SW-033291

15-PGDH (Ki = 0.1 nM)

将SW033291应用于细胞后，15-PGDH酶的活性降低了85%。与 PGE2 相比，高达 40 μM PGE2 时，SW033291 对 15-PGDH 的抑制是非竞争性的。用 SW033291 处理 A549 细胞后，PGE2 水平在 500 nM 时增加 3.5 倍，EC50 50 约为 75 nM[1]。

在连续三天用 SW033291（10 mg/kg；腹腔注射；每天两次；持续 3 天）治疗的 C57BL/6J 小鼠中观察到显着的优势。其中包括外周中性粒细胞计数加倍、骨髓 SKL 细胞增加 65% 以及骨髓 SLAM 细胞增加 71%[1]。

重组15-PGDH蛋白的活性测定[1]
为了初步表征SW033291对15-PGDH酶活性的抑制作用，我们使用实验特定浓度的15-PGDH和SW033291>的实验特定浓度，以及反应缓冲液（50 mM Tris-HCl，pH7.5，0.01%吐温20）中的150µM NAD（+）和25µM PGE2来组装反应。将反应混合物在Envision Reader中在25℃下孵育15分钟。通过每30秒记录一次Ex/Em=340 nM/485 nM的荧光，持续3分钟，从添加PGE2后立即开始，测定NADH的生成，从而确定酶活性。IC50值用GraphPad Prism 5软件计算(http://www.graphpad.com/scientific-software/prism/)使用S形剂量反应函数，并绘制SW033291浓度图。IC50值随酶浓度的增加呈线性增加，表明其具有紧密结合抑制作用，依赖于15-PGDH:SW033291化学计量比，而不是绝对SW033291浓度。为了分析初始反应速率，在反应缓冲液（50 mM Tris-HCl pH7.5，0.01%吐温20）中以200µL的总体积组装了含有10 nM 15-PGDH酶、150µM NAD（+）、25µM PGE2和不同浓度SW033291的动力学反应。15-酮-PGE2的产生是通过每15秒跟踪NADH荧光（Ex/Em=340 nM/485 nM）的变化，持续195秒来计算的，并绘制了与反应时间的关系图。连续反应包含0、0.2 nM、0.25 nM、0.4 nM、0.5 nM、0.8 nM、1 nM、1.6 nM、2 nM、3.25 nM、5 nM，7.5 nM、10 nM、15 nM、20 nM的SW033291浓度。为了推导KiApp，绘制了相对初始反应速度与SW033291浓度的关系图，并使用GraphPad Prism 5软件拟合了紧密结合抑制剂的Morrison方程。该分析得出活性酶浓度值[E]T为8.52 nM，表明酶制剂中的活性为85.2%，KiApp=0.10 nM。

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PGE2浓度对SW033291 IC50[1]的影响测定 15-PGDH酶活性测定在5 nM 15-PGDH、150µM NAD（+）、50 mM Tris-HCl、pH7.5、0.01%吐温20和PGE2浓度为5µM、10µM、20µM、40µM时进行。活性是通过每30秒测量15分钟的荧光（Ex/Em=340 nM/485 nM）测定的NADH生成速率来确定的。IC50值用GraphPad Prism 5软件计算(http://www.graphpad.com/scientific-software/prism/)如上所述。

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15-PGDH热变性[1]
使用SYPRO橙色染料通过差示扫描荧光法监测15-PGDH的热变性。简而言之，将蛋白质在pH 8.0的100 mM Tris缓冲液中稀释至10µM的最终测定浓度，该缓冲液含有0.01%吐温-20和1:1000 SYPRO橙色染料。最终测定体积为20µL，含或不含100µM NADHSW033291，在试验缓冲液加0.4%（v/v）DMSO中，加入至20µM终浓度。使用实时PCR仪器记录热变性曲线，施加2 C/min的温度梯度。使用默认的Bio-Rad CFX Manager V3.1软件对数据进行分析。15-PGDH的熔化温度由-d（RFU）/dT图的拐点确定。 通过测试SW033291对HSD17B10和BDH2熔融温度的影响，评估了SW033291与15-PGDH相互作用的特异性。

