]
Cannazza, Giuseppe; Carrozzo, Mm; Cazzato, As; Bretis, Im; Troisi, L; Parenti, Carlo; Braghiroli, Daniela; Guiducci, Stefania; Zoli, Michele

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous measurement of adenosine (ADE), dopamine (DA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) in mouse brain microdialysates. High method sensitivity (LLOQ of 0.05 nM) was achieved by optimization of chromatographic and mass spectrometric parameters. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. The LC–MS/MS method was successfully applied to evaluate the effect of the systemic administration of cocaine or amphetamine on the extracellular levels of ADE, DA, ACh and 5-HT in the mouse nucleus accumbens by microdialysis.

2011 - Evaluation of stereo and chemical stability of chiral compounds [Articolo su rivista]
Cannazza, Giuseppe; Battisti, UMBERTO MARIA; Carrozzo, Marina Maria; Brasili, Livio; Braghiroli, Daniela; Parenti, Carlo

A stopped-flow bidimensional recycle HPLC (sf-BD-rHPLC) configuration has been used to investigate simultaneously the stereo and chemical stability of labile chiral compounds. The single enantiomers of a racemate can be separated on chiral column (first dimension) and each one can be trapped in the achiral column (second dimension) that works as reactor.By filling the achiral column with the appropriate aqueous buffers it is possible to evaluate the stability of the trapped enantiomer toward aqueous buffer itself. It was possible to recycle the reaction products formed in the chiral column (first dimension) where they are separated by a second six valve port. The reaction rate constants were calculated for the different processes occurred in the achiral column by means of corresponding peak areas. The method was applied to a pharmacological active compound: (±)7-chloro-5-ethyl-3-methyl-3,4-dihydro-2H-benzo[1,2,4]thiadiazine 1,1-dioxide ((±)-1) to evaluate enantiostability and hydrolysis in conditions similar to those of biological fluid. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis and enantiomerization at the same temperature and in the same solvents used in sf-BD-rHPLC. The good agreement of the results obtained validate the novel procedure developed. Furthermore, the results generated off-line were used to determine the influence of solvents on the racemization of (±)-1.

2011 - Molecular modeling studies, synthesis, configurational stability and biological activity of 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide [Articolo su rivista]
Battisti, UMBERTO MARIA; Carrozzo, Marina Maria; Cannazza, Giuseppe; Puja, Giulia; Giulia, ; L., Troisi; Braghiroli, Daniela; Parenti, Carlo; K., Jozwiak

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPArs) has led to a search for new AMPAr positive modulators. Among them, 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (1) has attracted particular attention, because it is one of the most active benzothiadiazine–derived positive modulators of the AMPA receptor. It possesses two stereogenic centers, C3 and C6, thus it can exist as four stereoisomers. In this work, preliminary in silico studies suggested that 1 interacts stereoselectively with AMPArs. Single stereoisomers of 1 were prepared in order to evaluate their biological activity. However, studies regarding the configurational stability of the investigated compounds suggested a rapid epimerization at C3 in aqueous solvents, and we can expect the same reaction in vivo. Thus, electrophysiological experiments were performed on the two epimeric mixtures, (3∗,6R)- and (3∗,6S)- 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide, in order to evaluate their activities as positive allosteric modulators of AMPArs. The obtained data suggest that the (3∗,6S) epimeric mixture is the most active in positively modulating AMPArs, confirming in silico results.

2010 - Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer [Articolo su rivista]
Cannazza, Giuseppe; Carrozzo, Marina Maria; Battisti, UMBERTO MARIA; Braghiroli, Daniela; Parenti, Carlo; Troisi, Alessandro; Troisi, Luigino

Benzothiadiazines differently substituted at the sulfonamidic nitrogenatom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesizedand fully characterized. Enantioseparation of these compounds has revealedrapid on-column enantiomerization. The recently developed software DCXplorer hasbeen successfully applied to calculate enantiomerization kinetic parameters. Enantiomerizationbarriers of 3-phenyl substituted benzothiadiazines, calculated in this work, haveindicated a higher enantiomerization rate suggesting that the aromatic substituentexerts a strong effect on the enantiomerization process. Methyl substitution on N2 positionled to higher free energy barriers of enantiomerization, suggesting negative influenceof methyl in the N2 position on enantiomerization kinetics. However, methylationon N4 position increases the enantiomerization rates significantly. The results obtainedhave been employed to postulate an enantiomerization mechanism for chiral benzothiadiazinetype compounds.

