EC50: 0.82 μM (TGR5)[1]
TGR5 receptor (EC₅₀ = 0.82 μM, efficacy = 166%)
FXR receptor (EC₅₀ > 100 μM, efficacy = 18%) [1]
TGR5 receptor (selective and potent agonist, reported in Figure 1 as a 30-fold improvement over CA) [2]

A novel, highly effective, and selective TGR5 agonist with notable in vivo activity is called INT-777 [1]. In a cAMP-dependent way, INT-777 (3 μM) boosts ATP synthesis in the human enteroendocrine cell line NCI-H716 [2]. Adding INT-777 (10 μM) to the serosal side of distal colon segments stripped of serosal tissue results in a decrease in Isc and an increase in TEER. In preparations of the muco-submucosa that were treated with TTX and neuron-free, the impact of INT-777 on basal secretion was diminished [3].

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INT-777 dose-dependently induces GLP-1 release ex vivo in ileal explants isolated from high-fat-fed TGR5 transgenic mice. [1]
In STC-1 murine enteroendocrine cell line, bile acid-mediated activation of TGR5 leads to increased intracellular cAMP levels, triggering GLP-1 release. [1]
In enteroendocrine STC-1 cells, INT-777 triggered a dose-dependent increase in intracellular cAMP levels. This induction was abrogated upon reduction of TGR5 expression by a specific TGR5 shRNA. [2]
In human enteroendocrine NCI-H716 cells, INT-777 increased intracellular calcium influx in a TGR5- and cAMP-dependent manner. This effect was potentiated by TGR5 overexpression and inhibited by TGR5 RNA interference or adenylate cyclase inhibitor MDL-12330A. [2]
In NCI-H716 cells, INT-777 treatment increased GLP-1 release, an effect inhibited by MDL-12330A. [2]
In mouse enteroendocrine STC-1 cells, GLP-1 release triggered by INT-777 was enhanced by TGR5 overexpression and prevented by TGR5 RNA interference or MDL-12330A. [2]
In STC-1 cells, treatment with INT-777 resulted in a cAMP-dependent increase in cytochrome c oxidase (Cox) activity, cellular oxygen consumption, and ATP/ADP ratio. [2]
In primary brown adipocytes isolated from C57BL/6J mice, treatment with 3 μM INT-777 for 12 hours increased basal O₂ consumption, and the response to the uncoupling agent FCCP was more pronounced. [2]

INT-777 (1 μM/min/kg, oral) is a strong choleretic that favors the biliary-hepatic shunt channel with ducts in HF-fed TGR5-Tg men. It also inhibits the activation and subsequent binding of carboxy-CoA [1]. When given to TGR5-Tg mice that are fed a high-fat diet, INT-777 (30 mg/kg/day) enhances energy expenditure and decreases obesity and hepatic steatosis [2].

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In diet-induced obese mice, pharmacological targeting of TGR5 by INT-777 increases energy expenditure, resulting in reduced weight gain and adiposity. [1]
INT-777 improves liver function in high-fat-fed mice, reducing steatosis and fibrosis. [1]
In diet-induced obese (DIO) C57BL/6J mice, dietary intervention with INT-777 (30 mg/kg/day) for 10 weeks significantly attenuated body weight gain (~15%), reduced fat mass, and decreased liver and fat pad mass compared to high-fat (HF)-fed controls. [2]
In DIO mice, INT-777 treatment increased energy expenditure (as measured by O₂ consumption and CO₂ production) and reduced the respiratory quotient during the dark period, consistent with increased fat oxidation. [2]
INT-777 treatment improved liver function in DIO mice, evidenced by reduced liver steatosis (assessed by oil-red-O staining and liver lipid content), lower plasma levels of liver enzymes, and absence of liver fibrosis (assessed by Sirius red staining). [2]
Plasma triglyceride and non-esterified fatty acid (NEFA) levels were significantly reduced in HF-fed mice treated with INT-777. [2]
In both DIO and db/db mice, treatment with INT-777 (30 mg/kg/day) robustly improved oral glucose tolerance and improved glucose-stimulated insulin secretion profiles. Fasting glucose and insulin levels were decreased. [2]
Hyperinsulinemic-euglycemic clamp studies in DIO mice treated with INT-777 showed normalized glucose infusion rates, hepatic glucose production, glucose disposal rates, and insulin-mediated suppression of glucose production. Insulin-stimulated glucose uptake was improved in liver and muscle. [2]
In TGR5 transgenic (TGR5-Tg) mice fed a high-fat diet, oral glucose tolerance was markedly improved compared to controls, associated with enhanced postprandial GLP-1 and insulin secretion. Pancreatic islets from TGR5-Tg mice showed higher insulin content and a more normal size distribution. [2]
In TGR5 deficient (TGR5⁻/⁻) mice fed a high-fat diet, glucose tolerance was impaired. The ability of INT-777 to improve glucose tolerance and insulin secretion was blunted in TGR5⁻/⁻ mice, confirming its TGR5-dependent action. [2]
Pre-administration of INT-777 (30 mg/kg) moderately increased GLP-1 release after a glucose challenge in TGR5⁺/⁺ mice, an effect markedly potentiated by co-administration of a DPP4 inhibitor. This effect was blunted in TGR5⁻/⁻ mice. [2]