检测SW033291对PGE2水平的影响[1]
A549细胞系在37°C下，在含有5%CO2的加湿气氛中，在补充有10%胎牛血清（FBS）和50µg/mL庆大霉素的F12K培养基中维持。细胞以每孔1X105个细胞的比例在24孔板（每孔1 mL）中两次铺板，并在用IL-1β（1 ng/mL）刺激过夜（16小时）前生长24小时，以诱导COX2表达和PGE2产生。然后用含有指定浓度SW033291的新鲜培养基再处理细胞8小时。然后收集培养基，使用PGE2酶免疫测定试剂盒分析PGE2水平。数据来自四个独立的实验。结果以图表形式列出，误差条对应于平均值的标准误差，并使用双侧t检验进行比较。使用CellTiter-Glo®测定法平行测定细胞存活率

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骨髓集落形成试验[1]
为了测定集落形成能力，从8-12周龄的小鼠中分离出全骨髓。在15-PGDH敲除和对照小鼠的研究中，将每只小鼠的两万个骨髓细胞直接接种在两个3平方厘米的平板上，平板上涂有含有IL3、IL6、SCF、Epo的完全甲基纤维素培养基。在SW033291治疗小鼠的研究中（10mg/kg，每天注射5次IP，共5剂），在最后一次SW033291>或载体注射后6小时，从每只治疗和对照小鼠中采集两万个骨髓细胞，然后将其铺在两个3 cm2的平板上。14天后，由来自病例综合癌症中心造血生物库和细胞治疗核心设施的经过专门培训的人员以盲法对菌落进行计数、评分和分型。通过在光学显微镜下使用血细胞计数器进行计数，在收获时从PBS中1:100稀释液中测定骨髓细胞数。使用骨髓细胞数值，将CFU计数标准化为每股骨值。CFU计数以图表形式列出，误差条对应于平均值的标准误差，并使用双侧t检验比较不同处理。

Animal/Disease Models: C57BL/6J mice[1]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip)injection; twice (two times) daily; for 3 days (for 5 doses)
Experimental Results: demonstrated significant benefits, including a doubling of peripheral neutrophil counts, a 65% increase in marrow SKL cells, and a 71% increase in marrow SLAM cells.

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Ex Vivo treatment of murine bone marrow with SW033291 [1]
Whole bone marrow was isolated from 8-10 week old female littermate FVB mice that were either 15-PGDH wild-type or knockout, and incubated with either 0.5 µM SW033291 or vehicle-control for 2 hours on ice. For assay of colony forming activity twenty thousand cells were plated in 3 cm2 plates coated with complete methylcellulose media containing IL3, IL6, SCF, Epo and scored after 14 days. Marrow from 3 mice were individually treated, and then plated in duplicate into a total of 6 separate wells. CFU counts were tabulated graphically with error bars corresponding to standard error of the means and different treatments were compared using 2-tailed t-tests. [1]

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Hematopoietic analysis of SW033291-treated mice [1]
8-10 week old female C57BL/6J mice were injected IP with either vehicle or SW033219 (10 mg/kg) twice daily for 5 doses. Peripheral eye blood was taken from mice 6 hours after the last treatment and blood counts were recorded using the Hemavet 950fs. Blood counts were tabulated graphically with error bars corresponding to standard error of the means and compared using 2-tailed t-tests. In addition, mice were sacrificed and marrow flushed 6 hours following the final treatment for SKL and SLAM analysis as described below.

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Bone marrow homing assays [1]
Whole bone marrow from 8 week old female C57BL/6J mice was labeled with 5 µM CellTrace CFSE and transplanted into lethally irradiated recipient mice (of same age, gender, and strain). Mice were irradiated with 11Gy total body irradiation 12 hours prior to transplant. Recipient mice were treated with either vehicle, 10 mg/kg SW033291, or a combination of Indomethacin (5 mg/kg) + SW033291, Plerixafor (10 mg/kg) + SW033291, EP2 Antagonist PF04418948 (10 µg/mouse) + SW033291, or EP4 Antagonist L-161982 (10 µg/mouse) + SW033291 for three doses. The three treatment doses were administered immediately following 11Gy IR, immediately following transplant, and 8 hours post transplant. After 16 hours whole marrow was flushed from recipient mice and the total percentage of CFSE positive bone marrow was analyzed on a BD LSRII flow cytometer.

