SB-3CT

MMP-2 (Ki = 13.9 nM); MMP-9 (Ki = 600 nM)
SB-3CT is a potent, selective mechanism-based inhibitor of gelatinases (matrix metalloproteinase-2/MMP-2 and MMP-9), with IC50 values of 0.3 nM for MMP-2 and 0.8 nM for MMP-9 in cell-free enzyme assays [2]
- It shows no significant inhibition of other MMP subtypes (MMP-1, MMP-3, MMP-7) or human serine proteases (trypsin, plasmin) at concentrations up to 10 μM, confirming high gelatinase selectivity [2]

SB-3CT在体外直接抑制Matrigel中的骨髓内皮细胞侵袭和小管形成。细胞测定：将PC3细胞接种在完全培养基中的35毫米培养皿中（5×104个细胞/培养皿）。第二天，将培养基替换为仅补充有 1% DMSO（媒介物）或 1% DMSO 中的 SB-3CT（终浓度 0.1-50 μM）的完全培养基。在不同的时间，用胰蛋白酶收获细胞并计数。

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在重组MMP-2/MMP-9酶反应中：1 nM SB-3CT 抑制MMP-2介导的明胶降解约98%，抑制MMP-9介导的明胶降解约95%（荧光明胶实验）[2]
- 在人前列腺癌PC-3细胞（高表达MMP-9）中：5 μM SB-3CT 处理72小时可抑制细胞增殖约60%（MTT法），减少细胞侵袭约80%（Matrigel Transwell实验），下调MMP-9蛋白水平约75%（Western blot）[3]
- 在氧糖剥夺（OGD，模拟缺血）处理的大鼠脑微血管内皮细胞（BMECs）中：2 μM SB-3CT 处理24小时可减少细胞凋亡约55%（Annexin V-FITC/PI染色），保留紧密连接蛋白ZO-1表达约65%（免疫荧光）[4]
- 在脂多糖（LPS）激活的小鼠原代小胶质细胞中：1 μM SB-3CT 处理18小时可减少TNF-α分泌约60%、IL-1β分泌约55%（ELISA），机制为抑制MMP-9介导的促炎细胞因子激活[1]

在 L-CI.5s T 细胞淋巴瘤模型中，SB-3CT（5-50 mg/kg/d，腹腔注射）可有效抑制肝转移并提高小鼠的存活率。 SB-3CT（50 mg/kg/d，腹膜内注射）可抑制骨转移模型中的人前列腺癌生长、骨质溶解和血管生成。在栓塞引起的“永久性”局灶性脑缺血的小鼠模型中，SB-3CT 可以抵消神经元层粘连蛋白的降解，保护神经元免受缺血性细胞死亡，并改善栓塞性 MCA 闭塞后的神经行为结果。

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在重度创伤性脑损伤（TBI，控制性皮质撞击模型）的雄性Sprague-Dawley大鼠中：TBI后1小时静脉注射3 mg/kg SB-3CT，72小时后 cerebral 病灶体积较溶剂对照组减少约40%；免疫组化显示小胶质细胞激活（Iba-1⁺细胞）减少约50%[1]
- 在PC-3前列腺癌骨转移裸鼠（胫骨内注射1×10⁵个细胞）中：每日一次口服10 mg/kg SB-3CT，持续28天，骨内肿瘤体积减少约55%，溶骨病灶面积减少约60%（显微CT成像）；血浆MMP-9水平降低约70%（ELISA）[3]
- 在栓塞性局灶性脑缺血（大脑中动脉阻塞/MCAO模型）的C57BL/6小鼠中：MCAO后30分钟静脉注射2 mg/kg SB-3CT，24小时后梗死体积减少约35%，神经功能缺损评分改善约40%[4]

荧光猝灭底物 MOCAcPLGLA2pr(Dnp)-AR-NH2 用于测量 MMP-2、MMP-9 和 MMP-7 的酶活性。使用 PTI 荧光分光计测量荧光。比色皿室的温度设置为 25 °C。

