Pyrazole-Enriched Cationic Nanoparticles Induced Early- and Late-Stage Apoptosis in Neuroblastoma Cells at Sub-Micromolar Concentrations

Neuroblastoma (NB) is a severe form of tumor occurring mainly in young children and originating from nerve cells found in the abdomen or next to the spine. NB needs more effective and safer treatments, as the chance of survival against the aggressive form of this disease are very small. Moreover, when current treatments are successful, they are often responsible for unpleasant health problems which compromise the future and life of surviving children. As reported, cationic macromolecules have previously been found to be active against bacteria as membrane disruptors by interacting with the negative constituents of the surface of cancer cells, analogously inducing depolarization and permeabilization, provoking lethal damage to the cytoplasmic membrane, and cause loss of cytoplasmic content and consequently, cell death. Here, aiming to develop new curative options for counteracting NB cells, pyrazole-loaded cationic nanoparticles (NPs) (BBB4-G4K and CB1H-P7 NPs), recently reported as antibacterial agents, were assayed against IMR 32 and SHSY 5Y NB cell lines. Particularly, while BBB4-G4K NPs demonstrated low cytotoxicity against both NB cell lines, CB1H-P7 NPs were remarkably cytotoxic against both IMR 32 and SHSY 5Y cells (IC50 = 0.43–0.54 µM), causing both early-stage (66–85%) and late-stage apoptosis (52–65%). Interestingly, in the nano-formulation of CB1H using P7 NPs, the anticancer effects of CB1H and P7 were increased by 54–57 and 2.5–4-times, respectively against IMR 32 cells, and by 53–61 and 1.3–2 times against SHSY 5Y cells. Additionally, based on the IC50 values, CB1H-P7 was also 1-12-fold more potent than fenretinide, an experimental retinoid derivative in a phase III clinical trial, with remarkable antineoplastic and chemopreventive properties. Collectively, due to these results and their good selectivity for cancer cells (selectivity indices = 2.8–3.3), CB1H-P7 NPs represent an excellent template material for developing new treatment options against NB

Mini-Tablets: A Valid Strategy to Combine Efficacy and Safety in Pediatrics

open6In the treatment of pediatric diseases, mass-produced dosage forms are often not suitable for children. Commercially available medicines are commonly manipulated and mixed with food by caregivers at home, or extemporaneous medications are routinely compounded in the hospital pharmacies to treat hospitalized children. Despite considerable efforts by regulatory agencies, the pediatric population is still exposed to questionable and potentially harmful practices. When designing medicines for children, the ability to fine-tune the dosage while ensuring the safety of the ingredients is of paramount importance. For these purposes solid formulations may represent a valid alternative to liquid formulations for their simpler formula and more stability, and, to overcome the problem of swelling ability, mini-tablets could be a practicable option. This review deals with the different approaches that may be applied to develop mini-tablets intended for pediatrics with a focus on the safety of excipients. Alongside the conventional method of compression, 3D printing appeared particularly appealing, as it allows to reduce the number of ingredients and to avoid both the mixing of powders and intermediate steps such as granulation. Therefore, this technique could be well adaptable to the daily galenic preparations of a hospital pharmacy, thus leading to a reduction of the common practice of off-label preparations.openZuccari, Guendalina; Alfei, Silvana; Marimpietri, Danilo; Iurilli, Valentina; Barabino, Paola; Marchitto, LeonardoZuccari, Guendalina; Alfei, Silvana; Marimpietri, Danilo; Iurilli, Valentina; Barabino, Paola; Marchitto, Leonard

Preparation and Characterization of Amorphous Solid Dispersions for the Solubilization of Fenretinide

Fenretinide (4-HPR), a retinoid derivative, has shown high antitumor activity, a low toxicological profile, and no induction of resistance. Despite these favorable features, the variability in oral absorption due to its low solubility combined with the high hepatic first pass effect strongly reduce clinical outcomes. To overcome the solubility and dissolution challenges of poorly water-soluble 4-HPR, we prepared a solid dispersion of the drug (4-HPR-P5) using a hydrophilic copolymer (P5) previously synthesized by our team as the solubilizing agent. The molecularly dispersed drug was obtained by antisolvent co-precipitation, an easy and up-scalable technique. A higher drug apparent solubility (1134-fold increase) and a markedly faster dissolution were obtained. In water, the colloidal dispersion showed a mean hydrodynamic diameter of 249 nm and positive zeta potential (+41.3 mV), confirming the suitability of the formulation for intravenous administration. The solid nanoparticles were also characterized by a high drug payload (37%), as was also evidenced by a chemometric-assisted Fourier transform infrared spectroscopy (FTIR) investigation. The 4-HPR-P5 exhibited antiproliferative activity, with IC50 values of 1.25 and 1.93 µM on IMR-32 and SH-SY5Y neuroblastoma cells, respectively. Our data confirmed that the 4-HPR-P5 formulation developed herein was able to increase drug apparent aqueous solubility and provide an extended release over time, thus suggesting that it represents an efficient approach to improve 4-HPR bioavailability

