FXR (EC50: 99 nM)
Farnesoid X Receptor (FXR)：Obeticholic acid (6-ECDCA/INT-747) is a potent and selective FXR agonist with an EC50 of 99 nM in binding assays. It shows high specificity for FXR over other nuclear receptors. [1]

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].

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- FXR activation and gene regulation：Obeticholic acid (1 μM) induces 3- to 5-fold upregulation of small heterodimer partner (SHP) and bile salt export pump (BSEP) mRNA in rat hepatocytes, while suppressing cholesterol 7α-hydroxylase (CYP7A1) and Na+/taurocholate cotransporting peptide (NTCP) expression by 70–80%. [2]
- Gut barrier function modulation：In intestinal epithelial cells, obeticholic acid (10 nM) enhances claudin-1 expression and reduces claudin-2 expression, improving transepithelial electrical resistance (TEER) by 40% compared to vehicle control. [4]

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.

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- Cholestasis protection in estrogen-induced model：Oral administration of obeticholic acid (5 mg/kg daily for 5 days) to rats with estrogen-induced cholestasis restores bile flow to 85% of normal levels, reduces hepatic bile acid accumulation by 60%, and decreases serum alkaline phosphatase (ALP) activity by 50%. [2]
- Gut barrier preservation in cholestatic rats：In bile duct-ligated (BDL) rats, obeticholic acid (5 mg/kg every 2 days for 10 days) reduces intestinal bacterial translocation to mesenteric lymph nodes by 50% and increases ileal TEER by 30%, correlating with attenuated interferon-γ (IFN-γ) expression in the gut. [4]
- Blood pressure regulation in Dahl rats：Daily oral obeticholic acid (10 mg/kg for 4 weeks) lowers systolic blood pressure by 18 mmHg in high-salt fed Dahl rats, associated with 2-fold upregulation of dimethylarginine dimethylaminohydrolase (DDAH) in the kidney. [3]

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]

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FXR agonist activity assay：
1. Recombinant human FXR ligand-binding domain is incubated with obeticholic acid (0.01–10 μM) and a fluorescent ligand displacement probe.
2. Binding affinity is determined by measuring fluorescence polarization, with EC50 values calculated from dose-response curves. [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].

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- Hepatocyte gene expression analysis：
1. Primary rat hepatocytes are treated with obeticholic acid (0.1–10 μM) for 24 hours.
2. Total RNA is extracted, and SHP, BSEP, CYP7A1, and NTCP mRNA levels are quantified by qRT-PCR. [2]

- Intestinal epithelial barrier assay：
1. Caco-2 cells are cultured on transwell inserts and treated with obeticholic acid (1–100 nM) for 48 hours.
2. TEER is measured using an epithelial voltohmmeter, and claudin-1/claudin-2 protein expression is analyzed by Western blot. [4]

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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- Estrogen-induced cholestasis model：
1. Female Sprague-Dawley rats receive 17α-ethynylestradiol (5 mg/kg) subcutaneously daily for 5 days to induce cholestasis.
2. Obeticholic acid (5 mg/kg) or vehicle is administered orally once daily during the estrogen treatment period.
3. Bile flow is measured via bile duct cannulation, and liver tissues are analyzed for bile acid content and gene expression. [2]

- Bile duct ligation (BDL) model：
1. Male Wistar rats undergo BDL surgery to induce cholestasis.
2. Starting 3 days post-surgery, obeticholic acid (5 mg/kg) or vehicle is administered by gavage every 2 days for 10 days.
3. Intestinal permeability is assessed by fluorescein isothiocyanate (FITC)-dextran assay, and bacterial translocation is quantified by culture of mesenteric lymph nodes. [4]

- Hypertensive Dahl rat model：
1. Dahl salt-sensitive rats are fed a high-salt diet (8% NaCl) for 4 weeks.
2. Obeticholic acid (10 mg/kg) or vehicle is administered orally daily.
3. Blood pressure is measured weekly via tail-cuff plethysmography, and renal DDAH activity is analyzed by spectrophotometry. [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.

Toxicity Summary
Signs and Symptoms of Overdose
For patients with primary biliary cholangitis, the administration of OCA at doses higher than the recommended maximum (ie, 25 mg or 50 mg once daily) results in dose-dependent hepatotoxicity, evidenced by elevations in ascites, portal hypertension, jaundice, and exacerbation of primary biliary cholangitis.

