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Cecilia RUSTICHELLI

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

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Pubblicazioni

2023 - Human microglia synthesize neurosteroids to cope with rotenone-induced oxidative stress [Articolo su rivista]
Lucchi, Chiara; Codeluppi, Alessandro; Filaferro, Monica; Vitale, Giovanni; Rustichelli, Cecilia; Avallone, Rossella; Mandrioli, Jessica; Biagini, Giuseppe
abstract

We obtained evidence that mouse BV2 microglia synthesize neurosteroids dynamically to modify neurosteroid levels in response to oxidative damage caused by rotenone. Here, we evaluated whether neurosteroids could be produced and altered in response to rotenone by the human microglial clone 3 (HMC3) cell line. To this aim, HMC3 cultures were exposed to rotenone (100 nM) and neurosteroids were measured in the culture medium by liquid chromatography with tandem mass spectrometry. Microglia reactivity was evaluated by measuring interleukin 6 (IL-6) levels, whereas cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After 24 hours (h), rotenone increased IL-6 and reactive oxygen species levels by approximately +37% over the baseline, without affecting cell viability; however, microglia viability was significantly reduced at 48 h (p < 0.01). These changes were accompanied by the downregulation of several neurosteroids, including pregnenolone, pregnenolone sulfate, 5α-dihydroprogesterone, and pregnanolone, except for allopregnanolone, which instead was remarkably increased (p < 0.05). Interestingly, treatment with exogenous allopregnanolone (1 nM) efficiently prevented the reduction in HMC3 cell viability. In conclusion, this is the first evidence that human microglia can produce allopregnanolone and that this neurosteroid is increasingly released in response to oxidative stress, to tentatively support the microglia's survival.

2023 - Nanoparticulate systems for the delivery of Palmitoylethanolamide to muscle cells [Poster]
Maretti, Eleonora; Molinari, Susanna; Gioia, Federica; Rustichelli, Cecilia; Leo, Eliana Grazia
abstract

2022 - Atogepant: an emerging treatment for migraine [Articolo su rivista]
Rustichelli, Cecilia; Avallone, Rossella; Ferrari, Anna
abstract

Introduction: Until recently, only non-specific and not always well-tolerated medications were available for migraine prophylaxis. Currently, specific drugs such as calcitonin gene-related peptide (CGRP) monoclonal antibodies and second-generation gepants are marketed for migraine treatment. Atogepant, an orally active small molecule, is a potent, selective antagonist of the CGRP receptor and is the only gepant authorized exclusively for episodic migraine prophylaxis in adults. Areas covered: Using literature obtained from PubMed, Scopus, Web of Science, Cochrane, and ClinicalTrials.gov (up to February 13rd, 2022), the authors summarize and evaluate the available data on atogepant for the prophylaxis of episodic migraine. Expert opinion: From pivotal trials, the efficacy and tolerability of atogepant in episodic migraine prophylaxis seem comparable to those of CGRP monoclonal antibodies, even if comparative studies have not been conducted. To date, limited information is available on atogepant, including the optimal dose and duration of therapy; hence, it is difficult to establish whether it could be a first-line drug for migraine prophylaxis. Furthermore, it is important to evaluate if atogepant use is associated with the risk of cardiovascular and cerebrovascular events, which could result from potent and persistent blockade of vasodilation by CGRP.

2022 - Design, Characterization, and In Vitro Assays on Muscle Cells of Endocannabinoid-like Molecule Loaded Lipid Nanoparticles for a Therapeutic Anti-Inflammatory Approach to Sarcopenia [Articolo su rivista]
Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Truzzi, Eleonora; Iannuccelli, Valentina; Leo, Eliana
abstract

Inflammatory processes play a key role in the pathogenesis of sarcopenia owing to their effects on the balance between muscle protein breakdown and synthesis. Palmitoylethanolamide (PEA), an endocannabinoid-like molecule, has been well documented for its anti-inflammatory properties, suggesting its possible beneficial use to counteract sarcopenia. The promising therapeutic effects of PEA are, however, impaired by its poor bioavailability. In order to overcome this limitation, the present study focused on the encapsulation of PEA in solid lipid nanoparticles (PEA-SLNs) in a perspective of a systemic administration. PEA-SLNs were characterized for their physico-chemical properties as well as cytotoxicity and cell internalization capacity on C2C12 myoblast cells. Their size was approximately 250 nm and the encapsulation efficiency reached 90%. Differential scanning calorimetry analyses demonstrated the amorphous state of PEA in the inner SLN matrix, which improved PEA dissolution, as observed in the in vitro assays. Despite the high internalization capacity observed with the flow cytometer (values between 85 and 94% after 14 h of incubation), the Nile Red labeled PEA-SLNs showed practically no toxicity towards myoblasts. Confocal analysis showed the presence of SLNs in the cytoplasm and not in the nucleus. These results suggest the potentiality provided by PEA-SLNs to obtain an innovative and side-effect-free tool in the medical treatment of sarcopenia

2022 - Palmitoylethanolamide-loaded Solid Lipid Nanoparticles for a therapeutic anti-inflammatory approach of Sarcopenia involving ECS [Relazione in Atti di Convegno]
Leo, Eliana Grazia; Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Iannuccelli, Valentina
abstract

2021 - Allopregnanolone and pregnanolone are reduced in the hippocampus of epileptic rats, but only allopregnanolone correlates with the seizure frequency [Articolo su rivista]
Lucchi, Chiara; Costa, ANNA MARIA; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Background: Neurosteroids modulate epileptic activity by interacting with the γ-aminobutyric acid type A receptor, but their brain levels are still undetermined. Objectives: We aimed to establish levels of neurosteroids in the neocortex and hippocampus by liquid chromatography–mass spectrometry in epileptic rats. Methods: Kainic acid-treated rats were continuously monitored up to 9 weeks to determine the seizure frequency by video electrocorticography (n=23), and compared to age-matched controls monitored in the same manner (n=11). Results: A reduction in allopregnanolone (-50%; p<0.05, Mann-Whitney test) and pregnanolone levels (-64%; p<0.01) was found in the hippocampus, whereas pregnenolone sulfate, pregnenolone, progesterone, and 5α-dihydroprogesterone were nonsignificantly reduced. No changes were found in the neocortex. Moreover, allopregnanolone (but not pregnanolone) levels were positively correlated with the seizure frequency (r2=0.4606, p<0.01). Conclusions: These findings indicate a selective reduction in hippocampal levels of 3α-reduced neurosteroids. This reduction was partially mitigated by seizures in the case of allopregnanolone.

2021 - Comparison of pregnenolone sulfate, pregnanolone and estradiol levels between patients with menstrually-related migraine and controls: an exploratory study [Articolo su rivista]
Rustichelli, Cecilia; Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Lo Castro, Flavia; Baraldi, Carlo; Tomasi, Aldo; Ferrari, Anna
abstract

Background Neurosteroids affect the balance between neuroexcitation and neuroinhibition but have been little studied in migraine. We compared the serum levels of pregnenolone sulfate, pregnanolone and estradiol in women with menstrually-related migraine and controls and analysed if a correlation existed between the levels of the three hormones and history of migraine and age. Methods Thirty women (mean age ± SD: 33.5 ± 7.1) with menstrually-related migraine (MM group) and 30 aged- matched controls (mean age ± SD: 30.9 ± 7.9) participated in the exploratory study. Pregnenolone sulfate and pregnanolone serum levels were analysed by liquid chromatography-tandem mass spectrometry, while estradiol levels by enzyme-linked immunosorbent assay. Results Serum levels of pregnenolone sulfate and pregnanolone were significantly lower in the MM group than in controls (pregnenolone sulfate: P = 0.0328; pregnanolone: P = 0.0271, Student’s t-test), while estradiol levels were similar. In MM group, pregnenolone sulfate serum levels were negatively correlated with history of migraine (R2 = 0.1369; P = 0.0482) and age (R2 = 0.2826, P = 0.0025) while pregnenolone sulfate levels were not age-related in the control group (R2 = 0.04436, P = 0.4337, linear regression analysis). Conclusion Low levels of both pregnanolone, a positive allosteric modulator of the GABAA receptor, and pregnenolone sulfate, a positive allosteric modulator of the NMDA receptor, involved in memory and learning, could contribute either to headache pain or the cognitive dysfunctions reported in migraine patients. Overall, our results agree with the hypothesis that migraine is a disorder associated with a loss of neurohormonal integrity, thus supporting the therapeutic potential of restoring low neurosteroid levels in migraine treatment.

2021 - Dehydroepiandrosterone sulfate, dehydroepiandrosterone, 5α-dihydroprogesterone and pregnenolone in women with migraine: analysis of serum levels and correlation with age, migraine years and frequency [Articolo su rivista]
Rustichelli, Cecilia; Monari, Emanuela; Avallone, Rossella; Bellei, Elisa; Bergamini, Stefania; Tomasi, Aldo; Ferrari, Anna
abstract

Migraine is a very painful, disabling and extremely common disorder among the world's adult population, especially women, and it is associated with a variety of comorbidities. Neuroactive steroids exhibit pleiotropic actions on the nervous system. Alterations in their peripheral and central levels could be involved in the pathogenesis, still not fully understood, of migraine and its comorbidities. The purpose of our exploratory study was to determine and compare the serum levels of dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 5α-dihydroprogesterone (DHP) and pregnenolone (PREGNE) between women suffering from migraine without aura (MO group, n=30) and age-matched non-headache women as controls (C group, n=30). Correlations with age, migraine years and frequency were also analyzed. The patients were enrolled at a headache centre; controls were patients’ contacts. Calibrators and serum samples were spiked with the internal standards (ISs) solution and treated to deplete proteins and phospholipids. The obtained extracts were evaporated to dryness, derivatized and analysed by LC-MS/MS in multiple reaction monitoring mode. Analytes’ levels were determined by interpolation on the regression curves, generated from the analyte quantifier ion peak area to the corresponding IS. MO group presented significantly lower levels of DHEAS, DHEA and DHP compared to C group (P <0.05, Student’t test) and the neurosteroid levels negatively correlated with years of migraine and migraine days/3 months (P <0.05, linear regression analysis). These results parallel to previous studies showing reduced serum levels of allopregnanolone and pregnenolone sulfate in women with migraine. The low serum levels found for both excitatory and inhibitory neurosteroids suggested that women with migraine might suffer from inadequate neuroprotection, anti-inflammation activity and pain modulation. These deficits might underlie the migraine chronification process and represent the link between migraine and its various comorbidities.

2021 - Dehydroepiandrosterone Sulfate, Dehydroepiandrosterone, 5α-Dihydroprogesterone and Pregnenolone: Serum Analysis and Correlation Between Migraine and Non-headache Control Females [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Monari, Emanuela; Avallone, Rossella; Bellei, Elisa; Bergamini, Stefania; Tomasi, Aldo; Ferrari, Anna
abstract

Migraine is a very painful and disabling disorder of the nervous system (NS) affecting about 10% of the world's adult population, especially women and it is associated with a variety of comorbidities [1, 2]. Neuroactive steroids have pleiotropic actions on the NS. Alterations in their peripheral and central levels could be involved in the pathogenesis, still not fully understood, of migraine and its comorbidities [3]. The purpose of our exploratory study (approved by Modena Ethical Committee) was to determine serum levels of dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 5α-dihydroprogesterone (DHP) and pregnenolone (PREGNE) in women suffering from migraine without aura (n=30) and age-matched non-headache control females (n=30). The patients were enrolled at the Headache Centre of Modena; controls were patients’ contacts. Calibrators and serum samples were spiked with the ISs solution and treated to deplete proteins and phospholipids. The obtained extracts were evaporated to dryness, derivatized and analysed by RP-LC-ESI-MS/MS in MRM mode. Analyte’s levels were determined by interpolation on the regression curves, generated from the analyte quantifier ion peak area to the corresponding IS. Migraine women presented significantly lower levels of DHEAS, DHEA and DHP compared to controls (P<0.05) and the found concentrations negatively correlated with migraine history, and migraine days in the last three months (P< 0.05). These results parallel to our previous studies showing reduced serum levels of allopregnanolone and pregnenolone sulfate in migraine women [4,5]. The low serum levels found for both inhibitory and excitatory neurosteroids indicate that migraine women may suffer from inadequate neuroprotection, anti-inflammation activity and pain modulation. These deficits could represent the link between migraine and its various comorbidities. References [1] Headache classification Committee of the International Headache Society (IHS). Cephalalgia 2018, 38,1–211. [2] Yin JH., Lin YK., Yang CP., et al. Headache 2021. doi: 10.1111/head.14106. Online ahead of print. [3] Yilmaz, C.; Karali, K.; Fodelianaki, G.; et al. Front Neuroendocrinol 2019, 55, 100788. [4] Rustichelli, C.; Bellei, E.; Bergamini, S.; et al. Cephalalgia 2020, 40, 1355-1362. [5] Rustichelli, C.; Bellei, E.; Bergamini, S.; et al. J Head Pain 2021, 22, 13.

2021 - Drug delivery strategies of Geraniol via nose-to-brain for the treatment of neuronal disorders [Abstract in Atti di Convegno]
Truzzi, Eleonora; Dalpiaz, Alessandro; Rustichelli, Cecilia; Ferraro, Luca; de Oliveira Junior Edilson, Ribeiro; Fogagnolo, Marco; Pavan, Barbara; Beggiato, Sarah; Lima, Eliana; Leo, Eliana
abstract

Parkinson’s disease (PD) is a neurodegenerative disorder which affects 1% of people over 65 years. Neuroinflammation and mitochondrial dysfunction play a central role in the disorder progression. Therefore, anti-oxidants, promoters of neurotrophic factor expression and mitochondrial rescuers could prevent the disease progression. Geraniol (GER) is a natural compound with demonstrated anti-oxidant and neuroprotective activities in PD models. However, its activity is impaired by a fast metabolism following oral administration, with a half-life of about 12 minutes. To overcome this limitation, nose-to-brain administration is a promising strategy because it allows the direct delivery of drugs beyond the blood-brain barrier avoiding the first-pass metabolism. However, GER is highly volatile and irritant. Thus, an appropriate delivery system for its nose-to-brain administration is required. Two different strategies were developed in order to achieve a successful nose to brain delivery. The first one aimed the encapsulation of pure GER and GER prodrug in solid lipid nanoparticles (SLNs), while the second one, displayed here, yearns to complex GER with cyclodextrins (CDs). The inclusion complexes were designed in order to reduce GER volatility and to obtain long-term stable formulations as powders.

2021 - In vivo β-carotene skin permeation modulated by Nanostructured Lipid Carriers [Articolo su rivista]
Maretti, E.; Leo, E.; Rustichelli, C.; Truzzi, E.; Siligardi, C.; Iannuccelli, V.
abstract

Nanostructured Lipid Carriers (NLC) were investigated with the purpose of promoting skin permeation of the highly lipophilic β-carotene (BC) across the stratum corneum (SC) barrier so that it may perform its antioxidant properties in photo-aging and epithelial skin cancer prevention. Two differently sized NLC samples were developed using stearic acid and squalene as lipid matrix and evaluated in comparison with Microstructured Lipid Carriers (MLC). The carriers were characterized for morphology, size, Z-potential, BC loading and release as well as physical state by means of DSC and XRPD analyses. In vivo penetration of the carriers was assessed on humans by determining BC concentrations within the SC stratum disjunctum and stratum compactum layers removed by means of the tape stripping test in comparison with pure BC. Unlike MLC and pure BC that were mostly retained within the outermost layers of the SC, the NLC sample having the smallest size (about 200 nm) has proved to penetrate more deeply into the SC barrier. Accordingly, the goal of providing β-carotene actions against oxidative damages within the looser skin viable tissues could be envisaged.

2021 - Nasal biocompatible powder of geraniol oil complexed with cyclodextrins for neurodegenerative diseases: Physicochemical characterization and in vivo evidences of nose to brain delivery [Articolo su rivista]
Truzzi, Eleonora; Rustichelli, Cecilia; de Oliveira Junior, Edilson Ribeiro; Ferraro, Luca; Maretti, Eleonora; Graziani, Daniel; Botti, Giada; Beggiato, Sarah; Iannuccelli, Valentina; Lima, Eliana Martins; Dalpiaz, Alessandro; Leo, Eliana
abstract

Recently, many studies have shown that plant metabolites, such as geraniol (GER), may exert anti-inflammatory effects in neurodegenerative diseases and, in particular, Parkinson's disease (PD) models. Unfortunately, delivering GER to the CNS via nose-to-brain is not feasible due to its irritant effects on the mucosae. Therefore, in the present study β-cyclodextrin (βCD) and its hydrophilic derivative hydroxypropyl-beta-cyclodextrin (HPβCD) were selected as potential carriers for GER nose-to-brain delivery. Inclusion complexes were formulated and the biocompatibility with nasal mucosae and drug bioavailability into cerebrospinal fluid (CSF) were studied in rats. It has been demonstrated by DTA, FT-IR and NMR analyses that both the CDs were able to form 1:1 GER-CD complexes, arising long-term stable powders after the freeze-drying process. GER-HPβCD-5 and GER-βCD-2 complexes exhibited comparable results, except for morphology and solubility, as demonstrated by SEM analysis and phase solubility study, respectively. Even though both complexes were able to directly and safely deliver GER to CNS, GER-βCD-2 displayed higher ability in releasing GER in the CSF. In conclusion, βCD complexes can be considered a very promising tool in delivering GER into the CNS via nose-to-brain route, preventing GER release into the bloodstream and ensuring the integrity of the nasal mucosa.

2021 - Rational Use of Lasmiditan for Acute Migraine Treatment in Adults: A Narrative Review [Articolo su rivista]
Ferrari, Anna; Rustichelli, Cecilia
abstract

Purpose: This narrative review provides an update on the research that led to the development of ditans and lasmiditan for the acute treatment of migraine in adults and discusses the potential advantages and disadvantages of lasmiditan in clinical use. Methods: The electronic databases PubMed, Scopus, and ClinicalTrials.gov were searched from database inception through January 9, 2021, to identify relevant studies. Search results were assessed for their overall relevance to this review. Findings: Because part of the effect of the triptans is mediated by the serotonin 1F receptors, which are not present in the smooth muscle, a pure agonist of these receptors, lasmiditan, was developed. Lasmiditan is hypothesized to act on antinociceptive pathways and inhibit the calcitonin gene-related peptide release. Lasmiditan was approved by the US Food and Drug Administration in 2019 based on the results of 2 pivotal trials that found a significant difference from placebo in the percentage of patients who achieved freedom from pain and most bothersome symptom at 2 h. The main concern of lasmiditan derives from its central nervous systemerelated adverse effects, mainly dizziness and paraesthesia, probably attributable to its high blood brain barrier penetration. These central nervous system adverse effects impair driving performance for hours and might be suboptimal for individuals with migraine who want to quickly stop the migraine attack to resume their activities as soon as possible. Implications: Despite the advantage of being beneficial in the acute treatment of migraine without vasocostrictive action, lasmiditan also presents limitations, in particular the central nervous system adverse effects. Moreover, head-to-head trials against triptans and gepants are indispensable to determine the better option for patients.

2021 - Relationship between delta rhythm, seizure occurrence and allopregnanolone hippocampal levels in epileptic rats exposed to the rebound effect. [Articolo su rivista]
Costa, A. M.; Lucchi, C.; Malkoç, A.; Rustichelli, C.; Biagini, G.
abstract

Abstract: Abrupt withdrawal from antiepileptic drugs is followed by increased occurrence of epileptic seizures, a phenomenon known as the “rebound effect”. By stopping treatment with levetiracetam (LEV 300 mg/kg/day, n = 15; vs saline, n = 15), we investigated the rebound effect in adult male Sprague-Dawley rats. LEV was continuously administered using osmotic minipumps, 7 weeks after the intraperitoneal administration of kainic acid (15 mg/kg). The effects of LEV were determined by comparing time intervals, treatments, and interactions between these main factors. Seizures were evaluated by video-electrocorticographic recordings and power band spectrum analysis. Furthermore, we assessed endogenous neurosteroid levels by liquid chromatography-electrospray- tandem mass spectrometry. LEV significantly reduced the percentage of rats experiencing seizures, reduced the seizure duration, and altered cerebral levels of neurosteroids. In the first week of LEV discontinuation, seizures increased abruptly up to 700% (p = 0.002, Tukey’s test). The power of delta band in the seizure postictal component was related to the seizure occurrence after LEV withdrawal (r2 = 0.73, p < 0.001). Notably, allopregnanolone hippocampal levels were positively related to the seizure occurrence (r2 = 0.51, p = 0.02) and to the power of delta band (r2 = 0.67, p = 0.004). These findings suggest a role for the seizure postictal component in the rebound effect, which involves an imbalance of hippocampal neurosteroid levels.

