Nuclear Factor-kappa B (NF-κB) pathway (inhibition of IκBα degradation, IKK activity, and NF-κB transcription) [1]

Erdosteine is an oral mucolytic drug that is utilized in many chronic respiratory disorders as an expectorant. By preventing NF-κB activation in LPS-stimulated murine macrophages, eugenosteine reduces inflammation. Erdosteine, however, does not prevent the phosphorylation of the Akt and MAPK pathways brought on by LPS. Cell viability was assessed in order to assess the harmful effects of erdosteine on macrophages. Erdosteine at 1, 10, or 100 μg/mL did not cause any observable cytotoxicity. In RAW 264, LPS (1 μg/mL) treatment caused IκBα degradation.7 cells, RAW 264, with the highest deterioration seen after 10 minutes.Erdosteine at the specified concentrations was pretreated for 6 hours on 7 cells, and after that, LPS (1 μg/mL) was added after 10 minutes. The baseline levels of IκBα were unaffected by the ergotoxine pretreatment. Baseline levels of IκBα were unaffected by treatment with DMSO alone in a volume equivalent to that used for erdosteine administration. After 10 minutes of LPS treatment, there was a drop in the quantity of IκBα. IκBα degradation can be successfully inhibited by pretreatment with erdosteine at the prescribed concentration and time [1].

---

In LPS-stimulated RAW 264.7 mouse macrophage cells, pretreatment with Erdosteine (10 μg/ml for 6 hours) inhibited the degradation of IκBα. This effect was concentration-dependent, with 10 μg/ml showing inhibition while 1 μg/ml did not. [1]
Erdosteine pretreatment (10 μg/ml for 6 hours) attenuated the LPS-induced nuclear translocation of the NF-κB p65 subunit, decreasing its level in the nuclear fraction and increasing it in the cytosolic fraction. [1]
In an immunoprecipitation kinase assay, Erdosteine (10 μg/ml) inhibited LPS-induced IKK activity, as measured by the phosphorylation of GST-IκBα. [1]
Using a dual-luciferase reporter assay, Erdosteine (10 μg/ml) inhibited LPS-induced NF-κB promoter transcriptional activity in RAW 264.7 cells. [1]
Erdosteine treatment (10 μg/ml) significantly suppressed the LPS-induced increase in IL-6 and IL-1β production in RAW 264.7 cell supernatants, as measured by ELISA. [1]
Erdosteine treatment (1, 10, 100 μg/ml) did not exhibit cytotoxicity in RAW 264.7 cells after 24 hours, as determined by an MTS assay. [1]
Erdosteine treatment (1, 10, 100 μg/ml) effectively attenuated the LPS-induced increase in intracellular reactive oxygen species (ROS) levels in RAW 264.7 cells in a concentration-dependent manner, as measured by DCFH-DA staining and flow cytometry. [1]
Erdosteine pretreatment (up to 100 μg/ml) did not inhibit LPS-induced phosphorylation of Akt, MEK, ERK, JNK, or p38 MAPK in RAW 264.7 cells, as determined by Western blotting. [1]

The following four groups of 26 male mice were created: Groups 1–4: control; Group 2–4: methotrexate (MTX) treatment; Group 5–6: MTX + Erdosteine treatment. On the first day of the trial, Groups 2 and 4 received a single oral dose of erdosteine every day for seven days, whereas Groups 3 and 4 received a single intraperitoneal injection of methotrexate. Upon completion of the study, the animals' testicles were extracted and weighed. Comparing the methotrexate group to the control group, there was a significant difference (p<0.05) in the total antioxidant capacity, total oxidative stress level, and myeloperoxidase activity. Between the methotrexate group and the control group, there was no difference in the levels of lipid peroxidation. In conclusion, methotrexate-induced toxicity to the testicles is effectively prevented by erdosteine. By causing spermatogenesis to develop in the seminiferous tubules, co-administration of erdosteine and methotrexate heals testicular injury [2].

---

In a mouse model of methotrexate (MTX)-induced testicular toxicity, oral administration of Erdosteine at 10 mg/kg/day for 7 days, starting on the same day as a single intraperitoneal injection of MTX (10 mg/kg), significantly reduced the MTX-induced elevation of testicular myeloperoxidase (MPO) activity. MPO activity in the MTX + Erdosteine group was 0.83 ± 0.05 U/g protein, compared to 1.50 ± 0.16 U/g protein in the MTX-alone group (p < 0.001). [2]
Erdosteine treatment significantly reduced the MTX-induced elevation of total oxidative stress (TOS) levels in testicular tissue. TOS levels in the MTX + Erdosteine group were 16 ± 2 mmol H2O2 equiv./g protein, compared to 33 ± 2 mmol H2O2 equiv./g protein in the MTX-alone group (p < 0.001). [2]
Erdosteine treatment did not significantly alter the MTX-induced elevation of total antioxidant capacity (TAC) levels in testicular tissue. TAC levels in the MTX + Erdosteine group were 1.40 ± 0.11 mmol Trolox equiv./g protein, compared to 1.38 ± 0.10 mmol Trolox equiv./g protein in the MTX-alone group (p > 0.05). [2]
Erdosteine treatment significantly reduced the oxidative stress index (OSI) compared to the MTX-alone group. The OSI in the MTX + Erdosteine group was 1.19 ± 0.07, compared to 2.23 ± 0.10 in the MTX-alone group (p = 0.006). [2]
Histological examination of testicular tissue showed that Erdosteine administration with MTX improved testicular injuries, such as seminiferous tubule atrophy, vacuolization, and cellular desquamation, which were clearly observed in mice treated with MTX alone. The appearance of spermatogenesis in seminiferous tubules was noted in the MTX + Erdosteine group. [2]

