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]

In addition to lowering 12-HETE in beta cells, ML355 prevents calcium mobilization and human platelet aggregation triggered by PAR-4 [1].

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1. ML355 dose-dependently inhibited human platelet aggregation induced by various platelet agonists. Washed human platelets were incubated with 25–100 µM of ML355 or DMSO for 5 min, and platelet aggregation was measured after stimulation with agonists like thrombin, PAR1-AP (1 µM), PAR4-AP (50 µM), and collagen (0.5 µg/mL). The inhibition was significant compared to the DMSO control (n=7 for thrombin-induced aggregation, n=4 for other agonists; , P <0.05; , P < 0.01) [3]
2. When combined with the COX-1 inhibitor ASA, ML355 exhibited stronger inhibition of human platelet aggregation than ASA alone. Washed human platelets were incubated with DMSO, 25 µM ML355, 100 µM ASA, or ML355 + ASA prior to aggregation assays [3]
3. ML355 treatment inhibited 12-HETE production in thrombin-stimulated human platelets. Human platelets (3×10⁸ platelets/mL) were treated with DMSO, 25 µM ML355, 100 µM ASA, or ML355 + ASA before activation by 0.25 nM and 0.5 nM thrombin. 12-HETE production (ng/1×10⁶ platelets) was determined by LC/MS/MS [3]
4. In ex vivo flow chamber assays under arterial shear conditions (1800/s), ML355 attenuated human platelet adhesion, aggregation, and thrombus formation over a collagen-coated surface. Human whole blood was pre-incubated with 25–100 µM of ML355 or DMSO for 5 min before perfusion for 4 min. The dynamics of platelet surface coverage were measured at 30-sec intervals, and the inhibition was significant compared to the DMSO control (n=8, P value determined by ANOVA analysis) [3]
5. In the presence of COX-1 inhibitor ASA, ML355 still inhibited platelet adhesion and aggregation in ex vivo flow chamber assays. Human whole blood was pre-incubated with DMSO, 25 µM ML355, 100 µM ASA, or ML355 + ASA prior to perfusion [3]
6. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro [3]

In comparison to WT controls, ML355 (1.88–30 mg/kg; ir; twice daily for two days) significantly reduces the formation of thrombus in mice at higher doses[3].

