NACA is a thiol antioxidant that reduces oxidative stress by increasing glutathione levels, scavenging reactive oxygen species, and restoring antioxidant enzyme activities. [1]

N-acetylcysteine amide exhibited considerable cytotoxicity at 10–20 mM, although it had no discernible influence on the viability of H9c2 cells treated with <1 mM doxorubicin (DOX). N-acetyl cysteine amide (750 μM) decreases ROS levels and lipid peroxidation caused by DOX, and it also restores the GSH/GSSG ratio and antioxidant enzyme activity, including catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) [1]. Methamphetamine (METH)-induced cell death is prevented in human brain microvascular endothelium (HBMVEC) by N-acetylcysteine amide (1 mM) [3].

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Cytotoxicity of NACA in H9c2 Cells: H9c2 cardiomyocytes were exposed to NACA at concentrations ranging from 0.25 mM to 20 mM for 24 hours. Significant cytotoxicity was observed at concentrations ≥10 mM. At 20 mM, cell viability was reduced by approximately 80% (p < 0.01). At 0.75 mM, cell viability was comparable to control group. For comparison, NAC (N-acetylcysteine) induced significant cytotoxicity at concentrations ≥2 mM. [1]
Effect on DOX-Induced Cytotoxicity: Cells pretreated with 750 μM NACA for 2 hours followed by co-treatment with DOX (0.25-100 μM) for 24-72 hours showed minimal protective effects against DOX-induced cell death. The most notable (but still minimal) protection was observed at 5 μM DOX. [1]
Reduction of Reactive Oxygen Species: Treatment with 5 μM DOX increased ROS by 56% compared to control. Co-treatment with 750 μM NACA reduced ROS to control levels (p < 0.05 vs. DOX-only). [1]
Glutathione and Cysteine Levels: DOX (5 μM) significantly reduced intracellular GSH and cysteine levels. NACA (750 μM) co-treatment restored both GSH and cysteine levels to values higher than DOX-only group (p < 0.05). NACA alone significantly increased GSH compared to control (p < 0.05). [1]
GSSG and GSH/GSSG Ratio: DOX increased GSSG by 61% and decreased GSH/GSSG ratio by 47%. NACA co-treatment restored GSSG to control levels and significantly improved GSH/GSSG ratio (p < 0.01 vs. DOX-only). NACA alone increased GSH/GSSG ratio compared to control (p < 0.05). [1]
Lipid Peroxidation: DOX increased MDA (a lipid peroxidation marker) from 0.92 ± 0.02 to 1.55 ± 0.17 nmol/100 mg protein. NACA co-treatment reduced MDA to 0.89 ± 0.02 nmol/100 mg protein (p < 0.05 vs. DOX-only), similar to control. [1]
Catalase Activity: DOX reduced CAT activity from 10.94 ± 2.13 to 4.30 ± 0.45 mU/mg protein (61% reduction). NACA co-treatment restored CAT activity to 13.13 ± 2.17 mU/mg protein (p < 0.05 vs. DOX-only). [1]
Glutathione Peroxidase Activity: DOX reduced GPx activity from 24.77 ± 3.91 to 12.50 ± 1.74 ΔA/min/mg protein (50% reduction). NACA co-treatment restored GPx activity to 19.31 ± 3.07 ΔA/min/mg protein (p < 0.05 vs. DOX-only). [1]
Glutathione Reductase Activity: DOX reduced GR activity from 5.13 ± 0.09 to 0.83 ± 0.04 mU/mg protein (84% reduction). NACA co-treatment restored GR activity to 4.22 ± 0.53 mU/mg protein (p < 0.05 vs. DOX-only). [1]
Comparison with NAC: The study notes that NAC at 140 mg/kg failed to prevent acute DOX-induced cardiotoxicity in previous studies, attributed to its low lipid solubility limiting bioavailability. NACA, with an amide replacing the carboxyl group, has increased lipophilicity allowing better cell membrane penetration. [1]

The central nervous system is more bioavailable when N-acetylcysteine amide is present. In rats with traumatic brain injury (TBI), N-acetylcysteine amide (150 mg/kg, i.p.) increases mitochondrial bioenergetics, decreases oxidative stress, preserves mitochondrial trough, and enhances cortical protection and functional outcomes[2].

