Search

Rationale: Phosphodiesterase-4 (PDE4) inhibitors have demonstrated increased efficacy in patients with chronic obstructive pulmonary disease who had chronic bronchitis or higher blood eosinophil counts. Further characterization of patients who are most likely to benefit is warranted. Objective: To identify determinants of response to the PDE4 inhibitor tanimilast. Methods: A PDE4 gene expression signature in blood was developed by unsupervised clustering of the ECLIPSE study dataset (ClinicalTrials.gov ID: NCT 00292552; Gene Expression Omnibus Series ID: GSE76705). The signature was further evaluated using blood and sputum transcriptome data from the BIOMARKER study (NCT 03004417; GSE133513), enabling validation of the association between PDE4 signaling and target biomarkers. Predictivity of the associated biomarkers against clinical response was then tested in the phase-2b PIONEER tanimilast study (NCT 02986321). Measurements and Main Results: The PDE4 gene expression signature developed in the ECLIPSE dataset classified subgroups of patients associated with different PDE4 signaling in the BIOMARKER cohort with an area under the receiver operator curve of 98%. In the BIOMARKER study, serumIL-8 was the only variable that was consistently associated with PDE4 signaling, with lower levels associated with higher PDE4 activity. In the PIONEER study, the exacerbation rate reduction mediated by tanimilast treatment increased up to twofold in patients with lower IL-8 levels; 36% versus 18%, reaching statistical significance at<20 pg/ml (P = 0.035). The combination with blood eosinophils>150 μl21 or chronic bronchitis provided further additive exacerbation rate reduction: 45% (P = 0.013) and 47% (P = 0.027), respectively. Conclusions: Using selected heterogeneous datasets, this analysis identifies IL-8 as an independent predictor of PDE4 inhibition, as tanimilast had a greater effect on exacerbation prevention in patients with chronic obstructive pulmonary disease who had lower baseline serum IL-8 levels. Testing of this biomarker in other datasets is warranted. [ABSTRACT FROM AUTHOR]

BACKGROUND: The use of PDO (protected designation of origin) and PGI (protected geographical indication) labels allows to protect and promote agricultural products characterized by unique features related to the place of origin and traditional know‐how. However, the presence of non‐authentic products in the market represents a fraud that can be tackled applying analytical techniques combined with chemometric analysis. In this study, we applied multi‐element and multi‐isotope analysis to characterize PDO and PGI apples cultivated in northern Italy, comparing them with Italian apples without labels of geographical indications. RESULTS: The multi‐element and multi‐isotope approach allowed to characterize the apples cultivated in northern Italy. Despite a significant effect of the sampling sites on the apple composition, the comparison of the multi‐chemical fingerprint of the apples significantly varied among cultivation areas. Results of this characterization were used to classify samples according to their cultivation area applying a linear discriminant analysis (LDA). Outputs of the LDA showed that correct sample classification can be successfully achieved (balanced accuracy > 96%). Moreover, using a selection of variables, it was possible to correctly classify samples also at regional level. CONCLUSION: The presented evidences indicate that the multi‐element and multi‐isotope fingerprint can be successfully applied to traceability studies. The combination of this characterization with chemometric tools allows the classification of Italian apples based on their origin both on a national and regional scale. This approach represents an interesting tool to enhance and protect PDO and PGI Italian products. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV200) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV200 higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase‐4‐inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32‐day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophilhigh ≥ 3%; eosinophillow < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty‐one genes were differentially expressed in primary samples (p‐adjusted for false discovery rate < 0.05); all up‐regulated in eosinophilhigh patients and functionally enriched for type‐2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophilhigh patients. These findings suggest that type‐2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials. [ABSTRACT FROM AUTHOR]

BACKGROUND Apples have a leading role in the Italian fruit sector, and high‐quality apples, including the Golden Delicious variety, are cultivated mainly in the Northern mountain districts. In the present study, Golden Delicious apples from PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication) cultivation districts were characterized according to their Sr isotope composition and compared with apples from other Northern Italian districts. RESULTS: Apples collected in two consecutive years (2017 and 2018) confirmed the low annual variability of the 87Sr/86Sr ratio. The isotope ratio of apples was highly correlated with that of the soil extracts of the respective orchards. Statistical differences were highlighted between cultivation districts. However, because similar geological features characterized some areas, their ratios overlapped and a complete separation of the districts was not possible. CONCLUSION: The 87Sr/86Sr ratio is an excellent marker for studies of food traceability because it retains the information about the place of origin. However, its strength is limited when comparing products from cultivation areas sharing similar geological features. In the perspective of geographical traceability, a multichemical characterization can overcome the limits of single‐parameter approach. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The experiment was conducted during two consecutive seasons (years 2016 and 2017) in an organic apple orchard of the cultivar Jonathan. Several biostimulants were tested (10 in total), including humic acids, macro and micro seaweed extracts, alfalfa protein hydrolysate, amino acids alone or in combination with zinc, B-group vitamins, chitosan and a commercial product containing silicon. Treatments were performed at weekly intervals, starting from the end of May until mid-August. The macroseaweed extract was effective in stimulate tree growth potential in both years, as shown by a significantly larger leaf area (+20% as compared to control) and by an higher chlorophyll content and leaf photosynthetic rate in year 2016. As for the yield performances and apples quality traits at harvest (average fruit weight, soluble solids content, titratable acidity, and flesh firmness), they were generally affected by the different climatic conditions that characterized the two growing seasons (year 2017 being characterized by higher maximal and average temperatures and by limited rainfalls at the beginning of the season). Treatments with macroseaweed extract, B-group vitamins and alfalfa protein hydrolysate were able to significantly improve the intensity and extension of the red coloration of apples at harvest. Correspondingly, the anthocyanin content in the skin of apples treated with the same biostimulants resulted significantly higher than control, highlighting the potential influence of these substances on the synthesis of secondary metabolites in apple. The incidence of physiological disorders was also monitored during apple storage period. Amino acids plus zinc application was effective in reducing (more than 50%) the incidence of the "Jonathan spot," the main post-harvest disorder for this cultivar. [ABSTRACT FROM AUTHOR]