The ability of compounds to activate TGR5 was assessed in CHO cells transiently transfected with human TGR5, using a cAMP-responsive element-driven luciferase reporter assay. [1]
FXR activity was evaluated in COS1 cells using a cell-based bioluminescence assay. [1]
Cytochrome c oxidase (Cox) activity was evaluated in STC-1 cells by following the oxidation of fully reduced cytochrome C at 550 nm. Cells were treated with INT-777 at indicated concentrations for 1 hour. Vehicle or the adenylate cyclase inhibitor MDL-12330A (1 μM) was added 15 minutes prior to treatment. [2]
cAMP production was measured in STC-1 cells transfected with control or mTGR5 shRNA and treated with indicated concentrations of INT-777. [2]

The experiments are carried out in STC-1 or NCI-H716 cells treated with vehicle (DMSO) or INT-777. INT-777 is assessed for its agonistic activity on TGR5. cAMP production is performed. Cytochrome C oxidase activity is evaluated by following the oxidation of fully reduced cytochrome C at 550 nm. ATP/ADP ratio and GLP-1 release is measured according to the manufacturers instruction. Primary brown adipocytes are prepared and ileal explants are prepared.

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TGR5-mediated GLP-1 release was assessed in STC-1 enteroendocrine cells. [1]
Ex vivo GLP-1 release was measured in ileal explants from TGR5 transgenic mice after exposure to INT-777. [1]
Intracellular calcium levels were quantified in NCI-H716 cells seeded in Matrigel-coated 96-well plates. After transfection with mock vector, hTGR5 expression vector, or hTGR5 siRNA for 72 hours, cells were washed and assayed for intracellular calcium using Fluo-4 AM dye according to the manufacturer's protocol. Cells were then treated with INT-777 (1 μM or 10 μM), and calcium flux was measured. [2]
GLP-1 release was measured in NCI-H716 and STC-1 cell culture supernatants after treatment with INT-777 (with or without glucose or inhibitors) according to the manufacturer's instructions. For STC-1 cells, a DPP4 inhibitor was added to the culture medium to prevent GLP-1 degradation. [2]
Cellular oxygen consumption was measured in STC-1 cells using an extracellular flux analyzer. Cells were treated with vehicle or adenylate cyclase inhibitor MDL-12330A, followed by treatment with 1 μM INT-777. [2]
ATP/ADP ratio was measured in STC-1 cell lysates after treatment with INT-777 according to the manufacturer's instructions. [2]
Ex vivo GLP-1 release was measured in ileal explants isolated from high-fat-fed TGR5-Tg and control mice. Explants were exposed for 1 hour to indicated concentrations of lithocholic acid (LCA). [2]

In bile fistula rat models, INT-777 was administered intravenously (femoral vein infusion) or intraduodenally at a dose of 1 μmol/min/kg body weight for 1 hour. Bile secretion parameters were collected and analyzed. [1]
In diet-induced obese mice, INT-777 was administered pharmacologically to assess effects on energy expenditure, body weight, adiposity, and liver histology. [1]
For dietary intervention studies in C57BL/6J mice, diet-induced obesity was induced by feeding 8-week-old mice a high-fat diet (60% calories from fat) for 14 weeks. INT-777 was then mixed with the high-fat diet at a dose sufficient to achieve an in vivo dose of 30 mg/kg/day for up to 10 weeks. Body weight, food intake, and metabolic parameters were monitored. [2]
In db/db mice, 14-week-old animals were fed a chow diet without or with INT-777 (30 mg/kg/day) mixed into the diet for 6 weeks. [2]
In TGR5⁺/⁺ and TGR5⁻/⁻ mice, high-fat diet feeding was initiated for 9 weeks. After a first oral glucose tolerance test (OGTT), the high-fat diet was supplemented with INT-777 at 30 mg/kg/day for 4 weeks, followed by a second OGTT. [2]
For acute GLP-1 secretion studies, TGR5⁺/⁺ and TGR5⁻/⁻ mice on a chow diet received an oral gavage of saline or INT-777 (30 mg/kg), alone or in combination with a DPP4 inhibitor (3 mg/kg), 30 minutes prior to an oral glucose challenge. Blood was collected for GLP-1 measurement. [2]
Hyperinsulinemic-euglycemic clamp studies were performed on DIO mice after 10 weeks of dietary intervention with INT-777. Mice were fasted, and a primed continuous infusion of insulin (10 mU/min/kg) was administered to maintain euglycemia. Tritiated glucose was infused to assess glucose kinetics, and ¹⁴C-2-deoxyglucose was injected to measure tissue-specific glucose uptake. [2]
TGR5 transgenic (TGR5-Tg) mice were generated by oocyte injection of a bacterial artificial chromosome (BAC) containing the TGR5 locus. TGR5 deficient (TGR5⁻/⁻) mice were generated by crossing mice with a floxed TGR5 allele with CMV-Cre transgenic mice. [2]