2010 - Epimerization and hydrolysis of 3,6-dimethyl-2,3,5,6-tetrahydro[1,2,4]thiadiazino[6,5,4-hi]indole 1,1-dioxide [Articolo su rivista]
Carrozzo, Marina Maria; Cannazza, Giuseppe; Battisti, UMBERTO MARIA; Braghiroli, Daniela; Troisi, Luigino; Parenti, Carlo

In this study, configurational and chemical stability of (R,R),(S,S),(R,S),(S,R)-3,6-dimethyl-2,3,5,6-tetrahydro[1,2,4]thiadiazino[6,5,4-hi]indole 1,1-dioxide (1) were investigated by dynamic and stopped-flow HPLC methods. Single epimeric mixtures (R,R),(R,S)-1 and (S,S),(S,R)-1 were obtained combining synthetic and chromatographic strategies. Separation of (R,R)-1 and (R,S)-1 was achieved by chiral chromatography and absolute configuration of eluted epimers has been assigned basing on molecular modelling calculations. Epimerization and hydrolysis of (R,R),(R,S)-1 have been studied by classical off-column, dynamic HPLC and stopped-flow HPLC methods. The influence of different parameters, such as temperature, pH and dielectric constant was evaluated. The data obtained indicate that (R,R),(R,S)-1 undergoes to a rapid epimerization in aqueous solvent and hydrolysis in acidic conditions. Moreover, epimerization and hydrolysis were investigated in presence of an artificial membrane and in physiological buffers (pH 2.2 and 7.0 at 37.5 °C) to simulate in vivo conditions.

2010 - Quantitative analysis of acetylcholine in rat brain microdialysates by liquid chromatography coupled with electrospray ionization tandem mass spectrometry [Articolo su rivista]
Carrozzo, Marina Maria; Cannazza, Giuseppe; Pinetti, Diego; DI VIESTI, Vittoria; Battisti, UMBERTO MARIA; Braghiroli, Daniela; Parenti, Carlo; Baraldi, Mario

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the quantitative analysis of acetylcholine in rat brain dialysates. The separation of acetylcholine (ACh), choline (Ch), acetyl-β-methylcholine (IS) from endogenous compounds and Ringer's salts was achieved with cation exchange chromatography. Optimization of chromatographic and mass spectrometry parameters were perfomed in order to improve sensitivity of the method. The limit of detection were 0.05 and 3.75 fmol on column with S/N ratio of 3:1 for ACh and Ch, respectively. The limit of quantitation (LOQ) for ACh and Ch measured in Ringer's solution were 0.05 nM (0.25 fmol) and 3.75 nM (18.75 fmol), respectively at S/N ratio of 10:1. Linearity of the method has been evaluated in the concentrations range between 0.05 and 5.00 nM and 3.75 and 200 nM for ACh and Ch respectively. The correlation coefficients were 0.999 and 0.995 for ACh and Ch respectively, indicating very good linearity. The LC/MS/MS method developed has been applied to evaluate the effect of oral administration of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21), a positive modulators of AMPA receptor, on the release of ACh in the rat prefrontal cortex by microdialysis.

2010 - Regioselective reduction of 3-substituted 2,3-dihydrobenzothiadiazines with borohydrides [Articolo su rivista]
Battisti, UMBERTO MARIA; Cannazza, Giuseppe; M. M., Carrozzo; Braghiroli, Daniela; Parenti, Carlo; F., Rosato; L., Troisi

A simple and efficient synthetic path for N-1 or N-2 alkyl-substituted 2-aminobenzenesulfonamides wasdeveloped based on regioselective reduction with NaBH3CN in different solvents.

2010 - Simultaneous Determination of Enantiomerizationand Hydrolysis Kinetic Parameters of ChiralN-Alkylbenzothiadiazine Derivatives [Articolo su rivista]
Carrozzo, Marina Maria; Cannazza, Giuseppe; Battisti, UMBERTO MARIA; Braghiroli, Daniela; Parenti, Carlo

On-column stopped flow multidimensional HPLC (sfMDHPLC) anddynamic high-performance liquid chromatography were applied to investigate the influenceof alkyl substituents at the sulfonamidic and amino moieties of benzothiadiazine1,1-dioxide derivatives on hydrolysis and enantiomerization rate constants. The dataobtained indicate the presence of pyrrolo substituent at the 3,4 positions on benzothiadiazinerings inhibits the hydrolysis, whereas the enantiomerization occurs in acidicmedium. Hydrolysis rates are quite similar for the two benzothiadiazines methyl substitutedto nitrogen at 2- and 4-positions. Conversely, enantiomerization rate of 4-N-methylsubstituted is significantly higher than 2-N-methyl substituted.

