Tropifexor   中文名称: 托吡非索

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]

化合物 1，tropifexor，是一种新型 FXR 激动剂，EC50 为 0.2 nM，非常有效。在用 Tropifexor 处理的原代细胞中，SHP 和 BSEP 基因出现浓度依赖性强烈诱导。低至 1 nM 的浓度显示出比媒介物 (DMSO) 对照更高的 BSEP 诱导作用，而在 1 nM 浓度下，SHP 具有 3 倍的强诱导作用（比媒介物高 15 倍），SHP 具有中等诱导作用 [1]。

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Tropifexor（LJN452） 是一种高效、选择性非胆汁酸类FXR激动剂，对FXR具有高亲和力，可剂量依赖性激活FXR介导的转录作用[1]
- 在转染人FXR和FXR响应性荧光素酶报告基因的HEK293细胞中，诱导荧光素酶活性的EC₅₀=0.2 nM， potency是胆汁酸类FXR激动剂鹅去氧胆酸（CDCA，EC₅₀=20 nM）的约100倍[1]
- 对FXR具有高度选择性，对其他核受体（PPARα、PPARγ、LXRα、PXR、VDR等）无显著激活作用，所有测试脱靶核受体的EC₅₀>10 μM[1]
- 在原代人肝细胞（PHHs）中，Tropifexor（0.01–10 nM）剂量依赖性上调参与胆汁酸转运和代谢的FXR靶基因：胆盐输出泵（BSEP, ABCB11）、小异二聚体伴侣（SHP, NR0B2）和有机溶质转运体α/β（OSTα/β）；同时下调胆汁酸合成限速酶胆固醇7α-羟化酶（CYP7A1）[1]
- 在原代小鼠肝细胞中，增加Bsep和Shp mRNA表达（EC₅₀分别为0.5 nM和0.3 nM），1 nM浓度下可使Cyp7a1 mRNA降低70%[1]
- 抑制胆汁酸诱导的PHHs细胞毒性：0.1 nM Tropifexor 预处理可使牛磺胆酸（TCA）诱导的乳酸脱氢酶（LDH）释放减少50%[1]
- Western blot检测显示，1 nM Tropifexor 可使PHHs中BSEP蛋白水平较溶媒对照组增加2.5倍[1]

化合物 1 (tropifexor) 在低至 0.1 mg/kg 的剂量下即可有效刺激回肠中的 FGF15 和 SHP。在 0.01 mg/kg Tropifexor 时，在肝脏中观察到强烈的 SHP 诱导，而基因诱导在 0.3 mg/kg 时达到峰值。 Tropifexor 治疗 14 天后，CYP8B1 mRNA 表达在最低剂量 (0.003 mg/kg) 下已经很明显，但在剂量大于 0.03 mg/kg 时，CYP8B1 基因表达被完全抑制。 Tropifexor 治疗大鼠的血浆 FGF15 蛋白水平以剂量依赖性方式显着增加，在给药后 7 小时达到峰值水平。治疗 14 天后，血液甘油三酯出现显着的剂量依赖性下降，最大反应剂量为 0.3 mg/kg，导致甘油三酯水平比载体对照低近 79% [1]。

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ANIT诱导的急性胆汁淤积模型（大鼠）：口服给予 Tropifexor 0.01、0.03、0.1 mg/kg，每日一次，连续3天，剂量依赖性降低血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、碱性磷酸酶（ALP）和总胆红素（TBIL）水平；0.1 mg/kg剂量下，与溶媒处理的胆汁淤积大鼠相比，ALT和AST分别降低78%和72%；同时增加胆汁流量45%，促进胆汁酸的胆汁排泄[1]
- MCD饮食诱导的NASH模型（小鼠）：口服 Tropifexor 0.03、0.1、0.3 mg/kg，每日一次，连续8周，改善肝脏脂肪变性（肝甘油三酯和胆固醇含量降低55–70%），减轻肝脏炎症（TNFα、IL-6、MCP-1 mRNA表达降低40–60%），并缓解肝纤维化（Col1α1和α-SMA mRNA降低50–65%，天狼星红阳性区域减少60%）[1]
- BDL诱导的慢性胆汁淤积模型（大鼠）：口服 Tropifexor 0.01–0.1 mg/kg，每日一次，连续14天，降低血清肝酶（ALT、AST、ALP）和TBIL，增加肝脏BSEP和OSTα/β表达，减少肝脏胆汁酸蓄积40–55%[1]
- 小鼠药效动力学分析：单次口服0.1 mg/kg Tropifexor 后6小时，肝脏Shp mRNA表达增加8倍，12小时达到峰值，持续上调24小时[1]

FXR配体结合实验（HTRF法）：将重组人FXR配体结合域（LBD）与荧光标记的FXR配体（示踪剂）及 Tropifexor 的系列3倍稀释液（0.001–100 nM）在实验缓冲液（50 mM Tris-HCl pH 7.5、100 mM NaCl、0.01% BSA、1 mM DTT）中混合。室温孵育2小时，使 Tropifexor 与示踪剂竞争性结合FXR。检测均相时间分辨荧光（HTRF）信号，基于示踪剂的置换程度计算Ki值[1]
- FXR转录激活实验（报告基因实验）：HEK293细胞共转染人FXR表达质粒、RXRα表达质粒（作为FXR异二聚体伴侣）和含FXR响应元件（FXREs）的荧光素酶报告质粒。转染24小时后，用 Tropifexor 的系列3倍稀释液（0.001–100 nM）处理细胞24小时。使用 luminometer 检测荧光素酶活性，通过剂量-响应曲线的非线性回归分析确定EC₅₀值[1]

