Cell Membrane Fragment-Wrapped Parenteral Nanoemulsions: A New Drug Delivery Tool to Target Gliomas.

Poor prognosis in high-grade gliomas is mainly due to fatal relapse after surgical resection in the absence of efficient chemotherapy, which is severely hampered by the blood-brain barrier. However, the leaky blood-brain-tumour barrier forms upon tumour growth and vascularization, allowing targeted nanocarrier-mediated drug delivery. The homotypic targeting ability of cell-membrane fragments obtained from cancer cells means that these fragments can be exploited to this aim. In this experimental work, injectable nanoemulsions, which have a long history of safe clinic usage, have been wrapped in glioma-cell membrane fragments via co-extrusion to give targeted, homogeneously sized, sterile formulations. These systems were then loaded with three different chemotherapeutics, in the form of hydrophobic ion pairs that can be released into the target site thanks to interactions with physiological components. The numerous assays performed in two-dimensional (2D) and three-dimensional (3D) cell models demonstrate that the proposed approach is a versatile drug-delivery platform with chemo-tactic properties towards glioma cells, with adhesive interactions between the target cell and the cell membrane fragments most likely being responsible for the effect. This approach's promising translational perspectives towards personalized nanomedicine mean that further in vivo studies are foreseen for the future.

Galactose: A Versatile Vector Unveiling the Potentials in Drug Delivery, Diagnostics, and Theranostics.

D-galactose, a simple natural compound, has been investigated as a powerful scaffold for drug delivery, diagnostics, and theranostics due to its distinctive properties and interactions with specific cell receptors. In the field of drug delivery, galactose functions as a ligand to selectively target cells expressing galactose receptors, such as hepatocytes, macrophages, and specific cancer cells. The direct attachment of galactose to the main drug or to drug-loaded nanoparticles or liposomes enhances cellular uptake, thereby improving drug delivery to the intended target cells. Galactose has also been found to be useful in diagnostics. Specifically, diagnostic tests based on galactose, such as the galactose elimination capacity test, are utilized to evaluate liver function and assess liver disease as well as hepatic functional reserve. Additionally, galactose-based theranostic agents can be designed by combining drug delivery and diagnostic capabilities. This review is an update of our previous review concerning the broad spectrum of possibilities for exploiting D-galactose as a vector for prodrug design and the synthetic strategies that allow its realization, jointly in diagnostics and theranostics, to highlight the versatility of this interesting vector.

Specific bioelectrical vectors pattern in individuals with sarcopenic obesity.

BACKGROUND: Sarcopenic obesity is a common condition in the elderly associated with excessive adiposity and low muscle mass and strength. AIMS: This study aims to establish a method for detecting bioelectrical characteristics in individuals with sarcopenic obesity through specific Bioelectrical Impedance Vector Analysis (specific BIVA), while considering the characteristics of individuals with healthy, sarcopenic, and obese conditions. METHODS: The sample was composed by 915 Italian adults over 50 years of age (men: 74.6 ± 8.8 y; women:76.3 ± 8.8 y) living in Sardinia (Italy). A dataset of 1590 US adults aged 21 - 49 years retrieved from the 2003 - 2004 National Health and Nutrition Examination Survey was also considered in a final step of the study. Anthropometric (stature, weight, waist, arm, and calf circumferences) and whole-body bioelectrical variables were taken. In the Italian sample, bioelectrical impedance was applied to estimate the relative content of fat mass and skeletal muscle mass. Groups with healthy body composition (NS-NO), or consistent with sarcopenia (S), sarcopenic obesity (S-O), and obesity (O) were defined based on the cut-offs suggested by European expert guidelines (EWGSOP2 and ESPEN-EASO). Specific BIVA was applied to compare groups and to identify the area for sarcopenic obesity within young-adults tolerance ellipses. The position of the specific vector of US individuals with S-O, selected on the basis of DXA measurements, was also considered. RESULTS: In both sexes of the Italian sample, the bioelectrical characteristics of the four groups were different (p < 0.001). The differences were mainly related to vector length, indicative of higher fat mass, which was longer in the O and S-O groups, and phase angle, a proxy of intracellular/extracellular water and muscle mass, lower in the sarcopenic groups. Bioelectrical vectors of the S-O group fell in the right quadrant, outside of the 95 % tolerance ellipses of young adults. The mean vector of the US sample with S-O fell in the same area. Within the S-O area, women had similar bioelectrical values, while men showed phase angle variability, which was related to the severity of the condition. CONCLUSIONS: Specific BIVA detects body composition peculiarities of individuals with sarcopenic obesity, thus allowing their diagnosis when associated with low handgrip strength values.