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1. Oral administration of ML355 in mice resulted in reasonable plasma drug levels, as confirmed by pharmacokinetic assessment. Mice were given 30 mg/kg of ML355 via oral gavage, and plasma concentrations were measured over time (mean and standard deviation, n=3 per time point) [3]
2. In the FeCl₃-induced mesenteric arteriole thrombosis model in mice, ML355 impaired thrombus growth and vessel occlusion. Wild-type (WT) mice were orally administered ML355 at doses of 15 mg/kg and 30 mg/kg (twice a day for 2 days), while control groups included WT mice treated with PEG and 12-LOX⁻/⁻ mice. Thrombus formation was recorded until complete vessel occlusion or stopped at 40 min if occlusion did not occur. The vessel occlusion time was significantly prolonged in ML355-treated groups compared to the WT control group (5 to 6 mice per group) [3]
3. In the laser-induced cremaster arteriole thrombosis model in mice, ML355 inhibited thrombus formation. WT mice were treated with ML355 at doses ranging from 1.88 mg/kg to 30 mg/kg (twice a day for 2 days). Platelet accumulation (green fluorescence) and fibrin formation (red fluorescence) were analyzed by changes in fluorescent intensity. Platelet recruitment was significantly inhibited at all ML355 doses above 1.88 mg/kg (P<0.001), while inhibition of fibrin was significant only at higher doses (15 mg/kg and 30 mg/kg, P<0.001) compared to the PEG-treated control (8–10 thrombi per mouse, 3 mice per group) [3]
4. When ML355 (15 mg/kg) was used in combination with ASA (100 mg/kg) in the laser-induced cremaster arteriole thrombosis model, the combination significantly inhibited platelet recruitment and platelet surface P-selectin expression in thrombi (P<0.001), whereas ASA alone only impaired platelet recruitment without significantly affecting P-selectin positive platelets [3]
5. In 12-LOX⁻/⁻ mice treated with ML355 (15 mg/kg, twice a day for 2 days) in the laser-induced cremaster arteriole thrombosis model, there was no significant difference in platelet accumulation and fibrin formation compared to 12-LOX⁻/⁻ mice treated with PEG (P>0.05), indicating that the inhibition of thrombus formation by ML355 requires platelet 12-LOX [3]
6. ML355 treatment had minimal effects on hemostasis in mice. In the laser ablation saphenous vein hemostasis model, WT mice treated with ML355 (15 mg/kg, twice a day for 2 days) showed attenuated platelet adhesion and accumulation (P<0.001) compared to WT mice or 12-LOX⁻/⁻ mice treated with PEG, but hemostatic plug formation was not impaired. Quantitative analysis of platelet accumulation and fibrin formation was performed at 30 sec, 5 min, and 10 min after laser ablation (2 independent injuries per mouse, 3 mice per group) [3]
7. In the laser-induced rupture of cremaster microvasculature model, ML355 treatment did not significantly increase bleeding. Fluorescein isothiocyanate-dextran (10,000 MW) was infused to visualize blood flow. The time required for the cessation of plasma dextran extravasation from arterioles and venules in ML355-treated WT mice was not significantly different from that in PEG-treated WT mice (P>0.05), while heparin-treated WT mice showed prolonged extravasation time (1–2 independent injuries per mouse, 3 mice per group) [3]
8. In the tail bleeding assay, WT mice treated with ML355 at doses of 3.5 mg/kg, 15 mg/kg, and 30 mg/kg (twice a day for 2 days) did not show significant increases in tail-bleeding time or total red blood cell loss compared to PEG-treated control mice (P>0.05). In contrast, 12-LOX⁻/⁻ mice had increased tail bleeding time (P <0.01) and blood loss (P <0.05) [3]

1. Platelet aggregation assay: Washed human platelets were prepared and adjusted to appropriate concentrations (e.g., 3×10⁸ platelets/mL for aggregation assays). The platelets were incubated with different concentrations of ML355 (25–100 µM) or DMSO for 5 min at room temperature. Then, various platelet agonists (thrombin, PAR1-AP, PAR4-AP, collagen) were added, and platelet aggregation was measured in real-time using appropriate instruments. For example, platelet aggregation of human platelets (3×10⁸ platelets/mL) was measured with a Chronolog Lumi-Aggregometer (model 700D) following the addition of PAR4-AP [3]
2. 12-HETE measurement assay in platelets: Human platelets were adjusted to a concentration of 3×10⁸ platelets/mL and treated with ML355, ASA, or their combination. The platelets were then activated with thrombin (0.25 nM or 0.5 nM). After incubation, the samples were processed, and 12-HETE production (expressed as ng/1×10⁶ platelets) was determined using LC/MS/MS [3]
3. Ex vivo flow chamber assay for platelet function under arterial shear: Human whole blood was pre-incubated with ML355 (25–100 µM) or DMSO for 5 min. The blood was then perfused over a collagen-coated surface at an arterial shear rate (1800/s) for 4 min. During perfusion, representative images of platelet adhesion and aggregation were captured at the end of each minute. The dynamics of platelet surface coverage were measured at 30-sec intervals to assess the effect of ML355 on platelet adhesion and thrombus formation [3]