2010 - Study on the racemization of synephrine by off-column chiral high-performance liquid chromatography [Articolo su rivista]
Pellati, Federica; Cannazza, Giuseppe; Benvenuti, Stefania

In this study, the racemization kinetic parameters of R-(-)-synephrine, the active phenethylamine alkaloid of Citrus aurantium L., were determined by means of an off-column HPLC method. Enantioseparation was carried out in different buffer solutions and solvents on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH, 100 mm x 4.0 mm i.d., 5 microm). The mobile phase was 10 mM sodium phosphate buffer (pH 6.0)-2-propanol (95:5, w/w), with 50 microM disodium EDTA, at 0.8 mL/min. The column was thermostatted at 20 degrees C and detection was set at 225 nm. The influence of pH value, ionic strength, temperature and addition of organic modifier on the rate constant, the half-life of racemization and the free energy barrier of racemization of R-(-)-synephrine were determined. Among the different chemical and physical parameters evaluated as affecting the racemization of naturally occurring R-(-)-synephrine, pH, temperature and addition of an organic co-solvent appear to have the strongest effect, while ionic strength does not exert a significant influence on the racemization rate. The results of the present study indicated that synephrine racemization is possible at high temperature at both acidic and basic pH values; therefore, the extraction procedure of R-(-)-synephrine from the plant material should be carried out under specific conditions to preserve the stereochemical integrity and the biological activity of this secondary metabolite.

2009 - A novel class of allosteric modulators of AMPA/Kainate receptors [Articolo su rivista]
Cannazza, Giuseppe; Krzysztof, Jozwiak; Parenti, Carlo; Braghiroli, Daniela; Carrozzo, Marina Maria; Puja, Giulia; Losi, Gabriele; Baraldi, Mario; Wolfgang, Lindner; Irving W., Wainer

The rapid hydrolysis in vivo of IDRA21 to 2-amino-5-chlorobenzensulfonamide has been demonstrated by microdialysis experiments. The IDRA21 metabolite possess in vitro a biological activity similar to that of IDRA21 itself. Taking 2-amino-5-chlorobenzensulfonamide as lead compound, a novel class of AMPAR positive allosteric modulators has been prepared.

2009 - On-line racemization by high-performance liquid chromatography [Articolo su rivista]
Cannazza, Giuseppe; Carrozzo, Marina Maria; Battisti, UMBERTO MARIA; Braghiroli, Daniela; Parenti, Carlo

An on-column stopped-flow bidimensional recycling HPLC procedure was developed to obtain an enantiomeric enrichment starting from a racemic mixture. The method developed was applied to two chiral compounds of pharmaceutical interest, (±)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide (1) and (±)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((±)IDRA21, (2)), since the pharmacological activity of the two benzothiadiazine derivatives investigated has been ascribed to only one enantiomer. Starting from a racemic mixture it was possible to obtain about 95% of pure enantiomer. The procedure was applied both in reverse-phase mode and in normal-phase mode. The scaled up and automatization of the novel analytical HPLC procedure represents a powerful tool to obtain pure enantiomer starting from racemic compounds without cumbersome stereoselective synthesis or expensive enantiopurification processes.

2009 - Study on the enantiomerization of synephrine by chiral high-performance liquid chromatography [Abstract in Atti di Convegno]
F. Pellati; G. Cannazza; G. Orlandini; S. Benvenuti

