ML355

The target of ML355 is platelet 12(S)-lipoxygenase (12-LOX), with potent inhibitory activity against this enzyme. It shows high selectivity over related lipoxygenases and cyclooxygenases, though specific IC50, Ki, or EC50 values for the target inhibition are not explicitly provided in the referenced literatures [3]

除了降低 β 细胞中的 12-HETE 之外，ML355 还可防止 PAR-4 引发的钙动员和人血小板聚集 [1]。

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1. ML355可剂量依赖性地抑制多种血小板激动剂诱导的人血小板聚集。实验中，将洗涤后的人血小板与25-100μM的ML355或二甲基亚砜（DMSO）共同孵育5分钟，随后加入凝血酶、PAR1-AP（1μM）、PAR4-AP（50μM）、胶原蛋白（0.5μg/mL）等激动剂，检测血小板聚集情况。结果显示，与DMSO对照组相比，ML355对血小板聚集的抑制作用具有统计学意义（凝血酶诱导的聚集实验n=7，其他激动剂诱导的聚集实验n=4；，P<0.05；，P<0.01）[3]
2. 当ML355与环氧化酶1（COX-1）抑制剂阿司匹林（ASA）联用时，其对人血小板聚集的抑制效果强于单独使用ASA。实验时，将洗涤后的人血小板与DMSO、25μM ML355、100μM ASA或ML355+ASA共同孵育后，再进行血小板聚集检测[3]
3. ML355可抑制凝血酶刺激的人血小板中12-羟基二十碳四烯酸（12-HETE）的生成。将浓度为3×10⁸个/毫升的人血小板与DMSO、25μM ML355、100μM ASA或ML355+ASA共同处理后，用0.25nM和0.5nM的凝血酶激活血小板，随后通过液相色谱-串联质谱（LC/MS/MS）检测12-HETE的生成量（以ng/1×10⁶个血小板为单位）[3]
4. 在体外动脉剪切力条件（1800/s）下的流动腔实验中，ML355可减弱人血小板在胶原包被表面的黏附、聚集及血栓形成。将人全血与25-100μM的ML355或DMSO共同孵育5分钟后，在胶原包被表面灌注4分钟，每隔30秒检测血小板表面覆盖率的动态变化。方差分析（ANOVA）结果显示，与DMSO对照组相比，ML355对血小板功能的抑制作用具有统计学意义（n=8）[3]
5. 在体外流动腔实验中，即使存在COX-1抑制剂ASA，ML355仍能抑制血小板的黏附与聚集。实验时，将人全血与DMSO、25μM ML355、100μM ASA或ML355+ASA共同孵育后再进行灌注[3]
6. 有趣的是，在体外实验中，当血小板暴露于高浓度凝血酶后，ML355的抗血小板作用会被逆转[3]