2021 - Urinary proteomics reveals promising biomarkers in menstrually related and post-menopause migraine [Articolo su rivista]
Bellei, Elisa; Bergamini, Stefania; Rustichelli, Cecilia; Monari, Emanuela; DAL PORTO, Michele; Fiorini, Alessandro; Tomasi, Aldo; Ferrari, Anna
abstract

Abstract: Migraine is an invalidating neuro-vascular disorder largely spread in the world population. Currently, its pathophysiology is not yet completely understood. The purpose of this study was to investigate the urinary proteome of women suffering from menstrually-related migraine (MM) and post-menopause migraine (PM) in comparison with non-headache women as controls, to search potential biomarkers of these migraine sub-types. Urine samples were analysed by mono-dimensional gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2DE) coupled to liquid chromatography-mass spectrometry (LC-MS/MS). Twenty-one urinary proteins were found significantly dysregulated in MM and PM (p<0.05). STRING Analysis database revealed interaction between 15 proteins, that resulted mainly involved in immune and inflammatory response. Seven of the most considerable proteins were further quantified by Western-blot: protein S100A8 (S10A8), up-regulated in MM, uromodulin (UROM), alpha-1-microglobulin (AMBP), gelsolin (GELS) and prostaglandin-H2 D-isomerase (PTGDS), over-expressed in PM, apolipoprotein A-I (APOA1) and transthyretin (TTHY), respectively down- and up-regulated in both migraineur groups vs controls. These candidate biomarkers might be involved in the neurophysiological network of MM and PM, thus helping to better understand the pathophysiology of these migraine forms. If validated in large-scale studies, this protein cluster could become a distinctive target for clinical applications in migraine diagnosis and treatment.

2020 - Augmentation of endogenous neurosteroid synthesis alters experimental status epilepticus dynamics [Articolo su rivista]
Lucchi, C.; Costa, A. M.; Senn, L.; Messina, S.; Rustichelli, C.; Biagini, G.
abstract

Neurosteroids can modulate γ-aminobutyric acid type A receptor-mediated inhibitory currents. Recently, we discovered that the neurosteroids progesterone, 5α-dihydroprogesterone, allopregnanolone, and pregnanolone are reduced in the cerebrospinal fluid of patients with status epilepticus (SE). However, it is undetermined whether neurosteroids influence SE. For this reason, first we evaluated whether the inhibitor of adrenocortical steroid production trilostane (50 mg/kg) could modify the levels of neurosteroids in the hippocampus and neocortex, and we found a remarkable increase in pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone levels using liquid chromatography tandem mass spectrometry. Second, we characterized the dynamics of SE in the presence of the varied neurosteroidal milieu by a single intraperitoneal kainic acid (KA; 15 mg/kg) injection in trilostane-treated rats and their controls. Convulsions started in advance in the trilostane group, already appearing 90 minutes after the KA injection. In contrast to controls, convulsions prevalently developed as generalized seizures with loss of posture in the trilostane group. However, this effect was transient, and convulsions waned 2 hours before the control group. Moreover, electrocorticographic traces of convulsions were shorter in trilostane-treated rats, especially at the 180-minute (P < .001) and 210-minute (P < .01) time points. These findings indicate that endogenous neurosteroids remarkably modulate SE dynamics.

2020 - BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level [Articolo su rivista]
Avallone, Rossella; Lucchi, Chiara; Puja, Giulia; Codeluppi, Alessandro; Filaferro, Monica; Vitale, Giovanni; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly, rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases.

2020 - Chitosan/heparin polyelectrolyte complexes as ion-paring approach to encapsulate heparin in orally administrable SLN: In vitro evaluation [Articolo su rivista]
Maretti, E.; Pavan, B.; Rustichelli, C.; Montanari, M.; Dalpiaz, A.; Iannuccelli, V.; Leo, E.
abstract

Enhancing oral bioavailability of hydrophilic drugs by encapsulation in lipid-based nanocarriers, including Solid Lipid Nanoparticles (SLN), has been well documented. In this work, high molecular weight heparin was “insolubilized” by an “ion-paring” approach, forming Chitosan/Heparin Polyelectrolyte Complexes (PEC) to promote its encapsulation in SLN. Hybrid PEC-SLN, heparin-loaded SLN (H-SLN) as well as naked PEC were prepared and characterized regarding size, Z potential, morphology, drug loading and drug release. Physicochemical characterization of the nanoparticles was also performed by differential scanning calorimetry (DSC), and Fourier Transform Infra-Red (FTIR) analysis. FITC-labeled naked PEC along with Nile Red labeled PEC-SLN were assessed on CaCo-2 cells to study cytotoxicity as well as cell internalization ability by cytometric and confocal analysis. Transepithelial electrical resistance (TEER) was measured on NCM460 cell monolayers to evaluate whether chitosan may induce a modification of tight junctions’ integrity at epithelial level. Results showed that the minimum size of PEC (around 170 nm) was at pH 5.5 with a positive surface charge and after encapsulation in SLN produced hybrid PEC-SLN with a size of about 370 nm and a negative zeta potential. In comparison to both H-SLN and naked PEC, PEC-SLN were able to achieve a pH-controlled drug release and showed on CaCo-2 cells low toxicity and rapid internalization. Finally, TEER measurements highlighted that the hybrid nanocarriers were internalized without interference in the membrane resistance. Therefore, PEC-SLN could be considered valuable candidate for further in vivo investigations about the systemic bioavailability of oral heparin.

2020 - Nasal administration of nanoencapsulated geraniol/ursodeoxycholic acid conjugate: Towards a new approach for the management of Parkinson's disease [Articolo su rivista]
de Oliveira Junior, Edilson Ribeiro; Truzzi, Eleonora; Ferraro, Luca; Fogagnolo, Marco; Pavan, Barbara; Beggiato, Sarah; Rustichelli, Cecilia; Maretti, Eleonora; Lima, Eliana Martins; Leo, Eliana; Dalpiaz, Alessandro
abstract

The combined use of different therapeutic agents in the treatment of neurodegenerative disorders is a promising strategy to halt the disease progression. In this context, we aimed to combine the anti-inflammatory properties of geraniol (GER) with the mitochondrial rescue effects of ursodeoxycholic acid (UDCA) in a newly-synthesized prodrug, GER-UDCA, a potential candidate against Parkinson's disease (PD). GER-UDCA was successfully synthetized and characterized in vitro for its ability to release the active compounds in physiological environments. Because of its very poor solubility, GER-UDCA was entrapped into both lipid (SLNs) and polymeric (NPs) nanoparticles in order to explore nose-to-brain pathway towards brain targeting. Both GER-UDCA nanocarriers displayed size below 200 nm, negative zeta potential and the ability to increase the aqueous dissolution rate of the prodrug. As SLNs exhibited the higher GER-UDCA dissolution rate, this formulation was selected for the in vivo GER-UDCA brain targeting experiments. The nasal administration of GER-UDCA-SLNs (1 mg/kg of GER-UDCA) allowed to detect the prodrug in rat cerebrospinal fluid (concentration range = 1.1 to 4.65 μg/mL, 30–150 min after the administration), but not in the bloodstream, thus suggesting the direct nose to brain delivery of the prodrug. Finally, histopathological evaluation demonstrated that, in contrast to the pure GER, nasal administration of GER-UDCA-SLNs did not damage the structural integrity of the nasal mucosa. In conclusion, the present data suggest that GER-UDCA-SLNs could provide an effective and non-invasive approach to boost the access of GER and UDCA to the brain with low dosages.

2020 - Proteomic serum profile in menstrual-related and post menopause migraine [Articolo su rivista]
Bellei, Elisa; Rustichelli, Cecilia; Bergamini, Stefania; Monari, Emanuela; Baraldi, Carlo; Lo Castro, Flavia; Tomasi, Aldo; Ferrari, Anna
abstract

The aim of this pilot study was to analyze the serum proteomic profile of women suffering from menstrual-related migraine (MM group, n = 15) and migraine in post-menopause (PM group, n = 15) in comparison with non-headache control females (C group, n = 15). Serum samples were subjected to two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) analysis for protein identification. Based on 2D-gel maps and PDQuest 2-D software, 13 differentially expressed spots, corresponding to 12 unique proteins identified by Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight/tandem mass spectrometry (LC-ESI-QToF-MS/MS), were detected in the MM and PM groups vs C group. Five inflammatory and regulatory of vascular integrity proteins (prothrombin, serum amyloid P-component, Ig kappa chain C region, apolipoprotein A-I, serum amyloid A-4 protein) were found deregulated in both MM and PM groups compared to C group; MM group showed the upregulation of other inflammatory protein fragments (inter-alpha-trypsin inhibitor heavy chain H4 and complement C4-A) compared to C group; PM group, in comparison with C group, displayed a noteworthy upregulation of transthyretin and other deregulated proteins (tetranectin, alpha-1-antitrypsin, haptoglobin, apolipoprotein A-IV) playing a role in anti-inflammatory and reparative processes. In conclusion, proteomic analysis was able to reveal differences in protein expression between migraine sufferers and non-headache women; as in other neurological diseases characterized by neuroinflammation, the serum proteome of migraine women presents an abundance of proteins indicative of cellular damage, oxidative stress and inflammation. This relevant inflammatory status, if confirmed in larger series, could represent a target for menstrual-related migraine treatment.

2020 - Serum levels of allopregnanolone, progesterone and testosterone in menstrually-related and postmenopausal migraine: A cross-sectional study [Articolo su rivista]
Rustichelli, Cecilia; Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Baraldi, Carlo; Castro, Flavia Lo; Tomasi, Aldo; Ferrari, Anna
abstract

Background: Reduced blood or cerebrospinal fluid levels of allopregnanolone are involved in menstrual cycle-linked CNS disorders, such as catamenial epilepsy. This condition, like menstrually-related migraine, is characterized by severe, treatment-resistant attacks. We explored whether there were differences in allopregnanolone, progesterone and testosterone serum levels between women with menstrually-related migraine (MM, n¼30) or postmenopausal migraine without aura who had suffered from menstrually-related migraine during their fertile age (PM, n¼30) and non-headache control women in fertile age (FAC, n¼30) or post-menopause (PC, n¼30). Methods: Participants were women with migraine afferent to a headache centre; controls were female patients’ acquaintances. Serum samples obtained were analyzed by HPLC-ESI-MS/MS. Results: In menstrually-related migraine and postmenopausal migraine groups, allopregnanolone levels were lower than in the respective control groups (fertile age and post-menopause) (p<0.001, one-way analysis of variance followed by Tukey-Kramer post-hoc comparison test) while progesterone and testosterone levels were similar. By grouping together patients with migraine, allopregnanolone levels were inversely correlated with the number of years and days of migraine/ 3 months (p 0.005, linear regression analysis). Conclusion: Decreased GABAergic inhibition, due to low allopregnanolone serum levels, could contribute to menstrually-related migraine and persistence of migraine after menopause. For the management of these disorders, a rise in the GABAergic transmission by increasing inhibitory neurosteroids might represent a novel strategy.

2020 - Targeting pituitary adenylate cyclase-activating polypeptide (PACAP) with monoclonal antibodies in migraine prevention: a brief review [Articolo su rivista]
Rustichelli, Cecilia; Lo Castro, Flavia; Baraldi, Carlo; Ferrari, Anna
abstract

Introduction Interest is growing in the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its specific PAC1 receptor in migraine and in their antagonism as a strategy for migraine prevention. Areas covered We discuss and critically evaluate (i) the evidence of the role of PACAP in migraine pathophysiology and (ii) the first clinical trials in migraine prophylaxis with monoclonal antibodies AMG 301 and ALD1910 which act against PAC1 and PACAP38 respectively. We examined PubMed, Scopus, and ClinicalTrials.gov electronic databases to examine the relevant material. Expert opinion There is much proof of the ability of PACAP to cause migraine, but there is limited evidence that blocking PACAP or PAC1 receptor can prevent migraine. However, the potential of anti-PACAP antibodies in migraine prophylaxis is high. Theoretically, if these antibodies block the activation of the trigeminovascular system, they will prevent the onset of migraine attacks. There are still knowledge gaps in the role of PACAP in migraine and the risk/benefit ratio of anti-PACAP antibodies must be carefully studied.

2019 - Allopregnanolone serum levels in female migraineurs [Abstract in Rivista]
Rustichelli, C.; Bellei, E.; Bergamini, S.; Monari, E.; Lo Castro, F.; Baraldi, C.; Cainazzo, M. M.; Tomasi, A.; Ferrari, A.
abstract

Background: Migraine and epilepsy are similar brain disorders in many aspects and for both, a neuronal hyperexcitability has been hypothesized. Cyclic changes in ovarian hormones are involved in exacerbating both migraine and epilepsy during perimenstrual period, leading to menstrually-related migraine and catamenial epilepsy, respectively. Ovarian hormones and derived neurosteroids can regulate important functions in neurons and glial cells in the brain; in particular, progesterone reduces seizure susceptibility partly through its conversion to allopregnanolone, a potent positive allosteric modulator of the GABA-A receptor [1]. In spite of their neuroprotective potential [2], the role of neurosteroids in migraine has not been thoroughly investigated. Therefore, we determined serum levels of testosterone, progesterone and allopregnanolone in three groups: women suffering from menstrually-related migraine (n=30), post-menopausal women suffering from migraine without aura (n=30) and non-headache control females (n=20). Methods: The enrolled migraineurs were patients afferent to the Headache Centre of Modena University Hospital; the control females were friends or relatives of the above patients. All women gave their written consent and the Ethical Committee of the Province of Modena approved the study. The fasting blood specimens were processed and then analyzed by HPLC-ESI-MS/MS. Results: Testosterone and progesterone levels were significantly higher in both non-headache control females and women suffering from menstrually-related migraine compared to post-menopausal women suffering from migraine without aura (P <0.005, t-test). Conversely, serum allopregnanolone levels were significantly lower in both women suffering from menstrually-related migraine (0.051 ng/mL; SD: 0.018) and post-menopausal women suffering from migraine without aura (0.025 ng/mL; SD: 0.013), compared to non-headache control females (0.078 ng/mL; SD 0.036, P <0.005, t-test). Conclusion: Women suffering from migraine presented low serum levels of allopregnanolone, a neurosteroid that modulates GABAergic inhibition. Consequently, the reduced GABAergic inhibition could inadequately protect women suffering from migraine against inflammatory and algogenic stimuli. In particular, it could contribute to the severity and poor response to treatments of migraine attacks. According to our preliminary results, a raise in the GABAergic transmission achieved by drugs increasing the biosynthetic pathway of inhibitory neurosteroids or the use of synthetic analogs could represent a possible novel therapeutic strategy for migraine management. [1] Meletti S., et al J. Neurochem. 2018; 147:275-284. [2] Reddy D.S., et al. Trends Pharmacol Sci. 2016;37:543-561.

2019 - Intrapatient variability of the hair levels of pain medications in chronic migraine patients - a pilot study [Articolo su rivista]
Ferrari, Anna; Rustichelli, Cecilia; Baraldi, Carlo; Vandelli, Daniele; Verri, Patrizia; Marchesi, Filippo; Licata, Manuela
abstract

Monitoring of prescription pain medications is strongly recommended, considering their abuse liability. We used hair analysis for monitoring pain medications in 12 women with chronic migraine and drug overuse. Hair samples were collected at baseline and after 4 and 8 months and analyzed by liquid chromatography‐electrospray tandem mass spectrometry (LC‐MS/MS). Intrapatient variability of the hair level‐to‐dose ratio was < 20% in most cases. The hair analysis detected changes in the dose taken by the same patient over time, both occasionally and chronically. The agreement between the changes in hair drug levels and those of taken doses was excellent. Therefore, hair analysis appeared advantageous for long‐term monitoring of pain medications, which is otherwise difficult to determine in conventional matrices. In the same sample, multiple medications could be simultaneously determined, and with three samples, nine months of therapy were objectively documented.

2019 - Newly synthesized surfactants for surface mannosylation of respirable SLN assemblies to target macrophages in tuberculosis therapy [Articolo su rivista]
Maretti, Eleonora; Costantino, Luca; Buttini, Francesca; Rustichelli, Cecilia; Leo, Eliana Grazia; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

The present study reports about new Solid Lipid Nanoparticle assemblies (SLNas) loaded with rifampicin (RIF) surface-decorated with novel mannose derivatives, designed for anti-tuberculosis (TB) inhaled therapy by dry powder inhaler (DPI). Mannose is considered a relevant ligand to achieve active drug targeting being mannose receptors (MR) overexpressed on membranes of infected alveolar macrophages (AM), which are the preferred site of Mycobacterium tuberculosis. Surface decoration of SLNas was obtained by means of newly synthesized functionalizing compounds used as surfactants in the preparation of carriers. SLNas were fully characterized in vitro determining size, morphology, drug loading, drug release, surface mannosylation, cytotoxicity, macrophage internalization extent and ability to bind MR, and intracellular RIF concentration. Moreover, the influence of these new surface functionalizing agents on SLNas aerodynamic performance was assessed by measuring particle respirability features using Next Generation Impactor. SLNas exhibited suitable drug payload, in vitro release, and more efficient ability to enter macrophages (about 80%) compared to bare RIF (about 20%) and to non-functionalized SLNas (about 40%). The involvement of MR specific binding has been demonstrated by saturating MR of J774 cells causing a decrease of RIF intracellular concentration of about 40%. Furthermore, it is noteworthy that the surface-decoration of particles produced a poor cohesive powder with an adequate respirability (fine particle fraction ranging from about 30% to 50%). Therefore, the proposed SLNas may represent an encouraging opportunity in a perspective of an efficacious anti-TB inhaled therapy.