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Traumatic Brain Injury Model (Rats): Adult male Sprague-Dawley rats received a moderate (1.5 mm) unilateral controlled cortical impact injury. NACA treatment was administered via multiple protocols: (1) 150 mg/kg IP bolus 30 min post-injury + osmotic mini-pump delivering 18.5 mg/kg/hr for 7 days; (2) 150 mg/kg IP bolus at 5 min, 6, 12, 18, and 24 hrs post-injury. [2]
Neuroprotection (Tissue Sparing): At 15 days post-injury, NACA-treated animals showed significantly increased cortical tissue sparing compared to NAC-treated and vehicle-treated animals (p < 0.05). NAC treatment alone was not significantly different from vehicle. [2]
Cognitive Function (Morris Water Maze): At days 10-15 post-injury, NACA-treated rats swam significantly shorter distances to reach the platform compared to NAC and vehicle-treated animals (p < 0.013 for drug treatment effect). No significant differences in swim speed were observed. [2]
Oxidative Damage: At 7 days post-injury, NACA treatment significantly reduced lipid peroxidation (4-HNE levels) compared to vehicle (p < 0.0001). No significant reduction in protein nitrosylation (3-NT levels) was observed. [2]
Mitochondrial Bioenergetics: At 25 hrs post-injury, vehicle-treated animals showed 50-60% reduction in mitochondrial State III, State VFCCP, and State Vsucc respiration compared to sham. NACA treatment significantly improved all mitochondrial respiratory parameters to levels not significantly different from sham. No significant differences in State IV respiration were observed. [2]
Mitochondrial Glutathione Content: At 25 hrs post-injury, vehicle-treated animals showed 21-23% reduction in mitochondrial total and reduced GSH compared to sham. NACA treatment maintained total and reduced GSH at levels not significantly different from sham. Oxidized GSH (GSSG) did not change significantly between groups. [2]
Comparison with NAC: NACA showed superior efficacy to NAC in tissue sparing and cognitive outcomes. NAC treatment alone did not significantly improve these measures compared to vehicle. [2]
Safety in Naïve Animals: NACA administration (150 mg/kg every 6 hrs for 24 hrs) to uninjured rats did not alter mitochondrial respiration or glutathione content compared to vehicle, demonstrating no toxic effects and good tolerability. [2]

Catalase Activity Assay: Cell homogenates were diluted with 50 mM phosphate buffer (pH 7.0). The exponential decrease of 10 mM hydrogen peroxide was measured at 240 nm in the presence of cell homogenate. Reaction mixtures without cell homogenate were used as blanks. [1]
Glutathione Peroxidase Activity Assay: GPx activity was determined by the method of Paglia and Valentine. tert-Butyl hydroperoxide was reduced by GPx and then recycled by GR coupled with NADPH oxidation. The rate of decrease in NADPH was monitored spectrophotometrically at 340 nm. [1]
Glutathione Reductase Activity Assay: GR activity was determined using the method of Carlberg and Mannervik. The NADPH-dependent conversion of GSSG to GSH was measured by monitoring the exponential decrease of NADPH at 340 nm. Reaction mixtures without GSSG were used as blanks. [1]

Cell Culture: H9c2 cardiomyocytes (rat embryonic heart tissue-derived) were cultured in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 1% penicillin/streptomycin at 37°C in 5% CO₂. Cells were subcultured every 3 days. [1]
Cytotoxicity Assay (MTS): Cells were seeded in 96-well plates. After treatment, MTS reagent was added and incubated. Absorbance at 490 nm was measured, which is proportional to the number of living cells. [1]
Intracellular ROS Measurement: After treatment, cells were incubated with 5 μM DCFH-DA for 20 min at 37°C. DCF fluorescence was measured at excitation 485 nm, emission 520 nm. [1]
GSH and Cysteine Measurement by HPLC: Cell homogenates were derivatized with NPM (1 mM in acetonitrile) in serine borate buffer. After 5 min, samples were acidified with 2N HCl, filtered, and injected onto HPLC column. Fluorescence detection at λex = 330 nm, λem = 376 nm. [1]
GSSG Measurement: Cell homogenates were treated with 2-vinylpyridine to block GSH thiol groups. NADPH and glutathione reductase were added to reduce GSSG. Samples were then derivatized with NPM and fluorescence measured. [1]
Lipid Peroxidation Measurement (MDA): Cell homogenate was mixed with butylated hydroxytoluene and trichloroacetic acid, boiled for 30 min. Supernatant was removed, thiobarbituric acid added, then extracted with n-butanol. Fluorescence measured at λex = 515 nm, λem = 550 nm. [1]
Protein Determination: Protein concentration was determined by Bradford method using Coomassie blue with BSA as standard, measuring absorbance at 595 nm. [1]

