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Giuseppe CANNAZZA
Professore Associato
Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103
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Pubblicazioni
- “Nuovi modulatori allosterici positivi delrecettore AMPA”
[Brevetto]
U., Battisti; Cannazza, Giuseppe; Puja, Giulia; Carozzo, M.; Braghiroli, Daniela; Parenti, Carlo; Troisi, L.; Jozwiak, K.
abstract
brevetto
2023
- Enantioseparation of chiral phytocannabinoids in medicinal cannabis
[Articolo su rivista]
Russo, Fabiana; Tolomeo, Francesco; Vandelli, Maria Angela; Biagini, Giuseppe; Laganà, Aldo; Laura Capriotti, Anna; Cerrato, Andrea; Carbone, Luigi; Perrone, Elisabetta; Cavazzini, Alberto; Maiorano, Vincenzo; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract
The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.
2023
- Synthesis and pharmacological activity of the epimers of hexahydrocannabinol (HHC)
[Articolo su rivista]
Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Schmid, Martin; Luongo, Livio; Perrone, Michela; Ricciardi, Federica; Maione, Sabatino; Laganà, Aldo; Capriotti, Anna Laura; Gallo, Alfonso; Carbone, Luigi; Perrone, Elisabetta; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract
Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semisynthetic
derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two
epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples.
2022
- Antiseizure effects of cannabidiol leading to increased peroxisome proliferator-activated receptor gamma levels in the hippocampal CA3 subfield of epileptic rats
[Articolo su rivista]
Costa, ANNA MARIA; Russo, Fabiana; Senn, Lara; Ibatici, Davide; Cannazza, Giuseppe; Biagini, Giuseppe
abstract
We evaluated the effects of cannabidiol (CBD) on seizures and peroxisome proliferator activated receptor gamma (PPAR) levels in an animal model of temporal lobe epilepsy (TLE). Adult male Sprague-Dawley rats were continuously monitored by video-electrocorticography up to 10 weeks after an intraperitoneal kainic acid (15 mg/kg) injection. Sixty-seven days after the
induction of status epilepticus and the appearance of spontaneous recurrent seizures in all rats, CBD was dissolved in medium-chain triglyceride (MCT) oil and administered subcutaneously at 120 mg/kg (n = 10) or 12 mg/kg (n = 10), twice a day for three days. Similarly, the vehicle was administered to ten epileptic rats. Brain levels of PPAR immunoreactivity were compared to those
of six healthy controls. CBD at 120 mg/kg abolished the seizures in 50% of rats (p = 0.033 vs. pretreatment, Fisher’s exact test) and reduced total seizure duration (p < 0.05, Tukey Test) and occurrence (p < 0.05). PPAR levels increased with CBD in the hippocampal CA1 subfield and subiculum (p < 0.05 vs. controls, Holm–Šidák test), but only the highest dose increased the immunoreactivity in the hippocampal CA3 subfield (p < 0.001), perirhinal cortex, and amygdala (p < 0.05). Overall, these
results suggest that the antiseizure effects of CBD are associated with upregulation of PPAR in the hippocampal CA3 region.
2022
- BENZOTHIADIAZINES DERIVATIVES AS NOVEL ALLOSTERIC MODULATORS OF KAINIC ACID RECEPTORS
[Articolo su rivista]
Puja, G.; Ravazzini, F.; Losi, G.; Bardoni, R.; Battisti, U. M.; Citti, C.; Cannazza, G.
abstract
The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.
2022
- Cis-Δ9-tetrahydrocannabinolic acid occurrence in Cannabis sativa L.
[Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Kaczorova, Dominika; Vandelli, Maria Angela; Biagini, Giuseppe; Laganà, Aldo; Laura Capriotti, Anna; Paris, Roberta; Fulvio, Flavia; Carbone, Luigi; Perrone, Elisabetta; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract
Cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC). The cis isomer of Δ9-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ9-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ9-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp).
2022
- Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth
[Articolo su rivista]
Costantino, Luca; Ferrari, Stefania; Santucci, Matteo; MH Salo-Ahen, Outi; Carosati, Emanuele; Franchini, Silvia; Lauriola, Angela; Pozzi, Cecilia; Trande, Matteo; Gozzi, Gaia; Saxena, Puneet; Cannazza, Giuseppe; Losi, Lorena; Cardinale, Daniela; Venturelli, Alberto; Quotadamo, Antonio; Linciano, Pasquale; Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; Guerrini, Remo; Pacifico, Salvatore; Luciani, Rosaria; Genovese, Filippo; Henrich, Stefan; Alboni, Silvia; Santarem, Nuno; CORDEIRO DA SILVA, Anabela; Giovannetti, Elisa; J Peters, Godefridus; Pinton, Paolo; Rimessi, Alessandro; Cruciani, Gabriele; M Stroud, Robert; C Wade, Rebecca; Mangani, Stefano; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola
abstract
2022
- Gender differences in Anxious-depressive symptomatology,
Metabolic Syndrome and Colorectal Adenomas among
outpatients undergoing colonoscopy: a cross-sectional study
according to a PNEI perspective
[Articolo su rivista]
Rioli, Giulia; Mattei, Giorgio; Bonamici, Caterina; Mancini, Stefano; Alboni, Silvia; Cannazza, Giuseppe; Sena, Paola; Roncucci, Luca; Pingani, Luca; Ferrari, Silvia; Galeazzi, Gian Maria
abstract
2022
- HPLC-MS/MS method applied to an untargeted metabolomics approach for the diagnosis of “olive quick decline syndrome”
[Articolo su rivista]
Di Masi, S.; De Benedetto, G. E.; Malitesta, C.; Saponari, M.; Citti, C.; Cannazza, G.; Ciccarella, G.