2019 - Novel engineered lipid-based nanoparticles for pulmonary tuberculosis inhalation therapy [Poster]
Maretti, Eleonora; Truzzi, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Martins Lima, Eliana; Leite Nascimento, Thais; Siligardi, Cristina; Gualtieri, Eva Magdalena; Miselli, Paola; Buttini, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Priorities to achieve the WHO goal of ending tuberculosis (TB) epidemic by 2030 include new drug treatments to simplify and shorter conventional drug regimens. TB is caused by Mycobacterium tuberculosis residing and surviving inside alveolar macrophages (AM). Considering that 75-80% of cases of infection remain localized in the lungs, the easiest and most successful therapy could involve the inhalation route offering benefits in terms of patient’s autonomy and compliance, by-passing hepatic metabolism, reducing dose amount, dose frequency, and treatment duration, thus minimising the risk of drug-resistant mutants, toxicity, and side effects. Inhalable powder formulations of repurposed drugs entail engineering techniques such as micro- or nanoparticulate carriers enabling drug emission by Dry Powder Inhaler devices, deposition onto alveolar epithelia, and transport into AM. Within this context, Solid Lipid Nanoparticle assemblies (SLNas) loaded with rifampicin, a clinically useful anti-TB drug, were produced by processing accepted excipients for DPI formulations through an optimized methodology that avoids organic solvents and is suitable for a large-scale production. The prototypes were functionalized by means of newly synthesized AM receptor-specific targeting agents as the ligands anchored on SLNas surface without chemical reactions. In vitro and in vivo preclinical studies highlighted functionalized SLNas with adequate respirability performance, safety, AM internalization ability, and mice lung deposition in an encouraging perspective of a potential efficacious pulmonary TB therapy. This research was supported by a grant on the project “FAR interdisciplinare 2017” from the University of Modena and Reggio Emilia, Modena, Italy (PI Prof. Luca Costantino)

2019 - Proteomic serum profile of female migraineurs [Abstract in Rivista]
Bellei, E; Bergamini, S; Monari, E; Rustichelli, C; Baraldi, C; Lo Castro, F; Tomasi, A; Ferrari, A
abstract

Background: Migraine is considered a complex disease, a variable disorder of nervous system function that has a genetic background, yet the final phenotypic outcome largely depends on the individual’s environment and lifestyle. In particular, there is a clear relationship between menstruation cycle and the onset of migraine. In fact, over 50% of migraine women suffer from perimenstrual attacks, that are more serious, lasting and resistant to the treatment than non-menstrual migraine attacks [1]. We hypothesized that serum proteome analysis could help to identify potential biomarkers of menstrually-related migraine (MM) and post-menopausal migraine (PMM). Methods: We analyzed and compared the serum proteomic profile of three groups: women suffering from MM (n=15), post-menopausal women suffering from migraine without aura (n=15) and non-headache control females (n=14). The enrolled migraineurs were patients afferent to the Headache Centre of Modena University Hospital; the control females were friends or relatives of the above patients. All women gave their written consent and the Ethical Committee of Modena approved the study. Serum samples obtained from each study participant were subjected to bi-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MS) analysis for protein identification. The 2D-gel maps were examined by the PDQuest software, to detect the differentially expressed protein spots between the different groups [2]. Results: A total of 13 significantly different protein spots were revealed in migraine women compared to controls. Of these proteins, most (n=10) resulted increased in migraineurs vs controls, while only 3 proteins were decreased. Specifically, the greater expression differences involved the up-regulation of transthyretin in PMM and the down-regulation of apolipoprotein A1 in MM. Other proteins, such as prothrombin, serum amyloid P-component and Ig-k-chain C region, were found significantly over-expressed in migraine sufferers in comparison to controls, while one spot, recognized as serum amyloid A-4 protein, resulted decreased. Conclusion: The serum proteome of migraine women showed proteins characteristic of cell damage, oxidative stress and lipoperoxidation, as well as acute phase proteins and inflammation markers. This pilot study demonstrates the ability of proteomics to reveal differences in protein expression between women suffering from MM and post-menopausal women suffering from migraine without aura against non-headache women. Further analysis will be carried out to expand and confirm these preliminary results. [1] Calhoun A.H. Headache 2018; 58:626-630. [2] Bellei E., et al. Amino Acids 2011; 40:145-156.

2019 - Seizures of illicit substances for personal use in two Italian provinces: analysis of trends by type and purity from 2008 to 2017 [Articolo su rivista]
Verri, Patrizia; Rustichelli, Cecilia; Ferrari, Anna; Marchesi, Filippo; Baraldi, Carlo; Licata, Manuela; Vandelli, Daniele; Palazzoli, Federica; Potì, Francesco; Silingardi, Enrico
abstract

Background: The use of illicit substances represents one of the most difficult problems to confront in the health system. Drug use is a global problem but is not uniform throughout the world, within the same country and changes over time. Therefore, knowing the illicit substances that are used in a territory is essential to better organize health services in that specific geographical area. To this aim, we analysed 4200 samples confiscated from individuals who held them for personal use by police forces in the Italian provinces of Modena and Reggio Emilia from 2008 to 2017. Methods: The suspected samples were screened by gas-chromatography-mass spectrometry (GC-MS) and by liquid chromatography-tandem mass spectrometry (LC-MS/MS); all samples were subsequently analysed by gas chromatography-flame ionization detector (GC-FID) for quantitative analyses. Results: Cannabis was the most seized illicit substance (70.7%). Over the study period, the number of seizures of herb with a high content of Δ9-THC increased. The number of cocaine seizures remained stable (total 16.1%), but the median purity of seized cocaine increased to 75% in 2017. Heroin seizures decreased over time, but the median purity of seized heroin reached 16.8% in 2017. In almost all the years, heroin samples with a purity exceeding the 97.5 percentile were found. Especially from 2014, the range of seized substances increased and started to include synthetic cathinones, phenylethylamines, UR-144, LSD, psilocybe, prescription opioid and hypnotics. In two cases, tramadol together with tropicamide was seized. Most of the seizures involved male subjects and 82% of the seizures were from individuals younger than 35 years of age. Conclusions: The persistence of old illicit drugs and the rapid emergence of new psychoactive substances represented a serious challenge for public health in the studied Italian area. Some useful interventions might be: informing mainly young people about the possible complications of cannabis use; implementing standardized procedures to diagnose and treat cocaine-related emergencies in hospitals; increasing the distribution of naloxone to antagonize possible heroin overdoses; equipping laboratories to be able to identify the new psychoactive substances.

2019 - Self-assembled organogelators as artificial stratum corneum models: key-role parameters for skin permeation prediction [Articolo su rivista]
Maretti, Eleonora; Rustichelli, Cecilia; Miselli, Paola; Leo, Eliana Grazia; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

Self-assembled organogelators were explored as artificial stratum corneum (SC) models for the in vitro skin permeation assessment. Four SC models consisting of binary (organogels) or ternary (microemulsion-based organogels) mixtures were developed using stearic acid, tristearin, or sorbitan tristearate, at two different concentrations, gelled in squalene. The permeation of lipophilic butyl-methoxydibenzoylmethane and hydrophilic methylene blue as the permeant compounds across the SC models was compared with ex vivo experiments using excised porcine ear skin. A multi-analytical approach was adopted to provide detailed understanding about organogelator organization within the SC models and find possible parameters playing key-roles in SC permeation prediction. The SC models were investigated for gelling properties and microstructure. Parameters such as gel occurrence, organogelator concentration, and rheological properties appeared as negligible conditions for skin permeation prediction. Conversely, arrangement packing, interactions, and crystallinity extent of the self-assembled organogelator were found to play a fundamental role in the simulation of SC barrier function according to the permeant feature.

2019 - The Impact of Lipid Corona on Rifampicin Intramacrophagic Transport Using Inhaled Solid Lipid Nanoparticles Surface-Decorated with a Mannosylated Surfactant [Articolo su rivista]
Maretti, Eleonora; Rustichelli, Cecilia; Gualtieri, Eva Magdalena; Costantino, Luca; Siligardi, Cristina; Miselli, Paola; Buttini, Francesca; Montecchi, Monica; Leo, Eliana Grazia; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

The mimicking of physiological conditions is crucial for the success of accurate in vitro studies. For inhaled nanoparticles, which are designed for being deposited on alveolar epithelium and taken up by macrophages, it is relevant to investigate the interactions with pulmonary surfactant lining alveoli. As a matter of fact, the formation of a lipid corona layer around the nanoparticles could modulate the cell internalization and the fate of the transported drugs. Based on this concept, the present research focused on the interactions between pulmonary surfactant and Solid Lipid Nanoparticle assemblies (SLNas), loaded with rifampicin, an anti-tuberculosis drug. SLNas were functionalized with a synthesized mannosylated surfactant, both alone and in a blend with sodium taurocholate, to achieve an active targeting to mannose receptors present on alveolar macrophages (AM). Physico-chemical properties of the mannosylated SLNas satisfied the requirements relative to suitable respirability, drug payload, and AM active targeting. Our studies have shown that a lipid corona is formed around SLNas in the presence of Curosurf, a commercial substitute of the natural pulmonary surfactant. The lipid corona promoted an additional resistance to the drug diffusion for SLNas functionalized with the mannosylated surfactant and this improved drug retention within SLNas before AM phagocytosis takes place. Moreover, lipid corona formation did not modify the role of nanoparticle mannosylation towards the specific receptors on MH-S cell membrane.

2018 - Aerodynamic performance of lipid-based nanocarrier functionalized by novel mannose derivatives for Rifampicin intramacrophagic delivery [Abstract in Atti di Convegno]
Maretti, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Buttini, Francesca; Iannuccelli, Valentina
abstract

Introduction. Alveolar macrophages (AM) are sites of infection by pathogens such as Mycobacterium tuberculosis. On the basis of the presence of mannose receptors on AM membranes, mannose moieties have been considered as ligands for macrophage active targeting. Mannosylated derivatives were synthesized to decorate the surface of Rifampicin (RIF) loaded Solid Lipid Nanoparticle assemblies (SLNas) for an inhaled anti-tuberculosis (TB) therapy by Dry Powder Inhaler (DPI) device with the goal to relate surface functionality with respirability performance. Methods. Biocompatible lipid components such as fatty acids and their derivatives were processed using the melt emulsification technique. SLNas surface decoration was obtained by means of four newly synthesized mannose derivatives having surfactant effect. The obtained mannosylated carriers were examined for their intrinsic properties (size, morphology and shape, surface charge, bulk and tap density, aerodynamic diameter, physical state of the components, drug loading and in vitro release) associated to respirability features assessed by Next Generation Impactor (NGI). Mannosylation occurrence was investigated using X-ray Photoelectron Spectroscopy for Chemical Analysis. Furthermore, SLNas cytotoxicity, macrophage internalization, and ability to provide RIF intramacrophagic transport were evaluated on J774 cell line by MTT test, flow cytometry, and confocal microscopy. Results and Discussion. SLNas exhibited adequate drug payload, in vitro release, and cell internalization (Fig. 1, Confocal microscopy images of J774 cells incubated for 6 h with Nile Red labeled SLNas after blue nuclei and green lysosome staining) with consequent efficient RIF translocation inside macrophages (about 70%). A relevant increase in drug content within macrophages provided by SLNas compared to free RIF solution (Fig. 2, Intracellular RIF percentage inside J774 cell line) confirmed both RIF difficulty to diffuse across macrophage membranes and SLNas efficacy to promote drug transport into cells. Furthermore, it is noteworthy that the presence of these new surface-active agents reduced powder cohesiveness without impairing respirability (fine particle fraction ranging from 30% to 50%) (Fig. 3, SLNas aerodynamic distribution by NGI upon powder aerosolisation). SLNas proposed in this research can be prepared by a green-technology avoiding organic solvents. Thus, these new SLNas may represent an encouraging opportunity in a perspective of an efficacious anti-TB inhaled therapy.

2018 - Low levels of progesterone and derivatives in cerebrospinal fluid of patients affected by status epilepticus [Articolo su rivista]
Meletti, Stefano; Lucchi, Chiara; Monti, Giulia; Giovannini, Giada; Bedin, Roberta; Trenti, Tommaso; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neurosteroids such as allopregnanolone may play a role in epilepsy as positive modulators of inhibitory currents mediated by γ-aminobutyric acid type A (GABAA ) receptor. Indeed, these molecules have been consistently shown to be anticonvulsants in animal models, but their role is still unclear in patients. For this reason, we investigated neurosteroids in the cerebrospinal fluid (CSF) of patients with status epilepticus (SE) by liquid chromatography tandem-mass spectrometry. Patients were retrospectively identified within subjects who received a lumbar puncture in the 2007-2017 period. Seventy-three patients (median age 65, ranging from 13 to 94 years; 67% women) with SE were evaluated. Controls (n = 52, median age 53, ranging from 16 to 93 years; 65% women) were patients presenting with symptoms for which a lumbar puncture was required by clinical guidelines, and who were negative at the end of the diagnostic work-up. Progesterone was 64% lower in patients with SE (p < 0.001). With respect to progesterone, upstream pregnenolone sulfate and pregnenolone did not change. Instead, downstream 5α-dihydroprogesterone, pregnanolone and allopregnanolone were respectively 49% (p < 0.001), 21% (p < 0.01) and 37% (p < 0.001) lower than in controls. Duration or type of SE, age and sex did not consistently affect CSF neurosteroid levels in the SE cohort. Instead, pregnenolone sulfate (Spearman's ρ = 0.4335, p < 0.01), allopregnanolone (ρ = 0.4121, p < 0.05), and pregnanolone (ρ = 0.592, p < 0.001) levels significantly increased by ageing in controls. We conclude that neurosteroidogenesis is defective in patients with SE. This article is protected by copyright. All rights reserved.

2018 - Polymeric and Solid Lipid Nanoparticles for nose-to-brain delivery of geraniolursodeoxycholic acid conjugate: development and characterization studies [Abstract in Atti di Convegno]
Edilson Oliveira Junior, ; Truzzi, Eleonora; Maretti, Eleonora; Leo, Eliana Grazia; Dalpiaz, Alessandro; Rustichelli, Cecilia; Marco, Fogagnolo; Eliana, Lima
abstract

Neurodegenerative disorder treatment is a challenge mainly due to the difficulty of drug transport across the blood-brain barrier [1]. Intranasal administration of nanoparticles as carrier system may increase drug concentration into the brain [2]. Geraniol (GER) has demonstrated antioxidant and neuroprotective activities in Parkinson’s disease animal models [3]. However, due to its volatility, GER is hardly incorporated into freezedrying nanoparticles. On the other hand, GER-ursodeoxycholic acid conjugate (GER-UDCA) is a non-volatile derivative with high potentiality to be incorporated into nanocarriers. Therefore, in this work GER-UDCA-loaded Solid Lipid Nanoparticles (SLNs) and PLGA nanoparticles (NPs) intended for nose-to-brain delivery were developed and characterized. SLNs were prepared by emulsion/solvent evaporation method and NPs by nanoprecipitation method. Briefly, formulations were optimized considering various processing variables and nanoparticle characterization was performed in terms of morphology, size, surface charge, drug loading (DL%), encapsulation efficiency (EE%) and in vitro drug release. Finally, the stability of free and encapsulated GERUDCA was evaluated in enzymatic medium from rat liver homogenates. GER-UDCA-SLN and GER-UDCA-NPs showed spherical shape, mean size of 120/180 nm with polydispersity index < 0.2, and zeta potential around −22/-26 mV, respectively. After freeze-drying, the DL% was 6% for SLN and 12% for NPs with EE% values of 89.3% and 60.1%, respectively. Preliminary data regarding in vitro release of GER-UDCA from the nanoparticles evidenced a higher dissolution rate than the free drug, probably due to the increase of surface contact. Results in liver rat homogenate suggested a contribution of the nanoparticles in the stability of the prodrug in physiologic environments. In conclusion, these GER-UDCA-loaded nanocarriers demonstrated a possible application in further in vivo studies of nose-to-brain drug delivery.

2018 - Polypharmacy Among Headache Patients: A Cross-Sectional Study [Articolo su rivista]
Ferrari, Anna; Baraldi, Carlo; Licata, Manuela; Rustichelli, Cecilia
abstract

Background Polypharmacy can appropriately treat multiple chronic conditions, but it can also increase potential harm. Polypharmacy information for primary headaches is minimal, despite drugs being the main tools to manage headaches. Objective The aim was to evaluate the prevalence, characteristics and risk factors of polypharmacy in patients with primary headaches and examine whether these variables differ between episodic and chronic headache patients. Methods We analysed polypharmacy (simultaneous use of five or more medications), medication type, comorbidity, and risk factors in 300 patients (mean age 42.81 ± 13.21 years) with primary headaches, divided into episodic and chronic, afferent to a headache centre. Results Patients took an average of 4.37 medications. Polypharmacy was common in 40.7% of patients, and among chronic patients, it reached 58.8%. Most patients used medications (mainly nonsteroidal anti-inflammatory drugs; 73.5%) to treat acute headaches, and 30.4% of episodic and 64.7% of chronic sufferers underwent prophylactic treatment (P < 0.0001), mostly using antidepressants (77.3%). Up to 76.7% of the cohort was taking other medications, primarily for acid-related disorders (21.7%). Comorbidities were present in 59.7% of the cohort. Variables significantly associated with polypharmacy were comorbidities, prophylactic treatment, and triptans (P < 0.001). Conclusions Patients with primary headaches, mainly young adults, are exposed to high polypharmacy, comparable to that of the elderly. Because increased numbers of drugs increase the risk of adverse reactions, the many medications concomitantly taken by primary headache sufferers should be frequently reviewed.

2018 - Ridotta concentrazione del progesterone e di altri neurosteroidi nel liquor di pazienti in stato di male epilettico [Abstract in Atti di Convegno]
Biagini, G.; Lucchi, C.; Monti, G.; Giovannini, G.; Bedin, R.; Rustichelli, C.; Meletti, S.
abstract

Lo stato di male epilettico (SE) costituisce una grave emergenza neurologica. In tale condizione, mediante cromatografia liquida e spettrometria di massa, abbiamo misurato e confrontato i livelli di pregnenolone solfato, pregnenolone, progesterone, 5α-diidroprogesterone, pregnanolone e allopregnanolone nel liquor di pazienti affetti da SE e in una coorte di controllo. Sono stati studiati 73 pazienti ricoverati per SE, con età di 65 anni (intervallo di variazione da 13 a 94 anni), dei quali il 67% donne. I controlli esaminati, pari a 52, avevano età compresa tra 16 e 93 anni (mediana pari a 53), dei quali il 65% donne. I risultati sono stati analizzati con il test di Mann-Whitney. Abbiamo anche correlato le concentrazioni dei neurosteroidi e l’età, il genere, la durata ed il tipo di SE. Il progesterone è risultato essere diminuito del 64% nel liquor dei pazienti con SE (p < 0.001). I neurosteroidi pregnenolone solfato e pregnenolone, precursori del progesterone, sono risultati a livelli analoghi a quelli dei controlli. I derivati del progesterone, al contrario, in particolare il 5α-diidroprogesterone (-49%, p < 0.001), il pregnanolone (-21%, p = 0.005) e l’allopregnanolone (-37%, p < 0.001), sono risultati essere inferiori ai controlli. Le variazioni osservate nei livelli liquorali di neurosteroidi non hanno travato spiegazioni nelle differenze di genere, età o tipo di SE. Inoltre, non sono stati riscontrati effetti dovuti alla durata del SE. In conclusione, questi risultati indicano che vari neurosteroidi anticonvulsivanti sono presenti a ridotte concentrazioni liquorali nei pazienti che presentano un’attività epilettica protratta.

2018 - Self-assembling organogelators for artificial stratum corneum models: key-role parameters in skin permeation prediction [Poster]
Iannuccelli, V.; Maretti, E.; Rustichelli, C.; Miselli, P.; Truzzi, E.; Leo, E.
abstract

The development of in vitro methods to predict in vivo percutaneous absorption of bioactive molecules is a challenge to which the researchers are called in order to eliminate or reduce the pharmacological and toxicological tests on animal models. Artificial stratum corneum (SC) models obtained by self-assembled oganogelators were designed for skin permeation assessment of butyl methoxydibenzoylmethane (BMDBM, log Po/w = 4.68) and methylene blue (MB, log Po/w = 0.91). A multi-analytical approach was adopted to provide detailed understanding about the gelator organization within the models and find possible parameters playing a key-role in in vivo and ex vivo SC permeation prediction. The evaluation of in vitro skin permeation data compared with those obtained ex vivo and previously in vivo on humans for BMDBM showed good correlations vitro/ex vivo and vitro/vivo for both butyl BMDBM, as the lipophilic permeant, and MB, as the hydrophilic permeant, by using TS20 as well as both STS and ME models. With the aim of providing detailed understanding about the organogelator behaviour and organization within the models and find possible parameters playing a key-role in SC permeation prediction a multi-analytical approach was adopted. All the models did not flow upon tube tilting and could be described as gels, with the exception of STS10 model that appeared as thick liquid being gelator concentration lower than mgc value. Unlike SA and TS models that exhibited networks capable of immobilizing completely the solvent, STS and ME10 models revealed the syneresis phenomenon according to gelator concentration. The actual presence of water within STS aggregates (reverse micelles) of ME models was demonstrated by means of TG/DTA analysis showing two thermal events in the range of about 50-130°C related to removal of water molecules. Unlike the pure gelators, XRPD profiles from all the SC models exhibited a broad peak at about 20° 2θ indicating the presence of a networked structure of the gelators where the width of the peak at half maximum is dependent on the crystallinity of the sample, which in turn is dependent on non-covalent interactions amongst the gelator molecules responsible for the formation of an ordered structure. Intermolecular interactions also arisen from FT-IR spectra showing subsided ester group stretching in TS, STS, and ME models. Architectural arrangements of the organogelators within TS, STS, and ME models, as outlined by microscopy analyses, involved round or worm-like architectures of spherulitic clusters. Under polarized light, the occurrence of birefringence revealed the so-called “maltese crosses” in STS models that are characteristic of liquid crystals with lamellar structures. The results demonstrated the relevant role of both the arrangement of gelator packing and crystallinity extent in mimicking SC in vivo/ex vivo skin permeation of both lipophilic and hydrophilic compounds. These findings could account for the behaviour and development of other artificial skin models involving different materials for the skin permeation prediction.

