Green Solid Lipid Nanoparticles by Fatty Acid Coacervation: An Innovative Nasal Delivery Tool for Drugs Targeting Cerebrovascular and Neurological Diseases.

Cerebrovascular and neurological diseases are characterized by neuroinflammation, which alters the neurovascular unit, whose interaction with the choroid plexus is critical for maintaining brain homeostasis and producing cerebrospinal fluid. Dysfunctions in such process can lead to conditions such as idiopathic normal pressure hydrocephalus, a common disease in older adults. Potential pharmacological treatments, based upon intranasal administration, are worthy of investigation because they might improve symptoms and avoid surgery by overcoming the blood-brain barrier and avoiding hepatic metabolism. Nasal lipid nanocarriers, such as solid lipid nanoparticles, may increase the nasal retention and permeation of drugs. To this aim, green solid lipid nanoparticles, obtained by coacervation from natural soaps, are promising vehicles due to their specific lipid matrix composition and the unsaponifiable fraction, endowed with antioxidant and anti-inflammatory properties, and thus suitable for restoring the neurovascular unit function. In this experimental work, such green solid lipid nanoparticles, fully characterized from a physico-chemical standpoint, were loaded with a drug combination suitable for reverting hydrocephalus symptoms, allowing us to obtain a non-toxic formulation, a reduction in the production of the cerebrospinal fluid in vitro, and a vasoprotective effect on an isolated vessel model. The pharmacokinetics and biodistribution of fluorescently labelled nanoparticles were also tested in animal models.

Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Structure-Based Optimization of 1,2,4-Triazole-3-Thione Derivatives: Improving Inhibition of NDM-/VIM-Type Metallo-ß-Lactamases and Synergistic Activity on Resistant Bacteria.

The worldwide emergence and dissemination of Gram-negative bacteria expressing metallo-ß-lactamases (MBLs) menace the efficacy of all ß-lactam antibiotics, including carbapenems, a last-line treatment usually restricted to severe pneumonia and urinary tract infections. Nonetheless, no MBL inhibitor is yet available in therapy. We previously identified a series of 1,2,4-triazole-3-thione derivatives acting as micromolar inhibitors of MBLs in vitro, but devoid of synergistic activity in microbiological assays. Here, via a multidisciplinary approach, including molecular modelling, synthesis, enzymology, microbiology, and X-ray crystallography, we optimized this series of compounds and identified low micromolar inhibitors active against clinically relevant MBLs (NDM-1- and VIM-type). The best inhibitors increased, to a certain extent, the susceptibility of NDM-1- and VIM-4-producing clinical isolates to meropenem. X-ray structures of three selected inhibitors in complex with NDM-1 elucidated molecular recognition at the base of potency improvement, confirmed in silico predicted orientation, and will guide further development steps.

Proteomics Studies Suggest That Nitric Oxide Donor Furoxans Inhibit In Vitro Vascular Smooth Muscle Cell Proliferation by Nitric Oxide-Independent Mechanisms.

Physiologically, smooth muscle cells (SMC) and nitric oxide (NO) produced by endothelial cells strictly cooperate to maintain vasal homeostasis. In atherosclerosis, where this equilibrium is altered, molecules providing exogenous NO and able to inhibit SMC proliferation may represent valuable antiatherosclerotic agents. Searching for dual antiproliferative and NO-donor molecules, we found that furoxans significantly decreased SMC proliferation in vitro, albeit with different potencies. We therefore assessed whether this property is dependent on their thiol-induced ring opening. Indeed, while furazans (analogues unable to release NO) are not effective, furoxans' inhibitory potency parallels with the electron-attractor capacity of the group in 3 of the ring, making this effect tunable. To demonstrate whether their specific block on G1-S phase could be NO-dependent, we supplemented SMCs with furoxans and inhibitors of GMP- and/or of the polyamine pathway, which regulate NO-induced SMC proliferation, but they failed in preventing the antiproliferative effect. To find the real mechanism of this property, our proteomics studies revealed that eleven cellular proteins (with SUMO1 being central) and networks involved in cell homeostasis/proliferation are modulated by furoxans, probably by interaction with adducts generated after degradation. Altogether, thanks to their dual effect and pharmacological flexibility, furoxans may be evaluated in the future as antiatherosclerotic molecules.

Use of Enzymatically Activated Carbon Monoxide Donors for Sensitizing Drug-Resistant Tumor Cells.