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.

Body fluids and muscle changes in trail runners of various distances.

Background: This study aims to investigate body fluids and muscle changes evoked by different trail races using anthropometric, bioelectrical, and creatine kinase (CK) measurements. Methods: A total of 92 subjects (55 men, 37 women) participating in three different races of 14, 35, and 52 km were evaluated before (PRE) and after (POST) the races. Classic bioelectrical impedance vector analysis was applied at the whole-body level (WB-BIVA). Additionally, muscle-localized bioelectrical assessments (ML-BIVA) were performed in a subgroup of 11 men (in the quadriceps, hamstrings, and calves). PRE-POST differences and correlations between bioelectrical values and CK, running time and race distance were tested. Results: Changes in whole-body vectors and phase angles disclosed an inclination towards dehydration among men in the 14, 35, and 52 km groups (p < 0.001), as well as among women in the 35 and 52 km groups (p < 0.001). PRE Z/H was negatively correlated with running time in the 35 km men group and 14 km women group (r = -0.377, p = 0.048; r = -0.751, p = 0.001; respectively). POST Z/H was negatively correlated with running time in the 14 km women group (r = -0.593, p = 0.02). CK was positively correlated with distance in men and women (p < 0.001) and negatively correlated with reactance and vector length in the 14 km men group (p < 0.05). ML-BIVA echoed the same tendency as the WB-BIVA in the 35 and 52 km runners, with the most notable changes occurring in the calves (p < 0.001). Conclusions: WB-BIVA and CK measurements underscored a conspicuous trend towards post-race dehydration and muscle damage, displaying a weak association with performance. Notably, ML-BIVA detected substantial alterations primarily in the calves. The study underscores the utility of BIVA as a technique to assess athlete's body composition changes.

New bioelectrical impedance vector references and phase angle centile curves in 4,367 adults: The need for an urgent update after 30 years.

BACKGROUND & AIMS: The bioelectrical impedance vector analysis (BIVA) represents a qualitative analysis of body composition. The vector, defined by resistance (R) and reactance (Xc) standardized by stature, can be evaluated compared to the 50%,75%, and 95% tolerance ellipses representative of the reference populations. The tolerance ellipses for healthy adults have been provided in 1995 and were developed by mixing underage, adult, and elderly subjects, possibly misrepresenting the actual adult population. The current multicentric, cross-sectional study aimed to provide new tolerance ellipses specific for the general adult population and as a secondary aim to present centile curves for the bioelectrical phase angle. METHODS: R, Xc, and phase angle were measured in 2137 and 2230 males and females using phase-sensitive foot-to-hand analyzers at 50 kHz. A minimum of 35 subjects were included for each sex and age category from 18 to 65 years. RESULTS: The new mean vectors showed a leftward shift on the R-Xc graph with respect to the former reference values (males: F = 75.3; p < 0.001; females: F = 36.6, p < 0.001). The results provided new 3rd, 5th, 10th, 25th, 50th, 75th, 90th, 95th, and 97th percentile curves for phase angle, identifying time point phases of decrement (males: -0.03° per year at 33.0-51.0 years and -0.05° per year after 51 years; females: -0.03° per year from 37.2 to 57.9 years). CONCLUSIONS: Compared to the original references, the new data are characterized by a different distribution within the R-Xc graph with a higher phase angle. Thirty years after the BIVA invention, the current study presents new tolerance ellipses and phase angle reference values for the adult population.

Discovery of a novel 1,3,4-oxadiazol-2-one-based NLRP3 inhibitor as a pharmacological agent to mitigate cardiac and metabolic complications in an experimental model of diet-induced metaflammation.