2018 - The role of ethyl glucuronide in supporting medico-legal investigations: Analysis of this biomarker in different postmortem specimens from 21 selected autopsy cases [Articolo su rivista]
Santunione, Anna Laura; Verri, Patrizia; Marchesi, Filippo; Rustichelli, Cecilia; Palazzoli, Federica; Vandelli, Daniele; Licata, Manuela; Silingardi, Enrico
abstract

Ethanol determination in postmortem blood is one of the most frequently requested analyses in legal medicine and forensic toxicology. Ethyl glucuronide is a non-oxidative ethanol metabolite. It is also a useful marker of ante-mortem alcohol ingestion when ethanol itself has been completely eliminated from the body and could be considered in autopsy cases to obtain more reliable indications. The aim of the present study was to investigate the ethyl glucuronide distribution in postmortem specimens from autopsy cases found to be positive for ethanol. We presented 21 autopsy cases in which central blood, peripheral blood and liver samples were available. Specimens were analyzed for ethyl glucuronide by liquid chromatography tandem mass spectrometry; we also recorded postmortem interval, case history, cause of death, use of drugs, metabolic disorders if present, putrefaction if present, history of ethanol abuse and information about ethanol intake before death. Our aim was to evaluate and to compare the ethyl glucuronide levels in different matrices taken from the same subject in order to provide a better understanding of the interpretation of postmortem ethyl glucuronide concentrations.

2017 - Decreased allopregnanolone levels in cerebrospinal fluid obtained during status epilepticus [Articolo su rivista]
Meletti, Stefano; Lucchi, Chiara; Monti, Giulia; Giovannini, Giada; Bedin, Roberta; Trenti, Tommaso; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neuroactive steroids are increasingly considered as relevant modulators of neuronal activity. Especially allopregnanolone (AP) and pregnenolone sulfate (PS) have been shown to possess, respectively, anticonvulsant or proconvulsant properties. In view of the potential role of these steroids, we aimed at evaluating AP and PS levels in cerebrospinal fluid (CSF) and blood samples obtained from patients with status epilepticus (SE). To this purpose, we enrolled 41 patients affected by SE and 41 subjects investigated for nonepileptic neurologic disorders. Liquid chromatographic procedures coupled with electrospray tandem mass spectrometry and routine laboratory investigations were performed. Significantly lower AP levels were found in the CSF of patients affected by SE (-30%; p &lt; 0.05, Mann-Whitney test). Notably, AP was not detectable in 28 of 41 patients affected by SE (p &lt; 0.01 vs. controls, Fisher's exact test). In serum, AP levels did not differ in the two considered groups. Conversely, PS was present at similar levels in the investigated groups. Finally, differences in AP levels could not be explained by a variation in CSF albumin content. These findings indicate that AP is defective in the CSF of patients affected by SE. This phenomenon was not dependent on carriers for steroids, such as albumin.

2017 - Glutamate receptor antagonists with the potential for migraine treatment [Articolo su rivista]
Ferrari, Anna; Rustichelli, Cecilia; Baraldi, Carlo
abstract

Preclinical, clinical, and other (e.g., genetic) evidence support the concept that migraine susceptibility may at least partially result from a glutamatergic system disorder. Therefore, the receptors of the glutamatergic system are considered relatively new targets for investigational drugs to treat migraine. Investigational and established glutamate receptor antagonists (GluRAs) have been shown to possess antinociceptive properties in preclinical models of trigeminovascular nociception and have been evaluated in clinical trials. This review focuses on preclinical and clinical studies of GluRAs for the treatment of migraine. Areas covered: A PubMed database search (from 1987 to December 2016) and a review of published studies on GluRAs in migraine were conducted. Expert opinion: All published clinical trials of investigational GluRAs have been unsuccessful in establishing benefit for acute migraine treatment. Clinical trial results contrast with the preclinical data, suggesting that glutamate (Glu) does not play a decisive role after the attack has already been triggered. These antagonists may instead be useful for migraine prophylaxis. Improving patient care requires further investigating and critically analyzing the role of Glu in migraine, designing experimental models to study more receptors and their corresponding antagonists, and identifying biomarkers to facilitate trials designed to target specific subgroups of migraine patients.

2017 - Monitoring of adherence to headache treatments by means of hair analysis [Articolo su rivista]
Ferrari, Anna; Licata, Manuela; Rustichelli, Cecilia; Baraldi, Carlo; Vandelli, Daniele; Marchesi, Filippo; Palazzoli, Federica; Verri, Patrizia; Silingardi, Enrico
abstract

The aim of this study was to evaluate the potential of hair analysis to monitor medication adherence in headache patients undergoing chronic therapy. For this purpose, the following parameters were analyzed: the detection rate of 23 therapeutic drugs in headache patients' hair, the degree of agreement between the self-reported drug and the drug found in hair, and whether the levels found in hair reflected the drug intake reported by the patients.

2017 - Nuove molecole funzionalizzanti per il targeting ai macrofagi di vettori lipidici [Brevetto]
Iannuccelli, Valentina; Maretti, Eleonora; Costantino, Luca; Leo, Eliana; Rustichelli, Cecilia; Truzzi, Eleonora
abstract

La presente invenzione ha per oggetto molecole derivate dal mannosio ed il loro uso per direzionare carrier farmacologici in maniera specifica verso i macrofagi. L’invenzione consiste nella sintesi di nuove molecole derivate dal mannosio utilizzate come funzionalizzanti di superficie per vettori micro- e nano- particellari progettati per attività intra-5 macrofagica di farmaci somministrabili per via inalatoria.

2017 - Reduced steroidogenesis in patients with PCDH19-female limited epilepsy [Articolo su rivista]
Trivisano, M.; Lucchi, Chiara; Rustichelli, Cecilia; Terracciano, A.; Cusmai, R.; Ubertini, G. M.; Giannone, G.; Bertini, E.; Vigevano, F.; Gecz, J.; Biagini, Giuseppe; Specchio, N.
abstract

Patients affected by protocadherin 19 (PCDH19)–female limited epilepsy (PCDH19- FE) present a remarkable reduction in allopregnanolone blood levels. However, no information is available on other neuroactive steroids and the steroidogenic response to hormonal stimulation. For this reason, we evaluated allopregnanolone, pregnanolone, and pregnenolone sulfate by liquid chromatographic procedures coupled with electrospray tandem mass spectrometry in 12 unrelated patients and 15 agematched controls. Wealso tested cortisol, estradiol, progesterone, and 17OH-progesterone using standard immunoassays. Apart from estradiol and progesterone, all the considered hormones were evaluated in basal condition and after stimulation with adrenocorticotropic hormone (ACTH). A generalized decrease in blood levels of almost all measured neuroactive steroids was found. When considering sexual development, cortisol and pregnenolone sulfate basal levels were significantly reduced in postpubertal girls affected by PCDH19-FE. Of interest, ACTH administration did not recover pregnenolone sulfate serum levels but restored cortisol to control levels. In prepubertal girls with PCDH19-FE, by challenging adrenal function with ACTH we disclosed defects in the production of cortisol, pregnenolone sulfate, and 17OH-progesterone, which were not apparent in basal condition. These findings point to multiple defects in peripheral steroidogenesis associated with and potentially relevant to PCDH19-FE. Some of these defects could be addressed by stimulating adrenocortical activity.

2017 - Surface engineering of Solid Lipid Nanoparticle assemblies by methyl α-d-mannopyranoside for the active targeting to macrophages in anti-tuberculosis inhalation therapy [Articolo su rivista]
Maretti, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Leo, Eliana Grazia; Croce, Maria Antonietta; Francesca, Buttini; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

This study describes the development of new mannosylated Solid Lipid Nanoparticle assemblies (SLNas) delivering rifampicin for an inhaled treatment of tuberculosis. SLNas were surface engineered with mannose residues to recognize mannose receptors located on infected alveolar macrophages and facilitate cell internalization. Two sets of SLNas were produced by the melt emulsifying technique using biocompatible lipid components, i.e. cholesteryl myristate combined with palmitic acid (PA set) or tripalmitin (TP set), in the presence of the targeting moiety, methyl α-d-mannopyranoside. Mannosylated SLNas were examined for their physical properties, drug payloads and release, as well as respirability in terms of emitted dose and respirable fraction determined by Next Generation Impactor. The most appropriate formulations were assessed for mannosylation using FTIR, XPS, SEM coupled with EDX analysis, and wettability assay, in comparison with the respective non-functionalized SLNas. Besides, cytotoxicity and cell internalization ability were established on J774 murine macrophage cell line. Mannosylated SLNas exhibited physical properties suitable for alveolar macrophage passive targeting, adequate rifampicin payloads (10-15%), and feasible drug maintenance within SLNas along the respiratory tract before macrophage internalization. Despite respirability impaired by powder cohesiveness, surface mannosylation provided quicker macrophage phagocytosis, giving evidence of an active targeting promotion.

2016 - Allopregnanolone is reduced in patients with PCDH19-related epilepsy [Abstract in Rivista]
Trivisano, M; Lucchi, C; Terracciano, A; Rustichelli, C.; Cusmai, R; Ubertini, Gm; Giannone, G; Bertini, E; Vigevano, F; Gecz, J; Biagini, G; Specchio, N
abstract

Aim: The inherit base of PCDH19-related epilepsy suggests a hormonal involvement due to de-regulation of AKR1C1-3 genes, which encode for crucial steroid hormone-metabolizing enzymes. Both mRNA and protein levels of AKR1C3 have been reported to be decreased in a small number of PCDH19 mutated patients. Aim of this study is to verify allopregnanolone and other neuroactive steroids serum levels in PCDH19 mutated patients. Methods: We performed a prospective case-control study. We enrolled 12 patients affected by PCDH19-related epilepsy and 15 controls, age-and sex-matched. Controls were recruited among subjects evaluated for praecox puberty or hyperandrogenism. In both groups blood samples were taken at basal (T0) and 60 min after (ACTH) administration (T1). Laboratory methods: Quantitative analysis of neuroactive steroids in serum was performed by liquid chromatography-electrospray tandem mass spectrometry. Study population: median age of PCDH19-mutated patients was 8.3+5.7 years (range 2.5-18.9). Other than epilepsy, 6 patients had mental retardation. Controls were all females, age 9.2+4.0 years (range 6.1-17.9). All controls had a normal cognitive profile. Nine patients had a diagnosis of praecox puberty and 6 hyperandrogenism. Results: All neuroactive steroids resulted down produced in patients with PCDH19-related epilepsy rather than controls. At basal assessment (T0), allopregnanolone was 0.9+0.5 vs 0.18+0.9 ng/ml (p&lt;0.05); pregnanolone was 0.0+0.5 vs 0.3+0.5 ng/ml (p&gt;0.05), but pregnanolone solphate sulfate was 7.39+5.53 vs 75.41+52.69 ng/ml (p&lt;0.05). Also cortisol, progesterone and 17-OH progesterone resulted to be lowered in PCDH19-mutated patients compared with controls. After ACTH injection (T1), both neuroactive steroids and the other peripheral steroids, were confirmed to be lower compared with controls. ACTH test demonstrated a normal functioning of peripheral glands. Conclusion: We documented a down regulation of all steroidogenesis in PCDH19-related epilepsy. Particularly we found allopregnanolone and pregnaenolone solphate sulfate deficiency. Allopregnanolone is a GABA-A receptor modulators influencing the neuronal excitability, thus representing a realistic therapeutic target for PCDH19-related epilepsy.

2016 - Anti-TB inhalation therapy: Design of mannose-based functionalised Solid Lipid Microparticles for an active targeting to alveolar macrophages [Abstract in Atti di Convegno]
Maretti, Eleonora; Rustichelli, Cecilia; Costantino, Luca; Truzzi, Eleonora; Sacchetti, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Human tuberculosis (TB) is mainly a disease of the lung characterised by a long chronic stage of infection and progressive pathology that compromise the respiratory system. This is a curable infectious bacterial disease caused by the Mycobacterium tuberculosis (Mtb). TB therapies have exploited conventional routes of administration, such as oral and intramuscular1. The pulmonary route appears the most reasonable and effective way to target the alveolar macrophages (AM) and eradicate surviving Mtb at the primary infected site of TB, especially considering that 75-80% of cases remain localised in the lungs. The anti-TB therapy by inhalation offers benefits compared with the current treatment in terms of patient’s compliance improvement, reduction in dose amount and frequency, treatment duration and TB diffusion in other organs, thus minimising the risk of drug-resistant mutants, toxicity and side effects. For a direct intramacrophagic antitubercular therapy using Dry Powder Inhaler (DPI) devices, Solid Lipid Microparticles (SLM), produced using the melt emulsifying technique followed by freeze-drying, were developed to load rifampicin, a first-line antitubercular drug. In the present project, SLM were modified to improve drug loading level and release as well as AM targeting. Several biocompatible lipid components such as fatty acids and their derivatives, diglycerides and triglycerides, were processed using mixtures of biocompatible stabilisers (sodium taurocholate and methyl mannopyranoside) in order to obtain SLM with maximum efficiency in terms of drug loading and release in simulated lung fluid. Lipids in the liquid physical state embedded into SLM provided Microstructured Lipid Carriers (MLC) that are known to exhibit superior advantages over SLM such as enhanced drug loading capacity and prevention of drug expulsion intended to maximise the drug concentration at the primary site of TB infection. The obtained microcarriers were examined for their intrinsic properties such as size and size distribution, morphology and shape, surface charge, bulk and tap density, aerodynamic diameter, physical state of the components, wettability, drug loading and release. Macrophages, as is common knowledge, possess mannose-specific membrane receptors (MR) that can be recognised by carriers bearing mannose residues, facilitating their internalisation 2, 3.
Therefore, the functionalisation of SLM surface by mannose derivatives used as the co- stabiliser in the SLM formulation was used to achieve an active targeting. The actual presence of mannose on SLM surface was investigated by means of X-ray Photoelectron Spectroscopy for Chemical Analysis (XPS) and Energy Dispersive X-ray Analysis (EDX).

2016 - Development and validation of a liquid chromatography-tandem mass spectrometric assay for quantitative analyses of triptans in hair [Articolo su rivista]
Vandelli, Daniele; Palazzoli, Federica; Verri, Patrizia; Rustichelli, Cecilia; Marchesi, Filippo; Ferrari, Anna; Baraldi, Carlo; Giuliani, Enrico; Licata, Manuela; Silingardi, Enrico
abstract

Triptans are specific drugs widely used for acute treatment of migraine, being selective 5HT1B/1D receptor agonists. A proper assumption of triptans is very important for an effective treatment; nevertheless patients often underuse, misuse, overuse or use triptans inconsistently, i.e., not following the prescribed therapy. Drug analysis in hair can represent a powerful tool for monitoring the compliance of the patient to the therapy, since it can greatly increase the time-window of detection compared to analyses in biological fluids, such as plasma or urine. In the present study, a liquid chromatography-tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the quantitative analysis in human hair of five triptans commonly prescribed in Italy: almotriptan (AL), eletriptan (EP), rizatriptan (RIZ), sumatriptan (SUM) and zolmitriptan (ZP). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode SPE cartridges and analyzed by LC–MS/MS under gradient elution in positive multiple reaction monitoring (MRM) mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection (LOD) and lower limit of quantitation (LLOQ), accuracy, precision, carry-over, recovery, matrix effect and dilution integrity. The method was linear in the range 10–1000 pg/mg hair, with R2 values of at least 0.990; the validated LLOQ values were in the range 5–7 pg/mg hair. The method offered satisfactory precision (RSD &lt;10%), accuracy (90–110%) and recovery (&gt;85%) values. The validated procedure was applied on 147 authentic hair samples from subjects being treated in the Headache Centre of Modena University Hospital in order to verify the possibility of monitoring the corresponding hair levels for the taken triptans.

2016 - Endogenous Neurosteroids Levels are Decreased in CSF During Status Epilepticus [Abstract in Rivista]
Monti, G; Lucchi, C; Rustichelli, C; Giovannini, G; Meletti, S; Biagini, G
abstract

Purpose: Neuroactive steroids, such as allopregnanolone (AP), are positive allosteric modulators of GABA-A receptors enhancing both synaptic and extrasynaptic GABA-A mediated inhibition and might be effective in the treatment of benzodiazepine-resistant status epilepticus (SE). No data are available on AP levels in central nervous system (CNS) of patients suffering from SE. This lack of information hampers the possibility to correctly design a rational therapeutic approach to SE. We designed a study to evaluate AP in serum and cerebrospinal fluid (CSF) of patients affected by SE. Method: We retrospectively (2007–2015) evaluated blood and CSF samples from 41 adult patients with diagnosis of SE who received lumbar puncture at SE onset (median of 4 days) for clinical purposes and for whom a CNS infection was finally excluded. 41 subjects (matched for age and sex) that had negative results after lumbar puncture for suspected idiopathic intracranial hypertension, CNS infection, or inflammatory disease served as control group. Quantitative analysis of neurosteroids was performed by liquid chromatography-electrospray tandem mass spectrometry. Results: Serum AP levels did not revealed significant differences between controls and SE patients. However, CSF AP levels were decreased by approximately 30% (p &lt; 0.05, Mann-Whitney test) in patients compared with controls. Interestingly, linear regression did not reveal a relationship between serum and CSF levels for AP in controls (R2 = 0.06, p = 0.27). On the contrary, a significant relationship (R2 = 0.57, p = 0.003) was present in patients affected by SE. Conclusion: We demonstrated that endogenous allopregnenolone is significantly reduced during SE in CSF. Administration of AP to patients suffering of SE could be useful to reestablish AP level in the CNS. In addition, exogenously administered AP may result to have a privileged access to brain tissue by virtue of the higher permeability of the blood- brain barrier during SE.

2016 - Hair analysis for detection of triptans occasionally used or overused by migraine patients-a pilot study [Articolo su rivista]
Ferrari, Anna; Baraldi, Carlo; Licata, Manuela; Vandelli, Daniele; Marchesi, Filippo; Palazzoli, Federica; Verri, Patrizia; Rustichelli, Cecilia; Giuliani, Enrico; Silingardi, Enrico
abstract

Purpose The aim of this study is to evaluate the detection rate of almotriptan, eletriptan, frovatriptan, sumatriptan, rizatriptan, and zolmitriptan in the hair of migraineurs taking these drugs; the degree of agreement between type of self-reported triptan and triptan found in hair; if the concentrations in hair were related to the reported cumulative doses of triptans; and whether hair analysis was able to distinguish occasional use from the overuse of these drugs. Methods Out of 300 headache patients consecutively enrolled, we included 147 migraine patients who reported to have taken at least one dose of one triptan in the previous 3 months; 51 % of the patients overused triptans. A detailed pharmacological history and a sample of hair were collected for each patient. Hair samples were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) by a method that we developed. Results All the triptans could be detected in the hair of the patients. The agreement between type of self-reported triptan and type of triptan found in hair was from fair to good for frovatriptan and zolmitriptan and excellent for almotriptan, eletriptan, sumatriptan, and rizatriptan (P &lt; 0.01, Cohen’s kappa). The correlation between the reported quantities of triptan and hair concentrations was statistically significant for almotriptan, eletriptan, rizatriptan, and sumatriptan (P &lt; 0.01, Spearman’ s rank correlation coefficient). The accuracy of hair analysis in distinguishing occasionally users from overusers was high for almotriptan (ROC AUC = 0.9092), eletriptan (ROC AUC = 0.8721), rizatriptan (ROC AUC = 0.9724), and sumatriptan (ROC AUC = 0.9583). Conclusions Hair analysis can be a valuable system to discriminate occasional use from triptan overuse.

