Functional Characterization of the Semisynthetic Bile Acid Derivative INT-767, a Dual Farnesoid X Receptor and TGR5 Agonist

Abstract

Two dedicated receptors for bile acids (BAs) have been identified, the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, which represent attractive targets for the treatment of metabolic and chronic liver diseases. Previous work characterized 6␣-ethyl-3␣,7␣-dihydroxy-5␤-cholan-24-oic acid (INT-747), a potent and selective FXR agonist, as well as 6␣-ethyl-23(S)-methyl-3␣,7␣,12␣-trihydroxy-5␤-cholan-24-oic acid (INT-777), a potent and selective TGR5 agonist. Here we characterize 6␣-ethyl-3␣,7␣,23-trihydroxy-24-nor-5␤-cholan-23-sulfate sodium salt (INT-767), a novel semisynthetic 23-sulfate derivative of INT-747. INT-767 is a potent agonist for both FXR (mean EC 50 , 30 nM by PerkinElmer AlphaScreen assay) and TGR5 (mean EC 50 , 630 nM by time resolved-fluorescence resonance energy transfer), the first compound described so far to potently and selectively activate both BA receptors.INT-767 does not show cytotoxic effects in HepG2 cells, does not inhibit cytochrome P450 enzymes, is highly stable to phase I and II enzymatic modifications, and does not inhibit the human ether-a-go-go-related gene potassium channel. In line with its dual activity, INT-767 induces FXRdependent lipid uptake by adipocytes, with the beneficial effect of shuttling lipids from central hepatic to peripheral fat storage, and promotes TGR5-dependent glucagon-like peptide-1 secretion by enteroendocrine cells, a validated target in the treatment of type 2 diabetes. Moreover, INT-767 treatment markedly decreases cholesterol and triglyceride levels in diabetic db/db mice and in mice rendered diabetic by streptozotocin administration. Collectively, these preclinical results indicate that INT-767 is a safe and effective modulator of FXR and TGR5-dependent pathways, suggesting potential clinical applications in the treatment of liver and metabolic diseases.

FXR activation normalizes insulin sensitivity in visceral preadipocytes of a rabbit model of MetS

“…Indeed, we found that i) the treatment of the HFD rabbits with the selective TGR5 agonist INT-777 does not affect HFD-induced glucose intolerance and increased fasting glycemia; ii) the expression of TGR5 in the liver and VAT is markedly lower compared to FXR; iii) the expression of FXR primary response genes, SHP and CYP7A1, is respectively upregulated and downregulated by OCA treatment, as expected following FXR activation (Rizzo et al 2006). These data, together with the known 200-fold greater agonistic activity of OCA for FXR when compared with TGR5 (Rizzo et al 2010), support the view that all the observed OCA effects on HFD-induced MetS are selectively mediated by FXR activation.…”

“…No other major metabolites resulting from a 7-dehydroxylation process, glucuronides, and other polar metabolites were identified (Intercept Pharmaceuticals (New York, NY, USA), Internal Report 2011). Structure, potency, and selectivity toward other nuclear hormone receptors have been described previously (Pellicciari et al 2002, Rizzo et al 2010. Blood samples were obtained from marginal ear vein at baseline and at week 12 in all the groups.…”

“…OCA is a first-in-class FXR agonist, endowed with high binding affinity and potency for FXR (EC 50 0.1 mM), with a 200-fold lower activity for TGR5 (Rizzo et al 2010).…”

Bile Acid Receptor Activation Modulates Hepatic Monocyte Activity and Improves Nonalcoholic Fatty Liver Disease

“…Twelve-week-old db/db and db/m mice were fed for 6 weeks with either normal chow only or chow containing the semisynthetic dual FXR/TGR5 agonist 6␣-ethyl-24-nor-5␤-cholane-3␣,7␣,23-triol-23-sulfate sodium salt (INT-767, Intercept Pharmaceuticals, New York, NY) at 30 mg/kg of body weight (24). Alternatively, mice were injected intraperitoneally daily with 20 mg/kg INT-767 resuspended in 1% methyl cellulose.…”

Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances Cyclin D1 expression and promotes human endometrial cancer cell proliferation

“…Cells were plated in 6-well dishes with regular growth medium the day before transfection to 50-60% confluence. On the second day the medium was changed with SFM without P/S, and cells were transfected with validated ON-TARGET plus SMART pool of siRNA targeted human TGR-5 or FXR 37 or control siRNA, purchased from Thermo Scientific Dharmacon (sequence information is patented and protected by Dharmacon), or with RNA duplex of validate RNAi targeted human CREB mRNA sequence 5'-GGC UAA CAA UGG UAC CGA Utt-3', or with a stealth RNAi control (Ambion) to a final concentration of 50 nM using Lipofectamine 2000 as recommended by the manufacturer. After 5 h, the transfection medium was changed with SFM (MAPK and cyclin D1 analysis by immunoblotting) or 5% charcoal-treated FBS (cell proliferation by [ 3 H]thymidine incorporation) in order to avoid Lipofectamine 2000 toxicity and cells were exposed to CDCA as indicated in figures.…”