abstract
Olive quick decline syndrome (OQDS) is a disorder associated with bacterial infections caused by Xylella fastidiosa subsp. pauca ST53 in olive trees. Metabolic profile changes occurring in infected olive trees are still poorly investigated, but have the potential to unravel reliable biomarkers to be exploited for early diagnosis of infections. In this study, an untargeted metabolomic method using high-performance liquid chromatography coupled to quadrupole-time-of-flight high-resolution mass spectrometry (HPLC-ESI-Q-TOF-MS) was used to detect differences in samples (leaves) from healthy (Ctrl) and infected (Xf) olive trees. Both unsupervised and supervised data analysis clearly differentiated the groups. Different metabolites have been identified as potential specific biomarkers, and their characterization strongly suggests that metabolism of flavonoids and long-chain fatty acids is perturbed in Xf samples. In particular, a decrease in the defence capabilities of the host after Xf infection is proposed because of a significant dysregulation of some metabolites belonging to flavonoid family. Moreover, oleic acid is confirmed as a putative diffusible signal factor (DSF). This study provides new insights into the host-pathogen interactions and confirms LC-HRMS-based metabolomics as a powerful approach for disease-associated biomarkers discovery in plants. Graphical abstract: [Figure not available: see fulltext.]
2022
- Kynurenine and kynurenic acid: two human neuromodulators found in Cannabis sativa L
[Articolo su rivista]
Russo, Fabiana; Tolomeo, Francesco; Vandelli, Maria Angela; Biagini, Giuseppe; Paris, Roberta; Fulvio, Flavia; Laganà, Aldo; Capriotti, Anna Laura; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract
L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.
2021
- A review of hemp as food and nutritional supplement
[Articolo su rivista]
Cerino, P.; Buonerba, C.; Cannazza, G.; D'Auria, J.; Ottoni, E.; Fulgione, A.; Di Stasio, A.; Pierri, B.; Gallo, A.
abstract
The term "hemp"refers to Cannabis sativa cultivars grown for industrial purposes that are characterized by lower levels of tetrahydrocannabinol (THC), the active principle responsible for Cannabis psychotropic effects. Hemp is an extraordinary crop, with enormous social and economic value, since it can be used to produce food, textiles, clothing, biodegradable plastics, paper, paint, biofuel, and animal feed, as well as lighting oil. Various parts of the hemp plant represent a valuable source of food and ingredients for nutritional supplements. While hemp inflorescence is rich in nonpsychoactive, yet biologically active cannabinoids, such as cannabidiol (CBD), which exerts potent anxiolytic, spasmolytic, as well as anticonvulsant effects, hempseed has a pleasant nutty taste and represents a valuable source of essential amino acids and fatty acids, minerals, vitamins, and fibers. In addition, hempseed oil is a source of healthy polyunsaturated fatty acids, and hemp sprouts are rich in antioxidants. This review article aims to provide a comprehensive outlook from a multidisciplinary perspective on the scientific evidence supporting hemp beneficial properties when consumed as food or supplement. Marketing of hemp-derived products is subjected to diversified and complex regulations worldwide for several reasons, including the fact that CBD is also the active principal of pharmaceutical agents and that regulatory bodies in some cases ban Cannabis inflorescence regardless of its THC content. Some key regulatory aspects of such a complex scenario are also analyzed and discussed in this review article.
2021
- Analysis of sequence variability and transcriptional profile of cannabinoid synthase genes in cannabis sativa l. Chemotypes with a focus on cannabichromenic acid synthase
[Articolo su rivista]
Fulvio, F.; Paris, R.; Montanari, M.; Citti, C.; Cilento, V.; Bassolino, L.; Moschella, A.; Alberti, I.; Pecchioni, N.; Cannazza, G.; Mandolino, G.
abstract
Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.
2021
- Analytical Methodologies for Lipidomics in Hemp Plant
[Capitolo/Saggio]
Cerrato, A.; Capriotti, A. L.; Montone, C. M.; Aita, S. E.; Cannazza, G.; Citti, C.; Piovesana, S.; Aldo, L.
abstract
The chemical composition of Cannabis sativa L. has been extensively studied for tens of years, but little is known about its lipidome. This chapter describes an analytical workflow for polar lipid determination in hemp. After extraction, lipids are enriched and isolated by graphitized carbon black sorbent, and the isolated lipid is analyzed by liquid chromatography (LC) coupled with high resolution mass spectrometry, leading to identification of many lipid species. We have developed a semi-automated platform using commercially available Lipostar software for lipid identification. Our approach affords the identification of 189 polar lipids in hemp extract, including sulfolipids and phospholipids. The number of the identified lipid species is by far the highest ever reported for Cannabis sativa.
