A robust ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of 10 components in glutathione cycle.

Glutathione (GSH) is an important antioxidant that is generated and degraded via the GSH cycle. Quantification of the main components in the GSH cycle is necessary to evaluate the process of GSH. In this study, a robust ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 10 components (GSH; γ-glutamylcysteine; cysteinyl-glycine; n-acetylcysteine; homocysteine; cysteine; cystine; methionine; glutamate; pyroglutamic acid) in GSH cycle was developed. The approach was optimized in terms of derivative, chromatographic, and spectrometric conditions as well as sample preparation. The unstable thiol groups of GSH, γ-glutamylcysteine, cysteinyl-glycine, n-acetylcysteine, cysteine, and homocysteine were derivatized by n-ethylmaleimide. The derivatized and underivatized analytes were separated on an amino column with gradient elution. The method was further validated in terms of selectivity (no interference), linearity (R2 > 0.99), precision (% relative standard deviation [RSD%] range from 0.57 to 10.33), accuracy (% relative error [RE%] range from -3.42 to 10.92), stability (RSD% < 5.68, RE% range from -2.54 to 4.40), recovery (RSD% range from 1.87 to 7.87) and matrix effect (RSD% < 5.42). The validated method was applied to compare the components in the GSH cycle between normal and oxidative stress cells, which would be helpful in clarifying the effect of oxidative stress on the GSH cycle.

Journey from lab to clinic: Design, preclinical, and clinical development of systemic, targeted dendrimer-N-acetylcysteine (D-NAC) nanomedicines.

Drug discovery is challenging task with numerous obstacles in translating drug candidates into clinical products. Dendrimers are highly adaptable nanostructured polymers with significant potential to improve the chances of clinical success for drugs. Yet, dendrimer-based drug products are still in their infancy. However, Hydroxyl polyamidoamine (PAMAM) dendrimers showed significant promise in drug discovery efforts, owning their remarkable potential to selectively target and deliver drugs specifically to activated microglia and astrocytes at the site of brain injury in several preclinical models. After a decade's worth of academic research and pre-clinical efforts, the hydroxyl PAMAM dendrimer-N-acetyl cysteine conjugate (OP-101) nanomedicine has made a significant advancement in the field of nanomedicine and targeted delivery. The OP-101 conjugate, primarily developed and validated in academic labs, has now entered clinical trials as a potential treatment for hyperinflammation in hospitalized adults with severe COVID-19 through Ashvattha Therapeutics. This chapter, we delve into the journey of the hydroxyl PAMAM dendrimer-N-acetylcysteine (NAC) OP-101 formulation from the laboratory to the clinic. It will specifically focus on the design, synthesis, preclinical, and clinical development of OP-101, highlighting the potential it holds for the future of medicine and the positive Phase 2a results for treating severe COVID-19.

Lipid-polymer hybrid nanoparticles loaded with N-acetylcysteine for the modulation of neuroinflammatory biomarkers in human iPSC-derived PSEN2 (N141I) astrocytes as a model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment associated with the accumulation of beta-amyloid protein (Aß). Aß activates glial cells in the brain, increasing the secretion of proinflammatory cytokines, which leads to neuroinflammation and neuronal death. Currently, there are no effective treatments that cure or stop its progression; therefore, AD is considered a global health priority. The main limitations are the low drug bioavailability and impermeability of the blood-brain barrier (BBB). Fortunately, nanomedicine has emerged as a promising field for the development of new nanosystems for the controlled and targeted delivery of drugs to the brain. Therefore, in this work, lipid-polymer hybrid nanoparticles (LPHNPs) conjugated with transferrin (Tf) to facilitate crossing the BBB and loaded with N-acetylcysteine (NAC) for its anti-inflammatory effect were synthesized, and their physicochemical characterization was carried out. Subsequently, an in vitro model involving human astrocytes derived from induced pluripotent stem cells (iPSC) from an AD-diagnosed patient was developed, which was brought to a reactive state by stimulation with lipopolysaccharides (LPSs). The cell culture was treated with either Tf-conjugated LPHNPs loaded with NAC (NAC-Tf-LPHNPs) at 0.25 mg mL-1, or free NAC at 5 mM. The results showed that NAC-Tf-LPHNPs favorably modulated the expression of proinflammatory genes such as interleukin-1ß (IL-1ß), amyloid precursor protein (APP) and glial fibrillary acidic protein (GFAP). In addition, they reduced the secretion of the proinflammatory cytokines interleukin 6 (IL-6), IL-1ß and interferon-gamma (INF-γ). Results for both cases were compared to the group of cells that did not receive any treatment. In contrast, free NAC only had this effect on the expression of IL-1ß and the secretion of the cytokines IL-6 and INF-γ. These results indicate the potential of NAC-Tf-LPHNPs for AD treatment.