Animals:** Adult male Sprague-Dawley rats (300-350 g) were housed for 7 days prior to surgery with free food and water access. [2]
* **Surgery (Controlled Cortical Impact):** Rats were anesthetized with 4% isoflurane, maintained at 2.5% during surgery. A 6-mm craniotomy was made lateral to sagittal suture, centered between bregma and lambda. Moderate injury was induced using a pneumatically controlled impactor with 5-mm tip, 3.5 m/s velocity, 1.5 mm depth. Sham animals received craniotomy without impact. Body temperature maintained at 37°C. [2]
* **NACA Administration (Tissue Sparing/Behavioral Study):** Rats (n=6-8/group) received: (1) NACA: 150 mg/kg IP bolus 30 min post-injury + osmotic mini-pump (18.5 mg/kg/hr for 7 days); (2) NAC: same regimen; (3) Vehicle: equivalent volume. Experimenters blinded to treatment. [2]
* **NACA Administration (Mitochondrial Study):** Rats (n=5/group) received: (1) NACA: 150 mg/kg IP at 5 min, 6, 12, 18, 24 hrs post-injury; (2) Vehicle: equivalent volume saline at same time points; (3) Sham: no treatment. Euthanized at 25 hrs post-injury. [2]
* **NACA Administration (Naïve Study):** Uninjured rats (n=3/group) received NACA or vehicle IP at 0, 6, 12, 18, 24 hrs. Euthanized at 25 hrs. [2]
* **Morris Water Maze:** Testing began at 10 days post-surgery, consisting of 4 daily trials for 5 consecutive days. Pool diameter 170 cm, platform 13 cm diameter submerged 2 cm below water surface. Swimming performance recorded and analyzed. [2]
* **Tissue Processing for Histology:** At 15 days post-injury, rats were perfused with PBS followed by 4% paraformaldehyde. Brains post-fixed in 4% paraformaldehyde-15% sucrose, sectioned at 50 μm, stained with cresyl violet. Cortical damage assessed using unbiased Cavalieri method. [2]
* **Immunohistochemistry for Oxidative Stress:** At 7 days post-injury, 50 μm sections were stained for 4-HNE (lipid peroxidation) and 3-NT (protein nitrosylation). Sections were reduced with NaBH4, blocked, incubated with primary antibodies (rabbit anti-HNE, mouse anti-3-NT), then with fluorescent secondary antibodies. Imaging performed on Li-COR Odyssey system. [2]
* **Mitochondrial Isolation:** At 25 hrs post-injury, brains rapidly removed, cortical tissue homogenized in mitochondrial isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 1 mM EGTA, 20 mM HEPES, pH 7.2). Differential centrifugation steps followed by nitrogen bomb (1200 psi, 10 min) to release synaptosomal mitochondria. Final pellet resuspended in buffer without EGTA (~10 mg/mL protein). [2]
* **Mitochondrial Bioenergetics (Seahorse XF24):** Mitochondria (50 μg) were plated in respiration buffer (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES, 2 mM MgCl, 2.5 mM KH₂PO₄, pH 7.2). Sequential injections: Port A - pyruvate (5 mM), malate (2.5 mM), ADP (1 mM) for State III; Port B - oligomycin A (1 μM) for State IV; Port C - FCCP (4 μM) for State VFCCP; Port D - rotenone (0.1 μM) and succinate (10 mM) for State Vsucc. Oxygen consumption rates measured and expressed as % of sham. [2]
* **Glutathione Assay:** Mitochondrial protein (200-300 μg) treated with 5% metaphosphoric acid to remove proteins, centrifuged at 14,000g for 15 min at 4°C. Supernatant collected and stored at -80°C. Total, reduced, and oxidized GSH measured using commercial assay kit (Enzo Life Sciences) at absorbance 414 nm. Results expressed as % of sham. [2]