Synephrine, the active phenethylamine alkaloid of Citrus aurantium L., is a biologically active compound that has effects on human metabolism that could help to reduce fat mass in obese people, since it stimulates lipolysis, raises the metabolic rate and promotes the oxidation of fat through increased thermogenesis. In the light of this, C. aurantium extracts are commonly used in the formulation of phytotherapic products employed in the treatment of obesity. It is well known that synephrine is a chiral compound and its enantiomers have shown different pharmacological activity on alpha- and beta-adrenoreceptors. In particular, R-(−)-synephrine is from 1 to 2 orders of magnitude more active than its S-(+)-counterpart. R-(−)-Synephrine has been isolated and identified in Citrus fruits. However, a certain amount of S-(+)-synephrine has been detected in C. aurantium dry extracts and dietary supplements (1). The presence of S-(+)-synephrine in C. aurantium natural products has been attributed to a possible enantiomerization of R-(-)-synephrine during the industrial production of the fruit extracts, using a high temperature and a long period of refluxing. To evaluate the enantiomerization kinetic parameters of R-(-)-synephrine, an off-column HPLC method based on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH) was developed. Analyses were carried out on a Chiral-CBH column (100 × 4.0 mm i.d., 5 um), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 uM). The flow rate was 0.8 mL/min. The column was thermostatted at 20°C. Detection was set at 225 nm. The individual enantiomers of the studied compound were isolated by fractional crystallization of the diastereomeric salts and subsequent ion-exchange. The rate constants and the free energy barriers of enantiomerization were determined in different solvents and buffer solutions at pH 1-11. The results generated by the off-line method were used to determine the influence of solvents and pH values on the enantiomerization rate of (+) and (-)-synephrine and to gain further insight into the enantiomerization mechanism of chiral phenethylamine type alkaloids in relation to the pKa values.

2008 - Enantiomerization and hydrolysis of (±)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide by stopped-flow multidimensional high-performance liquid chromatography [Articolo su rivista]
Cannazza, Giuseppe; Carrozzo, Marina Maria; Braghiroli, Daniela; Parenti, Carlo

A novel stopped-flow multidimensional HPLC (sf-MD-HPLC) procedure has been developed to investigate simultaneously the effect of the pH on the enantiostability and hydrolysis of (±)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide [(±)IDRA21]. It was possible to determinate the rate constants and free energy barriers of enantiomerization and hydrolysis rate constants of (±)IDRA21, by using two chiral stationary phases (CSPs) and one achiral C18 column. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis at the same temperature and in the same buffers used in sf-MD-HPLC. The good agreement of the results obtained validate the sf-MD-HPLC procedure. Furthermore, hydrolysis rate constants of (±)IDRA21 were calculated in a series of buffers over a pH range of 1.20-10.60 at 37 °C in order to evaluate the influence of the pH on hydrolysis.

2008 - Enantiomerization of chiral 2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4] benzothiadiazine 5,5-dioxide by stopped-flow multidimensional HPLC [Articolo su rivista]
Cannazza, Giuseppe; Carrozzo, Marina Maria; Braghiroli, Daniela; Parenti, Carlo

An on-column stopped-flow multidimensional HPLC (sfMDHPLC) procedure using two chiral stationaryphases (CSPs) and one achiral C18 column was developed for the determination of rateconstants and free energy barriers of enantiomerization of (±)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide. Moreover, a stopped-flow HPLC (sfHPLC) method previouslydeveloped was applied to the determination of kinetic parameters of enantiomerization of the abovecompound in the presence of a CSP. The individual enantiomers of the studied compound were isolatedin parallel by preparative HPLC and the rate constants and free energy barriers of enantiomerizationweredetermined in different solvents (off-column method). The data obtained by sfMDHPLC, sfHPLC and offcolumnmethods were compared. The (S) enantiomer of the studied compound (S18986) was preparedby asymmetric synthesis and subsequently purified by preparative HPLC, followed by the determinationof rate constants and free energy barriers of enantiomerization in different buffer solutions at pH 2–9.3.

2008 - Enantioseparation of the antidepressant reboxetine [Articolo su rivista]
Cannazza, Giuseppe; Braghiroli, Daniela; Carrozzo, Marina Maria; Parenti, Carlo; Sabbioni, Cesare; Mandrioli, Roberto; Fanali, Salvatore; Raggi, Maria Augusta

The enantioseparation of reboxetine by HPLC was investigated using chiral stationary phases (CSPs) containingcellulose Tris(3,5-dimethylphenyl)carbamate on silica gel (Chiralcel OD column) as the chiralselector. Reversed phase HPLC was the technique of choice for the analytical enantioseparation of reboxetine,while the chiral semipreparative separation was obtained with the same CSP, but in normal phaseconditions. The effects of the mobile phase pH and composition on analytical retention, enantioselectivityand resolution were investigated. The best performance was obtained using a mobile phase composed of0.5Msodium perchlorate at pH 6 and acetonitrile in the 60/40 (v/v) ratio. The semipreparative separationhas allowed obtaining pure enantiomers, but required the preparation of reboxetine free base. Differentn-hexane/alcohol mixtures were tested as mobile phases, varying both the nature of the alcohol and itspercentage in the mobile phase. Different n-hexane/alcohol mixtures were tested as mobile phase andthe best results were obtained by using a mobile phase composed of n-hexane and 2-propanol (80:20,v/v).

