Cell lines

HEK cells

Preparation Method

HEK cells in chamber slides were treated with 1 μM GW4064 or ionomycin (I, 1 μM) for 30 minutes, and endogenous NFATc1 was detected by immunocytochemical analysis. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Reaction Conditions

1 μM, 30 minutes

Applications

Consequent to calcineurin activation, nuclear translocation of endogenous NFATc1 was enhanced by GW4064 in a microscopy-based assay.

Animal models

4-week-old female BALB/c-nude mice

Preparation Method

HT-29 cells (5 × 10⁶) in logarithmic growth phase were subcutaneously injected into the flanks of nude mice. When the palatable xenograft tumors were established, oxaliplatin (3 mg/kg) and GW4064 (15 mg/kg) as both single agents and in combination was injected intraperitoneally twice a week for 3 weeks. The tumor width (b) and length (a) were measured using the callipers every 3 days.

Dosage form

15 mg/kg, i.p.

Applications

Compared with using oxaliplatin or GW4064 only, combination of oxaliplatin and GW4064 in HT-29 cell line inhibited the tumor formation more significantly in vivo.

GW4064, as a synthetic FXR agonist, was used for treatment of cholestatic liver diseases, metabolic syndrome and alcoholic liver disease.^[1]

In vitro experiment it shown that the IC50 values of GW4064 in SW620 and HT-29 cells were 7.6 μM and 13.8 μM, respectively.^[2]In vitro efficacy test it indicated that the GW4064 response was concentration dependent (EC50 values after 24 hours of treatment were 0.012 μM and 0.015 μM, respectively) on CRE and NFAT-RE luciferases.^[3]GW4064 dose dependently enhanced the basal cAMP level with EC50 of 0.241 μM, and suppressed forskolin-induced cAMP accumulation with IC50 of 0.07 μM.^[3]

In vivo study it demonstrated that Rats in the treatment group received an interperitoneal GW4064 injection of 30 mg/kg every other day for 2 wk, GW4064 could correct BA dysmetabolism to alleviate hepatotoxicity in SBR animals. In the meanwhile, GW4064 intervention decreased the fecal bile excretion and elevated plasma/hepatic conjugated BA levels. It also increased the reabsorption of conjugated BAs by inducing apical sodium-dependent bile salt transporter expression in the ileum.^[4]In vivo, treatment with 30 mg/kg for 7 consecutive days intraperitoneally, GW4064 alleviated social deficits in BTBR mice and modulated selective aspects of the composition of the gut microbiota.^[5]Mice were injected 20 mg/kg GW4064 intraperitoneally result in that decreased hepatic inflammation in the LPS-induced murine liver injury model.^[6].

References:
[1]. A.S. Alawad, C. Levy. FXR agonists: from bench to bedside, a guide for clinicians Dig. Dis. Sci., 61 (12) (2016), pp. 3395-3404.
[2]. Guo J, et al. GW4064 enhances the chemosensitivity of colorectal cancer to oxaliplatin by inducing pyroptosis. Biochem Biophys Res Commun. 2021 Apr 9;548:60-66.
[3]. Singh N, et al. Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors. Mol Endocrinol. 2014 May;28(5):659-73.
[4]. Cao Y, et al. FXR agonist GW4064 improves liver and intestinal pathology and alters bile acid metabolism in rats undergoing small intestinal resection. Am J Physiol Gastrointest Liver Physiol. 2019 Aug 1;317(2):G108-G115.
[5]. Liu J, et al. GW4064 Alters Gut Microbiota Composition and Counteracts Autism-Associated Behaviors in BTBR T+tf/J Mice. Front Cell Infect Microbiol. 2022 Jun 22;12:911259.
[6]. Liu HM, et al. GW4064 attenuates lipopolysaccharide induced hepatic inflammation and apoptosis through inhibition of the Toll like receptor 4 mediated p38 mitogen activated protein kinase signaling pathway in mice. Int J Mol Med. 2018 Mar;41(3):1455-1462.