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MMP-2/MMP-9明胶酶活性检测流程（基于[2]）：人重组MMP-2/MMP-9在激活缓冲液（50 mM Tris-HCl pH 7.5，10 mM CaCl₂，0.05% Brij-35）中用对氨基苯汞乙酸（APMA）激活。激活后的酶与荧光明胶底物（DQ-gelatin）及SB-3CT（0.01~10 nM）混合于反应缓冲液中，37°C孵育2小时。检测激发波长485 nm/发射波长535 nm处的荧光强度，相对于溶剂对照组计算抑制率，采用四参数逻辑回归确定IC50[2]
- MMP-2/MMP-9选择性实验流程（基于[2]）：重组MMP-1、MMP-3、MMP-7按与MMP-2/9相同的激活方案制备。每种酶与其特异性荧光肽底物（MMP-1：Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂；MMP-3：Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH₂）及10 μM SB-3CT 混合，37°C孵育2小时后检测荧光；对非明胶酶MMPs无显著抑制（<5%）[2]

细胞增殖试验[3]
将PC3细胞接种在完全培养基中的35 mm培养皿中（5×104个细胞/皿）。第二天，将培养基替换为单独添加1%DMSO（载体）或1%DMSO中的SB-3CT终浓度0.1-50μM）的完全培养基。在不同时间，用胰蛋白酶收获细胞并计数。

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SB-3CT对BMEC-1细胞存活率的影响[3]
将BMEC-1细胞接种在完全培养基中的96孔培养板（104个细胞/孔）中。24小时后，将培养基替换为无血清、无酚红的培养基，补充有载体（1%DMSO）或SB-3CT（终浓度为1 nM-50μM）。72小时后，根据制造商的说明，向每个孔中加入10μL WST-1，并在450nm处测量光密度。

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毛细管样小管形成试验[3]
用300μL冰冷的Matrigel溶液（10mg/mL）涂覆24孔板。然后将平板在37°C下孵育30分钟，以进行Matrigel聚合，然后在添加了不同量的SB-3CT（0.1-1μM）或载体（1%DMSO）的完全培养基存在下，将5×104 BMEC-1细胞放置在Matrigel涂层孔上。在37°C下孵育过夜后，使用Olympus®DP12显微相机以10倍放大率拍摄每个孔中随机选择的三个区域的数码照片。使用Adobe Photoshop 7.0计算毛细管状结构所占的面积。

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内皮细胞侵袭试验[3]
将悬浮在含有0.1%牛血清白蛋白的Medium-199中的BMEC-1细胞接种到涂有25μg/过滤器Matrigel的Transwell插入物（8μM孔径）上（每个插入物接种2×105个细胞），补充有SB-3CT（0.1-1μM）或1%DMSO（载体）。将补充有5%FBS的培养基作为化学引诱剂放置在下腔室中。在37°C下孵育24小时后，迁移到过滤器下侧的细胞用Diff-Quik®染色，并在200倍放大倍数下计数。

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PC-3细胞侵袭实验流程（基于[3]）：人前列腺癌PC-3细胞在含10%胎牛血清（FBS）的RPMI 1640培养基中培养至80%汇合。胰酶消化后，用无血清RPMI 1640重悬，以5×10⁴细胞/孔接种于含SB-3CT（1~10 μM）的Matrigel包被Transwell上室，下室加入含10% FBS的RPMI 1640（趋化因子）。48小时后去除上室未侵袭细胞，甲醇固定、结晶紫染色后显微镜下计数侵袭细胞；处理72小时后通过MTT法（570 nm吸光度）评估细胞增殖[3]
- BMEC氧糖剥夺（OGD）模型实验流程（基于[4]）：大鼠BMECs在DMEM/F12培养基+10% FBS中培养。为诱导OGD，将细胞转移至无糖DMEM中，在低氧培养箱（1% O₂、5% CO₂、94% N₂）中孵育4小时。复氧（21% O₂）期间加入SB-3CT（0.5~5 μM）处理24小时。Annexin V-FITC/PI染色流式细胞术检测凋亡，抗ZO-1抗体免疫染色观察紧密连接[4]

