Carmine Ostacolo

Detailed polyphenolic profiling of Annurca apple (M. pumila Miller cv Annurca) by a combination of RP-UHPLC and HILIC, both hyphenated to IT-TOF mass spectrometry

Annurca apple, a Southern Italian cultivar, possesses not only a particular taste and flavor, different from other types of apple, but also several healthy properties. With the aim to thoroughly elucidate the polyphenolic profile of this variety, listed as Protected Geographical Indication product, an extensive qualitative profiling of Annurca apple polyphenolic peel extract was carried out, by employing a combination of ultra high performance reversed phase (RP-UHPLC) and hydrophilic liquid chromatography (HILIC) coupled to ion trap-time of flight (IT-TOF) mass spectrometry. A total of 63 compounds were tentatively identified, 25 of which not reported in Annurca apple extract so far. Furthermore, thanks to the different selectivity obtained with the HILIC, in combination with accurate mass measurements, an improved separation and detection of procyanidins, was obtained. Moreover, the obtained profiles were compared with those of a conventional variety, such as Red Delicious (RD), highlighting their differences. This work contributes to increase the knowledge about the polyphenolic fingerprint of this typical apple variety.

Tryptamine-Based Derivatives as Transient Receptor Potential Melastatin Type 8 (TRPM8) Channel Modulators

Pharmacological modulation of the transient receptor potential melastatin type 8 (TRPM8) is currently under investigation as a new approach for the treatment of pain and other diseases. In this study, a series of N-substituted tryptamines was prepared to explore the structural requirements determining TRPM8 modulation. Using a fluorescence-based screening assay, we identified two compounds acting as an activator (2-(1H-indol-3-yl)-N-(4-phenoxybenzyl)ethanamine, 21) or an inhibitor (N,N-dibenzyl-2-(1H-indol-3-yl)ethanamine, 12) of calcium influx in HEK293 cells. In patch-clamp recordings, compound 21 displayed a significantly higher potency (EC50 = 40 ± 4 μM) and a similar efficacy when compared to menthol; by contrast, compound 12 produced a concentration-dependent inhibition of menthol-induced TRPM8 currents (IC50 = 367 ± 24 nM). Molecular modeling studies using a homology model of a single rat TRPM8 subunit identified a putative binding site located between the VSD and the TRP box, disclosing differences in the binding modes for the agonist and the antagonist.

Transepithelial Corneal Cross-Linking With Vitamin E-Enhanced Riboflavin Solution and Abbreviated, Low-Dose UV-A: 24-Month Clinical Outcomes

Purpose: To report the clinical outcomes with 24-month follow-up of transepithelial cross-linking using a combination of a D-alpha-tocopheryl polyethylene-glycol 1000 succinate (vitamin E-TPGS)-enhanced riboflavin solution and abbreviated low fluence UV-A treatment. Methods: In a nonrandomized clinical trial, 25 corneas of 19 patients with topographically proven, progressive, mild to moderate keratoconus over the previous 6 months were cross-linked, and all patients were examined at 1, 3, 6, 12, and 24 months. The treatments were performed using a patented solution of riboflavin and vitamin E-TPGS, topically applied for 15 minutes, followed by two 5-minute UV-A treatments with separate doses both at fluence below 3 mW/cm2 that were based on preoperative central pachymetry. Results: During the 6-month pretreatment observation, the average Kmax increased by +1.99 ± 0.29 D (diopter). Postoperatively, the average Kmax decreased, changing by −0.55 ± 0.94 D, by −0.88 ± 1.02 D and by −1.01 ± 1.22 D at 6, 12, and 24 months. Postoperatively, Kmax decreased in 19, 20, and 20 of the 25 eyes at 6 months, 12 months, and 24 months, respectively. Refractive cylinder was decreased by 3 months postoperatively and afterward, changing by −1.35 ± 0.69 D at 24 months. Best spectacle-corrected visual acuity (BSCVA) improved at 6, 12, and 24 months, including an improvement of −0.19 ± 0.13 logarithm of the minimum angle of resolution units at 24 months. There was no reduction in endothelial cell count. No corneal abrasions occurred, and no bandage contact lenses or prescription analgesics were used during postoperative recovery. Conclusions: Transepithelial cross-linking using the riboflavin-vitamin E solution and brief, low-dose, pachymetry-dependent UV-A treatment safely stopped keratoconus progression.

