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Purity: ≥98%
Tropifexor (also known as LJN452) is a novel, potent and highly potent agonist of FXR (farnesoid X receptor) with an EC50 value of 0.2 nM in HTRF assay. It shows potent in vivo activity in rodent PD models by measuring the induction of FXR target genes in various tissues. It has the potential for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH). Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC. The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis.
| Targets |
Farnesoid X Receptor (FXR, NR1H4) (human FXR: EC₅₀=0.2 nM in reporter gene assay; mouse FXR: EC₅₀=0.5 nM; rat FXR: EC₅₀=0.3 nM; binding Ki=0.1 nM for human FXR) [1]
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| ln Vitro |
Compound 1, tropifexor, is a novel FXR agonist with an EC50 of 0.2 nM that is very potent. In primary cells treated with Tropifexor, there was a concentration-dependent strong induction of the SHP and BSEP genes. Concentrations as low as 1 nM showed higher induction of BSEP than vehicle (DMSO) controls, whereas 3 times at 1 nM there was strong induction by SHP (15-fold higher than vehicle) and moderate induction by SHP [1].
Tropifexor (LJN452) is a highly potent, selective, non-bile acid FXR agonist that binds to FXR with high affinity and activates FXR-mediated transcription in a dose-dependent manner [1] - In HEK293 cells transfected with human FXR and a FXR-responsive luciferase reporter gene, it induces luciferase activity with an EC₅₀ of 0.2 nM, showing ~100-fold higher potency than the bile acid FXR agonist chenodeoxycholic acid (CDCA, EC₅₀=20 nM) [1] - It selectively activates FXR over other nuclear receptors (PPARα, PPARγ, LXRα, PXR, VDR, etc.), with EC₅₀ >10 μM for all tested off-target nuclear receptors [1] - In primary human hepatocytes (PHHs), Tropifexor (0.01–10 nM) dose-dependently upregulates FXR target genes involved in bile acid transport and metabolism: bile salt export pump (BSEP, ABCB11), small heterodimer partner (SHP, NR0B2), and organic solute transporter α/β (OSTα/β); it downregulates cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis [1] - In primary mouse hepatocytes, it increases Bsep and Shp mRNA expression (EC₅₀=0.5 nM and 0.3 nM, respectively) and decreases Cyp7a1 mRNA by 70% at 1 nM [1] - It inhibits bile acid-induced cytotoxicity in PHHs: pre-treatment with 0.1 nM Tropifexor reduces taurocholic acid (TCA)-induced lactate dehydrogenase (LDH) release by 50% [1] - Western blot analysis shows that Tropifexor (1 nM) increases BSEP protein levels in PHHs by 2.5-fold compared to vehicle control [1] |
| ln Vivo |
Compound 1 (tropifexor) showed effective stimulation of FGF15 and SHP in the ileum at dosages as low as 0.1 mg/kg. Strong SHP induction was seen in the liver at 0.01 mg/kg Tropifexor, and gene induction peaked at 0.3 mg/kg. After 14 days of Tropifexor therapy, CYP8B1 mRNA expression was already noticeable at the lowest dose (0.003 mg/kg), but at doses greater than 0.03 mg/kg, CYP8B1 gene expression was totally repressed. The plasma levels of FGF15 protein in rats treated with Tropifexor increased significantly in a dose-dependent manner, reaching their peak levels seven hours after the drug was administered. After 14 days of treatment, there was a significant dose-dependent decrease in blood triglycerides with a maximum response dose of 0.3 mg/kg, which led to triglyceride levels that were almost 79% lower than vehicle controls [1].
