Bile acid derivative; The mechanism of action of THDCA involves multiple targets and signaling pathways. It primarily exerts anti-apoptotic effects by inhibiting the calcium-mediated apoptotic pathway and the activation of caspase-12. Regarding its anti-inflammatory properties, THDCA significantly reduces the activity and expression of myeloperoxidase (MPO) and decreases the levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Research suggests that its anti-inflammatory activity may be associated with the regulation of the nuclear factor kappa-B (NF-κB) signaling pathway or the activation of the G protein-coupled bile acid receptor (TGR5), thereby mediating the regulation of intestinal immune homeostasis.

In vitro studies have demonstrated that THDCA significantly promotes the proliferation of intestinal epithelial cells. In IEC-6 and Caco-2 cell models, THDCA at concentrations ranging from 0.05 to 1.00 mM stimulates cell proliferation in a dose-dependent manner, with a marked effect observed after 6 days of treatment. Cell cycle analysis revealed that treatment with 0.5 mM THDCA for 24 hours significantly increased the proportion of cells in the S phase while decreasing those in the G1 phase, indicating accelerated cell cycle progression. Mechanistic studies found that THDCA upregulates the mRNA and protein expression of the proto-oncogene c-myc, and this proliferation-inducing effect is partially dependent on c-myc activation. Furthermore, THDCA prevents apoptosis by blocking calcium-mediated apoptotic pathways and caspase-12 activation.

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This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties [2].

In in vivo animal models, THDCA demonstrates significant protective effects. In a mouse model of sepsis (induced by lipopolysaccharide), intravenous administration of THDCA at 0.5 mg/kg significantly improved survival rates, reduced liver and kidney damage, ameliorated systemic inflammation, and normalized blood pressure. In studies on inflammatory bowel disease using a TNBS-induced ulcerative colitis mouse model, THDCA alleviated colonic mucosal injury and inflammatory infiltration, an effect associated with the inhibition of MPO activity and the reduction of TNF-α and IL-6 levels. Additionally, as a bile acid, THDCA regulates bile salt and biliary lipid secretion in rats.

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The prevention of the hepatotoxic effects produced by intravenous infusion of taurochenodeoxycholic acid (TCDCA) by coinfusion with taurohyodeoxycholic acid (THDCA) was evaluated in bile fistula rats; the hepatoprotective effects of the latter were also compared with those of tauroursodeoxycholic acid (TUDCA). Rats infused with TCDCA at a dose of 8 micromol/min/kg showed reduced bile flow and calcium secretion, as well as increased biliary release of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). This was associated with a very low biliary secretion rate of TCDCA (approximately 1 micromol/min/kg). Simultaneous infusion of THDCA or TUDCA at the same dose preserved bile flow and almost totally abolished the pathological leakage of the two enzymes into bile. The effect was slightly more potent for THDCA. The maximum secretion rate of TCDCA increased to the highest value (8 micromol/min/kg) when coinfused with either of the two hepatoprotective bile acids (BA), which were efficiently and completely secreted in the bile, without metabolism. Calcium output was also restored and phospholipid (PL) secretion increased with respect to the control saline infusion. This increase was higher in the THDCA study. These data show that THDCA is highly effective in the prevention of hepatotoxicity induced by intravenous infusion of TCDCA by facilitating its biliary secretion and reducing its hepatic residence time; this was associated with selective stimulation of PL biliary secretion [1].

Currently, no detailed protocol for a non-cellular enzyme/receptor binding assay (e.g., surface plasmon resonance or radioligand binding assay) specifically for THDCA has been reported in the available literature. However, based on its potential role as an agonist for the TGR5 receptor, a standard protocol might involve incubating various concentrations of THDCA with purified TGR5 receptor protein or membrane fragments from TGR5-overexpressing cells in a buffer. A fluorescent or radiolabeled tracer (e.g., ³⁵S-GTPγS) could be used to assess receptor activation. Competition binding experiments would allow the calculation of the binding affinity (Ki or IC₅₀) of THDCA for the receptor. Non-specific binding is determined by adding an excess of unlabeled ligand (e.g., other bile acid analogs).