2021
- Folic Acid-Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting
[Articolo su rivista]
Marverti, Gaetano; Marraccini, Chiara; Martello, Andrea; D'Arca, Domenico; Pacifico, Salvatore; Guerrini, Remo; Spyrakis, Francesca; Gozzi, Gaia; Lauriola, Angela; Santucci, Matteo; Cannazza, Giuseppe; Tagliazucchi, Lorenzo; Cazzato, Addolorata Stefania; Losi, Lorena; Ferrari, Stefania; Ponterini, Glauco; Costi, Maria P
abstract
Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor alpha (FRalpha) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRalpha selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.
2021
- HPLC-UV-HRMS analysis of cannabigerovarin and cannabigerobutol, the two impurities of cannabigerol extracted from hemp
[Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Capriotti, Anna Laura; Laganà, Aldo; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract
A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in "pure" commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01-1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.
2021
- In-depth cannabis fatty acid profiling by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry
[Articolo su rivista]
Piovesana, S.; Aita, S. E.; Cannazza, G.; Capriotti, A. L.; Cavaliere, C.; Cerrato, A.; Guarnaccia, P.; Montone, C. M.; Lagana, A.
abstract
Industrial hemp (Cannabis sativa L.) represents an important plant, used for a variety of uses including pharmaceutical and nutraceutical purposes. As such, a detailed characterization of the composition of this plant could help future research to further exploit the beneficial effects of hemp compounds on the human health. Among the many compounds of hemp, fatty acids represent an interesting class of minor components, which has been overlooked so far. In this work, an untargeted approach based on liquid-chromatography coupled to a high-resolution mass spectrometry and a dedicated structure-based workflow for raw data interpretation was employed for the characterization of fatty acids from hemp inflorescences. A simple method, without any chemical derivatization, was developed for extraction and characterization of fatty acids leading to the tentative identification of 39 fatty acid species in the five hemp samples. A quantitative analysis on the untargeted data was initially performed, using peak areas as surrogate of analyte abundance for relative quantitation. Five fatty acids resulted the most abundant in all hemp samples, with ca. 90% of the total peak area. For these compounds a targeted quantitative method was validated, indicating that the most abundant ones were linolenic acid (1.39–7.95 mg g-1) and linoleic acid (1.04–7.87 mg g-1), followed by palmitic acid (3.74–6.08 mg g-1), oleic acid (0.91–4.73 mg g-1) and stearic acid (0.64–2.25 mg g-1).
2021
- Origin of Δ9-tetrahydrocannabinol impurity in synthetic cannabidiol
[Articolo su rivista]
Citti, C.; Russo, F.; Linciano, P.; Strallhofer, S. S.; Tolomeo, F.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.
abstract
Introduction: Cannabidiol (CBD), the nonintoxicating constituent of cannabis, is largely employed for pharmaceutical and cosmetic purposes. CBD can be extracted from the plant or chemically synthesized. Impurities of psychotropic cannabinoids Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC have been found in extracted CBD, thus hypothesizing a possible contamination from the plant. Materials and Methods: In this study, synthetic and extracted CBD samples were analyzed by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry and the parameters that can be responsible of the conversion of CBD into THC were evaluated by an accelerated stability test. Results: In synthetic and extracted CBD no trace of THC species was detected. In contrast, CBD samples stored in the dark at room temperature on the benchtop for 3 months showed the presence of such impurities. Experiments carried out under inert atmosphere in the absence of humidity or carbon dioxide led to no trace of THC over time even at high temperature. Conclusions: The results suggested that the copresence of carbon dioxide and water from the air could be the key for creating the acidic environment responsible for the cyclization of CBD. These findings suggest that it might be appropriate to review the storage conditions indicated on the label of commercially available CBD.
2021
- Oxidative stress and multi-organel damage induced by two novel phytocannabinoids, cbdb and cbdp, in breast cancer cells
[Articolo su rivista]
Salbini, M.; Quarta, A.; Russo, F.; Giudetti, A. M.; Citti, C.; Cannazza, G.; Gigli, G.; Vergara, D.; Gaballo, A.
abstract
Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).
2021
- Phytocannabinomics: Untargeted metabolomics as a tool for cannabis chemovar differentiation
[Articolo su rivista]
Cerrato, A.; Citti, C.; Cannazza, G.; Capriotti, A. L.; Cavaliere, C.; Grassi, G.; Marini, F.; Montone, C. M.; Paris, R.; Piovesana, S.; Lagana, A.
abstract
Cannabis sativa is traditionally classified according to five chemotypes based on the concentration of the main phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG). However, cannabis chemovars and varieties very often present similar concentrations of such phytocannabinoids but different chemical profiles, which is unavoidably translated into different pharmacological effects when used for therapeutic purposes. For this reason, a more refined approach is needed for chemovar distinction, which is described in this study and named phytocannabinomics. The classification was achieved by a comprehensive characterization of the phytocannabinoid composition, by liquid chromatography coupled to high-resolution mass spectrometry untargeted metabolomics for the detection of over a hundred phytocannabinoids, and data analysis by chemometrics for chemovars differentiation. The method was developed on fifty cannabis varieties, grown under the same conditions, and was validated to discriminate between the standard chemotypes by partial least squares discriminant analysis. Then, the method was extended to consider the entire chemical variety of the cannabis accessions, by an unsupervised approach based on the principal component analysis. The latter approach clearly indicated several new subgroups within the traditional classifications, which arise from a unique composition of the minor phytocannabinoids. The existence of these subgroups, which were never described before, is of critical importance for evaluating the pharmacological effects of cannabis chemovars.