Potent and Broad-Spectrum Bactericidal Activity of a Nanotechnologically Manipulated Novel Pyrazole

: The antimicrobial potency of the pyrazole nucleus is widely reported these days, and pyrazole derivatives represent excellent candidates for meeting the worldwide need for new antimicrobial compounds against multidrug-resistant (MDR) bacteria. Consequently, 3-(4-chlorophenyl)-5-(4-nitrophenylamino)-1H-pyrazole-4-carbonitrile (CR232), recently reported as a weak antiproliferative agent, was considered to this end. To overcome the CR232 water solubility issue and allow for the determination of reliable minimum inhibitory concentration values (MICs), we initially prepared water-soluble and clinically applicable CR232-loaded nanoparticles (CR232-G5K NPs), as previously reported. Here, CR232-G5K NPs have been tested on several clinically isolates of Gram-positive and Gram-negative species, including MDR strains. While for CR232 MICs 65 128 \ub5g/mL (376.8 \ub5M) were obtained, very low MICs (0.36-2.89 \ub5M) were observed for CR232-G5K NPs against all of the considered isolates, including colistin-resistant isolates of MDR Pseudomonas aeruginosa and Klebsiella pneumoniae carbapenemases (KPCs)-producing K. pneumoniae (0.72 \ub5M). Additionally, in time-kill experiments, CR232-G5K NPs displayed a rapid bactericidal activity with no significant regrowth after 24 h on all isolates tested, regardless of their difficult-to-treat resistance. Conjecturing a clinical use of CR232-G5K NPs, cytotoxicity experiments on human keratinocytes were performed, determining very favorable selectivity indices. Collectively, due to its physicochemical and biological properties, CR232-G5K NPs could represent a new potent weapon to treat infections sustained by broad spectrum MDR bacteria

Development of Medicines for Rare Pediatric Diseases

To date, approximately 7000 rare diseases exist, affecting between 6% and 8% of the global population and >30 million people in the European Union [...

The Role of Extracellular Vesicles in the Progression of Human Neuroblastoma

The long-underestimated role of extracellular vesicles in cancer is now reconsidered worldwide by basic and clinical scientists, who recently highlighted novel and crucial activities of these moieties. Extracellular vesicles are now considered as king transporters of specific cargoes, including molecular components of parent cells, thus mediating a wide variety of cellular activities both in normal and neoplastic tissues. Here, we discuss the multifunctional activities and underlying mechanisms of extracellular vesicles in neuroblastoma, the most frequent common extra-cranial tumor in childhood. The ability of extracellular vesicles to cross-talk with different cells in the tumor microenvironment and to modulate an anti-tumor immune response, tumorigenesis, tumor growth, metastasis and drug resistance will be pinpointed in detail. The results obtained on the role of extracellular vesicles may represent a panel of suggestions potentially useful in practice, due to their involvement in the response to chemotherapy, and, moreover, their ability to predict resistance to standard therapies—all issues of clinical relevance

Mutual Jellification of Two Bactericidal Cationic Polymers: Synthesis and Physicochemical Characterization of a New Two-Component Hydrogel

Here, a new two-component hydrogel (CP1OP2-Hgel) was developed, simply by dispersing in water two cationic bactericidal polymers (CP1 and OP2) effective against several multidrug-resistant (MDR) clinical isolates of the most relevant Gram-positive and Gram-negative species. Interestingly, while OP2 acts only as an antibacterial ingredient when in gel, CP1 works as both an antibacterial and a gelling agent. To verify whether it would be worthwhile to use CP1 and OP2 as bioactive ingredients of a new hydrogel supposed for a future treatment of skin infections, dose-dependent cytotoxicity studies with CP1 and OP2 were performed on human fibroblasts for 24 h, before preparing the formulation. Although a significant cytotoxicity at concentrations > 2 µM was evidenced for both polymers, selectivity indices (SIs) over 12 (CP1) and up to six (OP2) were determined, due to the powerful antibacterial properties of the two polymers, thus supporting the rationale for their formulation as a hydrogel. The chemical structure and morphology of CP1OP2-Hgel were investigated by PCA-assisted attenuated total reflectance (ATR) Fourier-transform infrared (FTIR) analysis and scanning electron microscopy (SEM), while its rheological properties were assessed by determining its dynamic viscosity. The cumulative weight loss and swelling percentage curves, the porosity, and the maximum swelling capability of CP1OP2-Hgel were also determined and reported. Overall, due to the potent bactericidal effects of CP1 and OP2 and their favorable selectivity indices against several MDR pathogens, good rheological properties, high porosity, and strong swelling capability, CP1OP2-Hgel may, in the future, become a new weapon for treating severe nosocomial skin infections or infected chronic wounds. Further investigations in this sense are currently being carried out