To assess the effect of Erdosteine on IKK activity, an in vitro immune complex kinase assay was performed. RAW 264.7 cells were pretreated with Erdosteine (10 μg/ml) for 6 hours and then stimulated with LPS (1 μg/ml) for 5 minutes. Cell extracts were prepared and immunoprecipitated with an anti-IKKα antibody. The kinase reaction was carried out by incubating the immunoprecipitated complex with a buffer containing recombinant GST-IκBα (0.5 mg) and [γ-³²P]ATP (10 μCi) at 30°C for 30 minutes. The reaction products were separated by SDS-PAGE, transferred to a membrane, and visualized by autoradiography. The results showed that Erdosteine inhibited LPS-induced IKK activity. [1]

Cell Viability (MTS Assay): RAW 264.7 cells (1 x 10⁴ cells/well) were incubated with various concentrations of Erdosteine (1, 10, 100 μg/ml) for 24 hours. MTS solution (10 μl of 5 mg/ml) was then added to each well and incubated for 45 minutes. Cells were lysed with isopropyl alcohol, and absorbance was measured at 540 nm to assess viability. No cytotoxicity was observed at the tested concentrations. [1]
ROS Measurement: RAW 264.7 cells were treated with varying concentrations of Erdosteine (1, 10, 100 μg/ml) and LPS (1 μg/ml) for 6 hours. Cells were then incubated with 15 μM DCFH-DA for 30 minutes at 37°C. Intracellular ROS levels were immediately analyzed by flow cytometry. Erdosteine attenuated the LPS-induced ROS increase in a concentration-dependent manner. [1]
Western Blotting for IκBα, NF-κB p65, and Kinases: RAW 264.7 cells were pretreated with Erdosteine (at indicated concentrations and times) and then stimulated with LPS (1 μg/ml). Total, cytosolic, or nuclear proteins were extracted. Proteins (30 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific primary antibodies (e.g., anti-IκBα, anti-NF-κB p65, anti-phospho-Akt, anti-phospho-MAPKs) and HRP-conjugated secondary antibodies. Bands were visualized using an ECL kit. Results showed that Erdosteine inhibited IκBα degradation and p65 nuclear translocation but did not affect Akt or MAPK phosphorylation. [1]
NF-κB Luciferase Reporter Assay: RAW 264.7 cells were co-transfected with an NF-κB firefly luciferase reporter vector and a Renilla luciferase control vector (pRL-TV) using Lipofectamine. After 48 hours, cells were treated with Erdosteine (10 μg/ml) for 6 hours, followed by LPS (1 μg/ml) stimulation for 6 hours. Luciferase activity was measured using a luminometer and the Dual-Luciferase Reporter Assay System. Firefly luciferase activity was normalized to Renilla activity. Erdosteine inhibited LPS-induced NF-κB transcriptional activity. [1]
ELISA for Cytokines: RAW 264.7 cells (3 x 10⁵ cells/well) were treated with Erdosteine (10 μg/ml) and LPS (1 μg/ml) for 24 hours. Supernatants were collected, and concentrations of IL-6 and IL-1β were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Erdosteine significantly suppressed LPS-induced IL-6 and IL-1β production. [1]

The study used 26 male C57BL/6 mice (8 weeks old, 20-30 g). They were randomly divided into four groups. The Erdosteine group (n=6) received Erdosteine orally by gavage at a dose of 10 mg/kg daily for 7 days. The MTX + Erdosteine group (n=7) received a single intraperitoneal injection of methotrexate (10 mg/kg) on the first day, followed by oral administration of Erdosteine (10 mg/kg) daily for 7 days, starting on the same day. At the end of the experiment, animals were sacrificed by cervical dislocation, and testes were removed for biochemical and histological analysis. [2]

---

The study used 26 male C57BL/6 mice (8 weeks old, 20-30 g). They were randomly divided into four groups. The Erdosteine group (n=6) received Erdosteine orally by gavage at a dose of 10 mg/kg daily for 7 days. The MTX + Erdosteine group (n=7) received a single intraperitoneal injection of methotrexate (10 mg/kg) on the first day, followed by oral administration of Erdosteine (10 mg/kg) daily for 7 days, starting on the same day. At the end of the experiment, animals were sacrificed by cervical dislocation, and testes were removed for biochemical and histological analysis. [2]

Erdosteine is quickly absorbed after oral administration and is rapidly transformed through first-pass metabolism to its biologically active metabolite, N-thiodiglycolyl-homocysteine (M1). [1]
Following an oral dose of 300 mg, the peak plasma concentration (Cmax) of Erdosteine is 1.26 ± 0.23 μg/ml, which is reached at a time (Tmax) of 1.18 ± 0.26 hours after administration. [1]

[1]. Anti-inflammatory Effect of Erdosteine in Lipopolysaccharide-Stimulated RAW 264.7 Cells. Inflammation. 2016 Aug;39(4):1573-81.