In-vitro and in-vivo evaluation of oligoethylene esters as dermal prodrugs of 18β-glycyrrhetic acid

Novel polyoxyethylene esters of 18 β‐glycyrrhetic acid (GA) were synthesized and evaluated as potential dermal prodrugs. The permeation of these prodrugs (1a‐e) was studied in‐vitro, using excised human skin membranes (SCE; stratum corneum/epidermis) mounted in Franz type cells, and in‐vivo, evaluating the ability of these compounds to inhibit methyl nicotinate (MN)‐induced skin erythema in healthy human subjects. All the esters synthesized showed a good water stability, while the enzymatic hydrolysis rate was significantly affected by the length of the polyoxyethylenic chain used as promoiety. In in‐vitro percutaneous absorption studies, only esters 1b and 1c (respectively triethylen‐ and tetraethylenglycol derivatives) showed an increased flux through SCE membranes compared with GA. Furthermore, we observed an appreciable and sustained in‐vivo topical anti‐inflammatory activity of esters 1b and 1c compared with the parent drug.

Structure‐Based Design of Microsomal Prostaglandin E2 Synthase‐1 (mPGES‐1) Inhibitors using a Virtual Fragment Growing Optimization Scheme

A small library of 2,3‐dihydroxybenzamide‐ and N‐(2,3‐dihydroxyphenyl)‐4‐sulfonamide‐based microsomal prostaglandin E2 synthase‐1 (mPGES‐1) inhibitors was identified following a step‐by‐step optimization of small aromatic fragments selected to interact in focused regions in the active site of mPGES‐1. During the virtual optimization process, the 2,3‐dihydroxybenzamide moiety was first selected as a backbone of the proposed new chemical entities; the identified compounds were then synthesized and biologically evaluated, identifying derivatives with very promising inhibitory activities in the micromolar range. Subsequent structure‐guided replacement of the 2,3‐dihydroxybenzamide by the N‐(2,3‐dihydroxyphenyl)sulfonamide moiety led to the identification of N‐(2,3‐dihydroxyphenyl)‐4‐biphenylsulfonamide (6), the most potent small molecule of the series (IC50=0.53±0.04 μm). The simple synthetic procedure and the possibility of enhancing the potency of this class of inhibitors through additional structural modifications pave the way for further development of new molecules with mPGES‐1‐inhibitory activity, with potential application as anti‐inflammatory and anticancer agents.

Nanoparticles prolong N-palmitoylethanolamide anti-inflammatory and analgesic effects in vivo

N-Palmitoylethanolamide showed great therapeutic potential in the treatment of inflammation and pain but its unfavourable pharmacokinetics properties will hinder its use in the clinical practice. A nanotechnology-based formulation was developed to enhance the probability of N-palmitoylethanolamide therapeutic success, especially in skin disease management. Lipid nanoparticles were produced and characterized to evaluate their mean size, ζ-potential, thermal behaviour, and morphology. The ability of N-palmitoylethanolamide to diffuse across the epidermis as well as anti-inflammatory and analgesic effects were investigated. Particles had a mean size of about 150 nm and a ζ-potential of -40 mV. DSC data confirmed the solid state of the matrix and the embedding of N-palmitoylethanolamide while electron microscopy have evidenced a peculiar internal structure (i.e., low-electrondense spherical objects within the matrix) that can be reliably ascribed to the presence of oil nanocompartments. Lipid nanoparticles increased N-palmitoylethanolamide percutaneous diffusion and prolonged the anti-inflammatory and analgesic effects in vivo. Lipid nanoparticles seem a good nanotechnology-based strategy to bring N-palmitoylethanolamide to clinics.