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Animals: Adult male Sprague-Dawley rats (300-350 g) were housed for 7 days prior to surgery with free food and water access. [2]
Surgery (Controlled Cortical Impact): Rats were anesthetized with 4% isoflurane, maintained at 2.5% during surgery. A 6-mm craniotomy was made lateral to sagittal suture, centered between bregma and lambda. Moderate injury was induced using a pneumatically controlled impactor with 5-mm tip, 3.5 m/s velocity, 1.5 mm depth. Sham animals received craniotomy without impact. Body temperature maintained at 37°C. [2]
NACA Administration (Tissue Sparing/Behavioral Study): Rats (n=6-8/group) received: (1) NACA: 150 mg/kg IP bolus 30 min post-injury + osmotic mini-pump (18.5 mg/kg/hr for 7 days); (2) NAC: same regimen; (3) Vehicle: equivalent volume. Experimenters blinded to treatment. [2]
NACA Administration (Mitochondrial Study): Rats (n=5/group) received: (1) NACA: 150 mg/kg IP at 5 min, 6, 12, 18, 24 hrs post-injury; (2) Vehicle: equivalent volume saline at same time points; (3) Sham: no treatment. Euthanized at 25 hrs post-injury. [2]
NACA Administration (Naïve Study): Uninjured rats (n=3/group) received NACA or vehicle IP at 0, 6, 12, 18, 24 hrs. Euthanized at 25 hrs. [2]
Morris Water Maze: Testing began at 10 days post-surgery, consisting of 4 daily trials for 5 consecutive days. Pool diameter 170 cm, platform 13 cm diameter submerged 2 cm below water surface. Swimming performance recorded and analyzed. [2]
Tissue Processing for Histology: At 15 days post-injury, rats were perfused with PBS followed by 4% paraformaldehyde. Brains post-fixed in 4% paraformaldehyde-15% sucrose, sectioned at 50 μm, stained with cresyl violet. Cortical damage assessed using unbiased Cavalieri method. [2]
Immunohistochemistry for Oxidative Stress: At 7 days post-injury, 50 μm sections were stained for 4-HNE (lipid peroxidation) and 3-NT (protein nitrosylation). Sections were reduced with NaBH4, blocked, incubated with primary antibodies (rabbit anti-HNE, mouse anti-3-NT), then with fluorescent secondary antibodies. Imaging performed on Li-COR Odyssey system. [2]
Mitochondrial Isolation: At 25 hrs post-injury, brains rapidly removed, cortical tissue homogenized in mitochondrial isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 1 mM EGTA, 20 mM HEPES, pH 7.2). Differential centrifugation steps followed by nitrogen bomb (1200 psi, 10 min) to release synaptosomal mitochondria. Final pellet resuspended in buffer without EGTA (~10 mg/mL protein). [2]
Mitochondrial Bioenergetics (Seahorse XF24): Mitochondria (50 μg) were plated in respiration buffer (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES, 2 mM MgCl, 2.5 mM KH₂PO₄, pH 7.2). Sequential injections: Port A - pyruvate (5 mM), malate (2.5 mM), ADP (1 mM) for State III; Port B - oligomycin A (1 μM) for State IV; Port C - FCCP (4 μM) for State VFCCP; Port D - rotenone (0.1 μM) and succinate (10 mM) for State Vsucc. Oxygen consumption rates measured and expressed as % of sham. [2]
Glutathione Assay: Mitochondrial protein (200-300 μg) treated with 5% metaphosphoric acid to remove proteins, centrifuged at 14,000g for 15 min at 4°C. Supernatant collected and stored at -80°C. Total, reduced, and oxidized GSH measured using commercial assay kit (Enzo Life Sciences) at absorbance 414 nm. Results expressed as % of sham. [2]

