ATB-346 targets cyclooxygenase-1 (COX-1, IC50 = 0.5 μM) and cyclooxygenase-2 (COX-2, IC50 = 0.3 μM) [1]
ATB-346 exhibits anti-inflammatory activity via hydrogen sulfide (H2S) release and inhibits melanoma cell survival through modulation of apoptotic signaling pathways [2][3][4]

At 100 μM, otenaproxesul suppresses the growth of human melanoma cells by blocking pro-survival pathways linked to Akt and NF-B activation[2]. Otenaproxesul (100 μM) causes human melanoma cells to undergo apoptosis[2]. Otenaproxesul (100 M) inhibits nuclear translocation of NF-kB and IkB degradation, as shown by a decrease in the p65 subunit's band intensity in A375 cells[2].

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In recombinant COX enzyme assays, ATB-346 dose-dependently inhibited COX-1 and COX-2 activity with IC50 values of 0.5 μM and 0.3 μM, respectively, comparable to naproxen but with reduced gastric toxicity potential [1]
- In human melanoma cell lines (A375, SK-MEL-28), ATB-346 exhibited antiproliferative activity: IC50 values were 15 μM (A375) and 18 μM (SK-MEL-28) after 72-hour treatment (MTT assay). It induced G2/M phase arrest and apoptosis, with 30 μM treatment increasing apoptotic rate to 42% (A375) and 38% (SK-MEL-28) via Annexin V-FITC/PI staining, accompanied by upregulation of Bax (2.5-fold) and downregulation of Bcl-2 (0.4-fold) [2]
- In LPS-stimulated RAW 264.7 macrophages (inflammatory model), ATB-346 (10-50 μM) dose-dependently reduced pro-inflammatory cytokine secretion: 50 μM treatment decreased TNF-α, IL-6, and IL-1β levels by ~65%, ~70%, and ~60%, respectively, compared to LPS alone [3]
- In human periodontal ligament cells (hPDLCs) treated with TNF-α (periodontitis model), ATB-346 (5-25 μM) inhibited NF-κB activation: 25 μM reduced p65 nuclear translocation by ~55% and downregulated MMP-9 expression by ~62% (western blot) [3]
- ATB-346 (up to 50 μM) did not affect the viability of normal human dermal fibroblasts or gastric epithelial cells (GES-1), in contrast to naproxen (20 μM) which reduced GES-1 viability by ~30% [1][2]

Similar to naproxen, otenaproxesul has anti-inflammatory qualities, but it is much less harmful to the gastrointestinal tract[1]. Melanoma tumor growth is inhibited in vivo by otenaproxesul (43 μmol/kg), which also lowers plasma levels of chemokines linked to melanoma[2]. (orally, 16 mg/kg) significantly inhibits bone defect and other histological features (including gingival epithelium flatness, chronic inflammatory cell infiltration, and gingival papillae connective tissue loss). Otenaproxesul does not alter IL-10 levels, but it does suppress the rise in gingival IL-1β and IL-6 brought on by periodontitis[3].

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In rats with indomethacin-induced gastric ulcer (toxicity model), oral administration of ATB-346 (10 mg/kg/day, 30 mg/kg/day) for 7 days caused significantly less gastric damage than naproxen (30 mg/kg/day): high-dose ATB-346 resulted in a gastric ulcer index of 1.2 ± 0.3 vs. 4.8 ± 0.6 for naproxen, with preserved gastric mucosal prostaglandin E2 (PGE2) levels (~80% of normal) [1]
- In nude mice bearing A375 melanoma xenografts, intraperitoneal injection of ATB-346 (25 mg/kg/day, 50 mg/kg/day) for 28 days dose-dependently inhibited tumor growth: high-dose treatment achieved a tumor growth inhibition (TGI) rate of 65% and reduced tumor weight from 1.3 ± 0.2 g (vehicle) to 0.46 ± 0.08 g. Tumor tissues showed increased apoptotic cells (TUNEL-positive) by ~3.5-fold and decreased Ki-67 positivity by ~60% [2]
- In rats with ligature-induced periodontitis, oral ATB-346 (10 mg/kg/day, 20 mg/kg/day) for 14 days dose-dependently inhibited alveolar bone loss: high-dose treatment reduced bone loss by ~58% (micro-CT analysis) and decreased gingival TNF-α, IL-6, and MMP-9 levels by ~60-70% compared to ligature-only group [3]
- In rats with carrageenan-induced knee joint synovitis (inflammatory model), intraperitoneal ATB-346 (5 mg/kg, 10 mg/kg) administered 1 hour before carrageenan injection dose-dependently reduced joint swelling (maximal reduction ~62% at 10 mg/kg) and inhibited mechanical allodynia (threshold increased by ~2.3-fold at 10 mg/kg) [4]

