ATB-346

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]

100 μM 的 otenaproxesul 通过阻断与 Akt 和 NF-B 激活相关的促生存途径来抑制人类黑色素瘤细胞的生长 [2]。 Otenaproxesul (100 μM) 导致人类黑色素瘤细胞凋亡[2]。 Otenaproxesul (100 M) 抑制 NF-kB 的核转位和 IkB 降解，如 A375 细胞中 p65 亚基带强度的降低所示[2]。

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在重组COX酶活性实验中，ATB-346 剂量依赖性抑制COX-1和COX-2活性，IC50值分别为0.5 μM和0.3 μM，与萘普生相当但胃毒性潜力降低[1]
- 在人黑色素瘤细胞系（A375、SK-MEL-28）中，ATB-346 表现出抗增殖活性：72小时MTT实验测得IC50值为15 μM（A375）和18 μM（SK-MEL-28）。该药物诱导G2/M期阻滞和凋亡，30 μM处理后A375和SK-MEL-28细胞凋亡率（Annexin V-FITC/PI染色）分别达42%和38%，伴随Bax上调（2.5倍）和Bcl-2下调（0.4倍）[2]
- 在LPS刺激的RAW 264.7巨噬细胞（炎症模型）中，ATB-346（10-50 μM）剂量依赖性减少促炎细胞因子分泌：50 μM处理较单独LPS组降低TNF-α、IL-6和IL-1β水平分别约65%、70%和60%[3]
- 在TNF-α处理的人牙周膜细胞（hPDLCs，牙周炎模型）中，ATB-346（5-25 μM）抑制NF-κB激活：25 μM时减少p65核转位约55%，下调MMP-9表达约62%（western blot检测）[3]
- ATB-346（浓度高达50 μM）对正常人真皮成纤维细胞或胃上皮细胞（GES-1）活力无影响，而萘普生（20 μM）降低GES-1活力约30%[1][2]

与萘普生相似，奥替普舒具有抗炎作用，但对胃肠道的危害要小得多[1]。 otenaproxesul (43 μmol/kg) 可在体内抑制黑色素瘤肿瘤的生长，同时还可降低与黑色素瘤相关的趋化因子的血浆水平[2]。 （口服，16 mg/kg）显着抑制骨缺损和其他组织学特征（包括牙龈上皮平坦度、慢性炎症细胞浸润和牙龈乳头结缔组织损失）。 Otenaproxesul 不会改变 IL-10 水平，但确实会抑制牙周炎引起的牙龈 IL-1β 和 IL-6 的升高[3]。

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在吲哚美辛诱导的大鼠胃溃疡模型中，口服ATB-346（10 mg/kg/天、30 mg/kg/天，连续7天）造成的胃损伤显著少于萘普生（30 mg/kg/天）：高剂量ATB-346 组胃溃疡指数为1.2 ± 0.3，而萘普生组为4.8 ± 0.6，胃黏膜前列腺素E2（PGE2）水平保持正常水平的约80%[1]
- 在携带A375黑色素瘤异种移植物的裸鼠中，腹腔注射ATB-346（25 mg/kg/天、50 mg/kg/天，连续28天）剂量依赖性抑制肿瘤生长：高剂量组肿瘤生长抑制（TGI）率达65%，肿瘤重量从溶媒组的1.3 ± 0.2 g降至0.46 ± 0.08 g。肿瘤组织中TUNEL阳性凋亡细胞增加约3.5倍，Ki-67阳性率降低约60%[2]
- 在结扎诱导的大鼠牙周炎模型中，口服ATB-346（10 mg/kg/天、20 mg/kg/天，连续14天）剂量依赖性抑制牙槽骨流失：高剂量组减少骨流失约58%（微计算机断层扫描分析），较单纯结扎组降低牙龈TNF-α、IL-6和MMP-9水平60-70%[3]
- 在角叉菜胶诱导的大鼠膝关节滑膜炎模型中，腹腔注射ATB-346（5 mg/kg、10 mg/kg）于角叉菜胶注射前1小时给药，剂量依赖性减轻关节肿胀（10 mg/kg时最大减少约62%），并抑制机械性痛觉过敏（10 mg/kg时痛阈增加约2.3倍）[4]

COX-1/COX-2酶活性抑制实验：将重组人COX-1和COX-2蛋白稀释于含花生四烯酸（底物）和过氧化氢的反应缓冲液中。向反应体系中加入系列稀释的ATB-346（0.01-10 μM），37°C孵育30分钟。加入盐酸终止反应，酶联免疫吸附实验（ELISA）定量前列腺素E2（PGE2，COX活性产物）生成量。通过PGE2抑制的量效曲线计算IC50值[1]
- H2S释放实验：ATB-346（10-100 μM）在磷酸盐缓冲液（pH 7.4）中37°C孵育，采用硫化物敏感电极每30分钟检测一次H2S释放量。计算累积H2S释放量以验证药物的时间依赖性H2S释放能力[1][2]