[2]. Beneficial effect of erdosteine on methotrexate-induced testicular toxicity in mice. Toxicol Ind Health. 2010 Aug;26(7):433-8.

[3]. Recipharm’s proprietary molecule Erdosteine recognised as COPD treatment.

Erdosteine is an N-acyl amino acid. Erdosteine is a mucus-dissolving drug, also known as a mucolytic. It is a thiol derivative used to treat chronic obstructive bronchitis and its infectious acute exacerbations. The drug contains a sulfhydryl group, released after first-pass metabolism in the liver. The metabolites are three active metabolites that, in addition to their mucolytic activity, also possess free radical scavenging activity. Erdosteine works by controlling mucus production and viscosity while enhancing mucociliary clearance. It also combats free radicals generated by cigarette smoke. Clinical trials have shown that erdosteine is safe and well-tolerated. Taking 300 mg of erdosteine twice daily reduces cough (including cough frequency and severity) and reduces sputum viscosity faster and more effectively than placebo, and is more effective at reducing sputum adhesion than taking 30 mg of ambroxol twice daily. In patients with acute exacerbations of chronic bronchitis, the combination of erdosteine and amoxicillin resulted in higher antibiotic concentrations in sputum and faster, more significant improvement in clinical symptoms compared to placebo. Erdosteine has a low incidence of adverse reactions, most of which are gastrointestinal and usually mild. Erdosteine is a homocysteine-derived thiol derivative with mucolytic and free radical scavenging properties. Erdosteine and its metabolites improve sputum expectoration by regulating mucus production and viscosity, promoting mucociliary clearance. The drug also inhibits chemically induced cough reflexes and protects lung tissue from smoking-induced damage by scavenging free radicals. Indications: For the treatment of chronic bronchitis in adults. Mechanism of Action: Erdosteine is an oral mucolytic. It belongs to the thiol derivative class and is used to treat symptoms of chronic obstructive bronchitis. Erdosteine contains thiol groups, which are released after first-pass metabolism in the liver. Its three active metabolites possess mucolytic and free radical scavenging activities. Erdosteine increases sputum expectoration by regulating airway mucus production and viscosity, while enhancing mucociliary clearance. Erdosteine also inhibits the effects of free radicals in cigarette smoke. Clinical studies in patients with chronic obstructive pulmonary disease (COPD) have shown that the drug is generally safe and well-tolerated. Twice-daily administration of 300 mg erdosteine, compared to placebo, provides faster and more effective relief of cough (including cough frequency and severity) and reduces sputum viscosity; compared to twice-daily administration of 30 mg ambroxol, it is more effective in reducing sputum adhesion. In patients with acute exacerbations of chronic bronchitis, erdosteine in combination with amoxicillin increases the concentration of antibiotics in sputum, resulting in faster and more significant symptom relief compared to placebo. Erdosteine has a low incidence of adverse reactions, most of which are gastrointestinal and usually mild.

---

Erdosteine is a mucolytic agent used for chronic pulmonary diseases. It produces an active metabolite (Met 1) that contains an SH group, which is responsible for its antioxidant effects. [2]
In the context of this study, the protective effect of Erdosteine against MTX-induced testicular toxicity is attributed to its ability to inhibit neutrophil infiltration (as indicated by reduced MPO activity) and reduce oxidative stress (as indicated by reduced TOS and OSI). [2]
The study suggests that Erdosteine may have a clinical benefit in protecting against testicular damage in patients receiving MTX chemotherapy, but further research is needed. [2]

249.012

84611-23-4

Erdosteine-13C4;Erdosteine-d4

65632

White to off-white solid powder

1.5±0.1 g/cm3

590.4±50.0 °C at 760 mmHg

156-160°C

310.8±30.1 °C

0.0±3.6 mmHg at 25°C

1.615

-0.32

2

6

5

15

282

0

O=C(CSCC(NC1CCSC1=O)=O)O

QGFORSXNKQLDNO-UHFFFAOYSA-N

InChI=1S/C8H11NO4S2/c10-6(3-14-4-7(11)12)9-5-1-2-15-8(5)13/h5H,1-4H2,(H,9,10)(H,11,12)

2-[2-oxo-2-[(2-oxothiolan-3-yl)amino]ethyl]sulfanylacetic acid

Erdosteine PV144 PV 144 RV 144 RV144 PV-144

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)

DMSO : ~50 mg/mL (~200.55 mM)
H2O : ~6.67 mg/mL (~26.75 mM)

Solubility in Formulation 1: ≥ 3.25 mg/mL (13.04 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 3.25 mg/mL (13.04 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 32.5 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: ≥ 3.25 mg/mL (13.04 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 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 6.67 mg/mL (26.75 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.)