与 WT 对照相比，ML355（1.88–30 mg/kg；IR；每天两次，持续两天）在较高剂量下可显着减少小鼠血栓的形成[3]。

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1. 小鼠口服ML355后，血浆中可检测到一定水平的药物，药代动力学评估证实其具有合理的血药浓度。实验中，通过灌胃方式给予小鼠30mg/kg的ML355，在不同时间点采集血浆样本（每个时间点n=3），检测并分析血浆中药物浓度随时间的变化，结果以均值±标准差表示[3]
2. 在三氯化铁（FeCl₃）诱导的小鼠肠系膜小动脉血栓模型中，ML355可抑制血栓生长并延缓血管闭塞。野生型（WT）小鼠按15mg/kg和30mg/kg的剂量口服ML355（每日两次，连续2天），对照组包括经聚乙二醇（PEG）处理的WT小鼠和12-LOX基因敲除（12-LOX⁻/⁻）小鼠。观察并记录血栓形成过程，直至血管完全闭塞，若40分钟内未发生闭塞则停止记录。结果显示，与WT对照组相比，ML355处理组小鼠的血管闭塞时间显著延长（每组5-6只小鼠）[3]
3. 在激光诱导的小鼠提睾肌小动脉血栓模型中，ML355可抑制血栓形成。按1.88mg/kg至30mg/kg的剂量给WT小鼠口服ML355（每日两次，连续2天），通过荧光强度变化分析血小板聚集（绿色荧光标记）和纤维蛋白形成（红色荧光标记）情况。结果显示，剂量高于1.88mg/kg的ML355均可显著抑制血小板募集（P<0.001），而仅高剂量（15mg/kg和30mg/kg）的ML355对纤维蛋白形成的抑制作用具有统计学意义（P<0.001），每组3只小鼠，每只小鼠观察8-10个血栓[3]
4. 在激光诱导的小鼠提睾肌小动脉血栓模型中，将15mg/kg的ML355与100mg/kg的ASA联用，可显著抑制血小板募集及血栓中血小板表面P-选择素的表达（P<0.001），而单独使用ASA仅能抑制血小板募集，对P-选择素阳性血小板无显著影响[3]
5. 在激光诱导的小鼠提睾肌小动脉血栓模型中，给12-LOX⁻/⁻小鼠口服15mg/kg的ML355（每日两次，连续2天），其血小板聚集和纤维蛋白形成情况与经PEG处理的12-LOX⁻/⁻小鼠相比无显著差异（P>0.05），这表明ML355对血栓形成的抑制作用依赖于血小板中的12-LOX[3]
6. 在小鼠激光消融隐静脉止血模型中，ML355对止血功能影响较小。按15mg/kg的剂量给WT小鼠口服ML355（每日两次，连续2天），与经PEG处理的WT小鼠或12-LOX⁻/⁻小鼠相比，ML355处理组小鼠的血小板黏附与聚集虽有所减弱（P<0.001），但止血栓的形成未受影响。在激光消融后30秒、5分钟和10分钟时，对血小板聚集和纤维蛋白形成情况进行定量分析，每只小鼠进行2次独立损伤实验，每组3只小鼠[3]
7. 在激光诱导的小鼠提睾肌微血管破裂模型中，ML355处理不会显著增加出血风险。通过输注异硫氰酸荧光素-葡聚糖（分子量10,000）观察血流情况，结果显示，ML355处理的WT小鼠动脉和静脉中血浆葡聚糖外渗停止所需时间与PEG处理的WT小鼠相比无显著差异（P>0.05），而肝素处理的WT小鼠外渗停止时间明显延长，每只小鼠进行1-2次独立损伤实验，每组3只小鼠[3]
8. 在小鼠尾出血实验中，按3.5mg/kg、15mg/kg和30mg/kg的剂量给WT小鼠口服ML355（每日两次，连续2天），与PEG处理的对照组小鼠相比，ML355处理组小鼠的尾出血时间和总红细胞丢失量均无显著增加（P>0.05）；而12-LOX⁻/⁻小鼠的尾出血时间显著延长（P<0.01），红细胞丢失量显著增加（P<0.05）[3]

相关文献中未描述关于ML355的酶（或蛋白/受体）活性实验或靶点结合实验（如激酶活性实验、表面等离子体共振技术、等温滴定量热法、均相时间分辨荧光法等）相关信息[3]

1. 血小板聚集实验：制备洗涤后的人血小板，并调整至合适浓度（如用于聚集实验的浓度为3×10⁸个/毫升）。将血小板与不同浓度的ML355（25-100μM）或DMSO在室温下共同孵育5分钟，随后加入各种血小板激动剂（凝血酶、PAR1-AP、PAR4-AP、胶原蛋白），使用合适的仪器实时检测血小板聚集情况。例如，加入PAR4-AP后，用Chronolog Lumi-Aggregometer（700D型）实时检测人血小板（3×10⁸个/毫升）的聚集情况[3]
2. 血小板中12-HETE检测实验：将人血小板浓度调整为3×10⁸个/毫升，用ML355、ASA或二者的组合处理血小板，随后用凝血酶（0.25nM或0.5nM）激活血小板。孵育结束后，对样本进行处理，采用LC/MS/MS检测12-HETE的生成量（以ng/1×10⁶个血小板为单位）[3]
3. 动脉剪切力条件下血小板功能的体外流动腔实验：将人全血与ML355（25-100μM）或DMSO共同孵育5分钟，然后在动脉剪切力（1800/s）条件下，将血液在胶原包被表面灌注4分钟。灌注过程中，每分钟末拍摄血小板黏附与聚集的代表性图像，并每隔30秒检测血小板表面覆盖率的动态变化，以此评估ML355对血小板黏附及血栓形成的影响[3]

Animal/Disease Models: C57BL/6 mice[3]
Doses: 1.88 , 3.75, 7.5, 15, 30 mg/kg
Route of Administration: po (oral gavage); 2 times per day for two days
Experimental Results: The thrombus formation in mice was strongly inhibited by higher doses of ML355.