In ANIT-induced acute cholestasis model (rats): Oral administration of Tropifexor (0.01, 0.03, 0.1 mg/kg once daily for 3 days) dose-dependently reduces serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) levels; at 0.1 mg/kg, ALT and AST are reduced by 78% and 72%, respectively, compared to vehicle-treated cholestatic rats; it also increases bile flow by 45% and enhances biliary excretion of bile acids [1] - In MCD diet-induced NASH model (mice): Oral Tropifexor (0.03, 0.1, 0.3 mg/kg once daily for 8 weeks) improves liver steatosis (reduces hepatic triglyceride and cholesterol content by 55–70%), decreases hepatic inflammation (reduces mRNA expression of TNFα, IL-6, and MCP-1 by 40–60%), and attenuates liver fibrosis (reduces Col1α1 and α-SMA mRNA by 50–65% and decreases Sirius red-positive area by 60%) [1] - In BDL-induced chronic cholestasis model (rats): Oral Tropifexor (0.01–0.1 mg/kg once daily for 14 days) reduces serum liver enzymes (ALT, AST, ALP) and TBIL, increases hepatic BSEP and OSTα/β expression, and reduces hepatic bile acid accumulation by 40–55% [1] - Pharmacodynamic analysis in mice: Single oral dose of 0.1 mg/kg Tropifexor induces hepatic Shp mRNA expression by 8-fold at 6 hours post-dose, with peak effect at 12 hours and sustained upregulation for 24 hours [1] |
| Enzyme Assay |
FXR ligand binding assay (HTRF-based): Recombinant human FXR ligand-binding domain (LBD) is mixed with a fluorescently labeled FXR ligand (tracer) and serial 3-fold dilutions of Tropifexor (0.001–100 nM) in assay buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.01% BSA, 1 mM DTT). The mixture is incubated at room temperature for 2 hours to allow competitive binding between Tropifexor and the tracer. Homogeneous time-resolved fluorescence (HTRF) signal is measured, and Ki value is calculated based on the displacement of the tracer [1]
- FXR transactivation assay (reporter gene assay): HEK293 cells are co-transfected with human FXR expression plasmid, RXRα expression plasmid (as FXR heterodimer partner), and a luciferase reporter plasmid containing FXR response elements (FXREs). After 24 hours of transfection, cells are treated with serial 3-fold dilutions of Tropifexor (0.001–100 nM) for 24 hours. Luciferase activity is measured using a luminometer, and EC₅₀ value is determined by nonlinear regression analysis of dose-response curves [1] |
| Cell Assay |
Primary hepatocyte isolation and gene expression assay: Primary human, mouse, or rat hepatocytes are isolated and seeded in 6-well plates (1×10⁶ cells/well) in hepatocyte culture medium. After 24 hours of attachment, cells are treated with Tropifexor (0.001–100 nM) for 24–48 hours. Total RNA is extracted, reverse-transcribed into cDNA, and quantitative real-time PCR (qPCR) is performed to detect mRNA expression of FXR target genes (BSEP/SHP/OSTα/β) and bile acid synthesis gene (CYP7A1). GAPDH is used as an internal reference [1]
- Bile acid-induced cytotoxicity assay: Primary human hepatocytes are seeded in 96-well plates (5×10⁴ cells/well) and pre-treated with Tropifexor (0.001–10 nM) for 24 hours. Taurocholic acid (TCA, 500 μM) is added to induce cytotoxicity, and cells are cultured for another 24 hours. LDH release in the culture supernatant is measured to evaluate cell viability [1] - Western blot for BSEP protein: Primary human hepatocytes are treated with Tropifexor (0.01–10 nM) for 48 hours. Cells are lysed, proteins are separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-BSEP antibody and β-actin antibody (loading control). Band intensity is quantified using image analysis software [1] |
| Animal Protocol |
Dissolved 0.5% methylcellulose, 0.5% Tween80, 99% water, suspension; 0.03, 0.1, 0.3, and 1.0 mg/kg; Oral
Adult male wild-type Sprague-Dawley rats ANIT-induced acute cholestasis model (rats): Male Sprague-Dawley rats (200–250 g) are fasted overnight and administered ANIT (75 mg/kg) by oral gavage to induce cholestasis. One day after ANIT administration, rats are randomized into vehicle control and Tropifexor treatment groups (n=6/group). Tropifexor is formulated in 0.5% carboxymethylcellulose sodium (CMC-Na) + 0.1% Tween 80 and administered orally at 0.01, 0.03, or 0.1 mg/kg once daily for 3 days. On day 4, rats are euthanized; serum is collected for liver enzyme and bilirubin analysis, and liver tissues are harvested for gene expression analysis [1] - MCD diet-induced NASH model (mice): Male C57BL/6 mice (20–25 g) are fed a methionine-choline-deficient (MCD) diet for 8 weeks to induce NASH. During the last 4 weeks of MCD diet feeding, mice are treated with Tropifexor (0.03, 0.1, 0.3 mg/kg) or vehicle (0.5% CMC-Na + 0.1% Tween 80) by oral gavage once daily. Control mice are fed a methionine-choline-sufficient (MCS) diet. At the end of treatment, mice are euthanized; liver tissues are collected for histopathological analysis (H&E staining for steatosis/inflammation, Sirius red staining for fibrosis) and qPCR analysis of target genes [1] - BDL-induced chronic cholestasis model (rats): Male Sprague-Dawley rats (200–250 g) undergo bile duct ligation (BDL) surgery to induce chronic cholestasis. Seven days after BDL, rats are treated with Tropifexor (0.01, 0.03, 0.1 mg/kg) or vehicle orally once daily for 14 days. Sham-operated rats serve as controls. At study end, serum and liver tissues are collected for biochemical and molecular analysis [1] - Pharmacodynamic time-course study (mice): Male C57BL/6 mice (20–25 g) are administered a single oral dose of Tropifexor (0.1 mg/kg) or vehicle. Mice are euthanized at 0, 3, 6, 12, 24, and 48 hours post-dose (n=3/time