The present work was designed to compare the vitamin C (ascorbic acid, AsA) content of pulp, peel, and juice of 64 apple cultivars. These cultivars were carefully identified as ‘true to type’ by molecular genetic tools, grown in the same site under identical conditions and processed by a standardized protocol. Twenty-one of them, accounting for more than 95% of the apple production of South Tyrol, were chosen to represent the current market, 16 were old or local cultivars formerly grown in the area, and 27 were new cultivars, including 15 with scab resistance and 12 with red flesh fruit. For the determination of the AsA content, a new High-Performance Liquid Chromatography with Diode-Array Detection method was developed and validated. While old cultivars stood out for their high AsA content in pulp and peel, the red-fleshed cultivars are the ones maintaining most of their AsA content during processing. Our data thus suggest a potential for old and red-fleshed cultivars for healthy juices or further processed food components. [ABSTRACT FROM AUTHOR]

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization. [ABSTRACT FROM AUTHOR]

In this study, a metabolomic investigation was presented to correlate single polyphenolic compounds in apple pulp with quality characteristics such as antioxidant activity and content of phenolic compounds and anthocyanins in apple skin. Since the concentration of these compounds is influenced by environmental factors, the twenty-two apple cultivars originate from the same site. The polyphenolic compounds were analyzed by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). The antioxidant activity, phenolic content, and anthocyanins were evaluated on the sunny and the shady sides of apple skin by spectrometric assays. In old apple varieties, the measured parameters were higher than in the commercial and red-fleshed varieties. By contrast, the profile of flavan-3-ols and anthocyanins was variable amongst commercial and red-fleshed varieties. The partial least square (PLS) method was applied to investigate the association between the skin proprieties and the metabolic profile of the pulp. The highest coefficients of determination in prediction (Q2) were obtained for compounds quantified in old cultivars. These results provided information to define the old apple varieties as a reliable group based on the pathway of the antioxidant compounds and anthocyanins content. Our results show the possibility to find cultivars with promising health features based on their content of polyphenols suitable for commercialization or breeding. [ABSTRACT FROM AUTHOR]

Background: Although phosphodiesterase-4 (PDE4) inhibitors have been shown to reduce COPD exacerbation rate, their biological mechanism of action is not completely elucidated at the molecular level. We aimed to characterise the whole genome gene expression profile of the inhaled PDE4-inhibitor CHF6001 on top of triple therapy in sputum cells and whole blood of patients with COPD and chronic bronchitis.Methods: Whole genome gene expression analysis was carried out by microarray in 54 patients before and after 32 days treatment with CHF6001 800 and 1600 μg and placebo twice daily (BID) in a randomised crossover study.Results: CHF6001 had a strong effect in sputum, with 1471 and 2598 significantly differentially-expressed probe-sets relative to placebo (p-adjusted for False Discovery Rate < 0.05) with 800 and 1600 μg BID, respectively. Functional enrichment analysis showed significant modulation of key inflammatory pathways involved in cytokine activity, pathogen-associated-pattern-recognition activity, oxidative stress and vitamin D with associated inhibition of downstream inflammatory effectors. A large number of pro-inflammatory genes coding for cytokines and matrix-metalloproteinases were significantly differentially expressed for both doses; the majority (> 87%) were downregulated, including macrophage inflammatory protein-1-alpha and 1-beta, interleukin-27-beta, interleukin-12-beta, interleukin-32, tumour necrosis factor-alpha-induced-protein-8, ligand-superfamily-member-15, and matrix-metalloproteinases-7,12 and 14. The effect in blood was not significant.Conclusions: Inhaled PDE4 inhibition by CHF6001 on top of triple therapy in patients with COPD and chronic bronchitis significantly modulated key inflammatory targets and pathways in the lung but not in blood. Mechanistically these findings support a targeted effect in the lung while minimising unwanted systemic class-effects.Trial Registration: ClinicalTrial.gov, EudraCT, 2015-005550-35. Registered 15 July 2016. [ABSTRACT FROM AUTHOR]