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1. Pharmacokinetic study in mice: Mice were given a single oral dose of ML355 at 30 mg/kg via oral gavage. At different time points after administration, blood samples were collected from the mice (n=3 per time point) to separate plasma. The plasma concentrations of ML355 were measured using appropriate analytical methods, and the pharmacokinetic profile (concentrations over time) was generated, with results expressed as mean and standard deviation [3]
2. FeCl₃-induced mesenteric arteriole thrombosis model in mice:
- Grouping: WT mice were divided into control groups (treated with PEG) and ML355-treated groups (15 mg/kg and 30 mg/kg). Additionally, 12-LOX⁻/⁻ mice were included as another control group.
- Drug administration: ML355 was administered orally to WT mice twice a day for 2 days.
- Thrombosis induction: FeCl₃ was topically applied to the mesenteric arterioles of the mice to induce thrombosis.
- Observation and measurement: Representative images of platelet adhesion, aggregation, and thrombus formation were captured at different times after FeCl₃ injury. Thrombus formation was recorded until complete vessel occlusion or stopped at 40 min if occlusion did not occur. The vessel occlusion time was measured and compared among different groups (5 to 6 mice per group) [3]
3. Laser-induced cremaster arteriole thrombosis model in mice:
- Grouping: WT mice were divided into PEG-treated control group and ML355-treated groups (doses of 1.88 mg/kg, 15 mg/kg, 30 mg/kg). For combination studies, a group treated with ML355 (15 mg/kg) + ASA (100 mg/kg) and an ASA alone (100 mg/kg) group were included. 12-LOX⁻/⁻ mice were divided into PEG-treated and ML355 (15 mg/kg)-treated groups.
- Drug administration: ML355 was administered orally twice a day for 2 days; ASA was administered via appropriate routes as needed.
- Thrombosis induction: A laser was used to induce injury in the cremaster arterioles of the mice to trigger thrombosis.
- Observation and measurement: Platelet accumulation (labeled with green fluorescence) and fibrin formation (labeled with red fluorescence) in growing thrombi were observed, and the dynamics of fluorescent intensity were analyzed. The mean fluorescence intensity of platelets and fibrin was measured, and statistical comparisons were made among groups (8–10 thrombi per mouse, 3 mice per group) [3]
4. Laser ablation saphenous vein hemostasis model in mice:
- Grouping: WT mice were divided into PEG-treated control group and ML355 (15 mg/kg)-treated group; 12-LOX⁻/⁻ mice treated with PEG were used as another control group.
- Drug administration: ML355 was administered orally twice a day for 2 days.
- Hemostasis induction: Laser ablation was performed on the saphenous vein of the mice to create repeated vascular injuries.
- Observation and measurement: Representative images of platelet accumulation (green) and fibrin formation (red) within the hemostatic plug at the injured site were captured. Quantitative analysis of platelet accumulation and fibrin formation was conducted at 30 sec, 5 min, and 10 min after laser ablation (2 independent injuries per mouse, 3 mice per group) [3]
5. Laser-induced rupture of cremaster microvasculature model in mice:
- Grouping: WT mice were divided into PEG-treated control group, ML355 (15 mg/kg)-treated group, and heparin (25 U, intravenously injected 10 min before microscopy)-treated group.
- Drug administration: ML355 was administered orally twice a day for 2 days; heparin was injected intravenously as specified.
- Injury induction: A high-intensity laser pulse was used to puncture a hole in the cremaster muscle arteriole wall to induce plasma extravasation.
- Observation and measurement: Fluorescein isothiocyanate-dextran (10,000 MW) was infused to visualize blood flow (plasma shown in red). The time required for the cessation of plasma dextran extravasation from arterioles and venules was measured and compared among groups (1–2 independent injuries per mouse, 3 mice per group) [3]
6. Tail bleeding assay in mice:
- Grouping: WT mice were divided into PEG-treated control group and ML355-treated groups (3.5 mg/kg, 15 mg/kg, 30 mg/kg); 12-LOX⁻/⁻ mice were divided into untreated and ML355 (30 mg/kg)-treated groups.
- Drug administration: ML355 was administered orally twice a day for 2 days.
- Bleeding induction: The tails of the mice were subjected to appropriate injury to induce bleeding.
- Observation and measurement: Tail-bleeding time and total red blood cell loss were measured and compared among different groups [3]