2007 - Optimizing Cell Permeation of an Antibiotic Resistance Inhibitor for Improved Efficacy [Articolo su rivista]
A., Venturelli; Tondi, Donatella; Cancian, Laura; Morandi, Federica; Cannazza, Giuseppe; B., Segatore; Prati, Fabio; G., Amicosante; B. K., Shoichet; Costi, Maria Paola

Abstract:Benzo[b]thiophene-2-ylboronic acid, 1, is a 27 nM inhibitor of the class C -lactamase AmpC and potentiates the activity of -lactam antibiotics in bacteria that express this and related enzymes. As is often true, the potency of compound 1 against the enzymes is much attenuated in cell culture against Gram negative bacteria, where the minimum inhibitor concentration of compound 1 is in the mid-micromolar range. Here, we modulated the properties of this lead to enhance its ability to cross the membrane, using a combination of X-ray crystallography, structure-based design, and application of physical models of outer membrane crossing. This strategy led us to derivatives with substantially improved permeability. Also, the greater solubility of these compounds allowed us to measure their efficacy at higher concentrations than with the lead 1, leading to higher maximum potentiation of the antibiotic effect of ceftazidime on resistant bacteria.

2007 - Sintesi, separazione enantiomerica e studi di stabilità di derivati diidropirrolo-benzotiadiazinici [Abstract in Atti di Convegno]
Cannazza G.; Carrozzo M.; Rustichelli C.; Parenti C.; Braghiroli D.

Risoluzione cromatografica preparativa di benzotiadiazine enantiomeriche e studi di stabilità

2006 - Energy barrier determination of enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds by enantioselective stopped-flow HPLC [Articolo su rivista]
Cannazza, Giuseppe; Braghiroli, Daniela; Iuliani, Piera; Parenti, Carlo

The synthesis and enantioseparation of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide derivatives are reported herein. A HPLC stopped-flow procedure was applied to the determination of rate constants and free energy barriers of enantiomerization of the compounds synthesized in the presence of achiral stationary phase. The individual enantiomers of the studied compounds were isolated in parallel by preparative HPLC on a Chiraspher NT column. Rate constants and free energy barriers of enantiomerization were determined in the mobile phase. The results were used to determine the influence of the chiral stationary phase on the enantiomerization process.

2005 - Detection of levodopa, dopamine and its metabolites in rat striatum dialysates following peripheral administration of L-DOPA prodrugs by mean of HPLC-EC [Articolo su rivista]
Cannazza, Giuseppe; A., Di Stefano; B., Mosciatti; Braghiroli, Daniela; Baraldi, Mario; F., Pinnen; P., Sozio; Benatti, Cristina; Parenti, Carlo

A high performance liquid chromatography (HPLC) method was developed to detected simultaneously L-dihydroxyphenylalanine (L-DOPA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum dilaysates following oral administration of L-DOPA or its prodrugs. The chromatographic system uses a reversed-phase C-IS column with electrochemical detection at +0.30 V. Mobile phase consisted of 0.05 M citric acid. sodium EDTA 50 muM, sodium octylsulphonate 0.4 nM at pH of 2.9 and 8%, methanol (v/v) at a flow rate of 1 ml/min. The calibration curves were linear over the concentration range of 10 nm to 100 muM and the lower limits of detections were 125 fmol for L-DOPA. 50 fmol for DOPAC, 250 fmol for DA and 150 fmol for HVA at signal noise to ratio of 3. The repeatability (or intra-day precision), expressed by the relative standard deviation. were better than 4%. The construction of microdialysis probes has been described. The in vitro relative recoveries of each microdialysis probe were. evaluated and the results show that they are similar and reproducible for all the analytes with CVs from 1 to 4%. The HPLC-EC method was applied to detect the extracellular levels Of L-DOPA. DA. DOPAC and HVA in the striatum dialysates of freely moving rats after oral administration of six new potential L-DOPA prodrugs.