2016 - Hair testing in clinical setting: simultaneous determination of 50 psychoactive drugs and metabolites in headache patients by LC tandem MS [Articolo su rivista]
Licata, Manuela; Rustichelli, Cecilia; Palazzoli, Federica; Ferrari, Anna; Baraldi, Carlo; Vandelli, Daniele; Verri, Patrizia; Marchesi, Filippo; Silingardi, Enrico
abstract

Headache patients suffering from recurrent attacks are a population at risk of overuse and abuse of analgesic medications. Associated with triptans, the first-line drugs recommended for the acute treatment, these patients usually take other medications such as opioids analgesics for the attack treatment, antidepressants and antiepileptics for prophylaxis treatment and benzodiazepines, non-benzodiazepine hypnotics and antipsychotics for the treatment of comorbidities. Regular and frequent use of triptans, like of any other symptomatic analgesic, can cause chronic headache and medication-overuse headache (MOH). In these circumstances, a detoxification treatment is necessary and therefore the monitoring and follow-up of the patients are crucial to the success of the treatment. In the present study, a LC tandem MS method has been developed for the identification of 50 psychoactive drugs in human hair, including triptans, benzodiazepines and metabolites, analgesics, antiepileptic, antidepressants and metabolites, a non-benzodiazepine hypnotic (z-drug),antipsychotics and metabolites. Hair samples were decontaminated, pulverized and incubated overnight in methanol; the extracts were then purified by a new and rapid QuEChERS procedure and analyzed by LC-MS/MS under gradient elution with positive ionization MRM mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection and lower limit of quantitation, precision and accuracy, carry-over, matrix effect, recovery and dilution integrity. The validated procedure has been applied to 234 real hair samples collected from headache patients with known type and dosage of the taken drugs; the obtained data could be of interest to evaluate the xenobiotic concentrations in patients with known therapy.

2016 - Medicinal Chemistry drives drug targeted delivery: a successful interplay [Abstract in Atti di Convegno]
Costantino, Luca; Maretti, Eleonora; Rustichelli, Cecilia; Truzzi, Eleonora; Sacchetti, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Targeted drug delivery is object of an intense research. A medicinal chemistry approach allowed us to modify an opioid peptide in order to remove the opioid activity and retain the ability to cross the blood-brain barrier. Polyester-based nanoparticles (Np) surface-decorated with this peptide were shown to be able to deliver loperamide (a model drug) into CNS, as well as cholesterol (for the treatment of Huntington’s disease) and albumin (a model of a cargo protein). With a view of clinical translation, however, polyester Np seems to be not well suited for CNS diseases. Thus, we decided to use our peptide in alternative ways other than as a conjugate with the carboxyl group of the polyester PLGA, starting material for the production of the Np. We develop a new synthetic procedure that allow the conjugation of the peptide with substrates containing hydroxyl groups, less reactive than the carboxy group of the polyester (we considered as a substrate the poly(vinyl alcohol)), as well as a linker that could be inserted between a cargo and the peptide targeting moiety. At the same time, we moved towards lipidic carriers, a kind of delivery agents already clinically available. To gain experience on these carriers, we designed rifampicin-loaded Solid Lipid Nanoparticle assemblies (SLNas), for a direct intramacrophagic antitubercular therapy using Dry Powder Inhaler (DPI) devices and, in the first instance, methyl mannopyranoside as targeting ligand, able to interact with mannose receptors present on macrophages. Results obtained will be presented and discussed.

2016 - Solid Lipid Nanoparticle assemblies (SLNas) for an anti-TB inhalation treatment-A Design of Experiments approach to investigate the influence of pre-freezing conditions on the powder respirability [Articolo su rivista]
Maretti, Eleonora; Rustichelli, Cecilia; Romagnoli, Marcello; Balducci, Anna Giulia; Buttini, Francesca; Sacchetti, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

For direct intramacrophagic antitubercular therapy, pulmonary administration through Dry Powder Inhaler (DPI) devices is a reasonable option. For the achievement of efficacious aerosolisation, rifampicin-loaded Solid Lipid Nanoparticle assemblies (SLNas) were developed using the melt emulsifying technique followed by freeze-drying. Indeed, this drying method can cause freezing or drying stresses compromising powder respirability. It is the aim of this research to offer novel information regarding pre-freezing variables. These included type and concentration of cryoprotectants, pre-freezing temperature, and nanoparticle concentration in the suspension. In particular, the effects of such variables were observed at two main levels. First of all, on SLNas characteristics – i.e., size, polydispersity index, zeta-potential, circularity, density, and drug loading. Secondly, on powder respirability, taking into account aerodynamic diameter, emitted dose, and respirable fraction. Considering the complexity of the factors involved in a successful respirable powder, a Design of Experiments (DoE) approach was adopted as a statistical tool for evaluating the effect of pre-freezing conditions. Interestingly, the most favourable impact on powder respirability was exerted by quick-freezing combined with a certain grade of sample dilution before the pre-freezing step without the use of cryoprotectants. In such conditions, a very high SLNas respirable fraction (>50%) was achieved, along with acceptable yields in the final dry powder as well as a reduction of powder mass to be introduced into DPI capsules with benefits in terms of administered drug dose feasibility.

2016 - SURFACE ENGINEERING OF SOLID LIPID NANOASSEMBLIES FOR INHALED INTRAMACROPHAGIC ANTI-TB THERAPY [Abstract in Atti di Convegno]
Costantino, Luca; Maretti, Eleonora; Truzzi, Eleonora; Rustichelli, Cecilia; Leo, Eliana Grazia; Zapparoli, Mauro; Iannuccelli, Valentina
abstract

For an inhaled tuberculosis (TB) treatment, antibiotic aerosolization has to be produced by using drugs in their solid state administered by means of Dry Powder Inhaler (DPI) devices. In this regard, untreated drugs generally fail to reach alveolar epithelium and penetrate alveolar macrophages (AM) as the primary site of the infection.1 Therefore, the urgency to treat TB disease effectively may be addressed with approaches consisting of micro- or nanoparticulate carriers redeveloping existing drugs to reach the intended goal.2, 3 Specific modifications of the particulate carrier surface by conjugation with molecules that can specifically bind the receptors (active targeting) are expected to boost the particle avidity to cells increasing accumulation and intracellular uptake. Macrophages possess mannose-specific membrane receptors (MR) that can recognize and facilitate the internalization of carriers bearing mannose residues. In particular, the infected AM have an overexpression of MR.4 In the present study, surface engineered Solid Lipid Nanoparticle assemblies (SLNas) were developed as potential carriers of rifampicin, a first choice antitubercular drug, intended to maximize drug concentration at the primary site of TB infection. To increase specificity for macrophages and internalization potential, SLNas surface was functionalized by a mannosylated derivative to induce AM active targeting. Biocompatible lipid components such as fatty acids and their derivatives, diglycerides and triglycerides were processed by means of the melt emulsifying technique using biocompatible surfactants (sodium taurocholate and methyl mannopyranoside). Mannosylated SLNas were examined for their intrinsic properties (size and size distribution, shape, surface charge, bulk and tap density, aerodynamic diameter, porosity, flowability, physical state of the components). Powder breathability in terms of Emitted Dose and Fine Particle Fraction was assayed by Next Generation Impactor (NGI). This information on powder interparticle adhesion and deaggregation ability influencing powder dispersion and deposition onto alveolar epithelia. SLNas mannosylation was investigated by means of X-ray Photoelectron Spectroscopy for Chemical Analysis and Energy Dispersive X-ray Analysis. Prototypes of SLNas in terms of successful functionalization, optimal breathability and chemico-physical stability, were examined for cytotoxicity by MTT test on murine macrophages J774 cell lines.

2015 - Gastroretentive montmorillonite-tetracycline nanoclay for the treatment of Helicobacter pylori infection [Articolo su rivista]
Iannuccelli, Valentina; Maretti, Eleonora; Montorsi, Monia; Rustichelli, Cecilia; Sacchetti, Francesca; Leo, Eliana Grazia
abstract

The paper aims to explore the potential benefits provided by an organically modified montmorillonite (nanoclay) in the problematic management of the Helicobacter pylori gastric infection that is one of the most prevalent infectious diseases worldwide. Two nanoclay samples were produced by the intercalation of tetracycline (TC) into the interlayer of montmorillonite (MM) under two different pH reaction conditions (pH 3.0 and 8.7). MM/TC nanoclays were characterized by EDX, XRD, FTIR, DSC, drug adsorption extent, in vitro mucoadhesiveness and desorption in simulated gastric media. The reaction between MM and TC led to a complete MM cation (Na+ and Ca2+) exchange process, an increase of MM characteristic interlayer spacing as well as an involvement of NHR3+ group of TC, regardless of the reaction pH value. However, MM/TC nanoclay obtained under alkaline conditions provided a lower TC adsorption as well as a drug fraction weakly linked to MM in comparison with the nanoclay obtained in acidic conditions. Both the nanoclays exhibited good mucoadhesion properties to porcine mucin and TC desorption occurring mainly via a cation exchange process by H+ ions. Based on the results obtained, TC intercalation into MM nanoplatelets could represent a potential advantageous approach allowing the antibiotic to distribute homogeneously on the gastric mucosa, diffuse through the gastric mucus layer and achieve the microorganism localization.

2015 - Neurosteroids as Endogenous Modulators of Epileptic Seizures [Abstract in Atti di Convegno]
Biagini, Giuseppe; Rustichelli, Cecilia; Lucchi, Chiara; Meletti, Stefano
abstract

Neurosteroids are a family of steroidal compounds which produce a variety of biological effects, mainly by interacting with the γ-aminobutyric acid receptor A (GABAA). Among neurosteroids, allopregnanolone and tetrahydrodeoxycorticosterone are positive modulators of GABAA receptors that were shown to counteract seizures in animal models. However, it is still to be defined the role of neurosteroids both in patients affected by epilepsy and in animal models based on spontaneously recurrent seizures. By using finasteride, an irreversible inhibitor of the enzyme 5α-reductase, we found that allopregnanolone cerebral levels were significantly decreased in rats treated with the convulsant pilocarpine. Interestingly, this decrease was followed by enhanced epileptogenesis. We also described a case of antiepileptic drug resistance in a patient that was maintained on finasteride to treat a dermatological disorder: seizures ceased by withdrawing finasteride. These findings point to a modulatory role of allopregnanalone and other neurosteroids on epileptic seizures.

2015 - PROGETTAZIONE E OTTIMIZZAZIONE DI SISTEMI MICROPARTICELLARI PER LA VEICOLAZIONE AI MACROFAGI ALVEOLARI DI FARMACI ANTITUBERCOLARI PER VIA INALATORIA [Poster]
Maretti, Eleonora; Rustichelli, Cecilia; Romagnoli, Marcello; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

La tubercolosi (TBC), causata dal Mycobacterium tuberculosis, è una patologia infettiva trasmissibile per via aerea che interessa un terzo della popolazione mondiale e rappresenta il principale fattore di mortalità per le persone colpite da HIV. E’ stata dichiarata dall'OMS “a major global health problem” con una elevata incidenza e un aumento dei casi di farmacoresistenza (MDR-TB). La strategia del piano proposto dall’OMS per ridurre l’impatto della TBC comprende lo sviluppo di strumenti nuovi ed efficaci al fine di prevenire, individuare e trattare la patologia (WHO, 2014). I limiti dall’attuale terapia di tipo convenzionale, per via orale o parenterale, risiedono nell’elevato dosaggio, nel lungo periodo di trattamento e nei numerosi effetti collaterali che possono essere evitati mediante lo sviluppo di Drug Delivey Systems innovativi in grado di modulare l’azione di farmaci già in uso. Poiché la tubercolosi polmonare è caratterizzata dal coinvolgimento dei macrofagi alveolari nei quali i bacilli rimangono vitali, la somministrazione di farmaci anti-TBC direttamente ai polmoni mediante carrier microparticellari consentirebbe il vantaggioso targeting ai macrofagi alveolari di antibiotici impossibilitati a diffondere attraverso le membrane cellulari. Ciò nonostante, non esistono, ad oggi, farmaci anti-TBC somministrabili per via inalatoria approvati per l’uso umano. Ancora meno studiati, nella progettazione di microcarrier per questa terapia, nonostante i benefici in termini di biocompatibilità, risultano i materiali naturali, quali i lipidi. Tra questi, quelli in grado di generare superfici microparticellari cariche o contenenti molecole coinvolte nel processo endocitico potrebbero promuovere l’uptake macrofagico del farmaco. Al fine di ottenere una polvere biocompatibile che, una volta inalata mediante Dry Powder Inhaler (DPI), favorisca la captazione del farmaco da parte dei macrofagi alveolari, il presente studio è stato finalizzato allo sviluppo e ottimizzazione di microparticelle caratterizzate da dimensioni nell'ambito della respirabilità (0.5-5 µm) per la veicolazione di un farmaco di prima linea quale la rifampicina. A tale scopo, sono stati impiegati materiali naturali, biocompatibili e biodegradabili, quali lipidi solidi, trattati con metodologie eco-friendly, in assenza di solventi organici. Sono state, pertanto, sviluppate Solid Lipid Microparticles (SLM) di acido stearico stabilizzate con sodio taurocolato al fine di direzionare il chemioterapico ai macrofagi alveolari. Il microcarrier formulato è stato caratterizzato dal punto di vista chimico-fisico per determinarne morfologia, dimensioni, carica superficiale, densità, diametro aerodinamico, livello di caricamento e rilascio in vitro del farmaco, attività antimicrobica, frazione respirabile, citotossicità e capacità di internalizzazione su linee cellulari macrofagiche J774. Il microcarrier è risultato idoneo per proprietà aerodinamiche, citotossicità e internalizzazione da parte di macrofagi murini (Maretti, 2014). Inoltre, l’analisi di alcuni parametri coinvolti nel processo di liofilizzazione, quali temperatura di congelamento, presenza di crioprotettori e diluizione del campione, eseguita mediante uno studio statistico di Design of Experiment (DOE), ha evidenziato la loro rilevante influenza sulla frazione respirabile del prodotto finale permettendone l’ottimizzazione.

2015 - SOLID LIPID MICROPARTICLES FOR INHALED ANTI-TB THERAPY BY DPI: INFLUENCE OF THE PRODUCTION PROCESS ON DRUG STABILITY AND POWDER BREATHABILITY [Poster]
Maretti, Eleonora; Bellani, Martina; Rustichelli, Cecilia; Sacchetti, Francesca; Romagnoli, Marcello; Balducci, Anna Giulia; Buttini, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

According to the WHO global report, tuberculosis (TB) remains one of the world’s deadliest communicable diseases. The current therapy involves three/four drug oral regimen, long-term therapy, high and frequent doses so producing several side-effects [1]. To overcome these drawbacks and improve treatment efficacy, the new strategies could involve new formulation design for old drugs. Among these, the shortest-term goal is represented by Drug Delivery Systems (DDS). In this latter context, considering that 75-80% of TB cases remain localized in the lungs, pulmonary route appears the most strategical route [2]. For an efficient drug delivery by Dry Powder Inhaler (DPI) device, several powder properties (particle microsize, irregular shape, low tap density, surface charge, weak adhesion between particles, good flowability) contribute to determining powder aerodynamic performance and, consequently, deposition onto alveolar epithelium, and phagocytosis by alveolar macrophages [3]. Based on these assumptions, Solid Lipid Microparticles (SLM), known to be biocompatible, biodegradable and physically stable were designed as the carrier for rifampicin (RIF). The present research focused on the evaluation of both RIF stability during the production phases and the role of variables relating to freeze-drying process (freezing conditions, sample dilution, cryoprotectants) affecting the powder aerosolization. Considering the complexity of the factors involved in a successful breathable powder, a statistical Design of Experiments (DOE) was adopted to study the critical variables that influence the final product. SLM were obtained by the melt emulsification technique under sonication by using stearic acid and sodium taurocholate as lipid and surfactant, respectively [4]. Loaded SLM were prepared by adding RIF in the melted lipid. All the emulsions were rapidly cooled to room temperature providing SLM that were purified by dialysis and freeze-dried. Freeze-drying was carried out following dilution with water, mixture with cryoprotectant (trehalose or mannitol), and lowering temperature of freezing. SLM exhibited an irregular shape and the following value ranges: size (470 - 1700 nm), PDI (0.32 – 0.94), circularity (0.43 – 0.66), bulk density (0.02 – 0.24 g/cm3), tapped density (0.04 – 0.31 g/cm3) and drug loading level (11.85 – 15.88%). The Emitted Dose and the Fine Particle Mass were between 92.1-103.4% and 0.9-6.83 mg, respectively. DOE approach highlighted the combination of the water dilution before freezing with the cryoprotectant use as the most important parameter to obtain a highly breathable powder as well as the influence of water dilution and freezing temperature on SLM breathability. For a powder to be inhaled by a DPI device, many parameters can guarantee quality and efficacy of the product. The analysis performed demonstrated RIF stability and proved to be efficient in distinguishing the major contribution factors on the final product and identifying the key factors that are helpful for the improvement of SLM production process.

2015 - Tramadol chronic abuse: An evidence from hair analysis by LC tandem MS [Articolo su rivista]
Verri, Patrizia; Rustichelli, Cecilia; Palazzoli, Federica; Vandelli, Daniele; Marchesi, Filippo; Ferrari, Anna; Licata, Manuela
abstract

Hair analysis, as complementary matrix, has expanded across the spectrum of toxicological investigations for misuse drug monitoring. Hair has become an important matrix for drug analysis, owing to the possibility to detect target analytes for long time periods, depending on hair length. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitation of tramadol, a widely used centrally-acting analgesic, and its main metabolites in hair (ODMT, NDMT, NOT). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode solid phase cartridges and analyzed by LC-MS/MS in positive ionization mode monitoring two transitions per analyte. The procedure was fully validated in terms of linearity, limit of detection and lower limit of quantitation (LLOQ), accuracy, precision, recovery, matrix effect and selectivity. The linear regression analysis was calibrated by deuterated internal standards; for all analytes, responses were linear over the range 0.04-40.00 ng/mg hair, with R2 values of at least 0.995. The method offered satisfactory precision (RSD &lt; 10%), accuracy (90-110%) and recovery (&gt;90%) values. The found LLOQ values for tramadol and metabolites were in the range 0.010-0.030 ng/mg hair. The proposed procedure was successfully applied to quantify tramadol and metabolites in real hair samples submitted to our laboratory: three cases of tramadol assumption within the therapeutic dosage (3 x 2 segments) and one case of tramadol abuse in a binge pattern (8 segments). The ranges found for TRAM, ODMT, NDMT and NOT were markedly higher in the abuse case (63.42-107.30, 3.76-6.26, 24.88-45.66, 0.22-1.18 ng/mg hair, respectively) compared to the other case reports (3.29-20.12, 0.28-1.87, 0.45-4.32, 0.07-0.80 ng/mg, respectively); also the values of NMDT/ODMT ratio differed significantly. According to the obtained data, we hypothesized that the binge pattern may influence the metabolites' to parent drug concentration ratios; therefore this parameter could represent a target assessment tool to monitor abuse cases.

2013 - Neurosteroids And Epileptogenesis [Articolo su rivista]
Biagini, Giuseppe; Rustichelli, Cecilia; Curia, Giulia; Vinet, Jonathan; Lucchi, Chiara; Pugnaghi, Matteo; Meletti, Stefano
abstract

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its upregulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus (SE) induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.

2013 - Neurosteroids and epileptogenesis. [Abstract in Atti di Convegno]
Biagini, Giuseppe; Rustichelli, Cecilia; Curia, Giulia; Lucchi, Chiara; Pugnaghi, Matteo; Meletti, Stefano; Avoli, M.
abstract

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its up-regulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through the administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.