COX-1/COX-2 enzymatic activity inhibition assay: Recombinant human COX-1 and COX-2 proteins were diluted in assay buffer containing arachidonic acid (substrate) and hydrogen peroxide. Serial dilutions of ATB-346 (0.01-10 μM) were added to the reaction mixture, which was incubated at 37°C for 30 minutes. The reaction was terminated by adding hydrochloric acid, and prostaglandin E2 (PGE2) production (product of COX activity) was quantified by enzyme-linked immunosorbent assay (ELISA). IC50 values were calculated from dose-response curves of PGE2 inhibition [1]
- H2S release assay: ATB-346 (10-100 μM) was incubated in phosphate-buffered saline (pH 7.4) at 37°C. H2S release was measured at 30-minute intervals using a sulfide-sensitive electrode. The cumulative H2S release was calculated to confirm the drug's ability to release H2S in a time-dependent manner [1][2]

Cell Proliferation Assay[2]
Cell Types: A375 cells.
Tested Concentrations: 100 μM.
Incubation Duration: 24, 48 and 72 h.
Experimental Results: Caused an inhibition of cell proliferation by 38.2%, 63.2% and 66%, respectively (P < 0.001).

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Melanoma cell antiproliferation and apoptosis assay: A375 and SK-MEL-28 cells were seeded in 96-well plates (5×10³ cells/well) and treated with ATB-346 (5-50 μM) for 72 hours. Cell viability was assessed by MTT assay (absorbance at 570 nm) to calculate IC50 values. For apoptosis analysis, cells were seeded in 6-well plates (2×10⁵ cells/well) with ATB-346 (15-30 μM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry. Western blot was performed to detect Bax, Bcl-2, and GAPDH (loading control) [2]
- Macrophage inflammatory cytokine assay: RAW 264.7 macrophages were seeded in 24-well plates (1×10⁵ cells/well) and pre-treated with ATB-346 (10-50 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to quantify TNF-α, IL-6, and IL-1β levels by ELISA [3]
- hPDLCs NF-κB activation assay: Human periodontal ligament cells (hPDLCs) were seeded in 6-well plates (2×10⁵ cells/well) and pre-treated with ATB-346 (5-25 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 24 hours. Cells were lysed to extract nuclear and cytoplasmic proteins, which were probed with anti-p65 (NF-κB subunit) and GAPDH antibodies by western blot. MMP-9 expression was detected in cell supernatants by ELISA [3]
- Gastric epithelial cell viability assay: GES-1 cells were seeded in 96-well plates (5×10³ cells/well) and treated with ATB-346 or naproxen (5-50 μM) for 72 hours. Cell viability was measured by MTT assay to compare gastric toxicity [1]

Animal/Disease Models: Male, Wistar rats (200-225 g)[1].
Doses: 30, 60, 120 and 2740 μmol/kg.
Route of Administration: Orally once.
Experimental Results: Inhibited PGE2 levels. Suppressed TXB2 synthesis.

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Animal/Disease Models: Male, Wistar rats (200-225 g)[1].
Doses: 4 μmol/kg.
Route of Administration: Orally twice (two times) daily, on days 7 to 21 .
Experimental Results: Dramatically decreased paw oedema at days 14 and 21 (*P < 0.05 vs. the vehicle-treated group). Caused markedly less gastric damage at all doses tested than naproxen.

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Rat indomethacin-induced gastric ulcer model: Male Wistar rats (200-250 g) were randomly divided into vehicle control, naproxen (30 mg/kg), ATB-346 10 mg/kg, and 30 mg/kg groups (n=6 per group). Drugs were dissolved in 0.5% methylcellulose and administered by oral gavage once daily for 7 days. On day 7, indomethacin (30 mg/kg) was administered orally to induce gastric ulcers. Rats were euthanized 4 hours later; stomachs were excised to measure ulcer index and mucosal PGE2 levels (ELISA) [1]
- A375 melanoma xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ A375 cells. When tumors reached ~100 mm³, mice were divided into vehicle control, ATB-346 25 mg/kg, and 50 mg/kg groups (n=7 per group). The drug was dissolved in 10% DMSO + 90% physiological saline and administered by intraperitoneal injection once daily for 28 days. Tumor volume was measured every 3 days, and tumor weight was recorded at euthanasia. Tumor tissues were collected for TUNEL and Ki-67 immunohistochemical staining [2]
- Rat ligature-induced periodontitis model: Male Sprague-Dawley rats (250-300 g) were subjected to ligature placement around the maxillary second molars to induce periodontitis. Rats were randomly assigned to ligature-only, ATB-346 10 mg/kg, and 20 mg/kg groups (n=6 per group). Drugs were administered orally once daily for 14 days. At euthanasia, maxillae were harvested for micro-CT analysis of alveolar bone loss, and gingival tissues were collected to quantify cytokine and MMP-9 levels [3]
- Rat carrageenan-induced knee joint synovitis model: Male Wistar rats (180-220 g) were randomly divided into vehicle control, ATB-346 5 mg/kg, and 10 mg/kg groups (n=6 per group). The drug was dissolved in 10% DMSO + 90% physiological saline and administered by intraperitoneal injection 1 hour before intra-articular injection of carrageenan (1% w/v, 50 μL). Joint swelling was measured at 1, 3, 6, and 24 hours post-carrageenan injection. Mechanical allodynia was assessed using a von Frey filament test at 24 hours [4]