2021
- Recent applications of mass spectrometry for the characterization of cannabis and hemp phytocannabinoids: From targeted to untargeted analysis
[Articolo su rivista]
Capriotti, A. L.; Cannazza, G.; Catani, M.; Cavaliere, C.; Cavazzini, A.; Cerrato, A.; Citti, C.; Felletti, S.; Montone, C. M.; Piovesana, S.; Lagana, A.
abstract
This review is a collection of recent applications of mass spectrometry studies for the characterization of phytocannabinoids in cannabis and hemp plant material and related products. The focus is mostly on recent applications using mass spectrometry as detector, in hyphenation to typical separation techniques (i.e., liquid chromatography or gas chromatography), but also with less common couplings or by simple direct analysis. The papers are described starting from the most common approach for targeted quantitative analysis, with applications using low-resolution mass spectrometry equipment, but also with the introduction of high-resolution mass analyzers as the detectors. This reflects a common trend in this field, and introduces the most recent applications using high-resolution mass spectrometry for untargeted analysis. The different approaches used for untargeted analysis are then described, from simple retrospective analysis of compounds without pure standards, through untargeted metabolomics strategies, and suspect screening methods, which are the ones currently allowing to achieve the most detailed qualitative characterization of the entire phytocannabinoid composition, including minor compounds which are usually overlooked in targeted studies and in potency evaluation. These approaches also represent powerful strategies to answer questions on biological and pharmacological activity of cannabis, and provide a sound technology for improved classification of cannabis varieties. Finally, open challenges are discussed for future directions in the detailed study of complex phytocannabinoid mixtures.
2021
- Targeted and untargeted characterization of underivatized policosanols in hemp inflorescence by liquid chromatography-high resolution mass spectrometry
[Articolo su rivista]
Montone, C. M.; Aita, S. E.; Cannazza, G.; Cavaliere, C.; Cerrato, A.; Citti, C.; Mondello, L.; Piovesana, S.; Lagana, A.; Capriotti, A. L.
abstract
The paper describes the development of a targeted quantitative method for the analysis of policosanols in hemp inflorescence. Policosanols are long chain aliphatic alcohols, with carbon chains typically in the range 20–36, with interesting biological activities. These compounds are typically separated by gas chromatography and only a few methods employ liquid chromatography for policosanols. In both cases, methods always include the derivatization of policosanols. In this study, policosanols were separated by ultra-high performance liquid chromatography without any derivatization and detected using high resolution mass spectrometry by formation of lithiated adducts. The procedure was optimized and a quantitative method was validated for the most abundant policosanols (with C24, C26, C27, C28, and C30 chain lengths) in industrial hemp inflorescence extracts. The method was used for the quantitative analysis of policosanols in five hemp types. Hemp wax was found rich in these compounds, especially C26 and C28 policosanols, which may prove useful for revalorization of wax by-products. Finally, the acquired data were also used to expand the search to the untargeted qualitative analysis of policosanols using Compound Discoverer. The untargeted method allowed the annotation of underivatized policosanols up to C33.
2021
- Techno-economic study of a small scale gasifier applied to an indoor hemp farm: From energy savings to biochar effects on productivity
[Articolo su rivista]
Pedrazzi, S.; Santunione, G.; Mustone, M.; Cannazza, G.; Citti, C.; Francia, E.; Allesina, G.
abstract
The hemp market is fast growing due to demand for cannabidiol, nutraceutical and hemp fiber products. This work demonstrates the economical advantage of biomass gasification application to indoor hemp production. Gasifiers provide electrical energy, heat and biochar: these are highly valuable products for indoor growers where lights and thermal management are key costs of the business. Energy produced in an autonomous and renewable way increases the sustainability and in the facility. In this paper a small scale gasifier is fueled with certified “A1 plus” wood pellets to test its behavior and its biochar production rate. Biochar is used for hemp growing tests in an indoor hemp production facility. Results show how a 22 kW power plant is sufficient to guarantee almost complete sustainability in a 80 m2 facility. In the best case scenario where energy saving, biochar and thermal energy selling are considered, the gasifier investment has a payback time of about 3.5 years. At the end of the gasifier lifespan, the Net Present Value reaches 249 k€ considering a discount rate of 6%. Consequential results were also obtained from biochar application to pot growing substrates: there was a 7.7% increase in dry flower production and a 33.9% increase in total plant fresh biomass. Cannabinoids profiles resulted not affected by biochar application.
2021
- The novel heptyl phorolic acid cannabinoids content in different Cannabis sativa L. accessions
[Articolo su rivista]
Linciano, Pasquale; Russo, Fabiana; Citti, Cinzia; Tolomeo, Francesco; Paris, Roberta; Fulvio, Flavia; Pecchioni, Nicola; Vandelli, Maria Angela; Laganà, Aldo; Capriotti, Anna Laura; Biagini, Giuseppe; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe
abstract
The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol
(Δ9-THCP) raised a series of questions relating to the presence and abundance of these new
unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly
their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in
order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as
analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of
both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing
four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to highresolution
mass spectrometry-based method was applied for the putative identification of other heptyl homologs
of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic
acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA),
cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).
