FXR (EC50: 99 nM)

In rat hepatocytes, obeticholic acid (INT-747) elevates the expression of FXR-regulated genes[1]. Liver JNK-1 and JNK-2 expression is decreased by obeticholic acid (INT-747)[2]. In every examined strain, obeticholic acid (INT-747) at 256 μg/mL completely inhibits bacterial growth. After INT-747 is added to an intestinal epithelium of Caco-2 cells that has been exposed to IFN-γ, intestinal permeability is not changed[3].

The cholestasis caused by E217α was totally reversed by obeticholic acid (INT-747) (10 mg/kg/day). By boosting the relative abundance of β -MCA, TCDCA, and TDCA, the administration of obeticholic acid (INT-747) partially reverses the impairment in total bile acid secretion caused by E217α[1]. In the mice, obeticholic acid (INT-747)7 (10 mg/kg) and HS exacerbate lung congestion. In animals administered HS, INT-747 does not improve renal pathology[2]. In BDL rats, obeticholic acid (INT-747) (5 mg/kg) considerably improves survival. BDL rats treated with obeticholic acid (INT-747) show a substantial increase in the expression of pore-closing claudin-1 only in the ileum. ZO-1 is markedly up-regulated in the ileum in BDL rats treated with INT-747[3].

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Bacterial translocation (BTL) drives pathogenesis and complications of cirrhosis. Farnesoid X-activated receptor (FXR) is a key transcription regulator in hepatic and intestinal bile metabolism. We studied potential intestinal FXR dysfunction in a rat model of cholestatic liver injury and evaluated effects of obeticholic acid (INT-747), an FXR agonist, on gut permeability, inflammation, and BTL. Rats were gavaged with INT-747 or vehicle during 10 days after bile-duct ligation and then were assessed for changes in gut permeability, BTL, and tight-junction protein expression, immune cell recruitment, and cytokine expression in ileum, mesenteric lymph nodes, and spleen. Auxiliary in vitro BTL-mimicking experiments were performed with Transwell supports. Vehicle-treated bile duct-ligated rats exhibited decreased FXR pathway expression in both jejunum and ileum, in association with increased gut permeability through increased claudin-2 expression and related to local and systemic recruitment of natural killer cells resulting in increased interferon-γ expression and BTL. After INT-747 treatment, natural killer cells and interferon-γ expression markedly decreased, in association with normalized permeability selectively in ileum (up-regulated claudin-1 and occludin) and a significant reduction in BTL. In vitro, interferon-γ induced increased Escherichia coli translocation, which remained unaffected by INT-747. In experimental cholestasis, FXR agonism improved ileal barrier function by attenuating intestinal inflammation, leading to reduced BTL and thus demonstrating a crucial protective role for FXR in the gut-liver axis.

All new compounds were tested in an established cell-free ligand sensing assay, which measured the ligand-dependent recruitment of an SRC1 peptide to FXR by fluorescence resonance energy transfer.4 The results, reported in Table 1, show that obeticholic acid (INT-747; 6-ECDCA) (6b) is a very potent FXR agonist with an EC50 of 99 nM. Also, the 6α-MeCDCA (6a) and 6α-PrCDCA (6c) derivatives demonstrated good potency as FXR agonists, while the 6α-BnCDCA derivative (6d) was essentially inactive.
In a reporter gene, (hsp70EcRE)2-tk-LUC,6a assay employing the full length human FXR in HuH7 cells, 6-ECDCA (6b) was a potent full agonist with an EC50 of 85 nM (Figure 2). When tested across a standard panel (described in ref 6a) of nuclear receptor LBD-GAL4 chimeric receptors,4 1 μM 6b activated only the FXR(LBD)-GAL4 chimera (data not shown). No significant activation of other receptors was seen at 1 μM. Thus, 6b is a potent and selective steroidal FXR agonist.[1]

The exposure of rat hepatocytes to 1 microM 6-ECDCA caused a 3- to 5-fold induction of small heterodimer partner (Shp) and bile salt export pump (bsep) mRNA and 70 to 80% reduction of cholesterol 7alpha-hydroxylase (cyp7a1), oxysterol 12beta-hydroxylase (cyp8b1), and Na(+)/taurocholate cotransporting peptide (ntcp) [2].