2013 - Simultaneous determination of pregnenolone sulphate, dehydroepiandrosterone and allopregnanolone in rat brain areas by liquid chromatography-electrospray tandem mass spectrometry [Articolo su rivista]
Rustichelli, Cecilia; Pinetti, Diego; Lucchi, Chiara; Ravazzini, Federica; Puja, Giulia
abstract

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol-formic acid, purified by Hybrid-SPE cartridges and subjected to LC-ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p<0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain.

2012 - n-3 Dietary supplementation and lipid metabolism: Differences between vegetable- and fish-derived oils [Articolo su rivista]
Campioli, Enrico; Rustichelli, Cecilia; Avallone, Rossella
abstract

The effect of flaxseed oil rich in linolenic acid (ALA), and a mixed oil (flaxseed oil and fish oil) rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the lipid clearance and peroxisome proliferator activated receptors (PPARs) in liver and adipose tissue of rats fed for 30 days with the two oils was evaluated. The results showed that after treatment with the mixed oil the hematic triacylglycerol content was significantly decreased compared to control animals. Regarding the tissue distribution of the major omega-3 fatty acids, both oils were able to increase ALA, EPA, docosapentaenoic acid (DPA) in liver and adipose tissue; and DHA solely in the adipose tissue. Finally the treatment with either flaxseed or mixed oil increased hepatic PPAR-γ expression but only the mixed oil enhanced the hepatic expression of PPAR-α. No effect on adipose tissue PPAR-γ expression was observed with both oils’ treatment.

2012 - Polyunsaturated fatty acid levels in rat tissues after chronic treatment with dietetic oils [Articolo su rivista]
Rustichelli, Cecilia; Avallone, Rossella; Campioli, Enrico; Braghiroli, Daniela; Baraldi, Mario
abstract

BACKGROUND: The essential fatty acids can be helpful in the prevention of several pathologies. The purpose of this study was to quantify the major n-3 and n-6 fatty acids in tissues of rats fed with flaxseed oil and with a dietetic oil in order to evaluate how their chronic supplementation could influence the correspondent in vivo levels and to study the effectiveness of the dietetic oil compared to flaxseed oil. RESULTS: Fatty acids were successfully extracted from biological samples, subjected to derivatization procedure and analysed by high-performance liquid chromatography with UV detection under gradient elution mode. The developed method showed good linearity, precision and accuracy with recoveries ranging from 89% to 92%. Animals treated with flaxseed and dietetic oils showed enhanced levels of n-3 fatty acids compared to control groups, with a significant higher levels of EPA and DHA in the brain and in the adipose tissue of the dietetic groups compared to the flaxseed groups. CONCLUSION: The obtained data underline that the tested oils can effectively enhance the tissue levels of n-3 fatty acids and therefore they could be successfully used in the dietetic treatment of lipid related diseases.

2010 - EFFETTI DELLA SUPPLEMENTAZIONE DIETETICA CON OLIO DI LINO SULLA SINTESI DELLE KINURENINE IN ANIMALI CON IL MORBO DI HUNTINGTON. [Abstract in Atti di Convegno]
Avallone, Rossella; Rustichelli, Cecilia; M. F., Notarangelo; Campioli, Enrico
abstract

E’ stato ipotizzato che il pathway delle kinurenine abbia un ruolo importante nel morbo di Huntington, una patologia neurodegenerativa causata dall’espansione del tratto di poliglutamina della proteina huntingtina. Durante la fase iniziale della patologia, i livelli cerebrali di due metaboliti della via delle kinurenine, l’acido chinolinico e il suo precursore la 3-idrossi chinurenina sono risultati elevati. Inoltre in uno studio clinico è stato dimostrato che il trattamento con acido eicosapentenoico aveva effetti benefici sulla corea e sui sintomi motori.Lo scopo della ricerca è stato quello di valutare gli effetti della supplementazione di acidi grassi della serie omega 3 nell’Huntington studiando in particolare il pathway delle kinurenine.Topi transgenici con morbo di Huntington (R6/2 - HD) e topi wild-type (WT) sono stati alimentati cronicamente per un mese con olio di oliva (utilizzato come controllo) e con olio dietetico costituito da un’associazione di olio di lino, ricco in acido linolenico (ALA), olio di girasole e olio di pesce ricco in acido eicosapentenoico (EPA) e docosaesanoico (DHA).Nel cervello è stato determinato un significativo aumento (p<0,05) dei livelli di ALA, EPA e DHA nel topo WT trattato con olio dietetico rispetto al corrispondente trattato col olio di oliva: lo stesso andamento è stato determinato nel topo HD trattato con olio dietetico con un aumento significativo in EPA e DHA (p<0,01). Non è stata determinata nessuna variazione nel livelli di acido linoleico e di acido arachidonico né nei topi WT né in quelli HD. Nei topi HD è stato determinato un minore livello di DHA (p<0,01) e un maggiore livello di acido arachidonico (p<0,01) rispetto ai topi WT. Il profilo degli acidi grassi nel tessuto adiposo riflette quello cerebrale mentre nel fegato è stato misurato un aumento significativo di EPA e DHA sia nel topo WT che nel topo HD dopo trattamento cronico con olio dietetico. EPA e DHA sono maggiori nel topo HD (p<0,001) al WT (p<0,05).Per quanto riguarda gli esperimenti sul pathway delle kinurenine, l’3H-kinurenina è stata iniettata nello striato e si sono misurati i metaboliti prodotti mediante analisi HPLC. Non è stata determinata alcuna variazione nelle concentrazioni di acido chinurenico, acido chinolinico e 3-idrossi chinurenina.Concludendo il trattamento dietetico con olio arricchito in acidi grassi delle serie omega 3 ha mostrato benefici nella patologia di Huntington.

2010 - Novel antifouling agent zinc pyrithione: determination, acute toxicity and bioaccumulation in marine mussels (Mytilus galloprovincialis) [Articolo su rivista]
Marcheselli, Marco; Rustichelli, Cecilia; Mauri, Marina
abstract

Anti-fouling biocide zinc pyrithione (ZnPT) and its biological fate have received little attention because this compound was assumed not to be persistent in marine ecosystems.We developed an analytical procedure that proved to be efficient and very sensitive in extracting ZnPT and its main secondary products, zinc and ionized PT‾, from both seawater and biological samples, namely in gills and digestive gland of the bioindicator species Mytilus galloprovincialis. Short-term experiments were carried out to investigate ZnPT toxicity and bioaccumulation. The effects on survival and the tissue bioaccumulation of ZnPT and its secondary products were studied on adult mussels from a natural population, collected inside the harbour area of Porto Santo Stefano (Italy) and exposed to sublethal doses of the biocide up to 7 days.ZnPT was shown to be persistent in experimental seawater in the short term. A basal level of ZnPT and ionized PT‾ was detected in mussels, indicating that ZnPT availability in the sampling site is already high enough to induce a detectable accumulation in individuals of the native population. ZnPT rapidly accumulated in tissues of the exposed mussels, proportionately to both exposure concentration and time, identifying the gills and the digestive gland as important targets in the biological path of the contaminants.Even though the 7day-LC50 = 8.27 µM established here appears high with respect to reported ZnPT environmental concentrations, our results indicate that this biocide could represent a threat for marine organisms in coastal environments and that further investigations on its biological effects at sublethal doses are needed.

2009 - Alpha-Linolenic acid and Linoleic acid in serum and tissues after flaxseed (Linum usitatissimum) oil in vivo administration [Articolo su rivista]
Avallone, Rossella; Rustichelli, Cecilia; Campioli, Enrico; Notarangelo, Francesca Maria Pia; Braghiroli, Daniela; Baraldi, Mario
abstract

Essential fatty acids can be helpful in the prevention of several pathologies. The bioavailability of acute supplementation of different doses of flaxseed oil was studied by analyzing the level of alpha-linolenic acid (ALA) and linoleic acid (LA) in serum and tissues (adipose, liver) of rats tested at 2, 4, 8 and 16 h after the administration. The amount of flaxseed oil administered at increasing doses corresponded to 1, 2.5 and 5 g ALA/kg of body weight. The corresponding fatty acid methyl esters obtained via direct methylation were quantified using gas chromatography/mass spectrometry. Serum ALA level increased after 1 or 2.5 g/kg. ALA was increased in both adipose and liver tissue 4 h after the administration of 1 g/kg of flaxseed oil. There was no further increase by using a higher oil dosage. LA did not change in serum at the doses used.

2009 - HPLC analysis of n-3 and n-6 fatty acid levels in rat serum after chronic treatment with dietetic oils [Articolo su rivista]
Rustichelli, Cecilia; Avallone, Rossella; Campioli, Enrico; Braghiroli, Daniela; Parenti, Carlo; Baraldi, Mario
abstract

High-performance liquid chromatography HPLC analysis of the major fatty acids of the n-3 family and n-6 family in rat serum was carried out. To perform sample cleanup, existing techniques of lipid extraction were tested and modified to achieve maximal free fatty acids recovery in a reasonable time. Concerning chromatographic analyses, p-bromophenacyl bromide was used as ultraviolet (UV)-derivatizing agent followed by reversed-phase HPLC/UV under mobile phase gradient conditions. The optimized and validated procedure was then applied to rats fed with flaxseed oil and a combination of flaxseed, fish and sunflower oils, defined as "dietetic oil", in order to evaluate how their chronic supplementation can influence serum levels of n-3 and n-6 fatty acids.

2009 - HPLC determination of fatty acid levels in rats after chronic supplementation of flaxseed oil and dietetic oil. [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Avallone, Rossella; Campioli, Enrico; Braghiroli, Daniela; Baraldi, Mario
abstract

Essential fatty acids (EFAs) belonging to -3 and -6 families play an important role in vivo, supporting the cardiovascular, reproductive, immune and nervous system functions; an inappropriate balance of these EFAs contributes to the development of diseases, while a proper balance helps maintain and even improve health.The purpose of this research was to study the levels of the principal -3 (ALA: n-lino-lenic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosa-hexaenoic acid) and -6 (LA: linoleic acid; AA: arachidonic acid) fatty acids in rat serum and tissues (liver, brain, adipose tissue) after a chronic treatment with flaxseed oil and with a dietetic oil, containing flaxseed oil, fish oil and sunflowers oil. The treatment was planned in order to give to rats the same ALA amount.The biological samples were subjected to liquid-liquid extraction, followed by derivatisation with p-bromophenacyl bromide and then analysed by HPLC/UV on a C18 reversed-phase column under gradient mobile phase conditions.Rats treated with flaxseed oil and dietetic oil showed enhanced levels of n-3 fatty acids compared to the correspondent control group. In serum, ALA levels were slightly higher in the dietetic oil group than in the flaxseed oil group; this fact is probably due to an enhanced absorption and also a decreased metabolism of ALA. The EPA, DPA and DHA serum levels, higher in the dietetic than in the flaxseed group, could derive both from ALA through its metabolism and also from EPA and DHA contained in the fish oil. Brain and adipose tissue showed EPA and DHA levels higher in the dietetic than in the flaxseed group, as occurred for serum samples. Concerning liver samples, the treatment with flaxseed or dietetic oil caused an enhancement for ALA, EPA and DPA levels, while no variations were found for DHA compared to control groups.The data obtained underline that the tested oils can enhance the levels of -3 fatty acids and therefore they could be successfully used in the dietetic treatment of lipid related diseases.

2008 - Bioaccumulation and heat shock proteins in marine mussels (Mytilus galloprovincialis) exposed to zinc pyrithione [Abstract in Atti di Convegno]
Marcheselli, Marco; Ciotti, F.; Rustichelli, Cecilia; Mauri, Marina
abstract

We investigated the biological fate and the effects of the organic biocide zinc pyrithione (ZPT) in comparison to inorganic Zn on the marine mussel Mytilus galloprovincialis, commonly widespread on hard bottoms of confined habitats such as harbours and marinas, where ZPT may be released in consequence of the use of antifouling paints and also become a source of Zn contamination. In particular, our aim was to highlight the pattern of accumulation of Zn both in his organic and inorganic form in the gills and in the digestive gland of the mussels and to assess whether a short term exposure to ZPT at sublethal doses could lead to the activation of repair mechanisms as highlighted by an increased expression of the early stress indicators hsp27, hsp60 and hsp70. ZPT has been introduced as a replacement for organotin compounds, which have recently been banned from antifouling treatments, but it has received little attention due to the lack of sufficient analytical methods for its determination in environmental matrices. ZPT is marketed as being an environmentally neutral, non-persistent compound based on the fact that it easily photolyses and rapidly degrades when exposed to direct sunlight (t½<1 hour). However, the analytical methods so far used measure the disappearance of the original biocide and not a reduction in total toxicity. Few studies have examined the toxicity of ZPT to marine non-target organisms. ZPT resulted very toxic to the crustacean Nitocra spinipes, with LC50 varying between 180 and 340 μg/l, underlining that ZPT can potentially be highly toxic towards marine organisms other than the fouling ones. To minimize oxidative damage and to cope with stressors, eukaryotic cells utilize a variety of protective mechanisms, including the expression of an assortment of proteins which are collectively referred to as “stress proteins”. HSPs showed to be a useful biomarker, in particular to detect early effects due to low level- or short term-exposures. Because of his ubiquity the marine mussel Mytilus galloprovincialis is widely used as a sentinel organism for the assessment of pollution in coastal environments. Monitoring its wellness is extremely important when harmful xenobiotic compounds are suspected to reach non-target species, as a consequence of submarine maintenance works or aquaculture-related treatments.

2008 - Flaxseed oil: acute and chronic supplementation increases serum and tissue concentrations of omega fatty acids in rats [Abstract in Atti di Convegno]
Campioli, Enrico; Avallone, Rossella; Rustichelli, Cecilia; Notarangelo, Francesca Maria Pia; Braghiroli, Daniela; Baraldi, Mario
abstract

We studied the bioavailability of acute supplementation of scalar doses of flaxseed oil by analysing the level of Linolenic acid (ALA, -3) and Linoleic acid (-6) in serum and tissues (adipose, liver and brain) of rats tested at 2-4-8-16 h after the administration. The amount of flaxseed oil administered by oral rate was 1.9, 4.7, 9.5 mL/kg corresponding to 1, 2.5, 5 g ALA/kg. Two techniques of lipid extraction were investigated to achieve maximal free fatty acids recovery in a reasonably short time. The corresponding fatty acid methyl esters obtained with direct methylation with MeOH/HCl, were quantified by gas chromatography/mass spectrometry (GC/MS) technique. GC-MS analyses were performed on a Gas-Chromatograph Varian 3400 on a HP-INNOWAX column (30 m x 0.25 mm; 0.25 m film thickness). Mass spectra were acquired on a Finnigan MAT SSQ 710A mass spectrometer in the electron impact (EI) mode.Serum ALA levels at 1 g/kg after 2h in the flaxseed oil group increased by 70% from 0.067 ± 0.007 to 0.096 ± 0.008 mg/mL (P<0.001 Anova) whereas no significant increase occurred in the flaxseed oil group at 2.5 g/kg (0.142 ± 0.009) or at 5 g/kg after 2 h (0.140 ± 0.008) when compared with the value obtained after 1 g/kg. A statistically significant increase of ALA was found in adipose tissue and in liver 4 h after the administration of 1 g/kg of ALA whereas higher doses (2.5-5 g/kg) did not produce any significant changes. Concerning linoleic acid (-6) no significant increased concentrations were found in serum at the three doses studied confirming that flaxseed oil is mainly a source of -3 fatty acids.

2008 - La supplementazione di olio di lino aumenta la concentrazione plasmatica e tissutale dell'acido linolenico e cambia l'espressione dei recettori attivanti la proliferazione dei perossisomi (PPAR) [Abstract in Atti di Convegno]
Avallone, Rossella; Rustichelli, Cecilia; Campioli, Enrico; Baraldi, Mario
abstract

E’ stata studiata la biodisponibilità dell’olio di lino dopo supplementazione acuta analizzando i livelli di acido linolenico (ALA), acido linoleico, EPA, DPA e DHA nel siero e nei tessuti (adiposo, epatico e cerebrale) di ratti dopo 2-4-8-16 ore dalla somministrazione. La quantità di olio di lino somministrata è stata corrispondente a 1-2,5-5 g di ALA/kg. Gli esteri metilici degli acidi grassi ottenuti mediante metilazione diretta con MeOH/HCl, sono stati quantificati mediante GC/MS (GC Varian 3400/ Finnigan MAT SSQ 710 A) ad impatto elettronico a –70 eV su colonna capillare HP-INNOWAX (30 m x 0,25 mm DI x 0,25 m).I livelli serici di ALA nel gruppo trattato con olio di lino alla dose di 1 g/kg, dopo 2h aumentano del 70% andando da 0,067 ± 0,007 a 0,096 ± 0,008 mg/mL (P<0.001 Anova, post test Bonferroni) mentre nessuna variazione significativa è stata riscontrata nel gruppo trattato con 2,5 g/kg (0,142 ± 0,009) o con 5 g/kg dopo 2 h (0,140 ± 0,008). Un aumento significativo di ALA è stato determinato nel tessuto adiposo e nel fegato dopo 4 ore dalla somministrazione di 1 g/kg mentre le dosi maggiori (2,5-5 g/kg) non producono nessun cambiamento significativo. Per quanto riguarda l’acido linoleico non è stato determinato nessun aumento significativo confermando che l’olio di lino è soprattutto una fonte di acidi grassi della serie -3.Alla luce dei risultati ottenuti dopo trattamento acuto, si è reso necessario valutare la biodisponibilità dell’olio di lino dopo trattamento cronico di un mese alla dose ottimale di 1 g di ALA/kg. In questo caso la quantificazione degli acidi grassi è stata effettuata mediante HPLC/UV con una colonna a fase inversa C18 previa derivatizzazione con p-bromofenacil bromuro [1]. Si è potuto notare un significativo incremento nel siero e nel fegato dei valori di ALA, EPA e DPA in conformità con studi precedenti [2]. Non ci sono significative variazioni dei livelli serici di colesterolo totale, HDL, LDL mentre si nota una diminuzione significativa della trigliceridemia.E’ stata inoltre valutata mediante tecnica di Western blot l’espressione dei recettori PPAR nelle due isoforme α, e γ rispettivamente nel fegato e nel tessuto adiposo dopo trattamento cronico con olio di lino.Le bande relative a PPAR  e  e -actina sono state quantificate e l’espressione di PPAR è stata valutata come rapporto PPAR- e  /-actina.[1] Metha et al. J. Chromat. B 1998, 719: 9-23[2] Harper C.R. et al. J. Nutr. 2006, 136: 2844-2848.

2008 - Variazione degli acidi grassi W3 e W6 e plasticità del recettore attivante la proliferazione dei perossisomi (PPAR-a) dopo trattamento cronico con olio di lino e olio dietetico nel fegato. [Abstract in Atti di Convegno]
Campioli, Enrico; Avallone, Rossella; Rustichelli, Cecilia; Baraldi, Mario
abstract

Lo scopo del presente lavoro è stato di quantificare l’acido α-linolenico (ALA), eicosapentaenoico (EPA), docosapentaenoico (DPA), docosaesaenoico (DHA), linoleico e arachidonico nel fegato di ratti trattati cronicamente con olio di lino e con un’associazione di oli vegetali e pesce. Si è valutata inoltre, la plasticità dell’isoforma α del recettore PPAR nel fegato.I PPAR sono dei recettori nucleari, eterodimerizzati con il recettore per l’acido 9-cis retinoico [1]. Questi agiscono come fattori di trascrizione per geni coinvolti nel metabolismo. Molti composti si legano con alta affinità ai recettori PPARs, tra questi troviamo gli acidi grassi insaturi a lunga catena (acido linoleico e linolenico), acidi grassi ramificati, acidi grassi ossidati o coniugati, ed infine eicosanoidi, leucotrieni e fibrati.Il tessuto epatico degli animali trattati per un mese con i suddetti oli è stato omogeneizzato con una miscela estraente costituita da ispropanolo/eptano/acido solforico secondo la metodica descritta da Mehta et al. [2]. Dopo aver aggiunto uno standard interno costituito dai 6 acidi grassi da analizzare, i campioni sono stati portati a secco in corrente di azoto e derivatizzati secondo la metodica descritta da Puttmann et al. [3] per ottenere i corrispondenti p-bromofenacil bromuri derivati. I campioni così ottenuti sono stati analizzati mediante HPLC a 35°C, colonna RP-18 endcapped (125 x 4 mm; 5 μm), detector UV a 254 nm.Successivamente il tessuto è stato omogeneizzato in buffer di lisi RIPA, addizionato di inibitori delle proteasi e centrifugato. I campioni così ottenuti sono stati caricati su gel di poliacrilamide bis-tris 4-12% e sottoposti ad analisi di Western blot.Dall’analisi HPLC si è riscontrato per quanto riguarda l’olio di lino un aumento significativo di ALA (p<0,001; one way Anova, Bonferroni’s post test), EPA (p<0,05) e DPA (p<0,05). Per quanto riguarda l’olio dietetico si è avuto un aumento significativo di ALA ed EPA, mentre si è avuto una diminuzione significativa dell’acido linoleico (p< 0,01) e dell’acido arachidonico (p<0,001). I risultati dei Western blot condotti sul tessuto epatico non evidenziano un aumento dell’espressione recettoriale del PPAR-α nei ratti trattati con olio di lino ed olio dietetico.

