Bile acids (BAs) are important signaling molecules acting via the farnesoid X nuclear receptor (FXR) and the membrane G protein-coupled bile acid receptor 1 (GPBAR1). Besides deconjugation of BAs, the oxidoreductive enzymes of colonic bacteria and hepatocytes enable the conversion of BAs into their epimers or dehydrogenated forms. Obeticholic acid (OCA) is the first-in-class BA-derived FXR agonist approved for the treatment of primary biliary cholangitis.

Herein, a library of OCA derivatives, including 7-keto, 6-ethylidene derivatives and 3β-epimers, was synthetized and investigated in terms of interactions with FXR and GPBAR1 in transaction assays and evaluated for FXR target genes expression in human hepatocytes and C57BL/6 mice. The derivatives were further subjected to cell-free analysis employing in silico molecular docking and a TR-FRET assay. The conversion of the 3βhydroxy epimer and its pharmacokinetics in mice were studied using LC–MS.

We found that only the 3β-hydroxy epimer of OCA (3β-isoOCA) possesses significant activity to FXR in hepatic cells and mice. However, in a cell-free assay, 3β-isoOCA had about 9-times lower affinity to FXR than did OCA. We observed that 3β-isoOCA readily epimerizes to OCA in hepatocytes and murine liver. This conversion was significantly inhibited by the hydroxy-Δ⁵-steroid dehydrogenase inhibitor trilostane. In addition, we found that 3,7-dehydroobeticholic acid is a potent GPBAR1 agonist.

We conclude that 3β-isoOCA significantly activates FXR due to its epimerization to the more active OCA by hepatic metabolism. Other modifications as well as epimerization on the C3/C7 positions and the introduction of 6-ethylidene in the CDCA scaffold abrogate FXR agonism and alleviate GPBAR1 activation.

胆汁酸（BAs）是重要的信号分子，其通过法尼醇X核受体（FXR）和膜G蛋白偶联的胆汁酸受体1（GPBAR1）起作用。除了BAs的结合以外，结肠细菌和肝细胞的氧化还原酶还可以将BAs转化为其差向异构体或脱氢形式。奥贝胆酸（OCA）是首个获准用于治疗原发性胆源性胆管炎的BA衍生的FXR激动剂。

本文在交易分析中合成并研究了OCA衍生物库，包括7-酮，6-亚乙基衍生物和3β-受体，与FXR和GPBAR1相互作用，并评估了人肝细胞和C57BL /中的FXR靶基因表达。 6只小鼠。使用计算机分子对接和TR-FRET测定法进一步对衍生物进行无细胞分析。使用LC-MS研究了3β羟基差向异构体在小鼠中的转化及其药代动力学。

我们发现只有OCA的3β-羟基差向异构体（3β-isoOCA）在肝细胞和小鼠中对FXR具有显着的活性。然而，在无细胞试验中，3β-isoOCA对FXR的亲和力比OCA低约9倍。我们观察到3β-isoOCA易于在肝细胞和鼠肝中向OCA异构化。这种转换是由羟基Δ显著抑制⁵ -steroid脱氢酶抑制剂曲洛司坦。此外，我们发现3,7-脱氢奥贝胆酸是一种有效的GPBAR1激动剂。

我们得出的结论是3β-isoOCA由于其被肝代谢代谢为活性更强的OCA而显着激活FXR。其他修饰以及C3 / C7位置的差向异构化以及在CDCA支架中引入6-亚乙基消除了FXR激动作用并减轻了GPBAR1的活化。