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

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\n1. 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]
\n2. FeCl₃-induced mesenteric arteriole thrombosis model in mice:
\n - 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.
\n - Drug administration: ML355 was administered orally to WT mice twice a day for 2 days.
\n - Thrombosis induction: FeCl₃ was topically applied to the mesenteric arterioles of the mice to induce thrombosis.
\n - 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]
\n3. Laser-induced cremaster arteriole thrombosis model in mice:
\n - 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.
\n - Drug administration: ML355 was administered orally twice a day for 2 days; ASA was administered via appropriate routes as needed.
\n - Thrombosis induction: A laser was used to induce injury in the cremaster arterioles of the mice to trigger thrombosis.
\n - 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]
\n4. Laser ablation saphenous vein hemostasis model in mice:
\n - 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.
\n - Drug administration: ML355 was administered orally twice a day for 2 days.
\n - Hemostasis induction: Laser ablation was performed on the saphenous vein of the mice to create repeated vascular injuries.
\n - 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]
\n5. Laser-induced rupture of cremaster microvasculature model in mice:
\n - 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.
\n - Drug administration: ML355 was administered orally twice a day for 2 days; heparin was injected intravenously as specified.
\n - Injury induction: A high-intensity laser pulse was used to puncture a hole in the cremaster muscle arteriole wall to induce plasma extravasation.
\n - 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]
\n6. Tail bleeding assay in mice:
\n - 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.
\n - Drug administration: ML355 was administered orally twice a day for 2 days.
\n - Bleeding induction: The tails of the mice were subjected to appropriate injury to induce bleeding.
\n - Observation and measurement: Tail-bleeding time and total red blood cell loss were measured and compared among different groups [3]

After oral administration of ML355 to mice, the plasma drug concentration reached a reasonable level. In the pharmacokinetic study, mice were administered 30 mg/kg of ML355 by oral gavage, and the plasma ML355 concentration was measured at different time points (n=3 at each time point). The results are expressed as mean and standard deviation, but specific parameters such as absorption rate, volume of distribution, metabolic pathway, excretion rate, half-life and oral bioavailability were not clearly 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 compound formed by the condensation of the amino group of 2-aminobenzothiazole and the sulfonic acid group of 4-[(2-hydroxy-3-methoxybenzyl)amino]benzenesulfonic acid. It is a 12-lipoxygenase inhibitor developed by Veralox Therapeutics for the treatment of heparin-induced thrombocytopenia and thrombosis. It is an EC 1.13.11.31 (arachidonic acid 12-lipoxygenase) inhibitor and a platelet aggregation inhibitor. It belongs to the benzothiazole, sulfonamide, monomethoxybenzene, phenol, secondary amine, and substituted aniline classes. Its function is similar to that of 2-aminobenzothiazole.
VLX-1005, a 12-lipoxygenase inhibitor, is a selective small molecule 12-lipoxygenase (12-LOX) inhibitor with potential antiplatelet and antithrombotic activity. Following intravenous injection, the 12-LOX inhibitor VLX-1005 inhibits platelet 12-LOX. This modulates Fcγ receptor IIa (FcγRIIa; CD32a) signaling, inhibiting FcγRIIa-mediated platelet activation and aggregation, and reducing thrombus formation. Activation of the FcγRIIa receptor plays a crucial role in immune-mediated thrombosis, such as heparin-induced thrombocytopenia (HIT). 12-lipoxygenase (12-LOX) is an enzyme expressed in platelets that regulates the activity of FcγRIIa in platelets. ML355 is the first highly selective 12-LOX inhibitor. Appropriate platelet responsiveness is essential for maintaining hemostasis, but excessive platelet responsiveness can lead to obstructive thrombus formation. Platelet 12(S)-lipoxygenase (12-LOX) is highly expressed in platelets, and in vitro experiments have shown that it can regulate platelet function and thrombus formation, suggesting that it plays a key role in thrombus formation in vivo. Prior to this study, the ability to target 12-LOX pharmacologically in vivo had not been established [3]. 2. The study data strongly support that 12-LOX is a key determinant of platelet reactivity in vivo, and the use of ML355 to inhibit platelet 12-LOX may represent a new type of antiplatelet therapy because it inhibits thrombus formation and vascular occlusion in vivo while having minimal impact 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.66 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.66 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.66 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.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.66 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 5: 15mg/kg inDMSO:Solutol:PEG400:water; 5:10:20:65

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