N-Acetyl Cysteine-Decorated Nitric Oxide-Releasing Interface for Biomedical Applications.

Biomedical devices are vulnerable to infections and biofilm formation, leading to extended hospital stays, high expenditure, and increased mortality. Infections are clinically treated via the administration of systemic antibiotics, leading to the development of antibiotic resistance. A multimechanistic strategy is needed to design an effective biomaterial with broad-spectrum antibacterial potential. Recent approaches have investigated the fabrication of innately antimicrobial biomedical device surfaces in the hope of making the antibiotic treatment obsolete. Herein, we report a novel fabrication strategy combining antibacterial nitric oxide (NO) with an antibiofilm agent N-acetyl cysteine (NAC) on a polyvinyl chloride surface using polycationic polyethylenimine (PEI) as a linker. The designed biomaterial could release NO for at least 7 days with minimal NO donor leaching under physiological conditions. The proposed surface technology significantly reduced the viability of Gram-negative Escherichia coli (>97%) and Gram-positive Staphylococcus aureus (>99%) bacteria in both adhered and planktonic forms in a 24 h antibacterial assay. The composites also exhibited a significant reduction in biomass and extra polymeric substance accumulation in a dynamic environment over 72 h. Overall, these results indicate that the proposed combination of the NO donor with mucolytic NAC on a polymer surface efficiently resists microbial adhesion and can be used to prevent device-associated biofilm formation.

Impact of the Acetyl Group on Cysteine: A Study of N-Acetyl-Cysteine through Rotational Spectroscopy.

Herein, we report a spectroscopic study of N-acetyl-L-cysteine, an important antioxidant drug, using Fourier-transform microwave techniques and in isolated conditions. Two conformers are observed, where most stable structure adopts a cis disposition, and the second conformer has a lower abundance and adopts a trans disposition. The rotational constants and the barriers to methyl internal rotation are determined for each conformer, allowing a precise conformation identification. The results show that the cis form adopts an identical structure in the crystal, solution, and gas phases. Additionally, the structures are contrasted against those of cysteine.

Evaluation of 3D-Printed Solid Microneedles Coated with Electrosprayed Polymeric Nanoparticles for Simultaneous Delivery of Rivastigmine and N-Acetyl Cysteine.

In the current study, coated microneedle arrays were fabricated by means of digital light processing (DLP) printing. Three different shapes were designed, printed, and coated with PLGA particles containing two different actives. Rivastigmine (RIV) and N-acetyl-cysteine (NAC) were coformulated via electrohydrodynamic atomization (EHDA), and they were incorporated into the PLGA particles. The two actives are administered as a combined therapy for Alzheimer's disease. The printed arrays were evaluated regarding their ability to penetrate skin and their mechanical properties. Optical microscopy and scanning electron microscopy (SEM) were employed to further characterize the microneedle structure. Confocal laser microscopy studies were conducted to construct 3D imaging of the coating and to simulate the diffusion of the particles through artificial skin samples. Permeation studies were performed to investigate the transport of the drugs across human skin ex vivo. Subsequently, a series of tape strippings were performed in an attempt to examine the deposition of the APIs on and within the skin. Light microscopy and histological studies revealed no drastic effects on the membrane integrity of the stratum corneum. Finally, the cytocompatibility of the microneedles and their precursors was evaluated by measuring cell viability (MTT assay and live/dead staining) and membrane damages followed by LDH release.

LC-MS/MS determination of pyocyanin-N-acetyl cysteine adduct: application for understanding Pseudomonas aeruginosa virulence factor neutralization.

Combating Pseudomonas aeruginosa infection is challenging. It secretes pyocyanin (PCN) pigment that contributes to its virulence. Neutralizing PCN via reaction with thiol-containing compounds may represent a potential therapeutic option. This study investigates the neutralization reaction between PCN and N-acetyl cysteine (NAC) for bacterial inhibition and explores its mechanism of action. The neutralization adduct (PCN-NAC) was synthesized by reacting the purified PCN and NAC. The adduct was analyzed and its structure was elucidated. LC-MS/MS method was developed for the determination of PCN-NAC in P. aeruginosa cultures post-treatment with NAC (0-5 mg/mL). The corresponding anti-bacterial potential was estimated and compared to nanoparticles (NPs) alone and under stress conditions. In silico studies were performed to support explaining the mechanism of action. Results revealed that PCN-NAC was exclusively detected in NAC-treated cultures in a concentration-dependent manner. PCN-NAC concentration (230-915 µg/mL) was directly proportional to the reduction in the bacterial viable count (28.3% ± 7.1-87.5% ± 5.9) and outperformed all tested NPs, where chitosan NPs induced 56.9% ± 7.9 inhibition, followed by zinc NPs (49.4% ± 0.9) and gold NPs (17.8% ± 7.5) even post-exposure to different stress conditions. A concomitant reduction in PCN concentration was detected. In silico studies revealed possible interactions between key bacterial proteins and PCN-NAC rather than the NAC itself. These results pose NAC as a potential choice for the management of P. aeruginosa infection, where it neutralizes PCN via the formation of PCN-NAC adduct.

A novel method for the accurate quantification of two isomeric mercapturic acids of 1,3-dichlorobenzene in human urine using isotope dilution online-SPE-LC-MS/MS.

1,3-dichlorobenzene (1,3-DCB) is an aromatic solvent that might be formed during thermal decomposition of bis(2,4-dichlorobenzoyl)peroxide used as initiator in silicone rubber production with many workers exposed worldwide. During metabolism of 1,3-DCB, two isomeric mercapturic acids can be formed from ring oxidation of 1,3-DCB in the liver, namely 2,4-dichlorophenylmercapturic acid (24CPhMA) and 3,5-dichlorophenylmercapturic acid (35CPhMA). These urinary mercapturic acids might serve as biomarkers of the toxicologically relevant absorbed dose of 1,3-DCB and have not been determined so far. Thus, we were aimed to develop an analytical method for quantification of these biomarkers. Authentic standards of both mercapturic acids as well as deuterium-labelled analogues were self-synthesized. A method for the quantification of both CPhMAs in human urine using online-SPE LC/MS/MS was developed and validated with an LOQ of 0.1 ng mL-1 for both CPhMAs. The analytes were extracted from urine by online-SPE on a restricted access material phase, transferred to the analytical column and quantified by tandem mass spectrometry. Interday (n = 6) and Intraday (n = 10) precision for both CPhMAs ranged from 1.7 to 4.3 % with accuracies between 99.4 and 109.9 % at concentrations of 0.6 and 3 ng mL-1. We applied the method on post-shift urine samples of 16 workers of the silicone rubber industry with occupational exposure to 1,3-DCB. Both CPhMAs were above LOQ in 15 of 16 urine samples with median levels (range) for 24CPhMA and 35CPhMA of 1.64 ng mL-1 (<0.1 - 8.2 ng mL-1) and 3.98 ng mL-1 (0.36 - 24.1 ng mL-1), respectively. This is the first report on specific urinary mercapturic acids of 1,3-DCB in humans. Our results show that ring oxidation of 1,3-DCB is considered to be a toxicologically relevant metabolic pathway in humans. This might improve risk assessment of 1,3-DCB-emissions in silicone rubber industry.

A cautionary note on the use of N-acetylcysteine as a reactive oxygen species antagonist to assess copper mediated cell death.

A new form of cell death has recently been proposed involving copper-induced cell death, termed cuproptosis. This new form of cell death has been widely studied in relation to a novel class of copper ionophores, including elesclomol and disulfiram. However, the exact mechanism leading to cell death remains contentious. The oldest and most widely accepted biological mechanism is that the accumulated intracellular copper leads to excessive build-up of reactive oxygen species and that this is what ultimately leads to cell death. Most of this evidence is largely based on studies using N-acetylcysteine (NAC), an antioxidant, to relieve the oxidative stress and prevent cell death. However, here we have demonstrated using inductively coupled mass-spectrometry, that NAC pretreatment significantly reduces intracellular copper uptake triggered by the ionophores, elesclomol and disulfiram, suggesting that reduction in copper uptake, rather than the antioxidant activity of NAC, is responsible for the diminished cell death. We present further data showing that key mediators of reactive oxygen species are not upregulated in response to elesclomol treatment, and further that sensitivity of cancer cell lines to reactive oxygen species does not correlate with sensitivity to these copper ionophores. Our findings are in line with several recent studies proposing the mechanism of cuproptosis is instead via copper mediated aggregation of proteins, resulting in proteotoxic stress leading to cell death. Overall, it is vital to disseminate this key piece of information regarding NAC's activity on copper uptake since new research attributing the effect of NAC on copper ionophore activity to quenching of reactive oxygen species is being published regularly and our studies suggest their conclusions may be misleading.

In Vitro Permeability Study of Homotaurine Using a High-Performance Liquid Chromatography with Fluorescence Detection Pre-Column Derivatization Method.

Homotaurine (HOM) is considered a promising drug for the treatment of Alzheimer's and other neurodegenerative diseases. In the present work, a new high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (λex. = 340 nm and λem. = 455 nm) method was developed and validated for the study of substance permeability in the central nervous system (CNS). Analysis was performed on a RP-C18 column with a binary gradient elution system consisting of methanol-potassium phosphate buffer solution (pH = 7.0, 0.02 M) as mobile phase. Samples of homotaurine and histidine (internal standard) were initially derivatized with ortho-phthalaldehyde (OPA) (0.01 M), N-acetylcysteine (0.01 M) and borate buffer (pH = 10.5; 0.05 M). To ensure the stability and efficiency of the reaction, the presence of different nucleophilic reagents, namely (a) 2-mercaptoethanol (2-ME), (b) N-acetylcysteine (NAC), (c) tiopronin (Thiola), (d) 3-mercaptopropionic acid (3-MPA) and (e) captopril, was investigated. The method was validated (R2 = 0.9999, intra-day repeatability %RSD < 3.22%, inter-day precision %RSD = 1.83%, limits of detection 5.75 ng/mL and limits of quantification 17.43 ng/mL, recovery of five different concentrations 99.75-101.58%) and successfully applied to investigate the in vitro permeability of homotaurine using Franz diffusion cells. The apparent permeability (Papp) of HOM was compared with that of memantine, which is considered a potential therapeutic drug for various CNSs. Our study demonstrates that homotaurine exhibits superior permeability through the simulated blood-brain barrier compared to memantine, offering promising insights for enhanced drug delivery strategies targeting neurological conditions.

Long-circulating thiolated chitosan nanoparticles of nintedanib with N-acetyl cysteine for treating idiopathic pulmonary fibrosis: In vitro assessment of cytotoxicity, antioxidant, and antifibrotic potential.

Nintedanib (NIN) is one of the FDA-approved tyrosine kinase inhibitor drugs used to treat idiopathic pulmonary fibrosis (IPF). This study aimed to formulate a long-circulating injection of Nintedanib to treat bedridden patients with IPF. Nintedanib was incorporated into chitosan nanoparticles (NIN-NP) via the ionic gelation method, and N-acetyl cysteine (NAC), a known antioxidant and mucolytic agent, was added to the NIN-NP (NAC-NIN-NP). The lyophilized formulation had a particle size of 174 nm, a polydispersity index of 0.511, and a zeta potential of 18.6 mV. The spherical nanoparticles were observed in transmission electron microscopy, whereas field emission scanning electron microscopy showed irregular clusters of NP. The thiolation of the chitosan in NAC-NIN-NP was confirmed by ATR-FTIR and NMR, which improved drug release profiles showing >90 % drug release that was 2.42-folds greater than NIN-NP lasting for five days. The DPPH assay showed that adding NAC increased the % inhibition of oxidation in blank-NP (from 54.59 % to 87.17 %) and NIN-NP (58.65 %-89.19 %). The MTT assay on A549 cells showed 67.57 % cell viability by NAC-NIN-NP with an IC50 value of 28 µg/mL. The NAC formulation reduced hydroxyproline content (56.77 µg/mL) compared to NIN-NP (69.48 µg/mL) in WI-38 cell lines. Meanwhile, the healthy cells count with NAC-NIN-NP was higher (5.104 × 103) than with NIN-NP (4.878 × 103). In Hoechst staining, no significant damage to DNA was observed by the drug or formulation. Therefore, NAC-NIN-NP could be a promising treatment option for IPF patients and can be studied further clinically.

(E)-2-Benzylidenecyclanones: Part XVIII Study the Possible Link between Glutathione Reactivity and Cancer Cell Cytotoxic Effects of Some Cyclic Chalcone Analogs A Comparison of the Reactivity of the Open-Chain and the Seven-Membered Homologs.

Non-enzymatic thiol addition into the α,ß-unsaturated carbonyl system is associated with several biological effects. In vivo, the reactions can form small-molecule thiol (e.g., glutathione) or protein thiol adducts. The reaction of two synthetic (4'-methyl- and 4'-methoxy substituted) cyclic chalcone analogs with reduced glutathione (GSH) and N-acetylcysteine (NAC) was studied by (high-pressure liquid chromatography-ultraviolet spectroscopy) HPLC-UV method. The selected compounds displayed in vitro cancer cell cytotoxicity (IC50) of different orders of magnitude. The structure of the formed adducts was confirmed by (high-pressure liquid chromatography-mass spectrometry) HPLC-MS. The incubations were performed under three different pH conditions (pH 3.2/3.7, 6.3/6.8, and 8.0/7.4). The chalcones intrinsically reacted with both thiols under all incubation conditions. The initial rates and compositions of the final mixtures depended on the substitution and the pH. The frontier molecular orbitals and the Fukui function were carried out to investigate the effects on open-chain and seven-membered cyclic analogs. Furthermore, machine learning protocols were used to provide more insights into physicochemical properties and to support the different thiol-reactivity. HPLC analysis indicated diastereoselectivity of the reactions. The observed reactivities do not directly relate to the different in vitro cancer cell cytotoxicity of the compounds.

Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer-N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines.

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated N-acylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2-oxazoline)-NH2 (PEtOx-NH2) and characterized by 1H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer-NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer-NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future.

N-acetylcysteine functionalized chitosan oligosaccharide-palmitic acid conjugate enhances ophthalmic delivery of flurbiprofen and its mechanisms.

An N-acetylcysteine functionalized chitosan oligosaccharide-palmitic acid conjugate (NAC-COS-PA) with bioadhesive and permeation promoting properties was synthesized to enhance transocular drug delivery. Flurbiprofen (FB) loaded self-assembled NAC-COS-PA nanomicelles (NAC-COS-PA-FB) were prepared and the drug loading was 7.35 ± 0.32%. Human immortalized corneal epithelial (HCE-T) cell cytotoxicity and hen's egg test-chorioallantoic membrane assays confirmed that the conjugate had good biocompatibility. The transportation efficiency of coumarin-6 (C6) loaded nanomicelles in the HCE-T cell monolayer was approximately 1.97 times higher than that of free C6. Decreased intracellular Ca2+ concentration and cell membrane potential, increased cell membrane fluidity, and reversible changes in the F-actin cytoskeleton are presumed to be responsible for the enhanced drug permeation. NAC-COS-PA exhibited strong binding capacity with mucin and rabbit eyeball. In vivo pharmacokinetics indicated that the area under the curve (AUC0-6 h) and the maximum concentration (Cmax) of NAC-COS-PA-FB were approximately 1.92 and 2.44 times that of the FB solution, respectively. NAC-COS-PA-FB demonstrated the best in vivo anti-inflammatory efficacy compared to unfunctionalized nanomicelles (COS-PA-FB) and FB solution. Consequently, NAC-COS-PA appears to be a promising bioadhesive carrier for ophthalmic delivery.

Raman Optical Activity of N-Acetyl-L-Cysteine in Water and in Methanol: The "Clusters-in-a-Liquid" Model and ab Initio Molecular Dynamics Simulations.

Raman and Raman Optical Activity (ROA) spectra of N-acetyl-L-cysteine (NALC), a flexible chiral molecule, were measured in water and in methanol to evaluate the solvent effects. Two different solvation approaches, that is, the DFT based "clusters-in-a-liquid" solvent model and the ab initio molecular dynamics (AIMD) simulations, were applied to simulate the Raman and ROA spectra. Systematic conformational searches were carried out using a recently developed conformational searching tool, CREST, with the inclusion of polarizable continuum model of water and of methanol. The CREST candidates of NALC and the NALC-solvent complexes were re-optimized and their Raman and ROA simulations were done at the B3LYP-D3BJ/def2-TZVP and the B3LYP-aug-cc-pVDZ//cc-pVTZ levels. Also, AIMD simulations, which includes some anharmonic effects and all intermolecular interactions in solution, were performed. By empirically weighting the computed Raman and ROA spectra of each conformer, good agreements with the experimental data were achieved with both approaches, while AIMD offered some improvements in the carbonyl and in the low wavenumber regions over the static DFT approach. The pros and cons of these two different approaches for accounting the solvent effects on Raman and ROA of this flexible chiral system will also be discussed.