2020
- A new software-assisted analytical workflow based on high-resolution mass spectrometry for the systematic study of phenolic compounds in complex matrices
[Articolo su rivista]
Cerrato, A.; Cannazza, G.; Capriotti, A. L.; Citti, C.; La Barbera, G.; Lagana, A.; Montone, C. M.; Piovesana, S.; Cavaliere, C.
abstract
Polyphenols are a broad class of plant secondary metabolites which carry out several biological functions for plant growth and protection and are of great interest as nutraceuticals for their antioxidant properties. However, due to their structural variability and complexity, the mass-spectrometric analysis of polyphenol content in plant matrices is still an issue. In this work, a novel approach for the identification of several classes of polyphenol derivatives based on ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry was developed. First, mass-spectrometric parameters were optimized in order to obtain a large set of diagnostic product ions for their high-confidence identification. The software Compound Discoverer 3.0 was then implemented with a comprehensive database of 45,567 polyphenol derivatives and with mass-spectrometric data for their building blocks, resulting in a specific tool for the semi-automatic identification of flavonoids, anthocyanins, ellagitannins, proanthocyanidins and phenolic acids. The method was then applied to the identification of polyphenols in industrial hemp (Cannabis sativa), a matrix whose use is recently spreading for pharmaceutical and nutraceutical purposes, resulting in the identification of 147 compounds belonging to the classes of flavonoids, proanthocyanidins and phenolic acids. The proposed method is applicable to the polyphenol profiling of any plant matrix and it is not dependent on data in the literature for their identification, allowing the discovery of compounds which have been never identified before.
2020
- Application of calcium carbonate nanocarriers for controlled release of phytodrugs against Xylella fastidiosa pathogen
[Articolo su rivista]
Baldassarre, F.; De Stradis, A.; Altamura, G.; Vergaro, V.; Citti, C.; Cannazza, G.; Capodilupo, A. L.; Dini, L.; Ciccarella, G.
abstract
Calcium carbonate-based hollow or porous particles are one of the preferred carriers for fabrication of drug delivery systems. We have developed an eco-friendly method to produce calcium carbonate nanocrystals, which have shown biocompatibility and optimal capacity to across cell membrane in human cell lines providing new tools in cancer therapy. The success of drug delivery systems has paved the way for the development of systems for controlled release of agrochemicals. In this work, we exploited calcium carbonate nanocrystals as carriers for targeted release of phytodrugs investigating a potential control strategy for the pathogen Xylella fastidiosa. This pathogen is the causal agent of the Olive Quick Decline Syndrome that is an unprecedented emergency in Italy and potentially in the rest of Europe. We studied nanocrystals interactions with bacteria cells and the application in planta to verify olive plants uptake. Ultrastructural analysis by electron microscopy shown an alteration of bacteria wall following nanocrystals interaction. Nanocrystals were adsorbed from roots and they translocated in plants tissues. Calcium carbonate carriers were able to encapsulate efficiently two types of antimicrobial substances and the potential efficacy was tested in experiment under greenhouse conditions.
2020
- Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol
[Articolo su rivista]
Linciano, P.; Citti, C.; Russo, F.; Tolomeo, F.; Lagana, A.; Capriotti, A. L.; Luongo, L.; Iannotta, M.; Belardo, C.; Maione, S.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.
abstract
The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and Δ9-tetrahydrocannabinol (Δ9-THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and Δ9-THCB) and a heptyl-(in CBDP and Δ9-THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that fills the gap between the pentyl and heptyl homologs of CBD and Δ9-THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and Δ9-THCH have been already identified and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ9-tetrahydrocannabinol monomethyl ether (Δ9-THCM). The unambiguously identification in cannabis extract of the n-hexyl homologues of CBD and Δ9-THC different from the corresponding methylated isomers (CBDM, CBGM and Δ9-THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantification of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of Δ9-THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confirmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and Δ9-THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.
2020
- Improved identification of phytocannabinoids using a dedicated structure-based workflow
[Articolo su rivista]
Montone, C. M.; Cerrato, A.; Botta, B.; Cannazza, G.; Capriotti, A. L.; Cavaliere, C.; Citti, C.; Ghirga, F.; Piovesana, S.; Lagana, A.
abstract
Phytocannabinoids are a broad class of compounds uniquely synthesized by the various strains of Cannabis sativa. Up to date, most investigation on phytocannabinoids have been addressed to the most abundant species, Δ9-tetrahydrocannabinol and cannabidiol, for their well-known wide range of pharmaceutical activities. However, in the recent years a large number of minor constituents have been reported, whose role in cannabis pharmacological effects is of current scientific interest. With the purpose of gaining knowledge on major and minor species and furnishing a strategy for their untargeted analysis, in this study we present an innovative approach for comprehensively identifying phytocannabinoids based on high-resolution mass spectrometry in negative ion mode, which allows discrimination of the various isomeric species. For a faster and more reliable manual validation of the tandem mass spectra of known and still unknown species, an extensive database of phytocannabinoid derivatives was compiled and implemented on Compound Discoverer software for the setup of a dedicated data analysis tool. The method was applied to extracts of the Italian FM-2 medicinal cannabis, resulting in the identification of 121 phytocannabinoids, which is the highest number ever reported in a single analysis. Among those, many known and still unknown unconventional phytocannabinoids have been tentatively identified, another piece in the puzzle of unravelling the many uncharted applications of this matrix.
