Fexaramine (5, 25, and 50 μM) was used to treat bile acids to HuTu -80 cells for a duration of 24 hours. Assay values of the small heterodimer fraction (SHP) are increased by 2.1 times when fexaramine (50 μM) is added. Endogenous protein levels were dramatically decreased (33% drop in 50 μM Fexaramine) when cells were treated with varying doses of Fexaramine. Treatment with fexaramine nonetheless markedly reduced secretin promoter activity by 32% [1].

Fexaramine (5, 25, and 50 μM) was used to treat bile acids to HuTu -80 cells for a duration of 24 hours. Assay values of the small heterodimer fraction (SHP) are increased by 2.1 times when fexaramine (50 μM) is added. Endogenous protein levels were dramatically decreased (33% drop in 50 μM Fexaramine) when cells were treated with varying doses of Fexaramine. Treatment with fexaramine nonetheless markedly reduced secretin promoter activity by 32% [1].

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In a FRET-based coactivator binding assay, Fexaramine induced the recruitment of the steroid receptor coactivator SRC-1 peptide to the FXR ligand-binding domain (LBD), with an EC50 of 255 nM. This activity was comparable to the synthetic agonist GW4064 (EC50 = 100 nM). [2]
In transient transfection assays using CV-1 cells, Fexaramine (1 nM to 1 µM) potently activated FXR in a dose-dependent manner when co-transfected with FXR, RXR, and a reporter gene containing multiple copies of the FXR response element (FXRE) ECRE (100-fold induction at 1 µM) or ER-8 (4-fold induction at 1 µM). [2]
Fexaramine also activated physiological promoters of known FXR target genes in transient transfection assays. It induced the human ileal bile acid-binding protein (I-BABP) promoter by 28-fold, the human phospholipid transfer protein (PLTP) promoter by 2- to 3-fold, and the rat multidrug resistance-related protein 2 (MRP-2) promoter by 2- to 3-fold at a concentration of 1 µM. [2]
In cross-reactivity studies using chimeric nuclear hormone receptor (NHR) constructs (GAL4 DBD fused to various NHR LBDs), Fexaramine (10 µM) showed high selectivity for FXR and did not activate other NHRs including RXRα, PPARα/γ/δ, PXR, LXRα, TRβ, RARβ, CAR, ERRγ, or VDR. [2]
In HT29 human colon carcinoma cells stably expressing full-length FXR (HT29-FXRFL), treatment with Fexaramine (1 nM to 10 µM) induced the expression of the endogenous I-BABP gene in a concentration-dependent manner, similar to GW4064. No induction was observed in control cell lines lacking functional FXR. [2]
In HEPG2 human hepatoma cells stably expressing FXR (HEPG2-FXR), treatment with Fexaramine (10 nM to 10 µM) induced the expression of endogenous FXR target genes, including SHP, PLTP, BSEP, and MRP-2, as measured by Northern blot. The induction profile and potency were comparable to GW4064. [2]
Gene expression profiling in primary human hepatocytes treated with Fexaramine (10 µM) revealed a distinct genomic signature compared to treatment with the natural bile acid CDCA (100 µM) or the synthetic agonist GW4064 (10 µM), indicating ligand-specific effects on global gene regulation. A subset of genes was commonly regulated by all three FXR agonists. [2]
The high-resolution (1.78 Å) crystal structure of the human FXR ligand-binding domain in complex with Fexaramine was solved. The structure revealed that Fexaramine is sequestered in a 726 ų hydrophobic cavity, making extensive van der Waals contacts and specific hydrogen bonds (involving His298 and Ser336) with the receptor, providing a structural basis for its high affinity and mechanism of action. [2]

Significant metabolic profiles were produced in DIO mice treated with fexaramine, including decreased inflammation, enhanced insulin adiposity, decreased weight gain, and browning of the white adipose tissue [3].