In VivoCharacterization in an Animal Model of Cholestasis. The most potent derivative obeticholic acid (INT-747; 6-ECDCA) (6b) was selected for further characterization in an in vivo model of cholestasis. Male Wistar rats (225−300 g) were catheterized at the right jugular vein using PE-50 polyethylene tubing, and the abdomen was opened through a midline incision. The common bile duct was isolated and cannulated. Saline solution was infused via the external jugular vein at the same infusion rate used later for BA, until a steady state in the bile flow was reached (75 min). BA were then dissolved in saline solution with 2% bovine serum albumin, pH 7.4, and infused for 90 min followed by 60 min of saline infusion. During the treatment, bile samples were collected every 15 min and weighed in order to determine the bile flow. Two protocols were used. In the first protocol, rats were randomly assigned to receive one of the following BA:  LCA (2), 6-ECDCA (6b), or CDCA (1) at 1.0, 1.5, or 3 μmol/kg/min. 6-ECDCA (6b) was also infused at a higher dose (7 μmol/kg/min). In the second protocol, cholestasis was induced by intravenous infusion of LCA (2). Three groups of animals were treated as follows:  LCA (2) (3 μmol/kg/min) alone, LCA (2) plus 6-ECDCA (6b) (3 μmol/kg/min), or LCA (2) plus CDCA (1) (3 μmol/kg/min). The results are shown in Figure 3. Administration of LCA alone at a rate of 3 μmol/kg/min caused a dramatic reduction in bile flow and extensive necrosis of liver cells (Figure 4b, arrow).[1]

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In this study, researchers describe the effect of 6-ethyl chenodeoxycholic acid/obeticholic acid (INT-747; 6-ECDCA), a semisynthetic bile acid derivative and potent FXR ligand, in a model of cholestasis induced by 5-day administration of 17alpha-ethynylestradiol (E(2)17alpha) to rats. The exposure of rat hepatocytes to 1 microM 6-ECDCA caused a 3- to 5-fold induction of small heterodimer partner (Shp) and bile salt export pump (bsep) mRNA and 70 to 80% reduction of cholesterol 7alpha-hydroxylase (cyp7a1), oxysterol 12beta-hydroxylase (cyp8b1), and Na(+)/taurocholate cotransporting peptide (ntcp). In vivo administration of 6-ECDCA protects against cholestasis induced by E(2)17alpha. Thus, 6-ECDCA reverted bile flow impairment induced by E(2)17alpha, reduced secretion of cholic acid and deoxycholic acid, but increased muricholic acid and chenodeoxycholic acid secretion. In vivo administration of 6-ECDCA increased liver expression of Shp, bsep, multidrug resistance-associated protein-2, and multidrug resistance protein-2, whereas it reduced cyp7a1 and cyp8b1 and ntcp mRNA. These changes were reproduced by GW4064, a synthetic FXR ligand. In conclusion, by demonstrating that 6-ECDCA protects against E(2)17alpha cholestasis, the data support the notion that development of potent FXR ligands might represent a new approach for the treatment of cholestatic disorders.[2]

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In this study, researchers evaluated the in vivo effect of obeticholic acid (INT-747; 6-ECDCA) on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). The data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls. Conclusion: This study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.[3]

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Dissolved in saline; 7 μmol/kg/min; Infused at the right jugular vein using PE-50 polyethylene tubing

Rat cholestasis model

Absorption, Distribution and Excretion
Obeticholic acid is absorbed in the gastrointestinal tract. The Cmax of obeticholic acid occurs at approximately 1.5 hours after an oral dose and ranges from 28.8-53.7 ng/mL at doses of 5-10mg. The median Tmax for both the conjugates of obeticholic acid is about 10 hours. One product monograph reports a Tmax of 4.5h for both 5 and 10mg doses. The AUC ranged from 236.6-568.1 ng/h/mL with 5mg to 10 mg doses.
About 87% of an orally administered dose is accounted for in the feces. Less than 3% of the dose can be recovered in the urine.
The volume of distribution of obeticholic acid is 618 L.
Clearance information for obeticholic acid is not readily available in the literature.

