Messa a punto di formulazioni semisolide per il trattamento della psoriasi ungueale

La psoriasi ungueale è una patologia cronica che può coinvolgere sia le unghie delle mani che quelle dei piedi , colpisce l'80-90 % dei pazienti affetti da psoriasi della pelle e nell'1-5% dei casi rimane confinata a livello ungueale. In base alla gravità, può impattare fortemente sulla vita del paziente sia da un punto di vista estetico che funzionale influenzando la vita sociale e le attività lavorative. Numerosi studi hanno dimostrato come la terapia sistemica per la cura della psoriasi della pelle porti a notevoli miglioramenti anche alla condizione ungueale ma per ottenere un azione maggiore a livello locale e soprattutto nei casi in cui la patologia colpisca solo le unghie risulta importante avere a disposizione un trattamento topico efficace, nonostante l'ostacolo rappresentato dalla scarsa permeabilità della superficie ungueale ai farmaci. Lo scopo di questo studio è stata la messa a punto di un idrogel a base di Ciclosporina A al 5%, vista la sua ormai conclamata efficacia nel trattamento della psoriasi e l'assenza in commercio di formulazioni a base di Ciclosporina per applicazioni ungueali. Si è proceduto prima con la realizzazione di un idrogel non medicato e successivamente sono state testate varie formulazioni e procedure per l'inserimento del principio attivo nell'idrogel. Sul prodotto finale, sono stati eseguiti studi di rilascio del principio attivo in vitro e di caratterizzazione dell'idrogel (capacità di swelling e angolo di contatto)

Microsampling and enantioselective liquid chromatography coupled to mass spectrometry for chiral bioanalysis of novel psychoactive substances

In this paper, the development of efficient enantioselective HPLC methods for the analysis of five benzofuran-substituted phenethylamines, two substituted tryptamines, and three substituted cathinones is described. For the first time, reversed-phase (eluents made up with acidic water-methanol solutions) and polar-ionic (eluent made up with an acetonitrile-methanol solution incorporating both an acidic and a basic additive) conditions fully compatible with mass spectrometry (MS) detectors were applied with a chiral stationary phase (CSP) incorporating the (+)-(18-crown-6)-tetracarboxylic acid chiral selector. Enantioresolution was achieved for nine compounds with α and RS factors up to 1.32 and 5.12, respectively. Circular dichroism (CD) detection, CD spectroscopy in stopped-flow mode and quantum mechanical (QM) calculations were successfully employed to investigate the absolute stereochemistry of mephedrone, methylone and butylone and allowed to establish a (R)<(S) enantiomeric elution order for these compounds on the chosen CSP. Whole blood miniaturized samples collected by means of volumetric absorptive microsampling (VAMS) technology and fortified with the target analytes were extracted following an optimized protocol and effectively analysed by means of an ultra-high performance liquid chromatography-MS system. By this way a proof-of-concept procedure was applied, demonstrating the suitability of the method for quali-quantitative enantioselective assessment of the selected psychoactive substances in advanced biological microsamples. VAMS microsamplers including a polypropylene handle topped with a small tip of a polymeric porous material were used and allowed to volumetrically collect small aliquots of whole blood (10 μL) independently from its density. Highly appreciable volumetric accuracy (bias, in the -8.7-8.1% range) and precision (% CV, in the 2.8-5.9% range) turned out

Investigating in vitro amyloid peptide 1-42 aggregation: impact of higher molecular weight stable adducts

The self-assembly of amyloid peptides (Aβ), in particular Aβ1–42, into oligomers and fibrils is one of the main pathological events related to Alzheimer’s Disease (AD). Recent studies have demonstrated the ability of carbon monoxide releasing molecules (CORMs) to protect neurons and astrocytes from Aβ1-42 toxicity. In fact, CORMs are able to carry and release controlled levels of CO and are known to exert a wide range of anti-inflammatory and anti-apoptotic activities at physiologically relevant concentrations. In order to investigate the direct effects of CORMs on Aβ1–42, we studied the reactivity of CORM-2 and CORM-3 with Aβ1–42 in vitro and the potential inhibition of its aggregation by mass spectrometry (MS), as well as fluorescence and circular dichroism (CD) spectroscopies. The application of an electrospray ionisation-mass spectrometry (ESI-MS) method allowed the detection of stable Aβ1–42/CORMs adducts, involving the addition of the Ru(CO)2 portion of CORMs at histidine residues on the Aβ1-42 skeleton. Moreover, CORMs showed anti-aggregating properties through formation of stable adducts with Aβ1–42 as demonstrated by a thioflavin T (ThT) fluorescence assay and MS analysis. As a further proof, comparison of the CD spectra of Aβ1–42 recorded in the absence and in the presence of CORM-3 at 1:1 molar ratio showed the ability of CORM-3 to stabilize the peptide in its soluble, unordered conformation, thereby preventing its misfolding and aggregation. This multi-methodological investigation revealed novel interactions between Aβ1–42 and CORMs, contributing new insights into the proposed neuroprotective mechanisms mediated by CORMs and disclosing a new strategy to divert amyloid aggregation and toxicity