Oral administration of ML355 in mice showed reasonable plasma drug levels. In the pharmacokinetic study, mice were given 30 mg/kg of ML355 via oral gavage, and plasma concentrations of ML355 were measured at different time points (n=3 per time point). The results were expressed as mean and standard deviation, but specific parameters such as absorption rate, distribution volume, metabolism pathways, excretion rate, half-life, and oral bioavailability were not explicitly provided [3]

[1]. Synthesis and structure-activity relationship studies of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase. J Med Chem. 2014 Jan 23;57(2):495-506.

[2]. An ALOX12-12-HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion injury. Nat Med. 2018 Jan;24(1):73-83.

[3]. First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis. Arterioscler Thromb Vasc Biol. 2017 Oct;37(10):1828-1839.

ML355 is a sulfonamide resulting from the formal condensation of the amino group of 2-aminobenzothiazole with the sulfo group of 4-[(2-hydroxy-3-methoxybenzyl)amino]benzenesulfonic acid. It is an inhibitor of 12-lipoxygenase, being developed by Veralox Therapeutics for the treatment of heparin-induced thrombocytopenia and thrombosis. It has a role as an EC 1.13.11.31 (arachidonate 12-lipoxygenase) inhibitor and a platelet aggregation inhibitor. It is a member of benzothiazoles, a sulfonamide, a monomethoxybenzene, a member of phenols, a secondary amino compound and a substituted aniline. It is functionally related to a 2-aminobenzothiazole.
12-Lipoxygenase Inhibitor VLX-1005 is a selective small molecule inhibitor of 12-lipoxygenase (12-LOX), with potential anti-platelet and anti-thrombotic activities. Upon intravenous administration, 12-LOX inhibitor VLX-1005 inhibits platelet 12-LOX. This modulates Fc gamma receptor IIa (FcgRIIa; CD32a) signaling, inhibits FcgRIIa-mediated platelet activation and aggregation, and reduces thrombus formation. The activation of the FcgRIIa receptor plays an important role in immune-mediated thrombosis, such as heparin-induced thrombocytopenia (HIT). 12-LOX, an enzyme expressed in platelets, regulates FcgRIIa activity in the platelet.

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1. ML355 is the first highly selective 12-LOX inhibitor. Adequate platelet reactivity is essential for maintaining hemostasis, but excessive platelet reactivity can lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX) is highly expressed in platelets and has been shown to regulate platelet function and thrombosis ex vivo, suggesting a key role in in vivo thrombosis. Before this study, the ability to pharmacologically target 12-LOX in vivo had not been established [3]
2. The data from the studies strongly support that 12-LOX is a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy, as it impairs thrombus formation and vessel occlusion in vivo with minimal effects on hemostasis [3]

C21H19N3O4S2

441.52

441.081

1532593-30-8

70701426

White to gray solid powder

1.5±0.1 g/cm3

654.5±65.0 °C at 760 mmHg

349.6±34.3 °C

0.0±2.0 mmHg at 25°C

1.725

3.95

137

3

8

7

30

651

0

OWHBVKBNNRYMIN-UHFFFAOYSA-N

InChI=1S/C21H19N3O4S2/c1-28-18-7-4-5-14(20(18)25)13-22-15-9-11-16(12-10-15)30(26,27)24-21-23-17-6-2-3-8-19(17)29-21/h2-12,22,25H,13H2,1H3,(H,23,24)

N-(1,3-benzothiazol-2-yl)-4-[(2-hydroxy-3-methoxyphenyl)methylamino]benzenesulfonamide

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 (5.66 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.66 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.66 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (5.66 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 5 中的溶解度: 15mg/kg inDMSO:Solutol:PEG400:water; 5:10:20:65

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。