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Metabolism / Metabolites
The metabolism of obeticholic acid occurs in the liver. Obeticholic acid is conjugated with glycine or taurine, followed by secretion into bile. The conjugates are then absorbed in the small intestine and then re-enter the liver via enterohepatic circulation. The intestinal microbiota in the ileum converts conjugated obeticholic acid in a deconjugated form that may be either reabsorbed or eliminated. Glycine conjugates account for 13.8% of the metabolites and taurine conjugates account for 12.3%. Another metabolite, 3-glucuronide, may also be formed, but displays little pharmacological activity.

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Biological Half-Life
The biological half-life of obeticholic acid is reported to be 24 hours.

Protein Binding
Obeticholic acid and its metabolic conjugates are >99% plasma protein-bound.

[1]. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity. J Med Chem. 2002 Aug 15;45(17):3569-72.

[2]. Protective effects of 6-ethyl chenodeoxycholic acid, a farnesoid X receptor ligand, in estrogen-induced cholestasis. J Pharmacol Exp Ther. 2005 May;313(2):604-12.

[3]. FXR agonist INT-747 upregulates DDAH expression and enhances sensitivity in high-salt fed Dahl rats. PLoS One. 2013 Apr 4;8(4):e60653.

[4]. The FXR Agonist Obeticholic Acid Prevents Gut Barrier Dysfunction and Bacterial Translocation in Cholestatic Rats. Am J Pathol. 2015 Feb;185(2):409-19.

Pharmacodynamics
The activation of the FXR by obeticholic acid acts to reduce the synthesis of bile acids, inflammation, and the resulting hepatic fibrosis. This may increase the survival of patients with PBC, but to date, an association between obeticholic acid and survival in PBC has not been established.

C26H44O4

420.63

420.323

C, 74.24; H, 10.54; O, 15.21

459789-99-2

Obeticholic acid-d5;1992000-80-2;Obeticholic Acid-d4

447715

White to off-white solid powder

1.1±0.1 g/cm3

562.9±25.0 °C at 760 mmHg

108-110

308.3±19.7 °C

0.0±3.5 mmHg at 25°C

1.530

5.68

3

4

5

30

649

11

C[C@@]([C@]1([H])[C@@H](CC)[C@H]2O)(CC[C@@H](O)C1)[C@]3([H])[C@]2([H])[C@@](CC[C@]4([H])[C@H](C)CCC(O)=O)([H])[C@]4(C)CC3

ZXERDUOLZKYMJM-ZWECCWDJSA-N

InChI=1S/C26H44O4/c1-5-17-21-14-16(27)10-12-26(21,4)20-11-13-25(3)18(15(2)6-9-22(28)29)7-8-19(25)23(20)24(17)30/h15-21,23-24,27,30H,5-14H2,1-4H3,(H,28,29)/t15-,16-,17-,18-,19+,20+,21+,23+,24-,25-,26-/m1/s1

(4R)-4-[(3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

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)

Solubility in Formulation 1: ≥ 5 mg/mL (11.89 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (11.89 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 4.76 mg/mL (11.32 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 47.6 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 4: ≥ 2.5 mg/mL (5.94 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.5 mg/mL (5.94 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in 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 EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.5 mg/mL (5.94 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: ≥ 2.5 mg/mL (5.94 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 8: 5 mg/mL (11.89 mM) in 1% Methylcellulose(MC) (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.

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