Tanimilast is an inhaled phosphodiesterase-4 inhibitor currently in phase 3 clinical development for treating chronic obstructive pulmonary disease (COPD) and asthma. This trial aimed to characterize the pharmacokinetics, mass balance, and metabolite profiling of tanimilast. Eight healthy male volunteers received a single dose of non-radiolabeled tanimilast via powder inhaler (NEXThaler ® (3200μg)), followed by a concomitant intravenous (IV) infusion of a microtracer ([ 14 C]-tanimilast: 18.5μg and 500nCi). Plasma, whole blood, urine, and feces samples were collected up to 240 hours post-dose to quantify non-radiolabeled tanimilast, [ 14 C]-tanimilast, and total-[ 14 C]. The inhaled absolute bioavailability of tanimilast was found to be approximately 50%. Following IV administration of [ 14 C]-tanimilast, plasma clearance was 22 L/h, the steady-state volume of distribution was 201 L, and the half-life was shorter compared to inhaled administration (14 vs. 39 hours, respectively), suggesting that plasma elimination is limited by the absorption rate from the lungs. 79% (71% in feces; 8% in urine) of the IV dose was recovered in excreta as total-[ 14 C]. [ 14 C]-tanimilast was the major radioactive compound in plasma, while no recovery was observed in urine and only 0.3% was recovered in feces, indicating predominant elimination through metabolic route. Importantly, as far as no metabolites accounting for more than 10% of the circulating drug-related exposure in plasma or the administered dose in excreta were detected, no further qualification is required according to regulatory guidelines. This study design successfully characterized the absorption, distribution, and elimination of tanimilast, providing key pharmacokinetic parameters to support its clinical development and regulatory application. Significance Statement This trial investigates PK and ADME profile of tanimilast, an inhaled PDE4 inhibitor for COPD and asthma. Eight male volunteers received a dose of non-radiolabeled tanimilast via NEXThaler ® and a microtracer IV dose. Results show pivotal PK results for the characterization of tanimilast, excretion route and quantification of significant metabolites, facilitating streamlined clinical development and regulatory approval.

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl- sn -glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.

The 87 Sr/ 86 Sr ratio of horticultural products mostly derives from that of the bioavailable Sr fraction of the soil where they grow and, therefore, varies according to the local geolithological features. This study investigated the intra- and intertree variability of the 87 Sr/ 86 Sr ratio in two apple orchards in South Tyrol and its relation with the soil 87 Sr/ 86 Sr ratio. In both orchards, a moderate homogeneity of the 87 Sr/ 86 Sr ratio was observed among subsamples of the same tree part (shoot axes, leaves, apple peels, and pulps). Moreover, the 87 Sr/ 86 Sr ratio homogeneity among tree parts was high intratree and low intertree. The variability of the 87 Sr/ 86 Sr ratio within the tree and within the orchard is explained in light of the 87 Sr/ 86 Sr ratios of the soil. This 87 Sr/ 86 Sr variability within orchards does not preclude its use as a geographical tracer; however, this aspect should be evaluated to correctly design a sampling campaign or to generalize the results.

The 87 Sr/ 86 Sr ratio of a produce is generally linked with the soil geological features of the growing areas. This study aimed at assessing to which extent the addition of external Sr by agricultural practices, like irrigation and mineral nutrient supply, influences this relationship. In a first experiment, oat plants in two soils reflected the 87 Sr/ 86 Sr of the soil. However, this link was significantly altered at increasing levels of external Sr soil supplies. In a second experiment, apple trees transplanted in pots modified their original 87 Sr/ 86 Sr, which became progressively closer to the soil Sr isotope ratio. The addition of tap water and fertilizers, with different Sr isotopic signatures, slightly affected plant 87 Sr/ 86 Sr. Results confirm the potential of the 87 Sr/ 86 Sr ratio as a geographical tracer of agricultural commodities, but whenever the range of 87 Sr/ 86 Sr variability among soils from different geographical areas is narrow, the influence of external Sr-sources may smooth over these diversities.

Inhibition of adenosine A2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson's disease (PD). We previously identified the triazolo-9H-purine, ST1535, as a potent A(2A)R antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω-1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω-1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A(2A)R was determined. Two compounds, (2-(3,3-dimethylbutyl)-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-amine (3 b) and 4-(6-amino-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-2-yl)-2-methylbutan-2-ol (3 c), exhibited good affinity against A(2A)R (Ki =0.4 nM and 2 nM, respectively) and high in vitro metabolic stability (89.5% and 95.3% recovery, respectively, after incubation with HLM for two hours)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here, we report on a mechanistic investigation based on DFT calculations and kinetic measures aimed at determining the energetics related to the cysteine nucleophilic attack on nitrile-carrying compounds. Activation energies were found to correlate well with experimental kinetic measures of reactivity with cysteine in phosphate buffer. The agreement between computations and experiments points to this DFT-based approach as a tool for predicting both nitrile reactivity toward cysteines and the toxicity of nitriles as electrophile agents.