Inspired by the recent advancements in understanding the binding mode of sulfonylurea-based NLRP3 inhibitors to the NLRP3 sensor protein, we developed new NLRP3 inhibitors by replacing the central sulfonylurea moiety with different heterocycles. Computational studies evidenced that some of the designed compounds were able to maintain important interaction within the NACHT domain of the target protein similarly to the most active sulfonylurea-based NLRP3 inhibitors. Among the studied compounds, the 1,3,4-oxadiazol-2-one derivative 5 (INF200) showed the most promising results being able to prevent NLRP3-dependent pyroptosis triggered by LPS/ATP and LPS/MSU by 66.3 ± 6.6% and 61.6 ± 11.5% and to reduce IL-1ß release (35.5 ± 8.8% µM) at 10 µM in human macrophages. The selected compound INF200 (20 mg/kg/day) was then tested in an in vivo rat model of high-fat diet (HFD)-induced metaflammation to evaluate its beneficial cardiometabolic effects. INF200 significantly counteracted HFD-dependent "anthropometric" changes, improved glucose and lipid profiles, and attenuated systemic inflammation and biomarkers of cardiac dysfunction (particularly BNP). Hemodynamic evaluation on Langendorff model indicate that INF200 limited myocardial damage-dependent ischemia/reperfusion injury (IRI) by improving post-ischemic systolic recovery and attenuating cardiac contracture, infarct size, and LDH release, thus reversing the exacerbation of obesity-associated damage. Mechanistically, in post-ischemic hearts, IFN200 reduced IRI-dependent NLRP3 activation, inflammation, and oxidative stress. These results highlight the potential of the novel NLRP3 inhibitor, INF200, and its ability to reverse the unfavorable cardio-metabolic dysfunction associated with obesity.

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.

Body Composition and Strength Symmetry of Kettlebell Sport Athletes.

Kettlebell sport (KBs) is increasingly popular, but very few studies have been focused on this discipline. This research aims to investigate the effects of KBs on body composition, strength symmetry, and segmental body composition symmetry in a sample of Italian elite athletes. Data were collected from a sample of 16 athletes of both sexes (11 men and 5 women; 34.5 ± 9.0 years of age). Anthropometric (height, weight, arm, thigh, calf, and waist circumferences), hand grip strength, and total and segmental bioelectrical variables were taken. Body composition was analysed by using specific bioelectrical impedance vector analysis (BIVA). Paired t-tests and confidence ellipses were applied to analyse bilateral differences. Elite athletes of both sexes showed high values of phase angle, indicative of high body cell mass and quality and proxy of muscle mass. Hand grip strength and body composition were symmetrical, with the only exception of a higher %FM in the right leg (Zsp: t = 3.556; p = 0.003). In conclusion, this study suggests that KBs contributes to muscle mass improvement, body composition, and strength symmetry, especially in the upper body.

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.

Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein.

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAFV600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAFV600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC50 values in the 40-88 nM range. Selected compounds inhibited BRAFV600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.

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.

Fat-Free Mass Using Bioelectrical Impedance Analysis as an Alternative to Dual-Energy X-Ray Absorptiometry in Calculating Energy Availability in Female Adolescent Athletes.

Energy availability (EA) is calculated by subtracting exercise energy expenditure from energy intake, adjusted for fat-free mass (FFM) obtained using accurate methods, such as dual-energy X-ray absorptiometry (DXA). Unlike DXA, the bioelectrical impedance analysis (BIA) is low in cost, simple and easy to carry out. This study aimed to test the concordance between the calculation of EA using FFM values from four BIA predictive equations and FFM obtained using DXA in female adolescent athletes (n = 94), recruited via social media. Paired Student's t test, Wilcoxon test, Lin's concordance correlation coefficient, root mean square error, limits of agreement, and mean absolute percentage error were used to evaluate agreement between the FFM values obtained by the four SF-BIA predictive equations and DXA. Regression linear analysis was used to determine the relation between FFM values obtained using DXA and the BIA predictive equations. Standardized residuals of the FFM and EA were calculated considering DXA values as reference. The most appropriate model for the FFM (limits of agreement = 4.0/-2.6 kg, root mean square error = 1.9 kg, mean absolute percentage error = 4.34%, Lin's concordance correlation coefficient = .926) and EA (limits of agreement = 2.51/4.4 kcal·kg FFM-1·day-1, root mean square error = 1.8 kcal·kg FFM-1·day-1, mean absolute percentage error 4.24%, Lin's concordance correlation coefficient = .992) was the equation with sexual maturity as a variable, while the equation with the greatest age variability was the one with the lowest agreement. FFM-BIA predictive equations can be used to calculate EA of female adolescent athletes. However, the equation should be chosen considering sex, age, and maturation status. In the case of athletes, researchers should use equations developed for this group.