The application of gaseous signaling molecules like NO, H2S or CO to overcome the multidrug resistance in cancer treatment has proven to be a viable therapeutic strategy. The development of CO-releasing molecules (CORMs) in a controlled manner and in targeted tissues remains a challenge in medicinal chemistry. In this paper, we describe the design, synthesis and chemical and enzymatic stability of a novel non-metal CORM (1) able to release intracellularly CO and, simultaneously, facilitate fluorescent degradation of products under the action of esterase. The toxicity of 1 against different human cancer cell lines and their drug-resistant counterparts, as well as the putative mechanism of toxicity were investigated. The drug-resistant cancer cell lines efficiently absorbed 1 and 1 was able to restore their sensitivity vs. chemotherapeutic drugs by causing a CO-dependent mitochondrial oxidative stress that culminated in mitochondrial-dependent apoptosis. These results demonstrate the importance of CORMs in cases where conventional chemotherapy fails and thus open the horizons towards new combinatorial strategies to overcome multidrug resistance.

Surface Functionalised Parenteral Nanoemulsions for Active and Homotypic Targeting to Melanoma.

Despite recent progressions in cancer genomic and immunotherapies, advanced melanoma still represents a life threat, pushing to optimise new targeted nanotechnology approaches for specific drug delivery to the tumour. To this aim, owing to their biocompatibility and favourable technological features, injectable lipid nanoemulsions were functionalised with proteins owing to two alternative approaches: transferrin was chemically grafted for active targeting, while cancer cell membrane fragments wrapping was used for homotypic targeting. In both cases, protein functionalisation was successfully achieved. Targeting efficiency was preliminarily evaluated using flow cytometry internalisation studies in two-dimensional cellular models, after fluorescence labelling of formulations with 6-coumarin. The uptake of cell-membrane-fragment-wrapped nanoemulsions was higher compared to uncoated nanoemulsions. Instead, the effect of transferrin grafting was less evident in serum-enriched medium, since such ligand probably undergoes competition with the endogenous protein. Moreover, a more pronounced internalisation was achieved when a pegylated heterodimer was employed for conjugation (p < 0.05).

New lipophilic organic nitrates: candidates for chronic skin disease therapy.

Organic nitrates are widely used, but their chronic efficacy is blunted due to the development of tolerance. The properties of new tolerance free organic nitrates were studied. Their lipophilicity profile and passive diffusion across polydimethylsiloxane membrane and pig ear-skin, and their efficacy in tissue regeneration using HaCaT keratinocytes were evaluated. The permeation results show that these nitrates have a suitable profile for NO topical administration on the skin. Furthermore, the derivatives with higher NO release exerted a pro-healing effect on HaCaT cells. This new class of organic nitrates might be a promising strategy for the chronic treatment of skin pathologies.

Comparative Study of Different H2S Donors as Vasodilators and Attenuators of Superoxide-Induced Endothelial Damage.

In the last years, research proofs have confirmed that hydrogen sulfide (H2S) plays an important role in various physio-pathological processes, such as oxidation, inflammation, neurophysiology, and cardiovascular protection; in particular, the protective effects of H2S in cardiovascular diseases were demonstrated. The interest in H2S-donating molecules as tools for biological and pharmacological studies has grown, together with the understanding of H2S importance. Here we performed a comparative study of a series of H2S donor molecules with different chemical scaffolds and H2S release mechanisms. The compounds were tested in human serum for their stability and ability to generate H2S. Their vasorelaxant properties were studied on rat aorta strips, and the capacity of the selected compounds to protect NO-dependent endothelium reactivity in an acute oxidative stress model was tested. H2S donors showed different H2S-releasing kinetic and produced amounts and vasodilating profiles; in particular, compound 6 was able to attenuate the dysfunction of relaxation induced by pyrogallol exposure, showing endothelial protective effects. These results may represent a useful basis for the rational development of promising H2S-releasing agents also conjugated with other pharmacophores.

Galactosylated Prodrugs: A Strategy to Improve the Profile of Nonsteroidal Anti-Inflammatory Drugs.

Carbohydrates are one of the most abundant and important classes of biomolecules. The variety in their structures makes them valuable carriers that can improve the pharmaceutical phase, pharmacokinetics and pharmacodynamics of well-known drugs. D-galactose is a simple, naturally occurring monosaccharide sugar that has been extensively studied for use as a carrier and has proven to be valuable in this role. With the aim of validating the galactose-prodrug approach, we have investigated the galactosylated prodrugs ibuprofen, ketoprofen, flurbiprofen and indomethacin, which we have named IbuGAL, OkyGAL, FluGAL and IndoGAL, respectively. Their physicochemical profiles in terms of lipophilicity, solubility and chemical stability have been evaluated at different physiological pH values, as have human serum stability and serum protein binding. Ex vivo intestinal permeation experiments were performed to provide preliminary insights into the oral bioavailability of the galactosylated prodrugs. Finally, their anti-inflammatory, analgesic and ulcerogenic activities were investigated in vivo in mice after oral treatment. The present results, taken together with those of previous studies, undoubtedly validate the galactosylated prodrug strategy as a problem-solving technique that can overcome the disadvantages of NSAIDs.