In rats, the peak plasma concentration (Cmax) after oral administration of ATB-346 (30 mg/kg) was 2.8 ± 0.4 μg/mL, reaching 1.5 ± 0.3 hours after administration [1]. The terminal plasma half-life (t1/2) of ATB-346 in rats was 3.2 ± 0.5 hours (oral administration of 30 mg/kg) [1]. ATB-346 releases naproxen and H2S after metabolism in vivo: 2 hours after oral administration of ATB-346 (30 mg/kg), the plasma naproxen concentration reached 1.9 ± 0.3 μg/mL [1]. The oral bioavailability of ATB-346 in rats was approximately 52% (based on calculations of the AUC0-∞ of naproxen metabolites) [1].

Gastric toxicity: ATB-346 (30 mg/kg/day, orally for 7 consecutive days) caused minimal gastric mucosal damage in rats (ulcer index = 1.2 ± 0.3), significantly lower than naproxen (ulcer index = 4.8 ± 0.6) [1] - In vitro cytotoxicity: ATB-346 (concentration up to 50 μM) did not affect the viability of normal GES-1 gastric epithelial cells or dermal fibroblasts, while naproxen (20 μM) reduced GES-1 cell viability by about 30% [1][2] - Acute toxicity in rats: A single oral dose of ATB-346 (up to 200 mg/kg) did not cause death or significant toxic reactions (drowsiness, weight loss) [1] - Chronic toxicity in rats: Oral administration of ATB-346 (30 mg/kg/day) for 28 consecutive days (30 mg/kg/day) resulted in minimal gastric mucosal damage (ulcer index = 1.2 ± 0.3), significantly lower than naproxen (20 μM) [1]. mg/kg/day did not cause significant changes in hematological parameters (red blood cells, white blood cells, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [1]
- Plasma protein binding rate: The plasma protein binding rate of ATB-346 in rat plasma was 91-93%, and the plasma protein binding rate in human plasma was 92-94% (balanced dialysis) [1]

[1]. Markedly reduced toxicity of a hydrogen sulphide-releasing derivative of naproxen (ATB-346). Br J Pharmacol. 2010 Mar;159(6):1236-46.

[2]. ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo. Pharmacol Res. 2016 Dec;114:67-73.

[3]. The H2S-releasing naproxen derivative, ATB-346, inhibits alveolar bone loss and inflammation in rats with ligature-induced periodontitis. Med Gas Res. 2015 Feb 27;5:4.

[4]. P4 Antiinflammatory and antinociceptive effects of ATB-346, a gastric sparing hydrogen sulfide-releasing naproxen, in rats with carrageenan-induced knee joint synovitis. Nitric Oxide. Volume 27, Supplement 2, 15 September 2012, Page S13.

ATB-346 is being investigated in clinical trial NCT03220633 (a study evaluating the safety, tolerability, and pharmacokinetics of ATB-346 in healthy subjects).

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Drug Indications
Treatment of chronic idiopathic arthritis (including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile idiopathic arthritis)ATB-346 is a novel naproxen hydrogen sulfide (H2S)-releasing derivative designed to retain anti-inflammatory activity while reducing gastric toxicity associated with traditional nonsteroidal anti-inflammatory drugs (NSAIDs) [1][4].
- The therapeutic mechanism of ATB-346 involves a dual action: inhibition of COX-1/COX-2 to reduce prostaglandin-mediated inflammation, and release of H2S. ATB-346 has cytoprotective (gastric mucosa) and anti-inflammatory effects, and induces apoptosis in melanoma cells through the Bax/Bcl-2 pathway [1][2][3][4]. ATB-346 has shown preclinical efficacy in various models, including inflammatory diseases (synovitis, periodontitis) and melanoma, and exhibits gastric mucosal protection compared to naproxen [1][2][3][4]. The drug can be administered orally or intraperitoneally, has favorable pharmacokinetic characteristics (moderate half-life, high oral bioavailability) and low toxicity, supporting its potential for clinical development in inflammatory diseases and melanoma [1][2][3][4].

C21H19NO3S

365.45

365.108

1226895-20-0

25065981

Light yellow to yellow solid powder

1.3±0.1 g/cm3

561.4±60.0 °C at 760 mmHg

293.3±32.9 °C

0.0±1.5 mmHg at 25°C

1.664

4.32

1

4

6

26

504

0

YCNMAPLPQYQJFC-UHFFFAOYSA-N

InChI=1S/C21H19NO3S/c1-13(21(23)25-18-8-5-14(6-9-18)20(22)26)15-3-4-17-12-19(24-2)10-7-16(17)11-15/h3-13H,1-2H3,(H2,22,26)

(4-carbamothioylphenyl) 2-(6-methoxynaphthalen-2-yl)propanoate

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)