FXR (EC50 = 65 nM)

GW4064 treatment (1, 2.5, 5, 10 μM) decreased cell lipid accumulation. In a dose-dependent way, GW4064 therapy dramatically reduced the levels of CD36 protein generated by oleic acid. When combined, these findings imply that long-term GW4064 therapy can prevent hepatic fat buildup by blocking Cd36 expression [2].

In C57BL/6 mice, GW4064 prevents body weight increase brought on by high-fat (HFD) or high-fat, high-cholesterol diets. Reduced liver triglyceride and free fatty acid levels showed that GW4064 treatment dramatically prevented diet-induced hepatic steatosis in HFD mice. Without changing the expression of genes directly related to adipogenesis, GW4064 dramatically lowers the expression of the lipid transporter CD36. Treatment with GW4064 decreases inflammation in the liver while having no effect on white adipose tissue [2]. In rats treated with ANIT, therapy with GW4064 (30 mg/kg) led to a statistically significant reduction in serum ALT, AST, LDH, and ALP activity. The use of GW4064 also markedly lowered serum bile acid levels. Rats given GW4064 had lower bilirubin levels, although the difference was not statistically significant. Notably, TUDCA merely decreased LDH levels; GW4064 was more successful in lowering these liver damage markers [3].

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Purpose: To examine the effect of farnesoid X receptor (FXR) activation by its synthetic agonist, 3-[2-[2-Chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) on diet-induced obesity and hepatic steatosis. Methods: Fifteen week-old C57BL/6 mice fed with high-fat diet (HFD) or high-fat, high-cholesterol diet were treated by twice weekly injection of GW4064 (50 mg/kg) intraperitoneally or DMSO (carrier solution) for 6 weeks. Body weight, body composition and food intake were monitored weekly. Serum glucose and insulin levels and lipid content in the liver were measured at the end of study. Additionally, genes involved in lipogenesis, gluconeogenesis and inflammation were analyzed by real time PCR. CD36 protein level was detected by western blot. Results: Activation of FXR by GW4064 suppressed weight gain in C57BL/6 mice fed with either HFD or high-fat and high-cholesterol diet. GW4064 treatment of mice significantly repressed diet-induced hepatic steatosis as evidenced by lower triglyceride and free fatty acid level in the liver. Analysis of genes involved in lipid metabolism showed GW4064 markedly reduced lipid transporter Cd36 gene expression without affecting expression of genes that are directly involved in lipogenesis. GW4064 treatment attenuated hepatic inflammation while having no effect on white adipose tissue. In addition, activation of FXR by GW4064 avoided diet-induced hyperinsulinemia and hyperglycemia through decreasing the transcript levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pase), two key enzymes in gluconeogenesis. Conclusions: The results verify the important function of FXR in diet-induced obesity and suggest that FXR agonists are promising therapeutic agents for obesity-associated metabolic disorders. [2]

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We investigated whether the synthetic FXR agonist GW4064 could protect against cholestatic liver damage in rat models of extrahepatic and intrahepatic cholestasis. In the bile duct-ligation and alpha-naphthylisothiocyanate models of cholestasis, GW4064 treatment resulted in significant reductions in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, as well as other markers of liver damage. Rats that received GW4064 treatment also had decreased incidence and extent of necrosis, decreased inflammatory cell infiltration, and decreased bile duct proliferation. Analysis of gene expression in livers from GW4064-treated cholestatic rats revealed decreased expression of bile acid biosynthetic genes and increased expression of genes involved in bile acid transport, including the phospholipid flippase MDR2. The hepatoprotection seen in these animal models by the synthetic FXR agonist suggests FXR agonists may be useful in the treatment of cholestatic liver disease.[3]

Cell culture [2]
Mouse liver cells (BNL CL.2) were maintained in a humidified incubator under 5% CO2 at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. When cells were divided into six-well plates and reached ~90% confluence, sub-confluent cells were washed three times with phosphate buffered saline (PBS) and replaced with serum-free DMEM supplemented with 1% fatty acid-free BSA. Oleic acid (final concentration 500 μM) and GW4064 at various concentrations were added and incubated for 24 h. Cells were then fixed with 4% formaldehyde for Oil Red O staining or harvested for protein and western blot analysis.

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Primary culture of human hepatocytes. [3]
Primary human hepatocytes were cultured on Matrigel-coated six-well plates at a density of 1.5 × 106 cells per well. The culture media consisted of serum-free Williams’ E medium supplemented with 100 nM dexamethasone, 100 U/ml penicillin G, 100 μg/ml streptomycin, and ITS-G. Forty-eight hours after isolation, cells were treated for 12 or 48 hours with GW4064 or chenodeoxycholic acid (CDCA), which was added to the culture medium as 1,000× stock solutions in DMSO. Control cultures received vehicle (0.1% DMSO) alone. Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions. Differentially regulated genes were identified using CuraGen Corp. GeneCalling Technology and RTQ-PCR as described above. Sequences of the primers and probes used for RTQ-PCR are listed in Table 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.61 mM) 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 (4.61 mM) in 10% DMF 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: ≥ 2.5 mg/mL (4.61 mM) in 10% DMF 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (3.83 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 0.5% methylcellulose: 11 mg/mL

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