2007 - Flaxseed oil supplementation increases plasma and tissue concentrations of W3 fatty acids in rats. [Abstract in Atti di Convegno]
Notarangelo, Francesca Maria Pia; Avallone, Rossella; Rustichelli, Cecilia; Campioli, Enrico; Braghiroli, Daniela; Baraldi, Mario
abstract

-Linolenic acid (ALA) is a major dietary (-3) fatty acid. The essential fatty acids must be absorbed by food intake and play a very important role in the coagulation (inhibition of platelets aggregation) and in the inflammatory reaction (anti-inflammatory effects). In cardiovascular diseases, particularly in coronary diseases, studies demonstrated a decreased mortality in populations who eat an omega-3 rich diet or who take an omega-3 supplement.We studied the bioavailability of acute supplementation of scalar doses of flaxseed oil (Organic Oils–Perugia) by analysing the level of ALA (-3) and Linoleic acid (-6) in serum and tissues (adipose, liver and brain) of rats tested at 2-4-8-16 h after the administration. The amount of flaxseed oil administered by oral rate was 1.9, 4.7, 9.5 mL/kg corresponding to 1, 2.5, 5 g ALA/kg. Two techniques of lipid extraction were investigated to achieve maximal free fatty acids recovery in a reasonably short time. The corresponding fatty acid methyl esters obtained with direct methylation with MeOH/HCl, were quantified by gas chromatography/mass spectrometry (GC/MS) technique. GC-MS analyses were performed on a Gas-Chromatograph Varian 3400 on a HP-INNOWAX column (30 m x 0.25 mm; 0.25 m film thickness). Mass spectra were acquired on a Finnigan MAT SSQ 710A mass spectrometer in the electron impact (EI) mode with an ionization energy of 70 eV; the ion source temperature was 250°C, the filament current was 200 A, the conversion dynode power was –15.0 kV and electron multiplier voltage was 1500 V.Serum ALA levels at 1 g/kg after 2h in the flaxseed oil group (n=25) increased by 70% from 0.067 ± 0.007 to 0.096 ± 0.082 mg/mL (P<0.001 Anova) whereas no significant increase occurred in the flaxseed oil group at 2.5 g/kg (0.142 ± 0.071) or at 5 g/kg after 2 h (0.140 ± 0.106) when compared with the value obtained with 1 g/kg. ALA (1g/kg) significantly increased after 4 h in adipose tissue and in liver but also in this case at higher doses (2.5-5 g/kg) the concentration wasn’t increased. Concerning linoleic acid (-6) no significant increased concentrations were found in serum at the three doses studied confirming that flaxseed oil is a source of -3 fatty acids. These data suggested that there is a limiting step in the adsorption of these fatty acids and that there is no advantage to take more than 1 g/kg of ALA supplementation.

2007 - HPLC analysis of essential fatty acids in rat plasma [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Avallone, Rossella; Notarangelo, Francesca Maria Pia; Campioli, Enrico; Braghiroli, Daniela; Baraldi, Mario
abstract

Linoleic acid (n-6 family) and -Linolenic acid (n-3 family) are two polyunsaturated fatty acids (PUFAs) that are known as essential fatty acids and must be provided by diet.They play an important role in coagulation and inflammatory reactions; concerning cardiovascular diseases studies demonstrated a decreased mortality in populations who eat an omega-3 rich diet or who take an omega-3 supplement.This paper deals with HPLC analysis of linoleic acid and linolenic acid in rat plasma in order to investigate the effect of flaxseed oil supplementation on plasma levels of these essential fatty acids.HPLC procedures are recommended for accurately quantifying of thermally labile moieties like polyunsaturated fatty acids; nevertheless a derivatization step is required as these analytes are lacking both in chromophore and fluorophore group and cannot therefore be detected at sufficiently low concentrations.In this work, p-bromophenacyl bromide is used as UV-derivatizing agent followed by HPLC/UV on C18 reversed phase column. The mobile phase gradient was studied in order to obtain a complete separation for the anaytes and the internal standard from matrix components.Concerning plasma sample clean-up, existing techniques of lipid extraction were tested and modified to achieve maximal free fatty acids recovery in a reasonable time.Studies on rats fed with flaxseed oil are currently in progress in order to evaluate how the chronic supplementation can increase plasma levels of n-3 and n-6 fatty acids.

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 - Analisi GC-EI-MS di acidi grassi omega-3 e omega-6 in matrici complesse [Abstract in Atti di Convegno]
Avallone, Rossella; Braghiroli, Daniela; Rustichelli, Cecilia; Manzini, Daniela; Campioli, Enrico; Baraldi, Mario
abstract

Lo scopo del presente lavoro è stato quello di sviluppare un metodo semplice e rapido per l'analisi degli acidi grassi nel siero e nel tessuto dopo somministrazione acuta o cronica di olio di semi di lino.

2006 - Solid state characterization of chloramphenicol palmitate. Raman spectroscopy applied to pharmaceutical polymorphs [Articolo su rivista]
Gamberini, Maria Cristina; Baraldi, Cecilia; A., Tinti; Rustichelli, Cecilia; Ferioli, Valeria; Gamberini, Gianfranco
abstract

A pharmaceutical active compound, chloramphenicol palmitate, appears in three polymorphic forms, that can be observed at room temperature.The stable form A (biologically inactive modification), the meta-stable form B (active modification) and unstable form C were found to have distinct Raman spectra, with bands attributable to the different polymorphs.The use of hot-stage Raman microscopy (the direct coupling of Raman microscopy and hot-stage) is demonstrated for the drug substance chloramphenicol palmitate form C. All modifications of form C were produced and identified by hot-stage Raman microscopy. A close correlation of thermal and spectroscopic information was achieved by this combination of techniques.As reported in several pharmacopoeias, the content of form A should be less than 10%; therefore, a mixture of 10% (w/w) A in B was prepared, and the presence of the characteristic bands of form A after subtraction of the pure B was revealed. Moreover, mixtures between 2 and 12% (w/w) A in B were investigated and the intensity ratio (as peak area) I413–435/I1035–1158 as a function of A percentage has been demonstrated to show a linear trend. Other methods for the characterization of polymorphs were used: Fourier transform infrared spectroscopy (FT-IR), Diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD).

2005 - Direct HPLC enantioseparation of Cetirizine Hydrochloride on polysaccharide-based chiral stationary phases [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Gamberini, Maria Cristina; Baraldi, Cecilia; Ferioli, Valeria; Gamberini, Gianfranco
abstract

Cetirizine hydrochloride is a non-sedating H1-receptor antagonist used in patients whit urticaria and allergic rhinitis. It is frequently administrated as racemate; although recent data demonstrate that the antihistaminergic activity of the racemate is primarily due to the levo-enantiomer, while dextrocetirizine failed to demonstrate any significant activity. Moreover levocetirizine exhibits a more pharmacokinetic behaviour due to a lower volume of distribution and a slower renal clearance; therefore cetirizine has been recently marked as levo- enantiomer (Xyzal) in Europe. In order to study the simultaneous determination of (+)- and (-)- cetirizine hydrochloride we have developed direct separation methods by HPLC on polysaccharide-based chiral stationary phases. Satisfactory results were achieved on a Chiralcel OD-R column under gradient elution whit a mobile phase of acetonitrile and perchlorate buffer (0.3M; pH 3.30); these conditions allowed the complete enantioseparations (Rs= 1.65) of the analyte in times suitable for routine analyses. Complete separations in short analysis time (Rs= 1.61) were performed on a Chiralcel OD column under isocratic normal phases conditions (85% n-hexane and 15% ethanol, containing 0.15% trifluoroacetic acid). These conditions were applied to the determination of the stability of cetirizine hydrochloride in methanolic solutions and our studies revealed the formation of an achiral and a chiral degradation product. The developed procedures could be of interest for routine analyses and stereoselective investigations.

2005 - Raman Spectroscopy applied to polymorphism of Tamoxifene Citrate [Abstract in Atti di Convegno]
Gamberini, Maria Cristina; Rustichelli, Cecilia; Baraldi, Cecilia; Ferioli, Valeria; Gamberini, Gianfranco
abstract

Different strategies for a systematic study of polymorphism can be applied: XRPD, DSC, FT-IR and Raman spectroscopy. Unlike X-ray diffraction Raman microscopy, a non-destructive and non-invasive technique, can be applied to pure solids or dosage forms without any sample preparation. The ability of Raman spectroscopy to discriminate between polymorphs has been demonstrated: different crystal structures usually show intensity and frequenciey changes in the Raman spectrum particularly in the rather crowded area from ~ 500 to ~ 1700 cm-1, known as “fingerprint region” which contains the majority of the bands used to identify a particular material. The use of Raman microscopy has been demonstrated for Tamoxifen Citrate: its trans isomer is in fact well known for its endocrine activity as an antiestrogenic agent. Tamoxifen citrate exists in two polymorphic form A and B, Which were found to have a distinct micro Raman spectrum whith bands attributable to the different forms. Another possible form was found and its Raman spectrum is different from the others. Other methods for the characterization of the three forms were used: XRDP, DSC, ATR/FTIR. Because of its non-invasive character, by focusing the micro Raman laser in to a capillary tube the convertion at room temperature of a suspension in ethanol of form A to B has been observed. Finally, the sensitivity to polymorphism of Raman spectroscopy, which makes it an ideal candidate for studies of crystal forms of Pharmaceutical compounds, could be analysed.

2005 - Second Joint Italian-Swiss Meeting on Medicinal Chemistry , Italy, Modena 19-20 February 2005. [Relazione in Atti di Convegno]
Brasili, Livio; Benvenuti, Stefania; Costantino, Luca; Costi, Maria Paola; Philipp, Floersheim; Franchini, Silvia; Gamberini, Gianfranco; Parenti, Carlo; Rastelli, Giulio; Rustichelli, Cecilia; Tait, Annalisa; Tondi, Donatella
abstract

The Second Joint Italian-Swiss Meeting on Medicinal Chemistry (ITCHMC2005) was held in Modena, (Italy) from September 12 to 16, 2005, under the auspices of the European Federation of Medicinal Chemistry (EFMC).This year, the annual meeting of the Division of Medicinal Chemistry of the Italian Chemical Society was co-organized with the Division for Medicinal Chemistry of the Swiss Chemical Society and it followed the first, successful one held in Torino in September 1997. This important event in the field of medicinal chemistry brought together scientists from both academia and industry to discuss different aspects of modern medicinal chemistry. Top-ranking scientists from medicinal chemistry and clinical development had the opportunity to meet and discuss the following topics: Carbohydrate Chemistry in Drug Design, Nuclear Receptors, Progress in Design and Development of Protease Inhibitors, Progress in Oncology Research and finally, Pain and Neurodegenerative Diseases.IntroductionThe conference was attended by 300 scientists from 13 countries, with most of the participants from Italy and Switzerland. The meeting allowed an extensive exchange of information and widespread networking. Of the 134 posters on display, 19 were selected for short oral presentations and two were selected for the Farminidustria awards. The meeeting was organized in six sessions with 6 Plenary (PL), 16 Main Lectures (ML) and 19 short communications (SC).The scientific sessions were held at the Forum Guido Monzani, a modern complex with multifunctional facilities; the opening ceremony took place at Accadenia Militare di Modena, housed in the seventeenth century Palazzo Ducale.Modena, city of art, culture and prosperous economy, offered an exciting background for stimulating scientific interactions. Participants were mainly from academia and other research centers together with 46 pharmaceutical companies; among them, six presented their work, namely Novartis Basel , Roche Basel, Novartis East Hannover USA, IRBM Pomezia Italy, Santhera Pharmaceutical AG, Heidelberg, GlaxoSmithKline (GSK) Verona, S-IN Soluzioni Informatiche, Vicenza. The areas covered by the meeting were advanced medicinal chemistry including computational chemistry, established drug targets, libraries and screens, inhibitor design and clinical advances. There were two poster sessions, with presentations given mainly by young scientists.

2004 - Effects of α- and β-cyclodextrin complexation on the physico-chemical properties and antioxidant activity of some 3-hydroxyflavones [Articolo su rivista]
Ml, Calabro; S., Tommasini; P., Donato; D., Raneri; R., Stancanelli; P., Ficarra; R., Ficarra; C., Costa; S., Catania; Rustichelli, Cecilia; Gamberini, Gianfranco
abstract

Inclusion complexes of some flavonols (3-hydroxyflavone, morin and quercetin) have been obtained with alpha- and beta-cyclodextrins, by the co-evaporation method. Different analytical techniques (DSC, XRPD, FT-IR, H-1-NMR, UV-Vis) have been employed for a throughout investigation of the structural characteristics of such supramolecular aggregates, which exhibited distinct spectroscopic features and properties from both guest and host molecules. The stoichiometric ratios and stability constants describing the extent of formation of the complexes have been determined by phase-solubility studies; in all cases type-A(L) diagrams have been obtained (soluble 1: 1 complexes). The effect of molecular encapsulation on the flavonols antioxidant activity has been afterwards evaluated, by means of different biological assays (Bathophenanthroline test; Comet assay; Lipid peroxidation). Complexation with cyclodextrins further improved the antioxidant activity, increasing drugs solubility in the biological moiety.

2004 - Enantioselective analyses of antihistaminic drugs by high performance liquid chromatography [Articolo su rivista]
Rustichelli, Cecilia; Gamberini, Maria Cristina; V., Ferioli; Gamberini, Gianfranco
abstract

High-performance liquid chromatographic methods have been developed for stereoselective separations of terfenadine and its active metabolite fexofenadine.Satisfactory enantioseparations of racemic terfenadine were achieved on a Chiralcel column by normal phase elution. Analysis of racemic fexofenadine, as such, took a very long time and achieved poor enantioselectivity on this column; nevertheless, the analyte when derivatised with diazomethane was well resolved.Racemic fexofenadine was also derivatised using a chiral agent: R-(+)-1-phenylethylisocyanate and subjected to achiral LC on a reversed-phase analytical column. Complete enantioseparations were achieved in short analysis times; excess reagent eluted before the diastereoisomeric pair and did not interfere.

2003 - Derivatisation procedures for the chromatographic enantioseparation of fexofenadine hydrochloride [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Gamberini, Gianfranco; Ferioli, Valeria
abstract

The paper describes two different pre-column derivatisation procedures for the chromatographic enantioseparation of fexofenadine hydrochloride

2003 - Enantioselective analyses of antihistaminic drugs by high-performance liquid chromatography on chiral stationary phases [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Ferioli, Valeria; Gamberini, Gianfranco
abstract

The present communication describes enantioselective analyses of terfenadine and fexofenadine by HPLC on Chiralcel OD and Chirobiotic V columns.

2002 - Caratterizzazione di complessi di inclusione flavonoidi/ciclodestrine mediante DSC, Raggi X, DRIFT, UV-VIS [Abstract in Atti di Convegno]
R., Ficarra; P., Donato; S., Tommasini; Gamberini, Gianfranco; M. L., Calabrò; Rustichelli, Cecilia; P., Ficarra
abstract

Il presente lavoro riguarda la sintesi, l'isolamento e la caratterizzazione spettroscopica di complessi di inclusione di flavonoidi in alfa- e beta-ciclodestrine

2002 - Caratterizzazione di farmaci polimorfi: terfenadina [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Gamberini, Gianfranco; Ferioli, Valeria; R., Ficarra; R., Stancanelli
abstract

Lo studio riguarda la caratterizzazione dello stato solido dei diversi polimorfi della terfenadina ottenuti mediante idonee procedure di cristallizzazione

2002 - Carbamazepina: valutazioni chimico analitiche e tossicologico forensi in reperti biologici post mortali [Abstract in Atti di Convegno]
Gamberini, Gianfranco; Licata, Manuela; Rustichelli, Cecilia; Ferioli, Valeria
abstract

E' stato messo a punto un metodo GC-MSper l'analisi della cabamazepina in reperti post-mortali previa estrazione SPE e sililazione dell'estratto ottenuto. La procedura è stata applicata a casi reali per individuare il ruolo causale o concausale dell'analita nel determinismo del decesso.

2002 - Study of flavonoids/beta-cyclodextrins inclusion complexes by NMR, FT-IR, DSC, X-ray investigation [Articolo su rivista]
R., Ficarra; S., Tommasini; D., Raneri; M. L., Calabro; M. R., Di Bella; Rustichelli, Cecilia; Gamberini, Maria Cristina; P., Ficarra
abstract

Flavonoids are natural substances with a lot of biological activities, including the antioxidant one. Their use in pharmaceutical field is, however, limited by their aqueous insolubility. As the formation of the inclusion complexes can improve their solubility in water, the flavonoids hesperetin, hesperidin, naringenin and naringin have been complexed with beta-cyclodextrin (beta-CD) by the coprecipitation method and studied in solution and in solid state by NMR, FT-IR, differential scanning calorimetry and X-ray techniques, The effects of complexation on the chemical shifts of the internal and external protons of beta-CD in the presence of each flavonoid were observed. (C) 2002 Elsevier Science B.V. All rights reserved.

2001 - Enantiomeric separation of local anaesthetic drug by HPLC on chiral stationary phases [Articolo su rivista]
Rustichelli, Cecilia; Ferioli, Valeria; Gamberini, Gianfranco; R., Stancanelli
abstract

High-performance liquid chromatographic methods on chiral stationary phases have been developed for the stereoselective separations of the enantiomers of articaine hydrochloride and its metabolite articainic acid. Complete separations of articaine enantiomers have been achieved on a Chiralcel OD analytical column using mobile phases containing n-hexane and 2-propanol, while articainic acid enantiomers were resolved with n-hexane and ethanol. Good results were also obtained on a Chirobiotic V analytical column with 5% acetonitrile in triethylammonium acetate solution (7 mM; pH 6.5); the column showed good chemioselectivity and stereoselectivity, allowing simultaneous separation of both the enantiomeric pairs of articaine and articainic acid.

2001 - Enantiomeric separation of local anaesthetic drug by HPLC on chiral stationary phases [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Ferioli, Valeria; Pavesi, Giorgia; Gamberini, Gianfranco
abstract

The present communication deals with the stereoselective separations of the enantiomers of articaine hydrochloride and its metabolite articainic acid by HPLC on chiral stationary phases.

2001 - New combined treatment of hypermelanosis: analytical studies on efficacy and stability improvement [Articolo su rivista]
Ferioli, Valeria; Rustichelli, Cecilia; Pavesi, G.; Gamberini, G.
abstract

The aim of this work was to evaluate the synergybetween di¡erent lightening agents when associated;depigmenting activity was tested in vitro bymonitoring the appearance of dopachrome, an intermediatein the melanogenesis process. The resultsobtained were compared with the depigmentingactivity of each single compound, keeping the sameglobal concentration of inhibitor.Our studies showed that the combinationof hydroquinoneand kojic acid had a synergistic e¡ect, andthat the maximum inhibiting action was achievedwith an equimolecular mixture. This result couldserve inthe cosmetics ¢eld to prepare skin-lighteningformulations that are less irritant.We also investigated the feasibility of complexinghydroquinone with cyclodextrin and evaluated thee¡ectiveness of the complex obtained in the treatmentof hyperpigmentation.