Management of overdose
There is no antidote for OCA. The FDA recommends closely monitoring the patient and providing appropriate care during an overdose.
OCA should be administered only to patients who have an inadequate response or are intolerant to ursodeoxycholic acid alone.

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Hepatotoxicity
In multiple preregistration clinical trials, obeticholic acid was found to decrease serum enzyme elevations in a high proportion of patients with different liver diseases. Instances of paradoxical worsening of liver disease or further increases in serum ALT or AST were not reported. However, the product label for obeticholic acid includes warnings that serious liver related adverse events occurred more commonly with active therapy than with placebo treatment. In a pooled analysis of 3 placebo controlled trials in patients with primary biliary cholangitis, liver related adverse events were 5.2 per 100 patient exposure years with 10 mg and 2.4 with placebo. Even higher rates occurred with higher doses of obeticholic acid: 19.8 per 100 patient years for 25 mg daily and 54.5 for 50 mg daily. The clinical features, timing of onset, pattern of enzyme elevations and course of these events were not described in detail. Within a little over a year after approval of obeticholic acid as therapy for primary biliary cholangitis, the FDA published a warning letter stating that they had received notification of 19 deaths and 11 cases of severe liver injury in patients taking obeticholic acid, most but not all of whom had preexisting cirrhosis (Case 1). More recently, severe instances of hepatic decompensation have been reported in patients with both primary biliary cholangitis as well as primary sclerosing cholangitis, two similar chronic cholestatic liver diseases.
In patients with normal alkaline phosphatase levels, obeticholic therapy is associated with slight elevations in alkaline phosphatase, but without accompanying changes in serum aminotransferase levels, GGT or bilirubin, suggesting that the increases are due to alkaline phosphatase from other sources (bone, gastrointestinal tract). Therapy with OCA has been associated with development of pruritus in up to one-third of patients, but the appearance or worsening of itching is not usually associated with worsening of the underlying liver disease or increase in bilirubin or bile acid levels (other than OCA). Thus, obeticholic acid has apparent beneficial effects on liver test abnormalities, but has been linked to rare instances of worsening liver disease which may have clinical significance in patients with preexisting cirrhosis, particularly with use of higher doses of OCA.

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Adverse Effects
The most common adverse effects associated with OCA administration include pruritus, fatigue, and abdominal pain and discomfort. Other reported adverse effects include rash, oropharyngeal pain, dizziness, constipation, arthralgia, dyslipidemia, headache, eczema, depression, hypersensitivity reactions, and abnormal thyroid function. The incidence of pruritus has been shown to increase in a dose-dependent manner and is increased when OCA is used as monotherapy. However, if a patient undergoes OCA therapy for 3 months without pruritus, this adverse effect is unlikely to occur subsequently.
If pruritus does occur, it can be managed with bile acid sequestrants, antihistamines, dose reduction, or a temporary dosing interruption. Esophageal varices and ascites were also shown to occur during a 3-year interim analysis of patients in the POISE trial. Obeticholic acid is also associated with decreases in high-density lipoprotein cholesterol and triglycerides and increases in low-density lipoprotein (LDL) cholesterol. However, a double-blind, placebo-controlled study of patients with NASH showed that atorvastatin could be in combination with OCA to mitigate LDL changes. In patients with decompensated cirrhosis or Child-Pugh B or C hepatic impairment who receive more frequent dosing than the recommended starting dosage of 5 mg once weekly, hepatic decompensation and failure have been reported. Patients at risk for hepatic decompensation should be closely monitored while on OCA.
Dose-dependent liver-related adverse reactions such as jaundice, worsening ascites, portal hypertension, and primary biliary cholangitis flare were also reported in patients with doses of 10 to 50 mg (5 times the recommended dose.) A pooled analysis of 3 placebo-controlled trials involving patients with PBC revealed that liver-related adverse effects occurred at a rate of 5.2 per 100 patient exposure years (PEY) for the 10 mg dose versus 2.4 for the placebo group. Liver-related adverse effects were 19.8 per 100 PEY for the 25 mg group and 54.5 per 100 PEY for the 50 mg group. Monitoring the patient's liver function during OCA therapy and liver-related adverse reactions is vital. Patients who experience paradoxical worsening of liver disease, progressive elevation of liver enzymes, or evidence of hepatic decompensation should discontinue OCA. Patients with cirrhosis presenting with portal hypertension should also discontinue OCA.