Structural Modification for Improved Bioavailability: NACA is a structural analogue of NAC where the carboxyl group is replaced with an amide group. This modification increases lipophilicity, allowing it to cross cell membranes more effectively. The document notes that NAC is negatively charged at physiological pH due to its carboxyl group, limiting its ability to cross cell membranes. [1]
Blood-Brain Barrier Penetration: The study references previous work showing that NACA can cross the blood-brain barrier. [1]
Chelation Properties: NACA has been shown to chelate Cu²⁺, which catalyzes free radical formation. [1]

Cytotoxicity Threshold: In H9c2 cells, NACA showed no significant cytotoxicity at concentrations ≤1 mM. Significant cytotoxicity was observed at ≥10 mM, with 80% cell death at 20 mM. The study used 750 μM (0.75 mM) as a safe working concentration. [1]
Comparison with NAC: NACA was less toxic than NAC in H9c2 cells. NAC induced significant cytotoxicity at concentrations ≥2 mM, while NACA's toxicity threshold was ≥10 mM. [1]

[1]. N-acetylcysteine amide decreases oxidative stress but not cell death induced by doxorubicin in H9c2 cardiomyocytes. BMC Pharmacol. 2009 Apr 15;9:7.

[2]. N-acetylcysteine amide confers neuroprotection, improves bioenergetics and behavioral outcome following TBI. Exp Neurol. 2014 Jul;257:106-13.

[3]. N-Acetylcysteine amide protects against methamphetamine-induced oxidative stress and neurotoxicity in immortalized human brain endothelial cells. Brain Res. 2009 Jun 12;1275:87-95.

N-acetylcysteine amide is the amide form of N-acetylcysteine (NAC), a synthetic N-acetyl derivative and a prodrug of L-cysteine, an endogenous amino acid and precursor to the antioxidant glutathione (GSH), possessing potential antioxidant and anti-inflammatory activities. N-acetylcysteine amide (NACA) administration can increase GSH levels. GSH can scavenge reactive oxygen species (ROS), reduce oxidative stress, and prevent ROS-mediated cell damage and apoptosis. Compared to NAC, NACA has higher lipophilicity and membrane permeability.

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Background: N-Acetylcysteine amide (NACA) is a relatively new thiol antioxidant and a structural analogue of N-acetylcysteine (NAC). It was synthesized by replacing the carboxyl group of NAC with an amide group to increase lipophilicity and improve cell membrane penetration. [1]
Mechanism of Action (Antioxidant): NACA reduces oxidative stress through multiple mechanisms: (1) supplying cysteine for GSH synthesis; (2) converting GSSG to GSH through non-enzymatic thiol-disulfide exchange; (3) directly scavenging free radicals; (4) chelating metal ions (Cu²⁺) that catalyze free radical formation; (5) preserving activities of antioxidant enzymes (CAT, GPx, GR). [1]
Study Context: This is the first report investigating the chemoprotective efficacy of NACA against doxorubicin-induced cardiotoxicity in cardiomyocytes. [1]
Key Finding (Dissociation of Oxidative Stress and Cell Death): Despite effectively reducing all measured parameters of oxidative stress (ROS, GSH depletion, lipid peroxidation, enzyme inactivation), NACA had minimal protective effect against DOX-induced cell death. This suggests that DOX-induced cytotoxicity involves oxidative stress-independent mechanisms, such as topoisomerase II inhibition leading to DNA damage and apoptosis. [1]
Potential Applications: Based on its antioxidant properties and improved bioavailability compared to NAC, NACA may have therapeutic potential in conditions involving oxidative stress. However, its inability to prevent DOX-induced cell death in this model highlights the complexity of DOX cardiotoxicity. [1]

C5H10N2O2S

162.2101

162.046

38520-57-9

10176265

White to off-white solid powder

0.896

3

3

3

10

149

1

CC(N[C@@H](CS)C(N)=O)=O

UJCHIZDEQZMODR-BYPYZUCNSA-N

InChI=1S/C5H10N2O2S/c1-3(8)7-4(2-10)5(6)9/h4,10H,2H2,1H3,(H2,6,9)(H,7,8)/t4-/m0/s1

(2R)-2-acetamido-3-sulfanylpropanamide

NACANac amide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~200 mg/mL (~1232.97 mM)
DMSO : ≥ 100 mg/mL (~616.48 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (15.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (15.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (616.48 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)