2004 - Escitalopram: meccanismo d’azione e profili clinico [Articolo su rivista]


2004 - Evaluation of rat striatal L-dopa and DA concentration after intraperitoneal administration of L-dopa prodrugs in liposomal formulations [Articolo su rivista]
A., Di Stefano; M., Carafa; P., Sozio; F., Pinnen; Braghiroli, Daniela; G., Orlando; Cannazza, Giuseppe; M., Ricciutelli; C., Marianecci; E., Santucci

Parkinson´s disease is a neurodegenerative disease and its symptoms are relieved by administration of L-dopa (LD), which is converted by neuronal aromatic L-aminoacid decarboxylase (AADC), restoring dopamine (DA) levels in surviving neurons. In order to minimize unfavourable side effects, we studied new dimeric LD derivatives, as potential prodrugs for Parkinson´s therapeutic treatment. To improve the bioavailability of the synthesized prodrugs, they were encapsulated in unilamellar liposomes of dimiristoylphosphatidylcholine (DMPC) and cholesterol (CHOL). In vivo microdialysis was used to monitor the striatal LD and DA concentrations after i.p. administration of new delivery systems. Bioavailability evaluation was performed by means of the HPLC-EC method. The striatal levels of LD and DA were remarkably elevated after i.p. administration of liposomal formulation of prodrug (+)-1b ([(O,O-diacetyl)-L-dopa-methylester]-succinyldiamide). This formulation showed about 2.5-fold increase in the basal levels of DA in dialysate rat striatum, suggesting that liposomal formulation of (+)-1b significantly increases LD and DA concentrations with respect to equimolar administration of LD itself or free prodrug (+)-1b.

2003 - Chiralità e farmaci antidepressivi [Articolo su rivista]

Chiralità e Farmaci antidepressivi

2003 - Developing New beta-Lactamase Inhibitors Through Structure-Based Design and Pharmacokinetic Improvement [Abstract in Atti di Convegno]


2003 - High-performance liquid chromatographic method for the quantification of anthranilic and 3-hydroxyanthranilic acid in rat brain dialysate [Articolo su rivista]
Cannazza, Giuseppe; Baraldi, Mario; Braghiroli, Daniela; Tait, Annalisa; Parenti, Carlo

Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) have attracted considerable attention as two of the L-tryptophan kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of ANA and 3-HANA has been developed using reversed-phase high performance liquid chromatography (HPLC) with fluorimetric detection. The HPLC assay was carried out using a C-18 column (5 mum, 250 x 4.6 mm W.). The mobile phase consisted of a mixture of 25 mM sodium/acetic acid buffer (pH 5.5) and methanol (90:10 v/v). Fluorimetric detection at lambda(ex) = 316 nm and lambda(em) = 420 nm was used. The assay was applied to the measurement of ANA and 3-HANA acid in rat brain dialysate following administration Of L-tryptophan or L-kynurenine. 3-HANA and ANA levels were progressively increased during 90 min following administration Of L-tryptophan, then decreased progressively to basal levels. 3-HANA levels were significantly higher than ANA levels after L-kynurenine administration. These findings suggest that the assay developed should provide an improved means for investigation of neurobiology of kynurenine pathway. (C) 2003 Elsevier Science B.V. All rights reserved.

2002 - Separation of reboxetine enantiomers by means of capillary electrophoresis [Articolo su rivista]
RAGGI M., A; Mandrioli, R; Sabbioni, C; Parenti, Carlo; Cannazza, Giuseppe; Fanali, S.

The novel antidepressant reboxetine, a selective norepinephrine reuptake inhibitor, is increasingly used in the treatment of different forms of major depression. Reboxetine is a chiral compound, and is marketed as a racemic mixture of (R,R)- and (S,S)-reboxetine; however, the pharmacokinetic and toxicological profiles of the two enantiomers are rather different. For this reason, a simple capillary electrophoretic method for the separation of reboxetine enantiomers has been developed. Sulfobutyl ether-β-cyclodextrin was chosen as the chiral selector, and several parameters, such as cyclodextrin and buffer concentration, buffer pH and capillary temperature were investigated in order to obtain good separation and acceptable run times. Using an uncoated, fused-silica capillary (internal diameter 50 νm, total length 48.5 cm, effective length 40.0 cm) and a background electrolyte consisting of a pH 3.0, 100 mm phosphate buffer containing 1.25 mm cyclodextrin, reboxetine enantiomers were baseline separated (resolution > 4) with a voltage of 20 kV in less than 16 min. Since pure enantiomers of reboxetine were not available, they were obtained from the racemic powder by means of direct-phase, high-performance liquid chromatography and their identity confirmed by circular dichroism spectra.