Multitarget drug design strategy in Alzheimer’s disease: focus on cholinergic transmission and amyloid-β aggregation

Background: Alzheimer pathogenesis has been associated with a network of processes working simultaneously and synergistically. Over time, much interest has been focused on cholinergic transmission and its mutual interconnections with other active players of the disease. Besides the cholinesterase mainstay, the multifaceted interplay between nicotinic receptors and amyloid is actually considered to have a central role in neuroprotection. Thus, the multitarget drug-design strategy has emerged as a chance to face the disease network. Results: By exploiting the multitarget approach, the present study provides new molecules able to target the cholinergic pathway, by joining direct nicotinic receptor stimulation to acetylcholinesterase inhibition, and to inhibit Aβ aggregation. Conclusions: These new compounds emerged as a suitable starting point for a further optimization process

Evaluation of the effects of acetylcholinesterase inhibitors in the zebrafish touch-evoked response: quantitative vs. qualitative assessment

Background: The difficulty of finding new treatments for neurological diseases with great impact in our society like Alzheimer's disease can be ascribed in part to the complexity of the nervous system and the lack of quick and costeffective screening tools. Such tools could not only help to identify potential novel treatments, but could also be used to test environmental contaminants for their potential to cause neurotoxicity. It has been estimated that 5-10% of the anthropogenic chemicals are developmental neurotoxic (DNT) and exposure to DNT compounds has been linked to several neurological diseases. Within this study we were testing the applicability of a quick and cost-effective behavioural test using zebrafish embryos: the touch-evoked response assay, in this case, an assay evaluating the swimming response to a tap in the tail. Two acetylcholinesterase (AChE) inhibitors positive controls (paraoxon and huprine Y), as well as 10 huprine-derivative compounds were tested and the results were evaluated using 2 different methods, a quantitative and a qualitative one. Results: We could show that the methodology presented is able to detect behavioural effects of AChE inhibitors. A good correlation between the results obtained with the quantitative and the qualitative method was obtained (R2 = 0.84). Conclusions: Our proposed method enables combination of screening for new drugs with toxicity screening in a whole embryo model alternative to animal experimentation, thereby merging 2 drug development steps into one

N-1,2,3-triazole-isatin derivatives for cholinesterase and β-amyloid aggregation inhibition: A comprehensive bioassay study

Our goal was the evaluation of a series of N-1,2,3-triazole-isatin derivatives for multi-target activity which included cholinesterase (ChE) inhibition and β-amyloid (Aβ) peptide anti-aggregation. The compounds have shown considerable promise as butyrylcholinesterase (BuChE) inhibitors. Although the inhibition of eel acet- ylcholinesterase (eeAChE) was weak, the inhibitions against equine BuChE (eqBuChE) and human BuChE (hBuChE) were more significant with a best inhibition against eqBuChE of 0.46 μM. In some cases, these mo- lecules gave better inhibitions for hBuChE than eqBuChE. For greater insights into their mode of action, mole- cular docking studies were carried out, followed by STD-NMR validation. In addition, some of these compounds showed weak Aβ anti-aggregation activity. Hepatotoxicity studies showed that they were non-hepatoxic and neurotoxicity studies using neurite out- growth experiments led to the conclusion that these compounds are only weakly neurotoxic

Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

Background: Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods: Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results: CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions: When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform

Discovery of Dual Aβ/Tau Inhibitors and Evaluation of Their Therapeutic Effect on a Drosophila Model of Alzheimer's Disease

Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-β (Aβ) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aβ clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. Based on this, herein, we describe the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aβ and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aβ42 and Tau proteins. We then evaluated their neuronal toxicity and ability to cross the blood−brain barrier (BBB), together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 μM) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aβ42 expressing flies and generating a better outcome than doxycycline (50 μM). Moreover, 22 proved to be able to decrease Aβ42 aggregates in the brains of the flies. We conclude that bivalent small molecules based on 22 deserve further attention as hits for dual Aβ/Tau aggregation inhibition in A

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease