N-acetylcysteine Amide Research Articles

N-Acetylcysteine Amide Alleviates Cyclophosphamide-Induced Endothelial Damage

Rui He 1,2, Wenyi Zheng 1,2, Terra Slof 3, Eva Wärdell 4, Agneta Månsson-Broberg 4, Svante Norgren 5, Per Ljungman 2, Ying Zhao 1, 2 , Moustapha Hassan 1, 21.Experimental Cancer Medicine, Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Sweden2.Center of Allogeneic Stem Cell Transplantation (CAST) and Clinical Research Center, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden.3.Institute for Life Sciences and Chemistry, HU University of Applied Sciences Utrecht, Oudenoord 330, 3513 EX Utrecht, Netherlands4.Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.5.Children´s and Women´s Health Theme, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenAimThe current study aimed to investigate the protective role of the novel antioxidant N-acetylcysteine amide (NACA) in cyclophosphamide (Cy)-induced endothelial injury and to explore the underlying mechanism.BackgroundThe alkylating agent Cy is still one of the important corner stones in cancer treatment. Cy is used also as a part of the conditioning regimens prior to hematopoietic cell transplantation (HCT) and as a prophylactic treatment post transplantation in graft-versus-host disease (GVHD). Several studies have shown that high doses of Cy are associated with a number of adverse effects including alopecia, gonadal toxicity, hemorrhagic cystitis, carcinogenesis and late cardiovascular toxicity. Moreover, we have shown that treatment with high-dose Cy caused damage to the arterial endothelium, which might contribute to late cardiovascular disorders. Hitherto, there is no prophylactic strategy for Cy-induced endothelial toxicity. Oxidative stress is one of the main players in the pathogenesis of endothelial disorders and hence is an exploitable trait for targeted treatment.MethodsThe prophylactic efficacy of NACA was evaluated using human aortic endothelial cells (HAECs) and human umbilical endothelial cells (HUVECs). The efficacy of NACA was compared with the clinically approved derivative N-acetylcysteine (NAC). Cy is a prodrug that cannot be activated in the endothelial cells. Therefore, cells were incubated with Cy active metabolite, 4-hydroxycyclophosphamide (4-OH-Cy). In order to reveal the underling mechanism of action, we have investigated and evaluated the following parameters: alteration in cell morphology, cell death pathway, inhibition of DNA damage, variation of redox hemostasis, and neovascularization.ResultsWe found that pre-incubation with 5 mM NACA preserved the normal endothelial cell morphology. However, for the quantification of the obtained results, cell colonies were visualized using crystal violet staining while cell viability was determined using WST-1 assay. Moreover, NACA was found to decrease 4-OH-Cy-induced DNA damage and cell death via caspase-dependent apoptosis in both HAECs and HUVECs. In addition, NACA significantly inhibited 4-OH-Cy-induced oxidative stress by reducing the intracellular levels of reactive oxygen species (ROS) and by improving the cellular antioxidative capacity. Increased activities of catalase and superoxide dismutase (SOD) together with increased levels of reduced glutathione (GSH) were observed. Meanwhile, NACA pre-treatment rebalanced endothelial nitric oxide synthase (eNOS) and arginase I in HAECs, suggesting that NACA may be used as prophylactic strategy to avoid the impaired endothelium-dependent vasodilation caused by 4-OH-Cy. Moreover, data from tube formation assay indicated that NACA preserved the angiogenic capability of endothelial cells that was compromised by 4-OH-Cy through blockage of Notch signaling pathway. Our results showed that in contrast to NAC, its amide derivative NACA showed remarkably superior ability in alleviating the endothelial damage caused by 4-OH-Cy.ConclusionsIn summary, the present investigation showed that the novel antioxidant NACA could be applied to protect against 4-OH-Cy-induced endothelial cell dysfunction and death. Higher bioavailability and better cellular uptake make NACA to a better prophylactic agent compared to NAC. To translate these findings into the clinic, additional in vivo studies are warranted to confirm the protective potential of NACA. DisclosuresLjungman: Takeda: Consultancy, Other: Endpoint committee, speaker; Janssen: Other: Investigator; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy.

Continuous Monitoring Reveals Protective Effects of N‐Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit

Microphysiological systems mimic the in vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. In this study, the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2 min temporal resolution is reported. Its capabilities are showcased in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol-ene-epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell-substrate endothelial resistance monitoring allows for capturing the formation and breakdown of the BBB model, which consists of cocultured hiPSC-derived endothelial-like and astrocyte-like cells. Clear cellular disruption is observed when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide are reported. Using metabolomic network analysis reveals further drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like this opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

The SARS‐CoV‐2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike‐ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor‐binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol‐reactive compounds are able to inhibit viral entry. N‐acetyl cysteine amide, L‐ascorbic acid, JTT‐705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

Methamphetamine and HIV-Tat Protein Synergistically Induce Oxidative Stress and Blood-Brain Barrier Damage via Transient Receptor Potential Melastatin 2 Channel.

Synergistic impairment of the blood-brain barrier (BBB) induced by methamphetamine (METH) and HIV-Tat protein increases the risk of HIV-associated neurocognitive disorders (HAND) in HIV-positive METH abusers. Studies have shown that oxidative stress plays a vital role in METH- and HIV-Tat-induced damage to the BBB but have not clarified the mechanism. This study uses the human brain microvascular endothelial cell line hCMEC/D3 and tree shrews to investigate whether the transient receptor potential melastatin 2 (TRPM2) channel, a cellular effector of the oxidative stress, might regulate synergistic damage to the BBB caused by METH and HIV-Tat. We showed that METH and HIV-Tat damaged the BBB in vitro, producing abnormal cell morphology, increased apoptosis, reduced protein expression of the tight junctions (TJ) including Junctional adhesion molecule A (JAMA) and Occludin, and a junctional associated protein Zonula occludens 1 (ZO1), and increased the flux of sodium fluorescein (NaF) across the hCMEC/D3 cells monolayer. METH and HIV-Tat co-induced the oxidative stress response, reducing catalase (CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) activity, as well as increased reactive oxygen species (ROS) and malonaldehyde (MDA) level. Pretreatment with n-acetylcysteine amide (NACA) alleviated the oxidative stress response and BBB damage characterized by improving cell morphology, viability, apoptosis levels, TJ protein expression levels, and NaF flux. METH and HIV-Tat co-induced the activation and high protein expression of the TRPM2 channel, however, early intervention using 8-Bromoadenosine-5′-O-diphosphoribose (8-Br-ADPR), an inhibitor of TPRM2 channel, or TRPM2 gene knockdown attenuated the BBB damage. Oxidative stress inhibition reduced the activation and high protein expression of the TRPM2 channel in the in vitro model, which in turn reduced the oxidative stress response. Further, 8-Br-ADPR attenuated the effects of METH and HIV-Tat on the BBB in tree shrews—namely, down-regulated TJ protein expression and increased BBB permeability to Evans blue (EB) and NaF. In summary, the TRPM2 channel can regulate METH- and HIV-Tat-induced oxidative stress and BBB injury, giving the channel potential for developing drug interventions to reduce BBB injury and neuropsychiatric symptoms in HIV-infected METH abusers.

N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation

The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms in neurons remains difficult due to the limited access to human tissue. Using human induced pluripotent stem cells (hiPSCs), we generated functional dopaminergic (DA) neurons showing positive expression of dopaminergic markers TH and DAT, mature neuronal marker MAP2 and functional synaptic markers synaptophysin and PSD-95. These DA neurons were electrophysiologically characterized, and exhibited inward Na + currents, overshooting action potentials and spontaneous postsynaptic currents (sPSCs). POLG patient-specific DA neurons (POLG-DA neurons) manifested a phenotype that replicated the molecular and biochemical changes found in patient post-mortem brain samples namely loss of complex I and depletion of mtDNA. Compared to disease-free hiPSC-derived DA neurons, POLG-DA neurons exhibited loss of mitochondrial membrane potential, loss of complex I and loss of mtDNA and TFAM expression. POLG driven mitochondrial dysfunction also led to neuronal ROS overproduction and increased cellular senescence. This deficit was selectively rescued by treatment with N-acetylcysteine amide (NACA). In conclusion, our study illustrates the promise of hiPSC technology for assessing pathogenetic mechanisms associated with POLG disease, and that NACA can be a promising potential therapy for mitochondrial diseases such as those caused by POLG mutation.

Effects of N-Acetylcysteine and N-Acetylcysteine Amide on Erythrocyte Deformability and Oxidative Stress in a Rat Model of Lower Extremity Ischemia-Reperfusion Injury.

N-acetylcysteine (NAC) is an antioxidant which works as a free radical scavenger and antiapoptotic agent. N-acetylcysteine-amide (NACA) is a modified form of NAC containing an amide group instead of a carboxyl group of NAC. Our study aims to investigate the effectiveness of these two substances on erythrocyte deformability and oxidative stress in muscle tissue. Materials and Methods. A total of 24 Wistar albino rats were used in our study. The animals were randomly divided into five groups as control (n: 6), ischemia (n: 6), NAC (n: 6), and NACA (n: 6). In the ischemia, NAC, and NACA groups, 120 min of ischemia and 120 min of reperfusion were achieved by placing nontraumatic vascular clamps across the abdominal aorta. The NAC and NACA groups were administered an injection 30 min before ischemia (100 mg/kg NAC; 100 mg/kg NACA; intravenous). Blood samples were taken from the animals at the end of the ischemic period. The lower extremity gastrocnemius muscle was isolated and stored at −80 degrees to assess the total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) values and was analyzed. Results. The erythrocyte deformability index was found to be statistically significantly lower in rats treated with NAC and NACA before ischemia-reperfusion compared to the groups that received only ischemia-reperfusion. In addition, no statistically significant difference was found between the control group and the NAC and NACA groups. The groups receiving NAC and NACA before ischemia exhibited higher total antioxidative status and lower total oxidative status while the oxidative stress index was also lower. Conclusion. The results of our study demonstrated the protective effects of NAC and NACA on erythrocyte deformability and oxidative damage in skeletal muscle in lower extremity ischemia-reperfusion. NAC and NACA exhibited similar protective effects on oxidative damage and erythrocyte deformability.

Low doses of the neonicotinoid insecticide imidacloprid induce ROS triggering neurological and metabolic impairments in Drosophila

Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.

In silico allicin induced S-thioallylation of SARS-CoV-2 main protease

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused due to new coronavirus infection with 3716075 deaths across the world as reported by the World Health Organization (WHO). SARS-CoV-2 main protease (Mpro) plays a vital role in the replication of coronavirus and thus an attractive target for the screening of inhibitors for the therapy of COVID-19. The preclinical drugs ebselen and PX-12 are potent inhibitors of SARS-CoV-2 Mpro and covalently modifies the active site Cys-145 residue of Mpro through selenosulfide/disulfide. In the current report, using virtual screening methods, reactive sulfur species allicin is subjecting for covalent docking at the active site of SARS-CoV-2 Mpro using PX-12 as a benchmark reference compound. The results indicate that allicin induces dual S-thioallylation of Cys-145 and Cys-85/ Cys-156 residues of SARS-CoV-2 Mpro. Using density functional theory (DFT), Gibbs free energy change (DG) is calculated for the putative reactions between N-acetylcysteine amide thiol and allicin/allyl sulfenic acid. The overall reaction is exergonic and allyl disulfide of Cys-145 residue of Mpro is involved in a sulfur mediated hydrogen bond. The results indicate that allicin causes dual S-thioallylation of SARS-CoV-2 Mpro which may be of interest for treatment and attenuation of ongoing coronavirus infection.

Hepatocellular carcinoma-derived high mobility group box 1 triggers M2 macrophage polarization via a TLR2/NOX2/autophagy axis

In many human cancers, including hepatocellular carcinoma (HCC), high density of infiltrating tumor-associated macrophages (TAM) is associated with poor prognosis. Most TAMs express a M2 phenotype subsequently supporting tumor growth. How tumor cells polarize these TAMs to a pro-tumor M2 phenotype is still poorly understood. Our previous studies have revealed that a Toll-like receptor 2 (TLR2)-dependent autophagy triggered by hepatoma-derived factors down-regulates NF-κB p65 and drives M2 macrophage differentiation. However, the underlying mechanisms and potential hepatoma-derived TLR2 ligands are not clear. Here, we provide evidence to reveal that NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS) generation is crucial for HCC-induced autophagy, NF-κB p65 down-regulation and M2 phenotype polarization in primary macrophages. This NOX2-generated ROS production in abolished in TLR2-deficient macrophages. HCC-derived or recombinant high-mobility group box 1 (HMGB1) is able to trigger this TLR2-mediated M2 macrophage polarization. Blockage of HMGB1 and ROS by inhibitors, ethyl pyruvate and N-acetylcysteine amide, respectively, significantly reduces both M2 macrophage accumulation and liver nodule formation in HCC-bearing mice. Our findings uncover a HMGB1/TLR2/NOX2/autophagy axis to trigger M2 macrophage polarization in HCC that can be considered as a novel therapeutic target for treating HCC.

Effects of N-acetylcysteine amide on anxiety and stress behavior in zebrafish.

Anxiety disorders are highly prevalent and a leading cause of disability worldwide. Their etiology is related to stress, an adaptive response of the organism to restore homeostasis, in which oxidative stress and glutamatergic hyperactivity are involved. N-Acetylcysteine (NAC) is a multitarget approved drug proved to be beneficial in the treatment of various mental disorders. Nevertheless, NAC has low membrane permeability and poor bioavailability and its limited delivery to the brain may explain inconsistencies in the literature. N-Acetylcysteine amide (AD4) is a synthetic derivative of NAC in which the carboxyl group was modified to an amide. The amidation of AD4 improved lipophilicity and blood-brain barrier permeability and enhanced its antioxidant properties. The purpose of this study was to investigate the effects of AD4 on behavioral and biochemical parameters in zebrafish anxiety models. Neither AD4 nor NAC induced effects on locomotion and anxiety-related parameters in the novel tank test. However, in the light/dark test, AD4 (0.001 mg/L) increased the time spent in the lit side in a concentration 100 times lower than NAC (0.1 mg/L). In the acute restraint stress protocol, NAC and AD4 (0.001 mg/L) showed anxiolytic properties without meaningful effects on oxidative status. The study suggests that AD4 has anxiolytic effects in zebrafish with higher potency than the parent compound. Additional studies are warranted to characterize the anxiolytic profile of AD4 and its potential in the management of anxiety disorders.

Pharmacokinetic profile of N-acetylcysteine amide and its main metabolite in mice using new analytical method

N-acetylcysteine amide (NACA) is the amide derivative of N-acetylcysteine (NAC) that is rapidly converted to NAC after systemic administration. It has emerged as a promising thiol antioxidant for multiple indications; however, the pharmacokinetic property is yet unclear due to lack of an accurate quantification method. The present investigation aimed to develop an analytical method for simultaneous quantification of NACA and NAC in plasma. A new reagent (2-(methylsulfonyl)-5-phenyl-1,3,4-oxadiazole, MPOZ) was introduced for thiol stabilization during sample processing and storage. Further, we utilized tris (2-carboxyethyl) phosphine (TCEP) to reduce the oxidized forms of NACA and NAC. After derivatization, NACA-MPOZ and NAC-MPOZ were quantified using liquid chromatography–mass spectrometry (LC-MS). The new method was validated and found to have high specificity, linearity, accuracy, precision, and recovery for the quantification of NACA and NAC in plasma. Furthermore, the formed derivatives of NACA and NAC were stable for 48 h under different conditions. The method was utilized in pharmacokinetic study which showed that the bioavailability of NACA is significantly higher than NAC (67% and 15%, respectively). The pharmacokinetic of NACA obeyed a two-compartment open model. The glutathione (GSH)-replenishing capacity was found to be three to four-fold higher after the administration of NACA compared to that observed after the administration of NAC. In conclusion, the present method is simple, robust and reproducible, and can be utilized in both experimental and clinical studies. NACA might be considered as a prodrug for NAC. Furthermore, this is the first report describing the pharmacokinetics and bioavailability of NACA in mouse.

N-acetylcysteine Amide Ameliorates Blast-Induced Changes in Blood-Brain Barrier Integrity in Rats.

Traumatic brain injury resulting from exposure to blast overpressure (BOP) is associated with neuropathology including impairment of the blood-brain barrier (BBB). This study examined the effects of repeated exposure to primary BOP and post-blast treatment with an antioxidant, N-acetylcysteine amide (NACA) on the integrity of BBB. Anesthetized rats were exposed to three 110 kPa BOPs separated by 0.5 h. BBB integrity was examined in vivo via a cranial window allowing imaging of pial microcirculation by intravital microscopy. Tetramethylrhodamine isothiocyanate Dextran (TRITC-Dextran, mw = 40 kDa or 150 kDa) was injected intravenously 2.5 h after the first BOP exposure and the leakage of TRITC-Dextran from pial microvessels into the brain parenchyma was assessed. The animals were randomized into 6 groups (n = 5/group): four groups received 40 kDa TRITC-Dextran (BOP-40, sham-40, BOP-40 NACA, and sham-40 NACA), and two groups received 150 kDa TRITC-Dextran (BOP-150 and sham-150). NACA treated groups were administered NACA 2 h after the first BOP exposure. The rate of TRITC-Dextran leakage was significantly higher in BOP-40 than in sham-40 group. NACA treatment significantly reduced TRITC-Dextran leakage in BOP-40 NACA group and sham-40 NACA group presented the least amount of leakage. The rate of leakage in BOP-150 and sham-150 groups was comparable to sham-40 NACA and thus these groups were not assessed for the effects of NACA. Collectively, these data suggest that BBB integrity is compromised following BOP exposure and that NACA treatment at a single dose may significantly protect against blast-induced BBB breakdown.

Activation of Pregnane X Receptor Sensitizes Mice to Hemorrhagic Shock-Induced Liver Injury.

Hemorrhagic shock (HS) is a life-threatening condition associated with tissue hypoperfusion and often leads to injury of multiple organs including the liver. Pregnane X receptor (PXR) is a species-specific xenobiotic receptor that regulates the expression of drug-metabolizing enzymes (DMEs) such as the cytochrome P450 (CYP) 3A. Many clinical drugs, including those often prescribed to trauma patients, are known to activate PXR and induce CYP3A. The goal of this study is to determine whether PXR plays a role in the regulation of DMEs in the setting of HS and whether activation of PXR is beneficial or detrimental to HS-induced hepatic injury. PXR transgenic, knockout, and humanized mice were subject to HS, and the liver injury was assessed histologically and biochemically. The expression and/or activity of PXR and CYP3A were manipulated genetically or pharmacologically in order to determine their effects on HS-induced liver injury. Our results showed that genetic or pharmacological activation of PXR sensitized wild-type and hPXR/CYP3A4 humanized mice to HS-induced hepatic injury, whereas knockout of PXR protected mice from HS-induced liver injury. Mechanistically, the sensitizing effect of PXR activation was accounted for by PXR-responsive induction of CYP3A and increased oxidative stress in the liver. The sensitizing effect of PXR was attenuated by ablation or pharmacological inhibition of CYP3A, treatment with the antioxidant N-acetylcysteine amide, or treatment with a PXR antagonist. Conclusion: We have uncovered a function of PXR in HS-induced hepatic injury. Our results suggest that the unavoidable use of PXR-activating drugs in trauma patients has the potential to exacerbate HS-induced hepatic injury, which can be mitigated by the coadministration of antioxidative agents, CYP3A inhibitors, or PXR antagonists.

The protective effect of N-acetylcysteine amide against paraquat-inducedneurotoxicity

N-acetylcysteine amide (NACA) is a new antioxidant molecule with powerful radical scavenging properties. The aim of this study was to investigate neuroprotective effects of NACA against paraquat (PQ) toxicity in the midbrains of rats by using motor coordination tests and biochemical and histological analysis. Thirty adult Wistar albino rats were divided into three groups: Group 1: control (n = 10), Group 2: PQ (10 mg/kg) (n = 10), and Group 3: PQ (10 mg/kg) $+$ NACA (100 mg/kg) (n = 10). NACA was administrated intraperitoneally 30 min before PQ injection. Performance was measured for a period of 28 days. The rotarod and accelerod tests were performed prior to and after the experimental period. After the experimental period, rats were sacrificed and midbrain tissues were removed. According to biochemical data, malondialdehyde levels exhibited a significant increase (P $

Quantitation of free and total N-acetylcysteine amide and its metabolite N-acetylcysteine in human plasma using derivatization and electrospray LC-MS/MS

Studies of N-acetylcysteine amide (NACA) in nonclinical models have demonstrated various antioxidant, anti-apoptotic, anti-inflammatory and neuroprotective effects, and it is currently being developed as a treatment for retinitis pigmentosa. Sensitive LC-MS/MS methods were developed and validated to quantitate reduced and total NACA and its major metabolite, N-acetylcysteine (NAC), in human plasma to support clinical studies involving NACA. To trap and stabilize reduced NACA and NAC at the time of collection, whole blood was immediately treated with 2-chloro-1-methylpyridinium iodide (CMPI) to convert free thiols to 1-methylpyridinyl thioether derivatives. Plasma was harvested and frozen until samples were assayed using protein precipitation and an LC-MS/MS separation based on hydrophilic-interaction chromatography (HILIC). To process NACA and NAC present as disulfides, an intermediate portion of the extract was further subjected to reduction with tris(2-carboxyethyl) phosphine; the released thiols were then reacted with CMPI, extracted, and analyzed as before, to measure total thiols. The method for NACA and NAC, whether free/reduced or total, covered a range from 50 ng/mL to 50 μg/mL in human plasma and required a single 25 μL plasma sample. Up to 180 samples could be assayed in a single session. The inter-run mean bias and precision (%CV) were within ±5% for the free thiol method and within ±8.5% for the total thiol method. Benchtop, freeze/thaw, and long-term stability were evaluated and acceptable. The NAC/NACA method applied to a clinical study demonstrated incurred sample reproducibility of 95.5% for NAC and 99.1% for NACA.

N-acetylcysteine amide provides neuroprotection via Nrf2-ARE pathway in a mouse model of traumatic brain injury.

BackgroundIncreasing evidence demonstrate N-acetylcysteine amide (NACA) provides neuroprotection and attenuated oxidative stress in rats following traumatic brain injury (TBI). The nuclear factor erythroid 2-related factor 2 (Nrf2)–antioxidant response element (ARE) signal pathway is activated after TBI and provides a protective effect against TBI. However, the function and mechanism of NACA in mice after TBI remain unknown. This study was to evaluate the neuroprotection of NACA and the potential action of the Nrf2-ARE pathway in a weight-drop mouse model of TBI.Materials and methodsFour groups of animals were randomly divided into sham, TBI, TBI+vehicle, and TBI+NACA (100 mg/kg, administered intraperitoneally). The protein levels of Nrf2, heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase-1 (NQO1), cleaved caspase-3 and the mRNA levels of HO-1 and NQO1 were detected. The neurobehavior, neuronal degeneration, apoptosis and oxidative stress were also assessed.ResultsTreatment with NACA significantly improved neurologic status at days 1 and 3 following TBI. Moreover, NACA promoted Nrf2 activation a day after TBI. The protein and mRNA levels of HO-1 and NQO1 were upregulated by NACA. Meanwhile, NACA treatment significantly reduced the level of malondialdehyde (MDA) and enhanced the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), which indicated NACA attenuated oxidative stress following TBI. NACA prominently reduced the protein level of cleaved caspase-3 and TUNEL-positive cells, indicating its antiapoptotic effect. Additionally, Fluoro-Jade C staining showed NACA alleviated neuronal degeneration a day after TBI.ConclusionsOur study reveals that NACA potentially provides neuroprotection via the activation of the Nrf2-ARE signaling pathway after TBI in mice.

HIV-1 Tat and methamphetamine co-induced oxidative cellular injury is mitigated by N-acetylcysteine amide (NACA) through rectifying mTOR signaling

Methamphetamine (Meth) is an addictive psychostimulant whose abuse is intimately linked to increased risks for HIV-1 infection. Converging lines of evidence indicate that Meth also aggravates the symptoms of HIV-associated neurocognitive disorders (HAND), though the underlying mechanisms remain poorly understood. By using the lipophilic antioxidant N-acetylcysteine amide (NACA) as an interventional agent, we examined the roles of oxidative stress in autophagy and apoptosis induced by HIV-Tat (the transactivator of transcription), Meth or their combined treatment in human SH-SY5Y neuroblastoma cells and in the rat striatum. Oxidative stress was monitored in terms of the production of intracellular reactive oxygen species (ROS) and antioxidant reserves including glutathione peroxidase (GPx) and Cu,Zn-superoxide dismutase (SOD). NACA significantly reduced the level of ROS and restored GPx and SOD to levels comparable to that of normal control, implying a cytoprotective effect of NACA against oxidative stress elicited by Tat- and/or Meth. Protein expression of mammalian target of rapamycin (mTOR) was measured in SH-SY5Y cells and in the rat striatum to further explore the underlying mechanism of NACA protect against oxidative stress. The results support a beneficial effect of NACA in vivo and in vitro through rectification of the mTOR signaling pathway. Collectively, our study shows that NACA protects against Meth and/or Tat-induced cellular injury in vitro and in the rat striatum in vivo by attenuating oxidative stress, apoptosis and autophagy, at least in part, via modulation of mTOR signaling.

Systematic Review of Human and Animal Studies Examining the Efficacy and Safety of N-Acetylcysteine (NAC) and N-Acetylcysteine Amide (NACA) in Traumatic Brain Injury: Impact on Neurofunctional Outcome and Biomarkers of Oxidative Stress and Inflammation.

No new therapies for traumatic brain injury (TBI) have been officially translated into current practice. At the tissue and cellular level, both inflammatory and oxidative processes may be exacerbated post-injury and contribute to further brain damage. N-acetylcysteine (NAC) has the potential to downregulate both processes. This review focuses on the potential neuroprotective utility of NAC and N-acetylcysteine amide (NACA) post-TBI. Medline, Embase, Cochrane Library, and ClinicalTrials.gov were searched up to July 2017. Studies that examined clinical and laboratory effects of NAC and NACA post-TBI in human and animal studies were included. Risk of bias was assessed in human and animal studies according to the design of each study (randomized or not). The primary outcome assessed was the effect of NAC/NACA treatment on functional outcome, while secondary outcomes included the impact on biomarkers of inflammation and oxidation. Due to the clinical and methodological heterogeneity observed across studies, no meta-analyses were conducted. Our analyses revealed only three human trials, including two randomized controlled trials (RCTs) and 20 animal studies conducted using standardized animal models of brain injury. The two RCTs reported improvement in the functional outcome post-NAC/NACA administration. Overall, the evidence from animal studies is more robust and demonstrated substantial improvement of cognition and psychomotor performance following NAC/NACA use. Animal studies also reported significantly more cortical sparing, reduced apoptosis, and lower levels of biomarkers of inflammation and oxidative stress. No safety concerns were reported in any of the studies included in this analysis. Evidence from the animal literature demonstrates a robust association for the prophylactic application of NAC and NACA post-TBI with improved neurofunctional outcomes and downregulation of inflammatory and oxidative stress markers at the tissue level. While a growing body of scientific literature suggests putative beneficial effects of NAC/NACA treatment for TBI, the lack of well-designed and controlled clinical investigations, evaluating therapeutic outcomes, prognostic biomarkers, and safety profiles, limits definitive interpretation and recommendations for its application in humans at this time.

Cerebellum Susceptibility to Neonatal Asphyxia: Possible Protective Effects of N-Acetylcysteine Amide.

Background After perinatal asphyxia, the cerebellum presents more damage than previously suggested. Objectives To explore if the antioxidant N-acetylcysteine amide (NACA) could reduce cerebellar injury after hypoxia-reoxygenation in a neonatal pig model. Methods Twenty-four newborn pigs in two intervention groups were exposed to 8% oxygen and hypercapnia, until base excess fell to −20 mmol/l or the mean arterial blood pressure declined to <20 mmHg. After hypoxia, they received either NACA (NACA group, n = 12) or saline (vehicle-treated group, n = 12). One sham-operated group (n = 5) served as a control and was not subjected to hypoxia. Observation time after the end of hypoxia was 9.5 hours. Results The intranuclear proteolytic activity in Purkinje cells of asphyxiated vehicle-treated pigs was significantly higher than that in sham controls (p = 0.03). Treatment with NACA was associated with a trend to decreased intranuclear proteolytic activity (p = 0.08), There were significantly less mutations in the mtDNA of the NACA group compared with the vehicle-treated group, 2.0 × 10−4 (±2.0 × 10−4) versus 4.8 × 10−5(±3.6 × 10−4, p < 0.05). Conclusion We found a trend to lower proteolytic activity in the core of Purkinje cells and significantly reduced mutation rate of mtDNA in the NACA group, which may indicate a positive effect of NACA after neonatal hypoxia. Measuring the proteolytic activity in the nucleus of Purkinje cells could be used to assess the effect of different neuroprotective substances after perinatal asphyxia.

Protective Effect of N-Acetylcysteine Amide on Blast-Induced Increase in Intracranial Pressure in Rats.

Blast-induced traumatic brain injury is associated with acute and possibly chronic elevation of intracranial pressure (ICP). The outcome after TBI is dependent on the progression of complex processes which are mediated by oxidative stress. So far, no effective pharmacological protection against TBI exists. In this study, rats were exposed to a single or repetitive blast overpressure (BOP) at moderate intensities of 72 or 110 kPa in a compressed air-driven shock tube. The degree and duration of the increase in ICP were proportional to the intensity and frequency of the blast exposure(s). In most cases, a single dose of antioxidant N-acetylcysteine amide (NACA) (500 mg/kg) administered intravenously 2 h after exposure to BOP significantly attenuated blast-induced increase in ICP. A single dose of NACA was not effective in improving the outcome in the group of animals that were subjected to repetitive blast exposures at 110 kPa on the same day. In this group, two treatments with NACA at 2 and 4 h post-BOP exposure resulted in significant attenuation of elevated ICP. Treatment with NACA prior to BOP exposure completely prevented the elevation of ICP. The findings indicate that oxidative stress plays an important role in blast-induced elevated ICP as treatment with NACA-ameliorated ICP increase, which is frequently related to poor functional recovery after TBI.