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Drug-Drug Interactions
Bile acid binding resins (eg, cholestyramine, colestipol, colesevelam): Obeticholic acid absorption and effectiveness may be reduced if administered concurrently with bile acid binding resins. To minimize interaction, OCA should be taken at least 4 hours before or after these resins.
Warfarin: Coadministration of OCA with warfarin can lower the international normalized ratio (INR). Monitoring INR levels and adjusting warfarin dosage may be necessary to maintain therapeutic efficacy.
Inhibitors of bile salt efflux pump: Concomitant administration of OCA with bile salt efflux pump inhibitors (eg, cyclosporine) may lead to bile salt accumulation in the liver, potentially causing clinical symptoms. These combinations should be avoided. However, if BSEP inhibitors are necessary, serum transaminase and bilirubin levels should be monitored.
CYP1A2 substrate: Obeticholic acid can potentially increase the exposure of CYP1A2 substrates. Serum drug concentrations should be observed in CYP1A2 substrates with narrow therapeutic indexes, such as theophylline and tizanidine.

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.

- Mechanism of action：Obeticholic acid activates FXR to suppress bile acid synthesis (via SHP-mediated inhibition of CYP7A1) and enhance bile acid export (via BSEP upregulation), thereby reducing hepatic bile acid overload. [1][2]
- Therapeutic potential：Investigated for treating cholestatic liver diseases (e.g., primary biliary cholangitis) and improving gut-liver axis dysfunction in cholestasis. [2][4]
- Formulation：Administered orally as a suspension in DMSO/PEG 300/Tween-80/saline for preclinical studies. [2][4]
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.

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Obeticholic acid is a dihydroxy-5beta-cholanic acid that is chenodeoxycholic acid carrying an additional ethyl substituent at the 6alpha-position. A semi-synthetic bile acid which acts as a farnesoid X receptor agonist and is used for treatment of primary biliary cholangitis. It has a role as a farnesoid X receptor agonist and a hepatoprotective agent. It is a dihydroxy-5beta-cholanic acid, a 3alpha-hydroxy steroid and a 7alpha-hydroxy steroid. It is functionally related to a chenodeoxycholic acid.
Primary biliary cirrhosis, or PBC, is a progressive and chronic condition that leads to hepatic injury often resulting in end-stage liver failure that requires liver transplantation. Obeticholic acid is a farnesoid-X receptor (FXR) agonist used to treat this condition, possibly allowing for increased survival. In 2016, it was granted approval to treat primary biliary cholangitis in combination with [ursodeoxycholic acid], which was previously the mainstay treatment for this condition. In May 2021, the FDA updated its prescribing information to contraindicate the use of obeticholic acid in patients with PBC and advanced cirrhosis (e.g. those with portal hypertension or hepatic decompensation) due to a risk of liver failure, in some cases requiring liver transplantation. Obeticholic acid is currently being considered for FDA approval to treat fibrosis caused by non-alcoholic liver steatohepatitis (NASH). The NDA from Intercept Pharmaceuticals was approved in November 2019 and obeticholic acid is expected to be granted full approval for this indication in 2020.

Obeticholic acid is a Farnesoid X Receptor Agonist. The mechanism of action of obeticholic acid is as a Farnesoid X Receptor Agonist.
Obeticholic acid (OCA) is a synthetically modified bile acid and potent agonist of the farnesoid X nuclear receptor (FXR) that is used to treat liver diseases including primary biliary cholangitis. Obeticholic acid has not been linked to elevations in serum enzyme levels during therapy, but has been linked to an increased rate of severe liver related adverse events such as ascites, jaundice and liver failure.
Obeticholic Acid is an orally bioavailable semi-synthetic bile acid derivative and an agonist of the nuclear bile acid receptor farnesoid X receptor (FXR) that may be used to lower hepatic exposure to bile acids. Upon oral administration, obeticholic acid targets and binds to FXR expressed in the liver and intestine, activating FXR-mediated bile acid, inflammatory, fibrotic, and metabolic pathways. This suppresses the production of bile acid in the hepatocytes and increases bile acid transport out of the hepatocytes, thereby reducing hepatic exposure to bile acids. FXR plays an important role in bile acid homeostasis and is involved in hepatic and intestinal inflammation and liver fibrosis.
OBETICHOLIC ACID is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2016 and has 3 approved and 12 investigational indications. This drug has a black box warning from the FDA.

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.)