Mannose-anchored quaternized chitosan/thiolated carboxymethyl chitosan composite NPs as mucoadhesive carrier for drug delivery.

There are various challenges for the mucosal delivery of drug, which is largely attributed to the absence of effective drug carriers that can make delivery to mucosal sites. In the present study, we aimed to synthesize bifunctional mucoadhesive nanoparticles (NPs) that could be used for mucosal delivery. N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (M-N-2-HACC) was modified with D-mannose, and N-acetyl-L-cysteine (NAC) was immobilized on the carboxymethyl chitosan (N-CMCS). The electrostatic interaction between the two substances was used to produce mannose-modified thiolated chitosan NPs (M-N-2-HACC/N-CMCS NPs). The NPs showed a particle size of 196.72 ± 0.45 nm and zeta potential of 17.12 ± 0.50 mV. Moreover, it demonstrated high hydrophilicity, enduring drug release, stability, safety, and mucosal adhesion, which contributed to the effectiveness of mucosal administration. Additionally, the NPs could be instantly absorbed by macrophages. Collectively, these results suggested that M-N-2-HACC/N-CMCS NPs could be used as a promising candidate for mucosal delivery.

Where are we with the use of N-acetylcysteine as a preventive and adjuvant treatment for COVID-19?

OBJECTIVE: As N-acetylcysteine (NAC) is promising as a re-purposed drug for the adjunctive or supportive treatment of serious COVID-19, this article aimed to describe current evidence. MATERIALS AND METHODS: A search was performed in PubMed/Medline for "NAC", "viral Infection", COVID-19", oxidative stress", "inflammation", retrieving preclinical and clinical studies. RESULTS: NAC is a pleiotropic molecule with a dual antioxidant mechanism; it may neutralize free radicals and acts as a donor of cysteine, restoring the physiological pool of GSH. Serious COVID-19 patients have increased levels of reactive oxygen species (ROS) and free radicals and often present with glutathione depletion, which prompts a cytokine storm. NAC, which acts as a precursor of GSH inside cells, has been currently used in many conditions to restore or protect against GSH depletion and has a wide safety margin. In addition, NAC has anti-inflammatory activity independently of its antioxidant activity. CONCLUSIONS: Clinical and experimental data suggest that NAC may act on the mechanisms leading to the prothrombotic state observed in severe COVID-19.

The role of topical N-acetylcysteine in ocular therapeutics.

N-acetylcysteine (NAC) was first discovered as a mucolytic agent in 1960. We investigate the role of topical NAC in ocular therapeutics, including its mechanism of action, current applications, and adverse effects. A systematic search of peer-reviewed articles identified 106 references including in vitro, in vivo and clinical studies on the use of NAC in the treatment of ocular diseases. NAC can be synthetically manufactured, and its mechanisms of action include mucolysis, scavenging hydroxyl radicals, and modulation of inflammatory cascades. These unique properties contribute to the diverse applications of NAC, including its steroid-sparing potential. NAC has been used topically in the treatment of corneal wounds, chemical injuries, keratitis, dry eye disease and meibomian gland dysfunction. The clinical benefits of NAC are evident over a wide range of concentrations, the most common being 5-10% topical NAC applied four times daily. Adverse effects such as corneal necrosis are rare, but have been reported with higher doses. NAC also has potential applications in laser epithelial keratomileusis, diabetic eye disease, retinitis pigmentosa, senile nuclear cataracts, macular degeneration, and cigarette smoke-induced corneal damage. Recently, chitosan-NAC has been used as a nanocarrier for the topical administration of medications to the ocular surface. Owing to its potent antioxidant, anti-inflammatory and mucolytic properties, topical NAC has had extensive use in the treatment of ocular pathology.