INT-777 is highly stable in human fecal broth, with over 95% remaining unchanged after 12 hours of incubation, indicating its resistance to bacterial 7α-dehydroxylation. [1] It has a potent choleretic effect, with a high bile secretion rate and low binding reaction rate; after intravenous or duodenal infusion, over 90% is secreted into bile in an unbound form. [1] INT-777 has a relatively high albumin binding rate (62%), which may help it enter systemic circulation and target peripheral tissues. [1] High performance liquid chromatography analysis confirmed the presence of INT-777 in treated mouse plasma at concentrations comparable to those of endogenous bile acids and β-mouse bile acids. [2] INT-777 treatment did not significantly alter the overall composition of plasma bile acids or the expression profile of enzymes involved in bile acid synthesis. [2]

[1]. Discovery of 6alpha-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity. J Med Chem. 2009 Dec 24;52(24):7958-61.

[2]. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab. 2009 Sep;10(3):167-77.

[3]. Reduction of epithelial secretion in male rat distal colonic mucosa by bile acid receptor TGR5 agonist, INT-777: role of submucosal neurons. Neurogastroenterol Motil. 2016 Jun 3. doi: 10.1111/nmo.

[4]. INT-777, a bile acid receptor agonist, extenuates pancreatic acinar cells necrosis in a mouse model of acute pancreatitis. Biochem Biophys Res Commun. 2018 Sep 3;503(1):38-44.

INT-777 is a bile acid derivative modified with 6α-ethyl and 23(S)-methyl to enhance the selectivity and metabolic stability of TGR5. [1] It is a potent and selective TGR5 agonist with potential applications in the treatment of metabolic diseases such as diabetes, obesity, and non-alcoholic steatohepatitis. [1] The compound is not easily metabolized by intestinal bacteria and has good physicochemical properties, such as lower detergency and higher metabolic stability compared to natural bile acids. [1] INT-777 is a semi-synthetic bile acid derivative modified with 6α-ethyl and 23(S)-methyl to act as a potent and selective TGR5 agonist with no activity against FXR. [2]
Its mechanism of action involves the activation of TGR5, which leads to an increase in intracellular cAMP levels, thereby enhancing mitochondrial oxidative phosphorylation, increasing the ATP/ADP ratio, closing K_ATP channels, mobilizing calcium ions through calcium channels, and ultimately stimulating enteroendocrine L cells to release GLP-1. [2]
INT-777 activation of TGR5 can also increase energy expenditure in brown adipose tissue and muscle, thereby helping to reduce fat accumulation and improve metabolic status. [2]
This study suggests that targeting TGR5 with INT-777 is a promising strategy for treating diabetes, obesity, and related metabolic disorders such as non-alcoholic steatohepatitis (NASH). [2] The study showed that the effects of INT-777 in improving glucose tolerance, insulin sensitivity, and reducing hepatic steatosis depend on TGR5 expression, as these effects are absent or diminished in TGR5-deficient mice. [2]

C₂₇H₄₆O₅

450.6511

450.335

1199796-29-6

INT-777 R-enantiomer;1198786-98-9

45483949

White to yellow solid powder

4.33

4

5

5

32

710

13

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

NPBCMXATLRCCLF-IRRLEISYSA-N

InChi Code InChI=1S/C27H46O5/c1-6-17-20-12-16(28)9-10-26(20,4)21-13-22(29)27(5)18(14(2)11-15(3)25(31)32)7-8-19(27)23(21)24(17)30/h14-24,28-30H,6-13H2,1-5H3,(H,31,32)/t14-,15+,16-,17-,18-,19+,20+,21+,22+,23+,24-,26+,27-/m1/s1

(2S,4R)-4-((3R,5S,6R,7R,8R,9S,10S,12S,13R,14S,17R)-6-ethyl-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylpentanoic acid

INT-777 S-EMCAINT 777 6-EMCAINT777

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)

Ethanol :≥ 50 mg/mL (~110.95 mM)
DMSO : ≥ 31 mg/mL (~68.79 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.55 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 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 2: ≥ 2.5 mg/mL (5.55 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 3: ≥ 2.5 mg/mL (5.55 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 4: 5 mg/mL (11.10 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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