2020
- Is cannabidiol a scheduled controlled substance? Origin makes the difference
[Articolo su rivista]
Citti, C.; Linciano, P.; Cannazza, G.
abstract
Cannabidiol (CBD) is the main cannabinoid naturally occurring in hemp. It has recently attracted the attention of the scientific community because of its numerous pharmacological activities. However, its legal status changes depending on whether it is chemically synthesized or extracted from the plant: extracted CBD is a scheduled controlled substance, whereas synthetic CBD is not under control. In Europe, extracted CBD is excluded from the cosmetic ingredients of the CosIng database. Given the confusion surrounding these different forms of CBD, there is an urgent need for clarity to shed light from both a regulatory and a chemical point of view. The impurity profiles of synthetic and natural CBD are different and could currently represent the only means to distinguish the origin of this substance.
2020
- Isolation of a High-Affinity Cannabinoid for the Human CB1 Receptor from a Medicinal Cannabis sativa Variety: Δ9-Tetrahydrocannabutol, the Butyl Homologue of Δ9-Tetrahydrocannabinol
[Articolo su rivista]
Linciano, P.; Citti, C.; Luongo, L.; Belardo, C.; Maione, S.; Vandelli, M. A.; Forni, F.; Gigli, G.; Lagana, A.; Montone, C. M.; Cannazza, G.
abstract
The butyl homologues of Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabutol (Δ9-THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of Δ9-THC, Δ9-THCB, showed an affinity for the human CB1 (Ki = 15 nM) and CB2 receptors (Ki = 51 nM) comparable to that of (-)-trans-Δ9-THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on Δ9-THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of Δ9-THCB toward the CB1 receptor.
2020
- New insights in hemp chemical composition: a comprehensive polar lipidome characterization by combining solid phase enrichment, high-resolution mass spectrometry, and cheminformatics
[Articolo su rivista]
Antonelli, M.; Benedetti, B.; Cannazza, G.; Cerrato, A.; Citti, C.; Montone, C. M.; Piovesana, S.; Lagana, A.
abstract
The chemical composition of Cannabis sativa L. has been extensively investigated for several years; nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure was developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted of a simple extraction with a mixture of methanol and chloroform (1:1, v/v). The hemp extracts were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effects of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 51 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed the number of lipid identifications to be increased compared to the same procedure without enrichment, which scored 100 lipids. Finally, a semi-quantitative analysis permitted the hemp polar lipidome to be characterized. The results of this study allow knowledge of the hemp chemical composition to be improved with a detailed description of its phospho- and sulfolipid profiles. [Figure not available: see fulltext.]
2020
- Pitfalls in the analysis of phytocannabinoids in cannabis inflorescence
[Articolo su rivista]
Citti, Cinzia; Russo, Fabiana; Sgrò, Salvatore; Gallo, Alfonso; Zanotto, Antonio; Forni, Flavio; Vandelli, Maria Angela; Laganà, Aldo; Montone, Carmela Maria; Gigli, Giuseppe; Cannazza, Giuseppe
abstract
The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract.
2020
- Production and use of co-composted biochar as soil amendment for cannabis sativa sp. Growth
[Relazione in Atti di Convegno]
Santunionea, G.; Turi, E.; Paris, R.; Francia, E.; Montanari, M.; Cannazza, G.
abstract
Biochar is a carbonaceous by-product of thermochemical conversion of ligno-cellulosic biomass. Its application to soil positively influences various soil physico-chemical properties. Biochar high specific surface area and high micro and macro porosity raise the soil water retention and nutrients absorptivity from the soil, enhancing biomass yield. However, biochar itself contains low nutrients amount and its amendment properties could be improved through organic matter addition, rich in microelements and nutrients. This work studies the integration of fresh organic matter and biochar in co-composting biochar process in order to investigate co-composted biochar (hereby called COMBI) effects on soil amelioration compared to biochar only. Specifically, biochar used in this study is the result of thermochemical conversion of lingo-cellulosic biomass waste through PP30 30 kW gasification power plant. Green matter comes from CREA Institute in Anzola (Bologna) hemp fields: After the fibers harvest, the organic wastes was collected and co-composted with biochar (15% v/v) to achieve a mature COMBI. The co-composting biochar process has been carried out in a 105 L volume composter for 3 weeks. It has been mixed by turning the composter to allow oxygenation during organic matter degradation reactions. The temperature profile, the humidity and the C/N content were monitored during the maturation process of COMBI. Then, COMBI has been applied to Cannabis sativa sp. pot growth test (Finola cultivar), where the effects of no amendment soil was used for control plants (C), 5% v/v biochar only amendment (5% B), 10% and 20% v/v co-composting biochar (10% COMBI and 20% COMBI) amendment soil were investigated and compared. The biomass production of Finola plants, the flowers weight and THC-CBD content were analyzed and ANOVA statistical analysis was performed among the four groups of plants.
