GW-4064

FXR (EC50 = 65 nM)

GW4064 处理（1、2.5、5、10 μM）减少细胞脂质积累。 GW4064 疗法以剂量依赖性方式显着降低了油酸产生的 CD36 蛋白水平。综合起来，这些发现表明长期 GW4064 治疗可以通过阻断 Cd36 表达来预防肝脏脂肪堆积 [2]。

在 C57BL/6 小鼠中，GW4064 可防止高脂肪 (HFD) 或高脂肪、高胆固醇饮食引起的体重增加。肝脏甘油三酯和游离脂肪酸水平降低表明，GW4064 治疗可显着预防 HFD 小鼠饮食诱导的肝脂肪变性。在不改变与脂肪生成直接相关的基因表达的情况下，GW4064 显着降低脂质转运蛋白 CD36 的表达。 GW4064 治疗可减少肝脏炎症，同时对白色脂肪组织没有影响 [2]。在用 ANIT 治疗的大鼠中，用 GW4064 (30 mg/kg) 治疗导致血清 ALT、AST、LDH 和 ALP 活性显着降低，具有统计学意义。 GW4064 的使用还显着降低了血清胆汁酸水平。给予 GW4064 的大鼠胆红素水平较低，但差异不具有统计学意义。值得注意的是，TUDCA 仅降低 LDH 水平； GW4064 在降低这些肝损伤标志物方面更为成功[3]。

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目的：研究法尼醇X受体(FXR)合成激动剂3-[2-[2-氯-4-[[3-(2,6-二氯苯基)-5-(1-甲基乙基)-4-异恶唑基]甲氧基]苯基]乙烯基]苯甲酸（GW4064）对饮食诱导的肥胖及肝脏脂肪变性的调控作用。 方法：15周龄C57BL/6小鼠分别饲喂高脂饮食(HFD)或高脂高胆固醇饮食，腹腔注射GW4064（50 mg/kg）或DMSO（溶剂对照），每周2次，持续6周。每周监测体重、体成分及摄食量。实验结束时检测血清葡萄糖、胰岛素水平及肝脏脂质含量，并通过实时荧光定量PCR分析脂肪生成、糖异生和炎症相关基因表达，蛋白质印迹法检测CD36蛋白水平。 结果：GW4064激活FXR可显著抑制高脂或高脂高胆固醇饮食小鼠的体重增长。治疗组小鼠肝脏甘油三酯和游离脂肪酸水平降低，表明GW4064有效缓解饮食诱导的肝脏脂肪变性。脂代谢相关基因分析显示，GW4064显著降低脂质转运体Cd36基因表达，但不直接影响脂肪生成关键基因。该药物还能减轻肝脏炎症反应，但对白色脂肪组织无显著影响。此外，GW4064通过降低糖异生关键酶磷酸烯醇式丙酮酸羧激酶(Pepck)和葡萄糖-6-磷酸酶(G6pase)的转录水平，改善饮食诱导的高胰岛素血症和高血糖。 结论：结果证实FXR在饮食诱导肥胖中的重要作用，提示FXR激动剂有望成为肥胖相关代谢紊乱的治疗药物。[2]

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我们进一步探究了合成FXR激动剂GW4064对肝外和肝内胆汁淤积大鼠模型的保护作用。在胆管结扎和α-萘异硫氰酸酯诱导的胆汁淤积模型中，GW4064治疗组血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶、乳酸脱氢酶等肝损伤标志物显著降低。接受GW4064治疗的大鼠肝坏死程度和范围减轻，炎症细胞浸润减少，胆管增生受到抑制。基因表达分析显示，GW4064可下调胆汁酸合成相关基因表达，同时上调包括磷脂翻转酶MDR2在内的胆汁酸转运蛋白基因。合成FXR激动剂在动物模型中表现出的肝保护作用表明，该类药物或可用于胆汁淤积性肝病的治疗。[3]

细胞培养[2]
小鼠肝细胞(BNL CL.2)在37°C、5% CO2的湿润培养箱中，使用添加了10%胎牛血清(FBS)和1%青霉素/链霉素的杜尔贝科改良 Eagle 培养基(DMEM)进行培养。当细胞接种于六孔板并达到约90%融合度时，用磷酸盐缓冲液(PBS)洗涤亚融合状态细胞三次，更换为含1%无脂肪酸BSA的无血清DMEM培养基。分别添加油酸(终浓度500 μM)和不同浓度的GW4064，继续培养24小时。随后用4%甲醛固定细胞进行油红O染色，或收集细胞用于蛋白质提取及蛋白质印迹分析。

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人原代肝细胞培养[3]
原代人肝细胞以每孔1.5×10⁶个细胞的密度接种于基质胶包被的六孔板，使用含100 nM地塞米松、100 U/ml青霉素G、100 μg/ml链霉素和ITS-G的无血清Williams’ E培养基培养。分离48小时后，向培养基中加入GW4064或鹅去氧胆酸(CDCA)处理12或48小时（药物以DMSO配制成1000×储存液添加）。对照组仅添加等体积溶剂(0.1% DMSO)。按照制造商说明使用TRIzol试剂提取总RNA，差异表达基因通过CuraGen公司GeneCalling技术和实时定量PCR（方法同前）进行鉴定。实时定量PCR所用引物和探针序列见表1。

