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].

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].

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]

[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.

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.

---

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.

---

View More

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.

---

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.

---

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