2020
- Receptors and channels possibly mediating the effects of phytocannabinoids on seizures and epilepsy
[Articolo su rivista]
Senn, Lara; Cannazza, Giuseppe; Biagini, Giuseppe
abstract
Epilepsy contributes to approximately 1% of the global disease burden. By affecting
especially young children as well as older persons of all social and racial variety, epilepsy is a
present disorder worldwide. Currently, only 65% of epileptic patients can be successfully treated
with antiepileptic drugs. For this reason, alternative medicine receives more attention. Cannabis has
been cultivated for over 6000 years to treat pain and insomnia and used since the 19th century to
suppress epileptic seizures. The two best described phytocannabinoids, (−)-trans-Δ9-
tetrahydrocannabinol (THC) and cannabidiol (CBD) are claimed to have positive effects on different
neurological as well as neurodegenerative diseases, including epilepsy. There are different
cannabinoids which act through different types of receptors and channels, including the
cannabinoid receptor 1 and 2 (CB1, CB2), G protein-coupled receptor 55 (GPR55) and 18 (GPR18),
opioid receptor μ and δ, transient receptor potential vanilloid type 1 (TRPV1) and 2 (TRPV2), type
A γ-aminobutyric acid receptor (GABAAR) and voltage-gated sodium channels (VGSC). The
mechanisms and importance of the interaction between phytocannabinoids and their different sites
of action regarding epileptic seizures and their clinical value are described in this review.
2019
- A novel phytocannabinoid isolated from Cannabis sativa L. with an in vivo cannabimimetic activity higher than Δ9-tetrahydrocannabinol: Δ9-Tetrahydrocannabiphorol
[Articolo su rivista]
Citti, C.; Linciano, P.; Russo, F.; Luongo, L.; Iannotta, M.; Maione, S.; Lagana, A.; Capriotti, A. L.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.
abstract
(-)-Trans-Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is the main compound responsible for the intoxicant activity of Cannabis sativa L. The length of the side alkyl chain influences the biological activity of this cannabinoid. In particular, synthetic analogues of Delta(9)-THC with a longer side chain have shown cannabimimetic properties far higher than Delta(9)-THC itself. In the attempt to define the phytocannabinoids profile that characterizes a medicinal cannabis variety, a new phytocannabinoid with the same structure of Delta(9)-THC but with a seven-term alkyl side chain was identified. The natural compound was isolated and fully characterized and its stereochemical configuration was assigned by match with the same compound obtained by a stereoselective synthesis. This new phytocannabinoid has been called (-)-trans-Delta(9)-tetrahydrocannabiphorol (Delta(9)-THCP). Along with Delta(9)-THCP, the corresponding cannabidiol (CBD) homolog with seven-term side alkyl chain (CBDP) was also isolated and unambiguously identified by match with its synthetic counterpart. The binding activity of Delta(9)-THCP against human CB1 receptor in vitro (K-i = 1.2 nM) resulted similar to that of CP55940 (K-i = 0.9 nM), a potent full CB1 agonist. In the cannabinoid tetrad pharmacological test, Delta(9)-THCP induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating a THC-like cannabimimetic activity. The presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties difficult to explain by the presence of the sole Delta(9)-THC.
2019
- Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog
[Articolo su rivista]
Citti, C.; Linciano, P.; Forni, F.; Vandelli, M. A.; Gigli, G.; Lagana, A.; Cannazza, G.
abstract
Cannabidiol (CBD), one of the two major active principles present in Cannabis sativa, is gaining great interest among the scientific community for its pharmaceutical, nutraceutical and cosmetic applications. CBD can be prepared either by chemical synthesis or extraction from Cannabis sativa (hemp). The latter is more convenient from several points of view, including environmental and economic, but mainly for the absence of harmful organic solvents generally employed in the chemical synthesis. Although CBD produced by hemp extraction is the most widely employed, it carries two major impurities. The first one is the already known cannabidivarin (CBDV), whereas the second one is supposed to be the butyl analog of CBD with a four-term alkyl side chain. In this work, we report the isolation by semi-preparative liquid chromatography and the unambiguous identification of this second impurity. A comprehensive spectroscopic characterization, including NMR, UV, IR, circular dichroism and high-resolution mass spectrometry (HRMS), was carried out on this natural cannabinoid. In order to confirm its absolute configuration and chemical structure, the stereoisomer (1R,6R) of the supposed cannabinoid was synthesized and the physicochemical and spectroscopic properties, along with the stereochemistry, matched those of the natural isolated molecule. According to the International Nonproprietary Name, we suggested the name of cannabidibutol (CBDB) for this cannabinoid. Lastly, an HPLC-UV method was developed and validated for the qualitative and quantitative determination of CBDV and CBDB in samples of CBD extracted from hemp and produced according to Good Manufacturing Practices regulations for pharmaceutical and cosmetic use.
2019
- Cannabinoid profiling of hemp seed oil by liquid chromatography coupled to high-resolution mass spectrometry
[Articolo su rivista]
Citti, Cinzia; Linciano, Pasquale; Panseri, Sara; Vezzalini, Francesca; Forni, Flavio; Vandelli, Maria Angela; Cannazza, Giuseppe
abstract
Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.