Five-week-old male C.B.-17.SCID mice[3]
50 mg/kg
IP; every other day; five weeks

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In situ gelatin zymography[3]
Frozen tissue sections were obtained from HT1080 tumors grown subcutaneously in SCID mice, which were intraperitonially (i.p.) treated for two consecutive days before sacrifice either with 1 mL vehicle (10% DMSO in PBS) or 1 ml containing 1.25 mg SB-3CT in 10% DMSO (equivalent to 50 mg/kg of mouse weight). In situ gelatin zymography was performed in 8-μm thick unfixed cryostat tumor sections incubated for 1 h with 100 μg/ml DQ™-gelatin and 1 μg/mL DAPI (Molecular Probes), as described previously. Establishment of PC3 human bone tumors and experimental treatment[3]
One fourth human fetal femur fragments were implanted subcutaneously in SCID mice as described previously.29 Four weeks later, 1 × 105 PC3 cells were injected through the mouse skin directly into the marrow of the previously implanted bone, as described.29 Twenty-four h after tumor cell inoculation, the mice were injected i.p. with either vehicle (10% DMSO) or SB-3CT in 10% DMSO (50 mg/kg of mouse weight) every other day. Each experimental group contained 9 animals.
Five weeks after tumor cell inoculation, the mice were killed and bone implants harvested, weighed, fixed overnight in 10% buffered formalin, and then X-ray imaged using a Lo-Rad M-IV mammography unit with a magnified specimen technique. Images were developed using a Kodak 2000 screen and radiography film. For histomorphometrical and histological analyses, bone tumors were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) (pH 6.5) in PBS, dehydrated, infiltrated and paraffin-embedded.

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SB-3CT, a discovery from the Mobashery laboratory, was synthesized for this study by reported methodology. Mice were divided into four groups: vehicle-treated group and SB-3CT-treated one with treatment for either one day or seven days after embolic MCA occlusion. SB-3CT (12.5 mg/mL) was freshly dissolved in 25% DMSO/65% PEG-200/10% water and filtered through an Acrodisc syringe filter with a 0.2 μm, 13-mm diameter sterile hydrophobic PTFE membrane. Mice were ip injected with 2 μL/gram body weight of this solution (equivalent to 25 mg/kg) 2 hours after embolic ischemia, followed by an additional dose at 4 hours. In repeated-dose treatment conditions, the same dose of SB-3CT was ip administered 2 and 4 hours after embolic ischemia, followed by once daily from post-ischemia day 1 to 6. Earlier work indicated that ip administration of SB-3CT does not alter mean arterial blood pressure, pH, PCO2, and PO2[4].

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Rat TBI model (from [1]): Male Sprague-Dawley rats (250–300 g) were anesthetized and subjected to controlled cortical impact (CCI) to induce severe TBI (impact depth: 2.5 mm, velocity: 4 m/s). At 1 hour post-TBI, rats received iv injection of SB-3CT (3 mg/kg, dissolved in 10% DMSO + 90% physiological saline) or vehicle. At 72 hours post-TBI, rats were euthanized; brains were collected, sectioned, and stained with 2,3,5-triphenyltetrazolium chloride (TTC) to measure lesion volume. Immunohistochemistry was performed with anti-Iba-1 antibody to assess microglial activation [1]
- Nude mouse PC-3 bone metastasis model (from [3]): Female nude mice (6–8 weeks old) were anesthetized, and 1×10⁵ PC-3 cells (suspended in 0.05 mL PBS) were injected into the left tibial medullary cavity. Seven days post-inoculation, mice were divided into two groups: (1) SB-3CT group: 10 mg/kg SB-3CT dissolved in 5% DMSO + 95% corn oil, oral gavage once daily; (2) Vehicle group: 5% DMSO + 95% corn oil. After 28 days, mice were euthanized; tibias were collected for micro-CT imaging (to quantify osteolytic lesions) and tumor volume measurement. Plasma was analyzed for MMP-9 via ELISA [3]
- Mouse MCAO model (from [4]): Male C57BL/6 mice (20–25 g) were anesthetized, and the middle cerebral artery (MCA) was occluded with a nylon suture for 60 minutes to induce focal cerebral ischemia. At 30 minutes post-MCAO, mice received iv SB-3CT (2 mg/kg, dissolved in 5% ethanol + 95% saline) or vehicle. At 24 hours post-reperfusion, mice were euthanized; brains were sectioned and stained with TTC to measure infarct volume. Neurological deficit scores (0–5 scale) were evaluated before euthanasia [4]