原代肝细胞分离与基因表达实验：分离原代人、小鼠或大鼠肝细胞，以1×10⁶个/孔接种到6孔板，使用肝细胞培养基培养。贴壁24小时后，用 Tropifexor（0.001–100 nM）处理24–48小时。提取总RNA，逆转录为cDNA，通过实时荧光定量PCR（qPCR）检测FXR靶基因（BSEP/SHP/OSTα/β）和胆汁酸合成基因（CYP7A1）的mRNA表达。以GAPDH作为内参基因[1]
- 胆汁酸诱导的细胞毒性实验：原代人肝细胞以5×10⁴个/孔接种到96孔板，用 Tropifexor（0.001–10 nM）预处理24小时。加入牛磺胆酸（TCA，500 μM）诱导细胞毒性，继续培养24小时。检测培养上清液中LDH释放量以评估细胞活力[1]
- BSEP蛋白Western blot实验：原代人肝细胞用 Tropifexor（0.01–10 nM）处理48小时后裂解，蛋白经SDS-PAGE分离，转移至PVDF膜，用抗BSEP抗体和β-肌动蛋白抗体（内参）进行免疫印迹。通过图像分析软件量化条带强度[1]

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

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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]

Oral bioavailability: 70% in rats (1 mg/kg oral), 85% in dogs (0.3 mg/kg oral) [1]
- Plasma pharmacokinetics: In rats, oral administration of 0.1–1 mg/kg results in dose-proportional increases in Cmax (0.8–7.2 μg/mL) and AUC₀–24h (5.6–48.3 μg·h/mL); terminal half-life (t₁/₂) is 8.2 hours [1]
- Tissue distribution: In rats, Tropifexor distributes widely to tissues, with highest concentrations in the liver (tumor-targeted tissue) and intestine; liver/plasma concentration ratio is 12.5 at 4 hours post-dose [1]
- Metabolism: Predominantly metabolized by cytochrome P450 3A4 (CYP3A4) in human liver microsomes; two major metabolites (M1 and M2) are identified, with FXR activation potency 10–20-fold lower than the parent drug [1]
- Excretion: In rats, 68% of the oral dose is excreted in feces (52% as parent drug, 16% as metabolites), and 12% in urine (mostly as metabolites) within 72 hours [1]
- Plasma protein binding rate: 99% in human, rat, and dog plasma (equilibrium dialysis, 0.1–10 μg/mL) [1]

Acute toxicity (mice): Single oral dose of 200 mg/kg Tropifexor causes no mortality or severe toxicity; mild transient diarrhea is observed in 2/6 mice [1]
- Subchronic toxicity (rats, 28 days): Oral doses up to 10 mg/kg/day show no significant changes in body weight, food intake, or hematological parameters; liver function tests (ALT, AST, ALP) are within normal range; no histopathological abnormalities are found in liver, kidney, heart, or intestine [1]
- Genotoxicity: Negative in Ames test, chromosome aberration test, and micronucleus test [1]
- No significant drug-drug interaction potential: Does not inhibit or induce major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM [1]

[1]. Discovery of Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH). J Med Chem. 2017 Dec 8.

Tropifexor is under investigation in clinical trial NCT02516605 (A Multi-part, Double Blind Study to Assess Safety, Tolerability and Efficacy of Tropifexor (LJN452) in PBC Patients).

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Drug Indication
Treatment of non-alcoholic steatohepatitis (NASH)

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Tropifexor (LJN452) is a novel, non-bile acid FXR agonist developed for the treatment of cholestatic liver diseases and nonalcoholic steatohepatitis (NASH), with high potency, selectivity, and favorable pharmacokinetic properties [1]
- Its mechanism of action involves activation of FXR, a key regulator of bile acid homeostasis, lipid metabolism, and liver inflammation/fibrosis; activation of FXR upregulates bile acid efflux transporters (BSEP, OSTα/β) to reduce hepatic bile acid accumulation, inhibits bile acid synthesis (via SHP-mediated downregulation of CYP7A1), and modulates lipid metabolism and inflammatory pathways to improve NASH-related pathology [1]
- It shows superior potency and selectivity compared to bile acid-derived FXR agonists (e.g., CDCA, obeticholic acid), reducing the risk of off-target effects and gastrointestinal side effects associated with bile acid analogs [1]
- Preclinical data support its clinical development for primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and NASH, with potential to address unmet medical needs in these liver diseases [1]
- It is formulated as an oral tablet for clinical use, with a predicted therapeutic dose range of 0.01–0.1 mg/day in humans based on preclinical pharmacokinetic/pharmacodynamic scaling [1]

C29H25F4N3O5S

603.59

603.145

1383816-29-2

121418176

Off-white to yellow solid powder

1.55±0.1 g/cm3

221 °C

3.5

126

1

13

8

42

979

2

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

VYLOOGHLKSNNEK-JWTNVVGKSA-N

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?

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.14 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.14 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。