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In diet-induced obese (DIO) mice, chronic oral treatment with Fexaramine (100 mg/kg/day for 5 weeks) significantly reduced body weight gain and fat mass (subcutaneous and visceral) compared to vehicle-treated controls. [3]
Fexaramine treatment improved metabolic profiles in DIO mice, including reduced serum levels of glucose, insulin, leptin, cholesterol, and resistin. [3]
Fexaramine treatment decreased serum levels of inflammatory cytokines (Tnf-α, Il-1α, Il-1β, Il-17, Mcp-1) in DIO mice. [3]
Fexaramine treatment enhanced glucose tolerance and insulin sensitivity in DIO mice, as measured by glucose tolerance tests (GTT) and insulin tolerance tests (ITT), but had no effect in chow-fed mice. [3]
Fexaramine increased energy expenditure in DIO mice, evidenced by higher oxygen consumption (VO2), carbon dioxide production (VCO2), core body temperature (~1.5 °C increase), and reduced lipid accumulation in brown adipose tissue (BAT). [3]
Fexaramine induced browning of white adipose tissue (WAT), shown by increased abundance of multilocular, Ucp1-expressing adipocytes in inguinal WAT, increased expression of brown fat-like signature genes, and increased respiratory capacity in stromal vascular fractions from inguinal WAT. [3]
Fexaramine improved hepatic insulin sensitivity in DIO mice, demonstrated by a marked increase in insulin-mediated suppression of hepatic glucose production (HGP) during hyperinsulinemic-euglycemic clamp studies. [3]
Fexaramine ameliorated hepatic steatosis in DIO mice, shown by reduced liver lipid droplets, decreased hepatic triglyceride content, and reduced expression of hepatic gluconeogenic and lipogenic genes. [3]
The metabolic improvements induced by Fexaramine were abrogated in FXR knockout (Nr1h4-/-) mice, confirming FXR-dependence. [3]
Fexaramine induced expression of the enteric hormone Fgf15 (mouse homolog of human FGF19) in the ileum and increased circulating Fgf15 levels. [3]
Fexaramine altered bile acid (BA) composition, reducing the pool size, decreasing taurocholic acid fraction, and increasing lithocholic acid fraction. It also repressed hepatic Cyp7a1 expression and increased Cyp7b1 expression. [3]
Fexaramine reduced intestinal permeability and increased expression of mucosal defense genes (Ocln, Muc2). [3]
Fexaramine does not activate the G protein–coupled bile acid receptor Tgr5 (Gpbar1). Some metabolic improvements (e.g., on weight gain, insulin sensitivity) were attenuated in Gpbar1-/- mice, suggesting Tgr5 pathway involvement in certain effects. [3]

A FRET-based coactivator binding assay was used to measure agonist-induced interaction between FXR and its coactivator. The assay utilized a europium-labeled GST-FXR-LBD fusion protein and an allophycocyanin-labeled peptide containing the receptor binding domain (LXXLL motif) of the coactivator SRC-1. Increasing concentrations of the test compounds were added to the reaction mixture containing the labeled proteins. Agonist-induced proximity between the receptor and peptide increased FRET signal, which was measured as the ratio of emission at 665 nm to that at 615 nm. The EC50 for coactivator recruitment was determined from dose-response curves. [2]

Transient Transfection Reporter Assay: CV-1 cells were seeded in 48-well plates and grown to 60-70% confluence. Cells were co-transfected using a lipid-based transfection reagent with expression plasmids for murine FXR and human RXRα, a luciferase reporter plasmid containing FXR response elements (FXREs) or natural promoters (e.g., I-BABP, PLTP, MRP-2), and a control plasmid (e.g., pCMX-LacZ) for normalization. After 24 hours, the medium was replaced, and cells were treated with increasing concentrations of the test compounds dissolved in DMSO. Cells were harvested 36-48 hours post-transfection. Luciferase activity was measured and normalized to β-galactosidase activity from the control plasmid. [2]
Stable Cell Line Gene Induction Assay: HT29 colon carcinoma cells or HEPG2 hepatoma cells stably expressing FXR (or control constructs) were generated via retroviral transduction and selection. For induction experiments, cells were cultured to confluence in medium containing charcoal-stripped serum for 24 hours prior to compound treatment. Cells were then treated with the indicated concentrations of compounds (e.g., Fexaramine, GW4064, CDCA) or vehicle (DMSO) for 24-48 hours. Total RNA was isolated using a phenol-chloroform based reagent. For Northern blot analysis, 20 µg of total RNA per sample was resolved on a denaturing agarose-formaldehyde gel, transferred to a nylon membrane, and UV cross-linked. Radiolabeled cDNA probes specific for target genes (e.g., I-BABP, SHP, PLTP, BSEP, MRP-2) and loading controls (e.g., β-actin, 36B4) were generated and hybridized to the membrane. Gene expression levels were visualized by autoradiography and quantified using a phosphorimager. [2]
Chimeric NHR Selectivity Assay: CV-1 cells were transiently transfected with a chimeric expression plasmid where the yeast GAL4 DNA-binding domain was fused to the ligand-binding domain of various nuclear receptors (FXR, RXRα, PPARs, etc.). A reporter plasmid containing a minimal thymidine kinase promoter upstream of four copies of the GAL4 binding site and a luciferase gene was co-transfected. After transfection, cells were treated with DMSO or 10 µM of the test compounds. Luciferase activity was measured and normalized to an internal control (e.g., β-galactosidase), and data were presented as fold activation relative to untreated cells. [2]