ASSESSMENT OF FENRETINIDE IN MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR VESICLES

Fenretinide (N-(4-hydroxyphenyl)-retinamide, 4-HPR) is a synthetic retinoid with fewer adverse effects than natural retinoids, whose efficacy against ovarian, prostate, small cell lung, brain, neuroectodermal-derived tumors has been widely demonstrated in several in vitro and preclinical studies. Unfortunately, clinical responses both in adult and pediatric patients are often partial, revealing a limited activity of 4-HPR against existing disease [1]. The underlying causes of this slight therapeutic efficacy consist in its poor water solubility, low bioavailability (estimated at 16%) and high first-pass hepatic effect. All trials have employed oral gelatin capsules containing 4-HPR suspended in corn oil and polysorbate 80. The low plasma drug concentrations achievable and/or the poor patient compliance in taking the required number of capsules strongly limit the effectiveness of this strategy. As a result, new oral and intravenous formulations are required to enhance the bioavailability of 4-HPR. To this end, we herein propose a new 4-HPR formulation based on the use of mesenchymal stem cellsderived extracellular vesicles (EVs) as endogenous carriers for delivering 4-HPR. EVs are nonimmunogenic, biocompatible nanocarriers able to interact with multiple cell types within the immediate vicinity and remote. After isolation from human umbilical cord, the mesenchymal stem cells were treated with high doses of 4- HPR to achieve a passive drug loading. The resulting 4-HPR-EVs were collected, purified by ultracentrifugation and characterized for size, concentration and morphology by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA), using a Nano Sight 300 instrument. The vesicles showed a mean diameter of about 130 nm with spherical shape (Figure 1). The drug amount encapsulated into the vesicles was determined by HPLC. Briefly, prior 4-HPR quantification an extraction procedure was optimized and, to estimate the analyte recovery an internal standard was employed. Since for this purpose, N-(4-ethoxyphenyl)- retinamide (4-EPR) has been reported, we developed a new operator-friendly one-step procedure to synthetize highly pure 4-EPR in quantitative yield. After exposure of mesenchymal stem cells to 25 μM 4-HPR for 48 h, we obtained 1.8 x1011 EVs/mL corresponding the a final drug concentration of 12 μM. Studies are currently being conducted to determine the best drug loading conditions and the reproducibility of the protocol

Enhanced Antibacterial Activity of a Cationic Macromolecule by Its Complexation with a Weakly Active Pyrazole Derivative

Molecules containing the pyrazole nucleus are widely reported as promising candidates to develop new antimicrobial compounds against multidrug-resistant (MDR) bacteria, where available antibiotics may fail. Recently, aiming at improving the too-high minimum inhibitory concentrations (MICs) of a pyrazole hydrochloride salt (CB1H), CB1H-loaded nanoparticles (CB1H-P7 NPs) were developed using a potent cationic bactericidal macromolecule (P7) as polymer matrix. Here, CB1H-P7 NPs have been successfully tested on several clinical isolates of Gram-positive and Gram-negative species, including relevant MDR strains. CB1H-P7 NPs displayed very low MICs (0.6–4.8 µM), often two-fold lower than those of P7, on 34 out of 36 isolates tested. Upon complexation, the antibacterial effects of pristine CB1H were improved by 2–16.4-fold, and, unexpectedly, also the already potent antibacterial effects of P7 were 2–8 times improved against most of bacteria tested when complexed with CB1H. Time-killing experiments performed on selected species established that CB1H-P7 NPs were bactericidal against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Selectivity indices values up to 2.4, determined by cytotoxicity experiments on human keratinocytes, suggested that CB1H-P7 NPs could be promising for counteracting serious infections sustained by most of the isolates tested in this study