Acute Exercise with Moderate Hypoxia Reduces Arterial Oxygen Saturation and Cerebral Oxygenation without Affecting Hemodynamics in Physically Active Males.

Hemodynamic changes during exercise in acute hypoxia (AH) have not been completely elucidated. The present study aimed to investigate hemodynamics during an acute bout of mild, dynamic exercise during moderate normobaric AH. Twenty-two physically active, healthy males (average age; range 23-40 years) completed a cardiopulmonary test on a cycle ergometer to determine their maximum workload (Wmax). On separate days, participants performed two randomly assigned exercise tests (three minutes pedaling at 30% of Wmax): (1) during normoxia (NORMO), and (2) during normobaric AH at 13.5% inspired oxygen (HYPO). Hemodynamics were assessed with impedance cardiography, and peripheral arterial oxygen saturation (SatO2) and cerebral oxygenation (Cox) were measured by near-infrared spectroscopy. Hemodynamic responses (heart rate, stroke volume, cardiac output, mean arterial blood pressure, ventricular emptying rate, and ventricular filling rate) were not any different between NORMO and HYPO. However, the HYPO test significantly reduced both SatO2 (96.6 ± 3.3 vs. 83.0 ± 4.5%) and Cox (71.0 ± 6.6 vs. 62.8 ± 7.4 A.U.) when compared to the NORMO test. We conclude that an acute bout of mild exercise during acute moderate normobaric hypoxia does not induce significant changes in hemodynamics, although it can cause significant reductions in SatO2 and Cox.

Intranasal lipid nanocarriers: Uptake studies with fluorescently labeled formulations.

Drug delivery by the intranasal route allows both systemic absorption and non-invasive brain targeting, due to the unique connection provided by the olfactory and trigeminal nerves between the brain and the external environment. Lipid nanocarriers can improve intranasal drug delivery by enhancing bioadhesion to nasal mucosa, and by protecting the encapsulated drug from biological degradation and transport efflux proteins. In this study two different biocompatible lipid nanocarriers were compared: nanoemulsions and solid lipid nanoparticles. The nasal uptake was investigated by labeling the nanocarriers lipid matrix with two fluorescent probes, 6-coumarin and rhodamine B, both lipophilic, yet characterized by different water solubility, in order to mimic the behavior of hypothetic drug compounds. Ex vivo permeation, in vivo pharmacokinetics and biodistribution studies were performed. 6-coumarin, water insoluble and therefore integral with the lipid matrix, was taken up to a limited extent, within a long timeframe, but with a proportionally more pronounced brain accumulation. In nanoemulsions soluble rhodamine B showed a relevant systemic uptake, with good bioavailability, likely due to the prompt release of the probe at the nasal mucosa.

Systolic and diastolic function during cycling at the respiratory threshold between elderly and young healthy individuals.

The hemodynamic consequences of aging have been extensively investigated during maximal incremental exercise. However, less is known about the effects of aging on hemodynamics during submaximal steady-state exercise. The aim of the present investigation was to compare the hemodynamics of healthy elderly and young subjects during an exercise bout conducted at the gas threshold (GET) intensity. Two groups of healthy, physically active subjects were studied: the elderly group-EG (n = 11; > 60 years old) and the young group-YG (n = 13; < 35 years old). Both groups performed a 5-min rectangular exercise test at the GET intensity. Hemodynamics were measured using echocardiography. The main finding was that stroke volume responses were higher in the YG than the EG (72.5 ± 16.7 vs. 52.4 ± 8.4 ml, respectively). The increased stroke volume capacity in the YG was the consequence of a greater capacity to increase cardiac preload and contractility and, to a lesser extent, to reduce systemic vascular resistance. Importantly, the atrial contribution to ventricular diastolic filling was substantially higher in the YG when compared to the EG.