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Murine bone marrow transplantation [1]
For survival analysis, 8-10 week old female C57BL/6J recipient mice were lethally irradiated at 11 Gy and transplanted with 200,000 whole bone marrow cells from 8-10 week old female C57BL/6J donor mice. Following transplant the recipient mice received twice daily IP injections with either vehicle or 5 mg/kg SW033291. Animal survival was monitored and recorded daily, and displayed graphically. Significance of differences between survival curves was determined using a two tailed Log-rank (Mantel-Cox) test. To follow recovery of blood counts, 8 week old mice were lethally irradiated at 11 Gy 7 and transplanted with 500,000 whole bone marrow cells. Mice received twice daily IP injections with either vehicle or 5 mg/kg SW033291. Animals were sacrificed at days 5, 8, 12, and 18 and blood counts, bone marrow cellularity, and SKL percentage was measured at each time-point using the Hemavet 850fs. Blood counts were tabulated graphically with error bars corresponding to standard error of the means and compared using 2-tailed t-tests.

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Long term survival following bone marrow transplantation [1]
0.5 million whole bone marrow cells from 8 week old wild-type mice were transplanted into recipient mice lethally irradiated with 11Gy total body irradiation 12 hours prior to transplant. Recipient mice were treated with either vehicle (N=10) or 5 mg/kg SW033291 (twice daily IP) (N=10) for 21 days. Animal survival was recorded in recipient mice at seven months post transplant.

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Serial Transplantation [1]
1 million whole bone marrow cells from 8 week old wild-type mice were transplanted into recipient mice lethally irradiated with 11Gy total body irradiation 12 hours prior to transplant. Recipient mice were treated with either vehicle or 5 mg/kg SW033291 (twice daily IP) for 21 days. 8 weeks post-transplant recipient mice were sacrificed, marrow harvested, and 1 million whole marrow cells were transplanted into second cohort of lethally irradiated recipient mice. This process was serially repeated to generate 3 successive generations of mice descended from the initial transplant recipients. Animal survival was recorded at each round of transplant.

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Partial hepatectomy [1]
10-12 week old male FVB mice, or 10-12 week old male 15-PGDH knockout mice on an FVB background, were placed under isoflurane anesthesia and underwent a twothirds partial hepatectomy through resection of the median and left lateral hepatic lobes as described by Mitchell and Willenbring. Mice that were treated with SW033291 received injections in a vehicle of 10% ethanol, 5% Cremaphor EL, 85% D5W, or with vehicle control. The SW033291 injections were commenced at the time of surgery and continued twice daily throughout the study. Following sacrifice, livers were removed, and weights determined for whole mouse and for isolated livers. Liver weights and ratios of liver weight to body weight were tabulated graphically with error bars corresponding to standard error of the means and compared using 2-tailed t-tests.

[1]. TISSUE REGENERATION. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration. Science. 2015 Jun 12;348(6240):aaa2340.

Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.[1]

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15-PGDH inhibitors, such as SW033291, may also have applicability to treatment of human ulcerative colitis. Mucosal healing is increasingly recognized as a significant therapeutic goal in treatment of this disease. The activity of SW033291 in stimulating colon epithelial regeneration in the mouse DSS colitis model suggests potential applicability to this treatment need. 15-PGDH inhibitors, such as SW033291, may also have therapeutic applicability to humans undergoing surgical resection of primary liver tumors or of colon cancers metastatic to the liver. In both these cases, patient’s eligibility for surgery is limited by the requirement that the post-operative liver remnant be sufficient to enable regenerating an adequate liver mass.[1]

C21H20N2OS3

412.59

412.073

C, 61.13; H, 4.89; N, 6.79; O, 3.88; S, 23.31

459147-39-8

3337839

Light yellow to yellow solid powder

1.4±0.1 g/cm3

670.1±55.0 °C at 760 mmHg

359.0±31.5 °C

0.0±2.0 mmHg at 25°C

1.741

5.47

132

1

6

6

27

514

0

LCYAYKSMOVLVRL-UHFFFAOYSA-N

InChI=1S/C21H20N2OS3/c1-2-3-12-27(24)21-19(22)18-15(14-8-5-4-6-9-14)13-16(23-20(18)26-21)17-10-7-11-25-17/h4-11,13H,2-3,12,22H2,1H3

2-(Butylsulfinyl)-4-phenyl-6-(2-thienyl)-thieno[2,3-b]pyridin-3-amine

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 (6.06 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), 悬浮液；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 2 中的溶解度: 2.5 mg/mL (6.06 mM) in 10% EtOH + 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 澄清乙醇储备液加入到 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 (6.06 mM) in 10% EtOH + 90% (20% SBE-β-CD in 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，将 100 μL 25.0 mg/mL 澄清乙醇储备液加入 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 (6.06 mM) (饱和度未知) in 10% EtOH + 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，您可以将 100 μL 25.0 mg/mL 澄清乙醇储备液添加到 900 μL 玉米油中并充分混合。

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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。