Physico-Chemistry of Dinitrosyl Iron Complexes as a Determinant of Their Biological Activity.

In this article we minutely discuss the so-called "oxidative" mechanism of mononuclear form of dinitrosyl iron complexes (M-DNICs) formations proposed by the author. M-DNICs are proposed to be formed from their building material-neutral NO molecules, Fe2+ ions and anionic non-thiol (L-) and thiol (RS-) ligands based on the disproportionation reaction of NO molecules binding with divalent ion irons in pairs. Then a protonated form of nitroxyl anion (NO-) appearing in the reaction is released from this group and a neutral NO molecule is included instead. As a result, M-DNICs are produced. Their resonance structure is described as [(L-)2Fe2+(NO)(NO+)], in which nitrosyl ligands are represented by NO molecules and nitrosonium cations in equal proportions. Binding of hydroxyl ions with the latter causes conversion of these cations into nitrite anions at neutral pH values and therefore transformation of DNICs into the corresponding high-spin mononitrosyl iron complexes (MNICs) with the resonance structure described as [(L-)2Fe2+(NO)]. In case of replacing L- by thiol-containing ligands, which are characterized by high π-donor activity, electron density transferred from sulfur atoms to iron-dinitrosyl groups neutralizes the positive charge on nitrosonium cations, which prevents their hydrolysis, ensuring relatively a high stability of the corresponding M-DNICs with the resonance structure [(RS-)2Fe2+ (NO, NO+)]. Therefore, M-DNICs with thiol-containing ligands, as well as their binuclear analogs (B-DNICs, respective resonance structure [(RS-)2Fe2+2 (NO, NO+)2]), can serve donors of both NO and NO+. Experiments with solutions of B-DNICs with glutathione or N-acetyl-L-cysteine (B-DNIC-GSH or B-DNIC-NAC) showed that these complexes release both NO and NO+ in case of decomposition in the presence of acid or after oxidation of thiol-containing ligands in them. The level of released NO was measured via optical absorption intensity of NO in the gaseous phase, while the number of released nitrosonium cations was determined based on their inclusion in S-nitrosothiols or their conversion into nitrite anions. Biomedical research showed the ability of DNICs with thiol-containing ligands to be donors of NO and NO+ and produce various biological effects on living organisms. At the same time, NO molecules released from DNICs usually have a positive and regulatory effect on organisms, while nitrosonium cations have a negative and cytotoxic effect.

N-Acetyl-l-cysteine-Loaded Nanosystems as a Promising Therapeutic Approach Toward the Eradication of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are a major health concern, mainly due to their contribution to increased bacterial resistance to well-known antibiotics. The conventional treatment of biofilms represents a challenge, and frequently, eradication is not achieved with long-lasting administration of antibiotics. In this context, the present work proposes an innovative therapeutic approach that is focused on the encapsulation of N-acetyl-l-cysteine (NAC) into lipid nanoparticles (LNPs) functionalized with d-amino acids to target and disrupt bacterial biofilms. The optimized formulations presented a mean hydrodynamic diameter around 200 nm, a low polydispersity index, and a high loading capacity. These formulations were stable under storage conditions up to 6 months. In vitro biocompatibility studies showed a low cytotoxicity effect in fibroblasts and a low hemolytic activity in human red blood cells. Nevertheless, unloaded LNPs showed a higher hemolytic potential than NAC-loaded LNPs, which suggests a safer profile of the latter. The in vitro antibiofilm efficacy of the developed formulations was tested against Staphylococcus epidermidis (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) mature biofilms. The results showed that the NAC-loaded LNPs were ineffective against S. epidermidis biofilms, while a significant reduction of biofilm biomass and bacterial viability in P. aeruginosa biofilms were observed. In a more complex therapeutic approach, the LNPs were further combined with moxifloxacin, revealing a beneficial effect between the LNPs and the antibiotic against P. aeruginosa biofilms. Both alone and in combination with moxifloxacin, unloaded and NAC-loaded LNPs functionalized with d-amino acids showed a great potential to reduce bacterial viability, with no significant differences in the presence or absence of NAC. However, the presence of NAC in NAC-loaded functionalized LNPs shows a safer profile than the unloaded LNPs, which is beneficial for an in vivo application. Overall, the developed formulations present a potential therapeutic approach against P. aeruginosa biofilms, alone or in combination with antibiotics.