Synthesis, hydrolysis, and skin retention of amino acid esters of α-tocopherol

The aim of this work was to synthesize new pro-vitamins of alpha-tocopherol (VE) able to release another moiety such as an amino acid, in order to obtain a combined antioxidant and moisturizing effect upon topical application. The new derivatives were characterized and tested for sensitivity to chemical and enzymatic hydrolysis. Lipophilicity was estimated using Log capacity factor and skin retention was determined in vitro, using rabbit ear skin as barrier. Five molecules were synthesized using L-proline, L-serine, L-tyrosine, L-asparagine, and L-citrulline as amino acidic moiety. All pro-vitamins showed similar or lower lipophilicity than alpha-tocopheryl acetate (VEAc), taken as reference, and similar stability in aqueous solutions. All pro-vitamins showed to be sensitive to enzymatic hydrolysis. None of the pro-vitamins crossed the skin in significant amounts, whereas they accumulated into the skin, in both the dermis and the epidermis. They are more hydrophilic and more water-soluble than the currently used acetate.

Customized Corneal Cross-Linking—A Mathematical Model

Purpose: To improve the safety, reproducibility, and depth of effect of corneal cross-linking with the ultraviolet A (UV-A) exposure time and fluence customized according to the corneal thickness. Methods: Twelve human corneas were used for the experimental protocol. They were soaked using a transepithelial (EPI-ON) technique using riboflavin with the permeation enhancer vitamin E–tocopheryl polyethylene glycol succinate. The corneas were then placed on microscope slides and irradiated at 3 mW/cm2 for 30 minutes. The UV-A output parameters were measured to build a new equation describing the time-dependent loss of endothelial protection induced by riboflavin during cross-linking, as well as a pachymetry-dependent and exposure time-dependent prescription for input UV-A fluence. The proposed equation was used to establish graphs prescribing the maximum UV-A fluence input versus exposure time that always maintains corneal endothelium exposure below toxicity limits. Results: Analysis modifying the Lambert–Beer law for riboflavin oxidation leads to graphs of the maximum safe level of UV-A radiation fluence versus the time applied and thickness of the treated cornea. These graphs prescribe UV-A fluence levels below 1.8 mW/cm2 for corneas of thickness 540 &mgr;m down to 1.2 mW/cm2 for corneas of thickness 350 &mgr;m. Irradiation times are typically below 15 minutes. Conclusions: The experimental and mathematical analyses establish the basis for graphs that prescribe maximum safe fluence and UV-A exposure time for corneas of different thicknesses. Because this clinically tested protocol specifies a corneal surface clear of shielding riboflavin on the corneal surface during UV-A irradiation, it allows for shorter UV-A irradiation time and lower fluence than in the Dresden protocol.

Synthesis and antihypertensive action of new Imidazo[1,2-a]pyridine derivatives, non peptidic Angiotensin II receptor antagonists

The synthesis and antihypertensive activity of a group of imidazo[1,2-a]pyridine is described. New synthesized compound have been tested both in vivo and in vitro as antagonists on Angiotensin AT1 receptor, and compared to Losartan, used as reference drug. Binding assay an Angiotensin AT1 receptor were carried on as well. Compounds 6b and 6g showed a potent antihypertensive activity and an high affinity on AT1 receptor.

Fast Profiling of Natural Pigments in Different Spirulina (Arthrospira platensis) Dietary Supplements by DI-FT-ICR and Evaluation of their Antioxidant Potential by Pre-Column DPPH-UHPLC Assay

Arthrospira platensis, better known as Spirulina, is one of the most important microalgae species. This cyanobacterium possesses a rich metabolite pattern, including high amounts of natural pigments. In this study, we applied a combined strategy based on Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) and Ultra High-Performance Liquid Chromatography (UHPLC) for the qualitative/quantitative characterization of Spirulina pigments in three different commercial dietary supplements. FT-ICR was employed to elucidate the qualitative profile of Spirulina pigments, in both direct infusion mode (DIMS) and coupled to UHPLC. DIMS showed to be a very fast (4 min) and accurate (mass accuracy ≤ 0.01 ppm) tool. 51 pigments were tentatively identified. The profile revealed different classes, such as carotenes, xanthophylls and chlorophylls. Moreover, the antioxidant evaluation of the major compounds was assessed by pre-column reaction with the DPPH radical followed by fast UHPLC-PDA separation, highlighting the contribution of single analytes to the antioxidant potential of the entire pigment fraction. β-carotene, diadinoxanthin and diatoxanthin showed the highest scavenging activity. The method took 40 min per sample, comprising reaction. This strategy could represent a valid tool for the fast and comprehensive characterization of Spirulina pigments in dietary supplements, as well as in other microalgae-based products.