Animals and treatment [2]
All procedures performed on mice were approved by the Institutional Animal Care and Use Committee at the University of Georgia, Athens, Georgia. Fifteen-week-old male C57BL/6 mice were fed a high-fat diet with or without additional 0.2% cholesterol and received twice weekly injections of GW4064 (50 mg/kg, intra-peritoneal) or carrier solution [dimethyl sulfoxide (DMSO)] solution for 6 weeks. [2]

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Bile duct ligation model of cholestasis. [3]
Under isoflurane anesthesia and sterile surgical conditions, the common bile duct was ligated in three locations and transected between the two distal ligatures. Sham controls underwent laparotomy, without ligation of the bile duct. The rats received a single analgesic dose of oxymorphone following surgery. Twenty-four hours after laparotomy, groups of rats (n = 6) received intraperitoneal injections once daily for 4 days. Bile duct–ligated (BDL) rats were treated with 5 ml/kg corn oil as vehicle, 30 mg/kg GW4064 in corn oil, or 15 mg/kg TUDCA in corn oil. Sham-operated animals received 5 ml/kg corn oil vehicle. Four hours after the final dose, serum and livers were collected for analysis.

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ANIT-induced cholestasis. [3]
Once daily for 4 days, rats (n = 6–8) received intraperitoneal injections of vehicle, GW4064, or TUDCA, as described above. On day 2 of treatment, 4 hours after the intraperitoneal injection, the vehicle-, GW4064-, and TUDCA-treated groups received a single, orally administered, 50 mg/kg dose of ANIT in olive oil. A second set of vehicle-treated rats was given an oral dose of olive oil (5 ml/kg) in place of ANIT to serve as the normal control. Serum and liver samples were collected as outlined above, 4 hours after the final dose.

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Dissolved in 0.5% methyl cellulose; 0-100 mg/kg; Oral gavage

Fisher rats

[1]. Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064. Bioorg Med Chem Lett, 2008, 18(15), 4339-4343.

[2]. Synthetic FXR agonist GW4064 prevents diet-induced hepatic steatosis and resistance. Pharm Res. 2013 May;30(5):1447-57.

[3]. Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J Clin Invest. 2003 Dec;112(11):1678-87.

GW 4064 is a stilbenoid. Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.[1]

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In summary, we demonstrate in the current study that GW4064-mediated FXR activation ameliorates diet-induced obesity, suppresses hepatic lipid accumulation, and maintains glucose and lipid homeostasis in C57BL/6 mice. These results suggest that the pharmacological activation of FXR represents a promising therapeutic strategy for treatment of obesity-associated liver steatosis. [2]

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Since LCA does not accumulate to appreciable levels in the cholestatic rat, we believe the protective effects of GW4064 in the chronic BDL and ANIT models are not due to reversal of the effects of LCA antagonism of FXR activity. From our data it appears likely that induction of the canalicular transporters BSEP, MDR2/3, and MRP2 and repression of bile acid biosynthesis by GW4064 in the cholestatic liver provides a mechanism to decrease the concentration of toxic bile acids in the liver. The role of FXR in other liver cell types, such as cholangiocytes, has not yet been elucidated, so it cannot be ruled out that the hepatoprotection by GW4064 may also involve additional mechanisms that are currently undefined.[3]

C28H22CL3NO4

542.84

541.061

C, 61.95; H, 4.09; Cl, 19.59; N, 2.58; O, 11.79

278779-30-9

278779-30-9;

9893571

White to off-white solid powder

1.4±0.1 g/cm3

702.1±60.0 °C at 760 mmHg

378.4±32.9 °C

0.0±2.3 mmHg at 25°C

1.654

8.49

72.56

1

5

8

36

742

0

CC(C)C1=C(C(=NO1)C2=C(C=CC=C2Cl)Cl)COC3=CC(=C(C=C3)/C=C/C4=CC(=CC=C4)C(=O)O)Cl

BYTNEISLBIENSA-MDZDMXLPSA-N

InChI=1S/C28H22Cl3NO4/c1-16(2)27-21(26(32-36-27)25-22(29)7-4-8-23(25)30)15-35-20-12-11-18(24(31)14-20)10-9-17-5-3-6-19(13-17)28(33)34/h3-14,16H,15H2,1-2H3,(H,33,34)/b10-9+

3-[(E)-2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-propan-2-yl-1,2-oxazol-4-yl]methoxy]phenyl]ethenyl]benzoic acid

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 (4.61 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), 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 (4.61 mM) in 10% DMF 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。
*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 (4.61 mM) in 10% DMF 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (3.83 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 20.8 mg/mL澄清的DMSO储备液加入到900 μL玉米油中，混合均匀。

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配方 5 中的溶解度： 0.5%甲基纤维素： 11 mg/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。