2002 - Strong versus Weak Chiral Cation Exchangers: Comparative Evaluation for Enantiomer Separation of Chiral Bases by Nonaqueous CEC [Articolo su rivista]
E., Zarbl; M., Lämmerhofer; A., Woschek; F., Hammerschmidt; Parenti, Carlo; Cannazza, Giuseppe; W., Lindner

Novel enantioselective silica-supported strong and weak cation exchange (SCX and WCX) materials (3.5 μm particles) based on enantiomerically pure N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutanesulfonic acid and corresponding phosphonic acid as well as carboxylic acid structural analogs as chiral selectors have been evaluated for enantiomer separation of chiral bases by non-aqueous capillary electrochromatography (CEC). Capillary columns packed with these chiral stationary phases (CSPs) showed enantioselectivity in non-aqueous CEC towards a variety of chiral bases including amino alcohols such as β-sympathomimetics and β-blockers. Chromatographic and electrokinetic properties of the strong and weak chiral cation exchangers were evaluated comparatively in terms of their pH* profile, i.e. in terms of their dependence on the base-to-acid ratio of the background electrolyte. It turned out that the SCX type CSPs, and in particular the one based on the β-amino sulfonic acid show a broader window of applicable and suitable experimental conditions for CEC. For example, a strong and constant EOF was obtained on the sulfonic acid based CSP over the entire pH* range studied, while the EOF velocity of the carboxylic acid based CSP was slow under acidic conditions. In the separation of chiral bases, the ion-exchange retention mechanism dominated over electrophoretic migration under most conditions, especially on the SCX type CSPs. The SCX phases exhibited reasonable enantioselectivity over a wider pH* range, while the weak chiral cation exchanger (WCX type CSP) showed enantiomer separation capabilities for primary, secondary, and tertiary chiral amines only in the alkaline pH* range. Sulfonic and phosphonic acid based CSPs possess broad spectrum of applicability. For example, clenbuterol enantiomers were well baseline resolved both on sulfonic acid based CSP (α = 1.33, RS = 14.2) as well as phosphonic acid based CSP (α = 1.13, RS = 4.9). In contrast, under the same conditions the corresponding carboxylic acid CSP exhibited enantioselectivity α of 1.08 and resolution RS of 1.3 only.

2002 - Synthesis of 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/Kainate receptors [Articolo su rivista]
Braghiroli, Daniela; Puja, Giulia; Cannazza, Giuseppe; Tait, A; Parenti, Carlo; Losi, G; Baraldi, M.

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives were synthesized and evaluated for their activity as allosteric modulators of kainate-activated currents in primary cultures of cerebellar granule neurons. Substitution of different groups at the 3-position of the benzothiadiazine ring distinguished between positive and negative allosteric modulatory properties.

2001 - Changes in kynurenic, anthranilic and quinolinic acid concentration in rat brain tissue durino developement [Articolo su rivista]
Cannazza G.; Chiarugi A.; Parenti C.; Zanoli P.; Baraldi M.

Kynurenic, anthranilic, and quinolinic acid, brain tissue concentrations and indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were determined in rat brain, during pre- and postnatal development. Quinolinic acid brain tissue concentration was significantly increased at birth ascompared with the prenatal level, then it declined rapidly in the postnatal period. By the contrary,kynurenic and anthranilic acids brain tissue concentrations in rat brain were significantly lowerat birth as compared with those found prenatally; then kynurenic acid concentration decreasedin the first postnatal week and increased thereafter, while anthranilic acid concentration increased in the first postnatal week and decreased thereafter. Indoleamine 2,3-dioxygenase [EC 113.11.17] activity were found unchanged in pre and post natal rat brain. The described oppositechanges in quinolinic and kynurenic acids concentrations, occurring in pre- and postnatal period,despite the lack of knowledge on the precise role played by these compounds on the differentneurotransmitter systems in the brain, could be involved in brain ontogenetic development.