2019
- Chemical and spectroscopic characterization data of ‘cannabidibutol’, a novel cannabidiol butyl analog
[Articolo su rivista]
Citti, C.; Linciano, P.; Forni, F.; Vandelli, M. A.; Gigli, G.; Lagana, A.; Cannazza, G.
abstract
Cannabidibutol (CBDB), a novel butyl analog of cannabidiol, was identified as impurity of commercial cannabidiol (CBD) extracted from hemp (for full data and results interpretation see “Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog” Citti et al, 2019). The compound was isolated from a CBD sample and subject to a full characterization. First, a complete spectroscopic characterization was performed by Nuclear Magnetic Resonance (NMR): in particular, 1H-NMR, 13C-NMR, COSY, HSQC and HMBC, which were followed by UV absorption and circular dichroism (CD) spectra. In order to confirm the structural identity and stereochemistry of the compound, a stereoselective synthesis of the trans isomer (1R,6R) was carried out and all the chemical and spectroscopic properties were analyzed. The synthesized compound was characterized by NMR (1H-NMR, 13C-NMR, COSY, HSQC and HMBC), Infra-Red spectroscopy (IR), UV and CD absorption, matching the results obtained for the natural isolated compound. With the analytical standard in hand, a simple high-performance liquid chromatography method coupled to UV detection (HPLC-UV) was developed and validated in house in terms of linearity, accuracy, precision, dilution integrity and stability. The present data might be useful to any researcher or industry that may run into a very common impurity of CBD extracted from hemp, so it can be easily compared with their own experimental data.
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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.
abstract
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 (Ï-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 Ï-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 Ï-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
abstract
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 100Â g Lâ1) and organic extracts (at 0.5, 1, and 2Â mg mLâ1) 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 40Â g Lâ1for 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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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, Giuseppe; Gallesi, G; Castellana, Carmela Nives; Clementi, F; Zoli, Michele; Gotti, C; Braida, D.
abstract
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
abstract
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]
Sacchetti, Francesca; Marraccini, Chiara; Cannazza, Giuseppe; Iannuccelli, Valentina; Hanuskova, Miriam; Maretti, Eleonora; Costi, Maria Paola; Leo, Eliana Grazia
abstract
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; Puja, Giulia; Ravazzini, Federica; Falchicchio, Aurelia; Perrone, Serena; Citti, Cinzia; Jozwiak, Krzysztof; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino
abstract
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
abstract
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]
Sacchetti, Francesca; Cazzato, ADDOLORATA STEFANIA; Marraccini, Chiara; Cannazza, Giuseppe; Iannuccelli, Valentina; Maretti, Eleonora; Costi, Maria Paola; Leo, Eliana Grazia
abstract
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.
2014
- EVALUATION OF LR STABILITY BY LC CHIP Q-TOF AND
QUANTITATIVE DETERMINATION OF LR PEPTIDE CELL PENETRATION BY LC-MS/MS
[Articolo su rivista]
Cazzato, ADDOLORATA STEFANIA; Cannazza, Giuseppe; Ponterini, Glauco; Marraccini, Chiara; Pirondi, Silvia; Genovese, Filippo; Costi, Maria Paola
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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]
Alboni, Silvia; Benatti, Cristina; Claudia, Montanari; Benatti, Stefania; Tascedda, Fabio; Cannazza, Giuseppe; Pariante Carmine, M; Brunello, Nicoletta
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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
abstract
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]
Pellati, Federica; Cannazza, Giuseppe; Orlandini, Giulia; Benvenuti, Stefania
abstract
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
abstract
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
abstract
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
abstract
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]
Venturelli, A.; Tondi, Donatella; Cancian, Laura; Morandi, Federica; Cannazza, Giuseppe; Segatore, B.; Prati, Fabio; Amicosante, G.; Shoichet, B. K.; Costi, Maria Paola
abstract
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, Giuseppe; Carrozzo, Marina Maria; Rustichelli, Cecilia; Parenti, Carlo; Braghiroli, Daniela
abstract
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
abstract
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
abstract
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]
Brunello, Nicoletta; Cannazza, Giuseppe; Auguglia, E.
abstract
.
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
abstract
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]
Brunello, Nicoletta; Cannazza, Giuseppe
abstract
Chiralità e Farmaci antidepressivi
2003
- Developing New beta-Lactamase Inhibitors Through Structure-Based Design and Pharmacokinetic Improvement
[Abstract in Atti di Convegno]
Venturelli, Alberto; B., Shoichet; Prati, Fabio; Cannazza, Giuseppe; Costi, Maria Paola
abstract
-
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
abstract
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.
abstract
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
abstract
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.
abstract
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 during developement
[Articolo su rivista]
Cannazza, Giuseppe; Chiarugi, A.; Parenti, Carlo; Zanoli, Paola; Baraldi, Mario
abstract
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]
Cannazza, Giuseppe; Zanoli, Paola; Baraldi, Mario
abstract
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]
Cannazza, Giuseppe; Braghiroli, Daniela; Tait, Annalisa; Baraldi, Mario; Parenti, Carlo; W., Lindner
abstract
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]
Cannazza, Giuseppe; Braghiroli, Daniela; Baraldi, Mario; Parenti, Carlo
abstract
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
[Relazione in Atti di Convegno]
Avallone, Rossella; Zanoli, Paola; Corsi, Lorenzo; Cannazza, Giuseppe; Baraldi, Mario
abstract
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]
Gubitz, G; Vollmann, B; Cannazza, Giuseppe; Schimd, M. G.
abstract
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.