2000 - Analytical characterisation of hashish samples [Articolo su rivista]
Ferioli, Valeria; Rustichelli, Cecilia; G., Pavesi; Gamberini, Gianfranco
abstract

This report describes the analytical characterisation of two hashish samples to establish if they belong to a common lot, although their physical appearance is quite different The samples were extracted and subjected to HPLC and GC for the separation and quantitation of the main cannabinoids. GC-MS enabled the selective identification of minor cannabinoids and terpenes. Hashish samples were also mineralised and analysed for metal traces by atomic absorption spectrometry (AA).

2000 - Solid-state study of polymorphic drugs: carbamazepine [Articolo su rivista]
Rustichelli, Cecilia; Gamberini, Gianfranco; Ferioli, Valeria; Gamberini, Maria Cristina; R., Ficarra; S., Tommasini
abstract

Polymorphs of a compound have solid crystalline phases with different internal crystal lattices; in pharmaceuticals,differences due to polymorphism and pseudopolymorphism can affect bioavailability and effective clinical use. Theaim of this work was to obtain the different polymorphic modifications of the anticonvulsant drug, carbamazepine,and to characterise them by means of typical structure-sensitive analytical techniques, such as FT-IR spectroscopy,XRPD and DSC. Further investigations were also performed by Hot Stage FT-IR thermomicroscopy, whichpermitted the visible and spectroscopic characterisation of the polymorphic forms during heating. Our results confirmthe existence of three different polymorphic forms for anhydrous carbamazepine: Form III, the commercial one,Form I, obtained by heating Form III and Form II, crystallised from ethanolic solution. Substantial differences weredetected among the polymorphs with regard to solid-state properties. Moreover, Hot Stage FT-IR thermomicroscopyproved its analytical potential to characterise the drug’s polymorphism.

2000 - Study of β-blockers/β-cyclodextrins inclusion complex by NMR, DSC, X-ray and SEM investigation [Articolo su rivista]
R., Ficarra; P., Ficarra; MR DI, Bella; D., Raneri; S., Tommasini; Ml, Calabro'; Gamberini, Maria Cristina; Rustichelli, Cecilia
abstract

...

1999 - Analytical Characterisation of Hashish Samples [Abstract in Atti di Convegno]
Ferioli, Valeria; Rustichelli, Cecilia; G., Pavesi; Gamberini, Gianfranco
abstract

This work describes the chromatographic characterisation of two hashish samples in order to establish if they belong to a common lot.

1999 - Properties of the racemic species of verapamil hydrochloride and gallopamil hydrochloride [Articolo su rivista]
Rustichelli, Cecilia; Gamberini, Maria Cristina; Ferioli, Valeria; Gamberini, Gianfranco
abstract

It is well known that the stereoselective actions associated with the enantiomeric constituents of a racemic drug candiffer markedly in their pharmacodynamic or pharmacokinetic properties. Nevertheless, molecular chirality manifestsitself in the solid, that is, crystalline state. The aim of this work was to characterize the solid-state properties ofverapamil HCl and gallopamil HCl, two well-known chiral calcium channel antagonists. The characterization of thesolid state for the single enantiomers and equimolecular mixtures for both the calcium antagonists was performed bysolid-state techniques such as Fourier transform infrared (FT-IR spectroscopy), X-ray powder diffractometry (XRD)and differential scanning calorimetry (DSC). The FT-IR spectra and XRD of the single enantiomers are differentfrom those of the corresponding equimolecular mixture owing to their different crystalline structure. The thermalbehavior of the racemates and pure enantiomers were examined by DSC, and the resultant experimental andtheoretical binary phase diagrams are discussed. Spectroscopic solid-state techniques, such as FT-IR and XRD, areuseful in combination with thermal analysis for characterizing the racemic species of chiral drugs. The data obtainedprove that the equimolecular mixtures of both verapamil hydrochloride and gallopamil hydrochloride exist as racemiccompounds. Determination of the enantiomeric purity of the enantiomers and racemic compounds of both thecalcium antagonists analyzed was performed by DSC.

1999 - Solid-state study of polymorphic drugs: carbamazepine [Abstract in Atti di Convegno]
Rustichelli, Cecilia; G., Gamberini; V., Ferioli; Gamberini, Maria Cristina; R., Ficarra; S., Tommasini
abstract

Aim of this work was to obtain the different polymorphic modifications of anhydrous carbamazepine and to characterise them by means of typical structure-sensitive analytical techniques, such as Ft-IR, DRIFT, XRPD and DSC.

1999 - Structure of beta-blockers/beta cyclodextrin inclusion complex by NMR, X-Ray and DSC investigation [Abstract in Atti di Convegno]
P., Ficarra; S., Tommasini; M. R., Di Bella; D., Raneri; R., Ficarra; M. L., Calabrò; Gamberini, Maria Cristina; Rustichelli, Cecilia
abstract

The inclusion complexes between two beta-blockers, atenolol and celiprolol, and the beta-cyclodextrin has been investigated in solution and in the solid state using X-Ray, DSC and NMR.

1998 - Analysis of cannabinoids in fiber hemp plant varieties (Cannabis sativa L.) by high-performance liquid chromatography [Articolo su rivista]
Rustichelli, Cecilia; Ferioli, Valeria; Baraldi, Mario; Zanoli, Paola; Gamberini, Gianfranco
abstract

An analytical procedure was developed for the detection of neutral and acidic cannabinoids in herbal cannabis without the need of any preliminary derivatization. The method was used to assay cannabinoid content of over one hundred fiber hemp samples grown in different Italian localities and harvested at different maturation level degrees during the summer. No interferences were observed due to the vegetal matrix. The influence of genetic factors and environmental conditions on cannabinoid content is discussed; the results may be of interest to enhance potential of fiber hemp in compliance with law enforcement purposes.

1998 - Evaluation of synergic activity between depigmenting agents [Abstract in Atti di Convegno]
Ferioli, Valeria; Rustichelli, Cecilia; M. B., Virgili; Gamberini, Gianfranco
abstract

Aim of this work was to evaluate the presence of synergy between different whitening agents when associated. In particular, binary mixtures of the following compounds were examined: hydroquinone, kojic acid, vitamin C and its derivatives. Depigmenting activity was evaluated by monitoringthe appearance of dopachrome, an intermediate in melanogenesis process

1997 - Analytical investigations on active principles in aloe leaf exudate [Abstract in Atti di Convegno]
Benvenuti, Stefania; Rustichelli, Cecilia; Vampa, Gabriella; Ferioli, Valeria; G., Grassi; Melegari, Michele; P., Codeluppi; Gamberini, Gianfranco
abstract

The present work deals with the development of various procedures to estimate aloin and related compounds in aloe products by means of UV, HPTLC and HPLC procedures.

1997 - Profiling of hemp plant varieties by high-performance liquid chromatography [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Ferioli, Valeria; Baraldi, Mario; Zanoli, Paola; Gamberini, Gianfranco
abstract

This work describes a rapid HPLC procedure to determine cannabinoid pattern in a considerable number of cannabis samples of different origin and age. Peak identification was also established by HPLC-MS analyses. The obtained results show different chromatographic profiles in the plant extracts depending on the different experimental treatment and growing conditions.

1997 - Resolution of the enantiomers of verapamil and gallopamil by chiral liquid chromatography mass spectrometry [Articolo su rivista]
Rustichelli, Cecilia; Ferioli, Valeria; Gamberini, G.
abstract

An HPLC-CSP method has been developed for the separation of enantiomers of verapamil and gallopamil using the improved version of the alpha(1)-acid glycoprotein, chiral stationary phase as selector with a volatile mobile phase. The results of the investigation provide the conditions which allow direct coupling to a mass spectrometer. In addition, the simultaneous enantioseparation of both the enantiomeric pairs of verapamil and gallopamil was achieved.

1997 - Solid-state properties of calcium channel antagonists [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Ferioli, Valeria; L., Mazzi; Gamberini, Gianfranco
abstract

In the present paper are reported the characterisation of the solid-state properties of verapamil hydrochloride and gallopamil hydrochloride by means of sensitive analytical techniques such as DRIFT, XRPD and DSC

1996 - Determinazione dei contenuti di cannabinoidi in Cannabis Sativa L. mediante cromatografia liquida abbinata alla spettrometria di massa [Articolo su rivista]
Gamberini, Gianfranco; Rustichelli, Cecilia; Baraldi, Mario
abstract

Sono state approntate procedure di analisi cromatografica abbinata alla spetttrometria di massa per la determinazione quantitativa del contenuto di cannabinoidi in campioni di Cannabis. I risultati ottenuti si sono rivelati utili al fine di verificare le possibili relazioni tra il contenuto di Cannabonoidi (acidi e neutri) e la pianta in esame (cultivar, grado di maturazione, sesso).

1996 - Simultaneous separation and identification of hashish constituents by coupled liquid chromatography mass spectrometry (HPLC-MS) [Articolo su rivista]
Rustichelli, Cecilia; Ferioli, Valeria; Vezzalini, Francesca; Rossi, M. C.; Gamberini, G.
abstract

This paper reports the use of liquid chromatography for the separation and determination of the major cannabinoids extracted from hashish samples. The direct coupling to the mass spectrometer enables the selective identification both of neutral and acidic cannabinoids. The developed method does not require any preliminary derivatization and should, therefore, be of interest in forensic analysis for simple and unequivocal determination of hashish constituents.

1995 - ANALYSIS OF PYRITHIONES BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY [Articolo su rivista]
Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca; Gamberini, G.
abstract

An analytical procedure has been developed for the determination of zinc pyrithione in antidandruff formulations. Zinc pyrithione was converted into a stable copper complex and then analysed by reversed-phase HPLC. The proposed method allows the separation of the analyte from related pyrithiones and therefore is able to verify the compliance of cosmetic preparations with current legislation.

1995 - Determination of Zinc Pyrithione and related compounds in hair care formulations by HPLC [Abstract in Atti di Convegno]
Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca; Gamberini, Gianfranco; G., Lugli
abstract

An analytical procedure has been developed for the determination of Zinc Pirithione in antidandruff formulations. The analyte was converted into a stable copper complex and then analysed by RP-HPLC. Moreover the proposed method allows the separation of zinc pirithione from the related pirithiones

1995 - HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF DIHYDROXYACETONE AS ITS BIS-2,4-DINITROPHENYLHYDRAZONE DERIVATIVE [Articolo su rivista]
Ferioli, Valeria; Vezzalini, Francesca; Rustichelli, Cecilia; Gamberini, G.
abstract

A rapid and simple HPLC method for the identification and determination of dihydroxyacetone, a tanning agent widely used in a variety of preparations, is presented. The method consists of the analysis of the formulated products after a derivatization reaction which enhances the response and the detection limit of the chromatographic analysis. The characterization and the elucidation of the spacial geometry of the synthesized derivative are described.

1995 - HPLC analysis and structural characterisation of dihydroxyacetone as its bis-2,4-dinitrophenylhydrazone derivative [Abstract in Atti di Convegno]
Ferioli, Valeria; Vezzalini, Francesca; Rustichelli, Cecilia; Gamberini, Gianfranco; G., Lugli
abstract

A rapid and simple HPLC procedure for the determination of dihydroxyacetone has been performed; the method involves the analysis of the examined formulations after a derivatisation reaction with 2,4-dinitrophenylhydrazine in order to enhance the detection limit.

1995 - Investigations of Cannabis constituents by HPLC/MS [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Vezzalini, Francesca; Rossi, Maria Cecilia; Ferioli, Valeria; G., Lugli; Gamberini, Gianfranco
abstract

An analytical HPLC/MS procedure was developed for the analysis of major neutral and acidic Cannabis constituens without the need for a derivatisation step of any kind

1994 - CALORIMETRIC AND INFRARED SPECTROPHOTOMETRIC STUDY OF ETHAMBUTOL DIHYDROCHLORIDE [Articolo su rivista]
Gamberini, Gianfranco; Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca
abstract

The binary phase diagram of [S,S(+)]-ethambutol dihydrochloride, a chemotherapeutic drug, and its [R,R(-)]-enantiomer, has been determined by IR spectroscopy, X-ray podwer diffraction and differential scanning calorimetry, the equimolecular mixture of [S,S(+)]/[R,R(-)] corresponds to a racemic compound. The infrared spectra of enantiomers and racemic compound differ especially in the finger print region. The solid-solid transition and fusion enthalpies and entropies for the optically active compounds and the corresponding racemic compound have been determined. The stability of the racemic compound has been evaluated on the basis of free energy of formation. In this paper the results of calorimetric and spectroscopic analyses for the determination of enantiomeric purity of ethambutol dihydrochloride have been reported.

1994 - DETERMINATION OF AZELAIC ACID IN PHARMACEUTICALS AND COSMETICS BY RP-HPLC AFTER PRECOLUMN DERIVATIZATION [Articolo su rivista]
Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca; Gamberini, Gianfranco
abstract

This paper reports a RP-HPLC method for the determination in topics of azelaic acid, a keratolytic and anti-comedogenic agent widely used in the treatment of all types of acne. A derivatization step was needed prior to chromatographic analysis because the analyte is lacking in chromophore. A sample clean-up procedure by solid-phase extraction was also developed to analyse azelaic acid in complex matrices, such as pharmaceutical and cosmetic formulations.

1994 - DIRECT DETERMINATION OF NON-UV-ABSORBING COMPOUNDS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY [Articolo su rivista]
Ferioli, Valeria; Gamberini, Gianfranco; Rustichelli, Cecilia; Vezzalini, Francesca
abstract

A direct liquid chromatographic method with UV detection has been developed for the determination of ethambutol dihydrochloride, a chemotherapeutic drug lacking in chromophoric groups. It allows the simultaneous separation of [S,S(+)]-ethambutol from meso-[S,R(+/-)]-form and its synthetic precursor [S(+)]-2-amino-1-butanol, although it is not stereospecific for the separation of the [R,R(-)]-enantiomer. Important factors affecting the selectivity and efficiency of separation (CuSO4 concentration, temperature and addition of an organic modifier in the mobile phase) have been studied. Moreover the proposed method allows the quantitative determination of ethambutol dihydrochloride in pharmaceutical formulations in order to verify the agreement with the claimed content for the drug.

1993 - Calorimetric and infrared spectrophotometric study of (S,S)-2,2?-(1,2ethanediyldiimino)bis-1-butanol hydrochloride [Abstract in Atti di Convegno]
Gamberini, Gianfranco; Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca
abstract

This paper reports the results of calorimetric and IR analysis for the characterisation of the solid state and for the determination of the enantiomeric purity of Ethambutol Hydrochloride.

1993 - Determination of medium chain dicarboxylic acids in pharmaceuticals and cosmetics by SPE and RP-HPLC analysis [Abstract in Atti di Convegno]
Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca; Gamberini, Gianfranco
abstract

A method has been developed for the extraction and determination of azelaic acid in pharmaceutical and cosmetic formulations. The method includes sample clean-up using SPE extraction, preparation of UV-absorbing derivatives and RP-HPLC separation.

1993 - Direct determination of non-UV absorbing compounds by high-performance liquid chromatography [Abstract in Atti di Convegno]
Ferioli, Valeria; Gamberini, Gianfranco; Rustichelli, Cecilia; Vezzalini, Francesca
abstract

A direct liquid chromatographic method has been developed for the determination of Ethambutol Hydrochloride, allowing the separation of its enantiomeric couple from the meso form. The analyses were performed on a LEC-Chiralsil D-ValCu column and the analytes compounds were detected by UV absorption as their copper chelates.

1992 - A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE ANALYSIS OF ADENOSINE AND SOME METABOLITES IN THE BRAIN-TISSUE OF RATS [Articolo su rivista]
Gamberini, G; Ferioli, Valeria; Zanoli, Paola; Zeneroli, Maria Luisa; Rustichelli, Cecilia; Baraldi, Mario
abstract

An analytical procedure has been developed for the assay of adenosine and some metabolites in brain tissue extracts of rats. This paper reports the extraction method, the technique adopted to avoid enzymatic transformations and the chromatographic conditions for the identification and quantitation of these nucleosides and bases. Peaks in the chromatograms of brain tissue extracts were identified by retention times, absorbance ratios of reference compounds and by enzymatic peak-shift.

1992 - Assessment of adenosine and its metabolites in brain areas of rats with hepatic encephalopathy [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Gamberini, Gianfranco; Ferioli, Valeria; Baraldi, Mario; Zeneroli, Maria Luisa
abstract

The aim of the present work was to verify whether changes in purine levels were implicated in the development of the hepatic encephalopathy syndrome. To this aim, rats were treated with galactosamine, sacrificed and samples of their brain tissues were subjeted to extraction protocol and analysed by RP-HPLC

1992 - Chromatographic analysis of sympathomimetic amines after fluorescence derivatization with benzocoumarin-3-carboxylic acid chloride [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Ferioli, Valeria; Gamberini, Gianfranco; R., Wintersteiger; R., Kuttner
abstract

This work reports the use of a new fluorescent reagents in derivatization procedures prior toTLC and HPLC separations of sympathomimetic amines. Experiments were performed to optimize the reaction conditions /time, temperature, reagent amount, choice and amount of catalyst).

1992 - DETERMINATION OF BRONIDOX AND BRONOPOL IN COSMETIC PRODUCTS BY REVERSED PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY [Articolo su rivista]
Ferioli, Valeria; Vezzalini, Francesca; Rustichelli, Cecilia; Gamberini, G.
abstract

A rapid and simple HPLC method is described for the separation and quantitative determination of bronidox and bronopol used as preservatives and listed in the current EEC Council Directives on cosmetic products. The proposed method allows the routine detection of these preservatives in different cosmetic formulations with a view for verifying their complicance with Italian legislation.

1991 - Analysis of bronidox and Bronopol in cosmetic products by reversed phase high-performance liquid chromatography [Abstract in Atti di Convegno]
Ferioli, Valeria; Vezzalini, Francesca; Rustichelli, Cecilia; Gamberini, Gianfranco
abstract

This paper reports a rapid and simple HPLC method for the separation and quantitative determination of bronidox and bronopol used as preservatives; the developed method allows to verify the compliance of commercial products with current European Economic Community (EEC) Council Directive.

1990 - Separation of chiral compounds with alpha-acid glycoprotein as selector [Articolo su rivista]
Gamberini, Gianfranco; Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca
abstract

The present paper reports an optimized procedure for the efficient and fast HPLC-CSP separation of the enantiomeric pair of verapamil uusing a chiral column with alpha-acid glycoprotein as selector. The influence of temperature, pH, flow-rate and concentration of organic modifier on the chromatographic separations is shown.

1990 - Separation of chiral compounds with alpha-acid glycoprotein as selector [Abstract in Atti di Convegno]
Gamberini, Gianfranco; Ferioli, Valeria; Rustichelli, Cecilia; Vezzalini, Francesca
abstract

The present paper reports an optimized procedure for the efficient and fast HPLC separation of the enantiomeric pair of verapamil hydrochloride using an AGP chiral stationary phase. The influence of pH, flow-rate, temperature and concentration of organic modifier was investigated.

1989 - Studio dei profili di purezza di alcuni narcotici mediante cromatografia liquida a fase inversa [Abstract in Atti di Convegno]
Ferioli, Valeria; Gamberini, Gianfranco; Rustichelli, Cecilia
abstract

Il lavoro riguarda la messa a punto di un metodo HPLC per lo studio dei profili di purezza di morfina, codeina ed eroina e, per quanto riguarda l'eroina, per lo studio della stabilità mediante la determinazione delle costanti di velocità di idrolisi. La metodica si è rivelata idonea ed alternativa al metodo ufficiale FU IX.