2001 - Prenatal exposure to methyl mercury in rats: Focus on changes in kynurenine pathway, [Articolo su rivista]

Previous studies showed learning and memory deficits following prenatal exposure to methyl mercury (MMC) in rats. Considering the described dysfunction in several neurotransmission systems after MMC exposure, one can surmise that changes in the kynurenine pathway could also be involved in an altered brain functional development. Thus we focused our attention on the potential alteration in the production of tryptophan metabolites by prenatal MMC exposure. For this purpose, brains were removed, at postnatal days 21 and 60, from rats treated, at gestational day 8, with saline or a single dose of MMC (8 mg/kg). The levels of tryptophan, glutamic, aspartic, kynurenic, anthranilic, and quinolinic acids were determined in hippocampal tissues of both groups of rats. No change was detected in the concentration of aspartic, glutamic, and kynurenic acids in 21- and 60-day-old exposed rats in comparison with age-matched controls. On the contrary, at 21 days of age but not at 60 days, we found a very significant reduction of anthranilic acid and, in parallel, an increase of quinolinic acid levels in MMC-exposed rats in comparison with control animals. Finally in the same brain area, tryptophan levels were significantly increased both at 21 and 60 days of postnatal life. These results suggest that an imbalance in the kynurenine pathway could be involved in the toxic effects induced by MMC on brain development.

2001 - Studies of enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds [Articolo su rivista]
G. Cannazza; D. Braghiroli; A. Tait; M. Baraldi; C. Parenti; W. Lindner

An on-column HPLC procedure using a chiral stationary phase (CSP) was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)IDRA21. Subsequently, the HPLC method was applied for investigation of two structurally related chiral compounds. The individual enantiomers of the studied compounds were isolated in parallel by preparative HPLC and rate constants and free energy barriers of enantiomerization were determined in different solvents. The on-column enantiomerization data revealed that CSP induces different rate constants for the two enantiomers. The results generated off-line were used to determine the influence of solvents on the racemization of (+) and (-) IDRA21 and to gain further insight into the enantiomerization mechanism of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds. Chirality 13:94-101, 2001,

2000 - Chiral resolution of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide using high-performance liquid chromatography on cellulose-based chiral stationary phases [Articolo su rivista]
G. Cannazza; D. Braghiroli; M. Baraldi; C. Parenti

Analytical high-performance liquid chromatography (HPLC) methods using derivatized cellulose chiral stationary phases (CSPs) were developed for the separation of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-) IDRA21). in previous studies, (+/-) IDRA21 has been found to have an interesting inhibitory effect on the desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor and improve cognition in animals. This compound posses one chiral carbon atom, but very little information has been reported on the stereoselectivity of his activity. Therefore resolution of the enantiomers of this compound and subsequent identification of stereospecifity in his pharmacological actions are clearly matters of interest. The resolution were made under normal- and reversed-phase conditions using a mobile phase consisting of n-hexane:2-propanol (70/30, v/v) and water:acetonitrile (60/40, v/v) respectively, and a CSP of silica-based cellulose tris-3,5-dimethyl-phenylcarbammate (Chiralcel OD and Chiracel OD-R). The enantiomeric nature of eluates was confirmed by circular dichroism (CD) spectra. A baseline separation (R-s > 1.5) was obtained in both cases. Furthemore the isolation of optical isomers of (+/-) IDRA21 was perfomed using a semipreparative column packed with the same cellulose OD CSP. (C) 2000 Elsevier Science B.V. All rights reserved.

1996 - Benzodiazepine-like compounds and GABA in flower heads of Matricaria Chamomilla [Articolo su rivista]

Extracted and purified benzodiazepine like compounds from dried flwer heads of Matricaria chamomilla was investigated through radioligand binding assay on rat cerebellar membrane. Moreover intracerbroventrivular injection of purified active fraction produced a significant decrease of locomotor activity in rats.

1996 - Chiral resolution of dipeptides by ligand exchange chromatography on chemically bonded chiral phases, [Articolo su rivista]

This paper deals with studies on the optical resolution of glycyl-DL-amino acid dipeptides and diastereomeric dipeptides on three different chemically bonded chiral ligand exchange chromatography (LEC)-phases. The phases were prepared by binding L-proline or L-hydroxyproline to silica gel using different silanes as spacer. Using 10−5 M copper(H) sulface as a mobile phase, eleven glycyl-dipeptides were resolved, nearly all of them with baseline separations. Several dipeptides containing two stereogenic centres were at least partially resolved into the four stereoisomers.