Effect of Physical Training on Body Composition in Brazilian Military.

The military are selected on the basis of physical standards and are regularly involved in strong physical activities, also related to particular sports training. The aims of the study were to analyze the effect of a 7-month military training program on body composition variables and the suitability of specific 'bioelectrical impedance vector analysis' (spBIVA), compared to DXA, to detect the changes in body composition. A sample of 270 male Brazilian cadets (19.1 ± 1.1 years), composed of a group practicing military physical training routine only (MT = 155) and a group involved in a specific sport training (SMT = 115), were measured by body composition assessments (evaluated by means of DXA and spBIVA) at the beginning and the end of the military routine year. The effect of training on body composition was similar in SMT and MT groups, with an increase in LST. DXA and spBIVA were correlated, with specific resistance (Rsp) and reactance (Xcsp) positively related to fat mass (FM), FM%, LST, and lean soft tissue index (LSTI), and phase angle positively related to LST and LSTI. Body composition variations due to physical training were recognized by spBIVA: the increase in muscle mass was indicated by the phase angle and Xcsp increase, and the stability of FM% was consistent with the unchanged values of Rsp. Military training produced an increase in muscle mass, but no change in FM%, independently of the sample characteristics at baseline and the practice of additional sports. SpBIVA is a suitable technique for the assessment of body composition in military people.

Usability of classic and specific bioelectrical impedance vector analysis in measuring body composition of children.

In this study, we aimed to analyse the relationship between body composition and bioelectrical variables in children and adolescents. The sample was composed of 6801 individuals (4035 males; 2766 females) aged 8-20 years included in the National Health and Nutrition Examination Survey (NHANES) years 1999-2004. Classic and specific bioelectrical impedance vector analysis (BIVA) were applied and compared with dual-energy X-ray absorptiometry (DXA) for the evaluation of fat mass (FM) and fat-free mass (FFM), and bioimpedance spectroscopy (BIS) for the evaluation of intra-cellular water (ICW), extra-cellular water (ECW), and total body water (TBW). Fat-free mass index (FFMI) was calculated. Spearman's correlation, regression, and depth-depth analyses were applied. The evaluation of body composition with BIVA agreed well with that of DXA or BIS, independently of sex, age, and ethnicity: classic BIVA was mostly sensitive to differences in TBW, ECW/ICW, whereas specific BIVA to differences in %FM, FFMI, and ECW/ICW. The depth-depth analysis confirmed the associations of classic BIVA (coeff. 0.500, p < 0.001), and specific BIVA (coeff. 0.512, p < 0.001), also considering the significant effect of age (p < 0.001). In classic BIVA the association was slightly stronger in females (by 0.03, p = 0.042) and among Blacks (0.06, p = 0.002), whereas in specific BIVA it was stronger by 0.06 (p < 0.001) in females and similar among ethnic groups. The combined use of the two BIVA approaches represents a valuable tool for complete evaluation of body composition in growth studies, for the prevention and monitoring of malnutrition, and the monitoring of the performance in young athletes.

Multitarget Antioxidant NO-Donor Organic Nitrates: A Novel Approach to Overcome Nitrates Tolerance, an Ex Vivo Study.

Chronic use of glyceryl trinitrate (GTN) is limited by serious side effects, such as tolerance and endothelial dysfunction of coronary and resistance arteries. Although GTN is used as a drug since more than 130 years, the mechanisms of the vasodilatory effects and of tolerance development to organic nitrates are still incompletely elucidated. New synthesized organic nitrates with and without antioxidant properties were characterized for their ex vivo tolerance profile, in order to investigate the oxidative stress hypothesis of nitrate tolerance. The organic nitrates studied showed different vasodilation and tolerance profiles, probably due to the ability or inability of the compounds to interact with the aldehyde dehydrogenase-2 enzyme (ALDH-2) involved in bioactivation. Furthermore, nitrooxy derivatives endowed with antioxidant properties did not determine the onset of tolerance, even if bioactivated by ALDH-2. The results of this study could be further evidence of the involvement of ALDH-2 in the development of nitrate tolerance. Moreover, the behavior of organic nitrates with antioxidant properties supports the hypothesis of the involvement of ROS in inactivating ALDH-2.