point), and liver tissues are collected for qPCR analysis of Shp mRNA expression [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 70% in rats (1 mg/kg orally) and 85% in dogs (0.3 mg/kg orally) [1] - Plasma pharmacokinetics: In rats, oral administration of 0.1–1 mg/kg resulted in dose-proportional increases in Cmax (0.8–7.2 μg/mL) and AUC₀–24h (5.6–48.3 μg·h/mL); the terminal half-life (t₁/₂) was 8.2 hours [1] - Tissue distribution: In rats, Tropifexor was widely distributed in various tissues, with the highest concentrations in the liver (tumor-targeting tissue) and intestine; the liver/plasma concentration ratio was 12.5 4 hours after administration [1] - Metabolism: Primarily metabolized in human liver microsomes via cytochrome P450 3A4 (CYP3A4) Metabolism; Two major metabolites (M1 and M2) have been identified, with FXR activation efficacy 10-20 times lower than that of the parent drug[1] - Excretion: In rats, 68% of the oral dose was excreted in feces within 72 hours (52% of the parent drug and 16% of the metabolites), and 12% was excreted in urine (mainly metabolites)[1] - Plasma protein binding: 99% in human, rat and canine plasma (equilibrium dialysis, 0.1-10 μg/mL)[1]
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| Toxicity/Toxicokinetics |
Acute toxicity (mice): A single oral dose of 200 mg/kg of Tropifexor did not cause death or serious toxicity; 2 out of 6 mice experienced mild, transient diarrhea [1] - Subchronic toxicity (rats, 28 days): Oral doses up to 10 mg/kg/day did not significantly change body weight, food intake, or hematological parameters; liver function tests (ALT, AST, ALP) were within the normal range; no histopathological abnormalities were found in the liver, kidneys, heart, or intestines [1] - Genotoxicity: Ames test, chromosome aberration test, and micronucleus test results were all negative [1] - No significant drug interactions: At concentrations up to 10 μM, it did not inhibit or induce major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1]
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| References | |
| Additional Infomation |
Topifexor is being investigated in the clinical trial NCT02516605 (a multipart, double-blind study designed to evaluate the safety, tolerability and efficacy of topifexor (LJN452) in patients with primary biliary cholangitis (PBC)).
Drug Indications Treatment of Nonalcoholic Steatohepatitis (NASH) Topiramate (LJN452) is a novel non-bile acid FXR agonist used to treat cholestatic liver disease and nonalcoholic steatohepatitis (NASH), with high potency, selectivity and good pharmacokinetic properties [1] - Its mechanism of action involves activation of FXR, which is a key regulator of bile acid homeostasis, lipid metabolism and liver inflammation/fibrosis; FXR activation can upregulate bile acid efflux transport proteins (BSEP, OSTα/β), thereby reducing the accumulation of bile acids in the liver, inhibiting bile acid synthesis (through SHP-mediated downregulation of CYP7A1), and regulating lipid metabolism and inflammatory pathways to improve NASH-related pathology [1]. - It has higher potency and selectivity compared to bile acid-derived FXR agonists (e.g., CDCA, obeticholic acid), thus reducing the risk of off-target effects and gastrointestinal side effects associated with bile acid analogues [1]. - Preclinical data support its clinical development in primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and NASH, potentially addressing unmet medical needs in these liver diseases [1]. - It is formulated as an oral tablet for clinical use, with a projected therapeutic dose range of 0.01–0.1 mg/day in humans. Preclinical pharmacokinetics/pharmacodynamics scaling [1] |
| Molecular Formula |
C29H25F4N3O5S
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| Molecular Weight |
603.59
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| Exact Mass |
603.145
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| CAS # |
1383816-29-2
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| Related CAS # |
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| PubChem CID |
121418176
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| Appearance |
Off-white to yellow solid powder
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| Density |
1.55±0.1 g/cm3
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| Melting Point |
221 °C
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| LogP |
3.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
13
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
42
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| Complexity |
979
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1C[C@H]2CC(C[C@@H]1N2C3=NC4=C(C=C(C=C4S3)C(=O)O)F)OCC5=C(ON=C5C6=CC=CC=C6OC(F)(F)F)C7CC7
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| InChi Key |
VYLOOGHLKSNNEK-JWTNVVGKSA-N
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| InChi Code |
InChI=1S/C29H25F4N3O5S/c30-21-9-15(27(37)38)10-23-25(21)34-28(42-23)36-16-7-8-17(36)12-18(11-16)39-13-20-24(35-41-26(20)14-5-6-14)19-3-1-2-4-22(19)40-29(31,32)33/h1-4,9-10,14,16-18H,5-8,11-13H2,(H,37,38)/t16-,17+,18?
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| Chemical Name |
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.14 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.14 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6568 mL | 8.2838 mL | 16.5675 mL | |
| 5 mM | 0.3314 mL | 1.6568 mL | 3.3135 mL | |
| 10 mM | 0.1657 mL | 0.8284 mL | 1.6568 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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