细胞增殖测定[2]
细胞类型： A375 细胞。
测试浓度：100 μM。
孵化时间：24、48 和 72 小时。
实验结果：对细胞增殖的抑制作用分别为38.2%、63.2%和66%（P < 0.001）。

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黑色素瘤细胞抗增殖及凋亡实验：A375和SK-MEL-28细胞以5×10³个细胞/孔接种到96孔板中，用ATB-346（5-50 μM）处理72小时。MTT法（570 nm吸光度）评估细胞活力并计算IC50值。凋亡分析中，细胞以2×10⁵个细胞/孔接种到6孔板，用ATB-346（15-30 μM）处理48小时，Annexin V-FITC/PI染色后流式细胞术分析。Western blot检测Bax、Bcl-2和GAPDH（内参）[2]
- 巨噬细胞炎症因子实验：RAW 264.7巨噬细胞以1×10⁵个细胞/孔接种到24孔板中，ATB-346（10-50 μM）预处理1小时后，用LPS（1 μg/mL）刺激24小时。收集培养上清，ELISA法定量TNF-α、IL-6和IL-1β水平[3]
- hPDLCs NF-κB激活实验：人牙周膜细胞（hPDLCs）以2×10⁵个细胞/孔接种到6孔板中，ATB-346（5-25 μM）预处理1小时后，用TNF-α（10 ng/mL）刺激24小时。裂解细胞提取核蛋白和胞质蛋白，western blot检测抗p65（NF-κB亚基）和GAPDH抗体。ELISA法检测细胞上清中MMP-9表达[3]
- 胃上皮细胞活力实验：GES-1细胞以5×10³个细胞/孔接种到96孔板中，用ATB-346或萘普生（5-50 μM）处理72小时。MTT法检测细胞活力以比较胃毒性[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, oral administration of ATB-346 (30 mg/kg) resulted in a peak plasma concentration (Cmax) of 2.8 ± 0.4 μg/mL at 1.5 ± 0.3 hours post-dosing [1]
- Terminal plasma half-life (t1/2) of ATB-346 in rats was 3.2 ± 0.5 hours (oral 30 mg/kg) [1]
- ATB-346 is metabolized to release naproxen and H2S in vivo: plasma naproxen concentration reached 1.9 ± 0.3 μg/mL at 2 hours after oral ATB-346 (30 mg/kg) administration [1]
- Oral bioavailability of ATB-346 in rats was ~52% (calculated based on AUC0-∞ of naproxen metabolite) [1]

Gastric toxicity: ATB-346 (30 mg/kg/day, oral for 7 days) induced minimal gastric mucosal damage (ulcer index = 1.2 ± 0.3) in rats, significantly lower than naproxen (ulcer index = 4.8 ± 0.6) [1]
- In vitro cytotoxicity: ATB-346 (up to 50 μM) did not affect viability of normal GES-1 gastric epithelial cells or dermal fibroblasts, while naproxen (20 μM) reduced GES-1 viability by ~30% [1][2]
- Acute toxicity in rats: Single oral administration of ATB-346 up to 200 mg/kg did not cause mortality or overt toxicity (lethargy, weight loss) [1]
- Chronic toxicity in rats: Repeated oral administration of ATB-346 (30 mg/kg/day for 28 days) did not induce significant changes in hematological parameters (RBC, WBC, platelets) or serum biochemical markers (ALT, AST, creatinine, BUN) [1]
- Plasma protein binding: ATB-346 exhibited plasma protein binding of 91-93% in rat plasma and 92-94% in human plasma (equilibrium 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 under investigation in clinical trial NCT03220633 (Study To Assess Safety, Tolerability And PK Of ATB-346 In Healthy Subjects).

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Drug Indication
Treatment of chronic idiopathic arthritis (including rheumatoid arthritis , psoriatic arthritis , ankylosing spondylarthritis and juvenile idiopathic arthritis )

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ATB-346 is a novel hydrogen sulfide (H2S)-releasing derivative of naproxen, designed to retain anti-inflammatory activity while reducing gastric toxicity associated with traditional non-steroidal anti-inflammatory drugs (NSAIDs) [1][4]
- The therapeutic mechanism of ATB-346 involves dual actions: inhibition of COX-1/COX-2 to reduce prostaglandin-mediated inflammation, and release of H2S to exert cytoprotective (gastric mucosa) and anti-inflammatory effects, as well as induce apoptosis in melanoma cells via Bax/Bcl-2 pathway modulation [1][2][3][4]
- ATB-346 has demonstrated preclinical efficacy in multiple models: inflammatory conditions (synovitis, periodontitis), melanoma, and exhibits gastric-sparing properties compared to naproxen [1][2][3][4]
- The drug is administered via oral or intraperitoneal routes, with favorable pharmacokinetic profiles (moderate half-life, good 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

93.64

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.84 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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