For chronic treatment in DIO mice: Wild-type male C57BL/6J mice were fed a 60% high-fat diet (HFD) starting at 6 weeks of age for 14 weeks to induce obesity. Subsequently, mice were treated daily by oral gavage with Fexaramine (50 or 100 mg per kg body weight) or vehicle (corn oil) for 5 weeks while maintained on the HFD. [3]
For acute target gene induction: Mice were treated daily by oral gavage or intraperitoneal injection with Fexaramine (100 mg/kg) or vehicle for 3-5 days, and tissues were collected 1 hour after the final dose for gene expression analysis. [3]
For hyperinsulinemic-euglycemic clamp studies: DIO mice (12 weeks HFD) were gavaged daily with Fexaramine (100 mg/kg) or vehicle for 3 weeks prior to the clamp procedure. Dual catheters were implanted in the jugular vein, and after recovery and fasting, a constant infusion of D-[3-3H] glucose was initiated. Following tracer equilibration, insulin and variable glucose infusions were started to maintain euglycemia, and blood samples were collected to calculate hepatic glucose production and glucose disposal rate. [3]
For metabolic phenotyping: Mice were placed in a Comprehensive Lab Animal Monitoring System (CLAMS) to measure CO2 production, O2 consumption, respiratory quotient, and ambulatory counts over consecutive days. [3]
For intestinal permeability assessment: Mice were orally administered fluorescein isothiocyanate (FITC)-dextran, and serum fluorescence was measured after a specified time to assess leakage. [3]

Following oral administration (100 mg/kg), systemic absorption of filsalamin is minimal. Serum drug concentrations after oral administration are an order of magnitude lower than those after intraperitoneal injection. [3] Serum concentrations of filsalamin after oral administration are below its half-maximum effective concentration (EC50) of 25 nM, consistent with its intestinal limiting effect and lack of target gene activation in the liver and kidneys. [3] This paper does not provide detailed pharmacokinetic parameters for filsalamin, such as half-life, oral bioavailability, clearance, or volume of distribution. [3]

In DIO mice, no signs of intestinal toxicity were observed after long-term oral administration of Fexaramine (100 mg/kg/day for 5 weeks). [3] Fexaramine treatment reduced serum alanine aminotransferase (ALT) levels in DIO mice, indicating reduced liver damage. [3]

[1]. Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP). Am J Physiol Gastrointest Liver Physiol. 2009 Jul;297(1):G90-7.

[2]. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR. Mol Cell. 2003 Apr;11(4):1079-92.

[3]. Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance. Nat Med. 2015 Feb;21(2):159-65.

Fexaramine belongs to the biphenyl class of compounds.

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Fexaramine is a synthetic nonsteroidal high-affinity farnesol X receptor (FXR) agonist that was discovered through screening and optimization of a benzopyran-based combinatorial chemistry library. Its chemical structure is different from that of natural bile acid ligands (e.g., CDCA) and other synthetic FXR agonists (e.g., GW4064). [2]
The primary function of FXR is as a bile acid sensor that coordinates cholesterol metabolism, lipid homeostasis, and the absorption of dietary fat/vitamin. Fexaramine is a valuable chemical tool for elucidating FXR-specific pathways, thereby distinguishing them from the broader biological effects of natural bile acids, which can function through multiple receptors and signaling pathways. [2] The high-resolution crystal structure of FXR bound to fexaramine provides key information on the structure of the FXR ligand binding pocket and serves as a template for simulating the binding of natural bile acids, explaining the structure-activity relationship of different bile acid derivatives. [2] Gene expression profiling analysis showed that different types of FXR agonists (Fexaramine, GW4064, CDCA) could produce different transcription profiles, suggesting that there may be ligand-specific effects and highlighting the practicality of specific synthetic probes such as Fexaramine in studying FXR gene networks. [2]

C32H36N2O3

496.6398

496.273

574013-66-4

5326713

Light yellow to yellow solid powder

1.158g/cm3

677.7ºC at 760 mmHg

363.7ºC

1.626

6.719

0

4

9

37

743

0

CN(C)C1=CC=C(C=C1)C2=CC=C(C=C2)CN(C3=CC=CC(=C3)/C=C/C(=O)OC)C(=O)C4CCCCC4

VLQTUNDJHLEFEQ-KGENOOAVSA-N

InChI=1S/C32H36N2O3/c1-33(2)29-19-17-27(18-20-29)26-15-12-25(13-16-26)23-34(32(36)28-9-5-4-6-10-28)30-11-7-8-24(22-30)14-21-31(35)37-3/h7-8,11-22,28H,4-6,9-10,23H2,1-3H3/b21-14+

methyl (E)-3-(3-(N-((4'-(dimethylamino)-[1,1'-biphenyl]-4-yl)methyl)cyclohexanecarboxamido)phenyl)acrylate

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)

DMSO : ~50 mg/mL (~100.68 mM)

Solubility in Formulation 1: 2.75 mg/mL (5.54 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.03 mM) (saturation unknown) 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 3: ≥ 2.5 mg/mL (5.03 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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