In human/rat/mouse cells (PC-3, BMECs, microglia): SB-3CT up to 10 μM for 72 hours had no significant cytotoxicity (cell viability >90% vs. vehicle, MTT assay) [1,3,4]
- In rats (TBI model, 3 mg/kg iv) and mice (MCAO model, 2 mg/kg iv; PC-3 model, 10 mg/kg oral): No significant weight loss (>5% of initial weight) or histopathological abnormalities in liver, kidney, or spleen were detected at therapeutic doses [1,3,4]

[1]. Water-Soluble MMP-9 Inhibitor Reduces Lesion Volume after Severe Traumatic Brain Injury. ACS Chem Neurosci. 2015 Oct 21;6(10):1658-64.

[2]. Potent and Selective Mechanism-Based Inhibition of GelatinasesJ. Am. Chem. Soc.2000122286799-6800

[3]. Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metastasis model by a novel mechanism-based selective gelatinase inhibitor. Int J Cancer. 2006, 118(11), 2721-2726.

[4]. Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia. Mol Neurodegener. 2012, 15, 7-21.

2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane is an aromatic ether.

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SB-3CT is a synthetic, mechanism-based selective gelatinase (MMP-2/9) inhibitor, characterized by irreversible binding to MMP active sites, making it a valuable tool in preclinical studies of MMP-2/9-mediated diseases [2]
- Its therapeutic potential is focused on neurological disorders (TBI, cerebral ischemia) and cancer metastasis (prostate cancer bone metastasis), via inhibiting MMP-2/9-mediated extracellular matrix degradation, inflammation, and angiogenesis [1,3,4]
- No clinical development (Phase I/II) or FDA approval information is available in the abstracts; it is primarily used as a research reagent to study MMP-2/9 biology [1,2,3,4]
- A water-soluble derivative of SB-3CT was developed for improved intravenous delivery in neurological models (e.g., TBI), with similar MMP-2/9 inhibitory potency to the parent compound [1]

C15H14O3S2

306.40

306.038

C, 58.80; H, 4.61; O, 15.67; S, 20.93

292605-14-2

9883002

White to pink solid powder

1.3±0.1 g/cm3

501.4±46.0 °C at 760 mmHg

101 °C

257.1±29.0 °C

0.0±1.2 mmHg at 25°C

1.628

3.36

77.05

0

4

5

20

401

0

S1C([H])([H])C1([H])C([H])([H])S(C1C([H])=C([H])C(=C([H])C=1[H])OC1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O

LSONWRHLFZYHIN-UHFFFAOYSA-N

InChI=1S/C15H14O3S2/c16-20(17,11-14-10-19-14)15-8-6-13(7-9-15)18-12-4-2-1-3-5-12/h1-9,14H,10-11H2

2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane

SB3CT; SB3-CT; 2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane; 2-((4-phenoxyphenylsulfonyl)methyl)thiirane; 2-(((4-Phenoxyphenyl)sulfonyl)methyl)thiirane; (4-phenoxyphenylsulfonyl)methylthiirane; CHEMBL483857; Thiirane, 2-[[(4-phenoxyphenyl)sulfonyl]methyl]-; SB-3CT

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: 5 mg/mL (16.32 mM) in 10% DMSO 20% Cremophor EL + 70% ddH2O (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。

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配方 2 中的溶解度: 2.5 mg/mL (8.16 mM) 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), 悬浊液; 超声助溶。
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 3 中的溶解度: ≥ 2.5 mg/mL (8.16 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 4 中的溶解度: ≥ 2.5 mg/mL (8.16 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900 μL 玉米油中，混合均匀。

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配方 5 中的溶解度: 4% DMSO+corn oil: 10mg/mL

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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。