In vitro cell-based assays for THDCA are typically conducted using intestinal epithelial cell lines, such as IEC-6 or Caco-2. For a cell proliferation assay: Cells are cultured in medium containing 10% fetal bovine serum until 70-80% confluence, then trypsinized and seeded into 96-well plates (approximately 1×10⁴ cells/well). After overnight adhesion, the medium is replaced with fresh medium containing various concentrations of THDCA (e.g., 0, 0.05, 0.50, 1.00 mM), with 3-6 replicate wells per concentration. Cells are incubated at 37°C in a 5% CO₂ incubator for 1 to 6 days. At specific time points (e.g., days 1, 2, 4, 6), cell viability is measured using the CCK-8 or MTT assay, and absorbance is read at 450 nm (CCK-8) or 570 nm (MTT) using a microplate reader. For cell cycle analysis, cells treated with 0.5 mM THDCA for 24 hours are harvested, fixed, stained with propidium iodide (PI), and analyzed by flow cytometry.

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The cytolytic effect of Taurohyodeoxycholic acid/THDCA and TUDCA on HepG2 cells was tested by incubating the cells with media containing THDCA or TUDCA or DCA at a final concentration ranging from 50 to 800 μmol/l for 3, 6, 12, 24, 48 and 72 h. To evaluate the hepatoprotective effect of THDCA and TUDCA, mixtures of 350 μmol/l DCA with increasing concentrations of either Taurohyodeoxycholic acid/THDCA or TUDCA (50, 100, 200 and 400 μmol/l) were incubated with HepG2 cells for 24 and 48 h. Enzyme release at each incubation time was compared to that induced by DCA alone at 350 μmol/l and 750 μmol/l.[2]

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Evaluation of hemolytic properties of bile salts on human erythrocytes: Hemolytic properties of different bile salts were evaluated on fresh human red blood cells obtained from normal healthy subjects, after centrifugation of venous samples at 5000 rpm for 10 min. Erythrocytes were washed three times in phosphate-buffered saline, resuspended to the original blood volume in Tris buffer (10 mmol Tris, 130 mmol NaCl, 10 mmol glucose, pH 7.4) and incubated in multiwell microplates with increasing concentrations of different bile salts (Taurohyodeoxycholic acid/THDCA, TUDCA, UDCA, CA CDCA, DCA, final concentration of the testing solutions: 25, 50, 100, 200, 400, 800 μmol/l, 0.5, 1, 2, 4 and 6 mmol/l) dissolved in 5 mmol/l Tris buffer (plus 0.15 mol/l NaCl and containing 100 mg/dl glucose, pH 7.4, at 37°C). For concentrations ranging from 25 to 800 μmol/l times of exposure were 50 min, 18 and 24 h, while for concentrations greater than 800 μmol/l only 50 min incubation time was used. Total hemoglobin content was determined in red blood cell supernatant after centrifugation of microplates at 15 000 rpm for 20 min at 4°C, and expressed as percent of that induced by NH4Cl (100% hemolysis) [2].

Using THDCA in a mouse model of sepsis as an example: C57BL/6N mice are used to establish a sepsis model via intraperitoneal injection of lipopolysaccharide (LPS). Experimental groups typically include a normal control group, a model group (LPS + vehicle), and a THDCA-treated group. In the treatment group, THDCA is administered intravenously at 0.5 mg/kg, either 30 minutes or 24 hours after LPS injection. Animal survival rates are monitored at various time points (e.g., up to 24 hours) post-LPS injection. At the end of the experiment, blood samples are collected for biochemical analysis (e.g., ALT, AST, creatinine, BUN) and cytokine measurement (e.g., TNF-α, IL-6). Liver, kidney, and lung tissues are also harvested; one portion is processed for H&E staining to assess histopathological damage, while another portion is used to measure MPO activity and inflammatory factor expression.

Systematic toxicology data specifically for THDCA are lacking in the available literature. However, important safety references can be drawn from a Phase I clinical trial of its analog, TDCA (HY209), in healthy volunteers. In that trial, following a single intravenous administration of TDCA at doses ranging from 0.1 to 1.6 mg/kg, all adverse events were mild, and no serious adverse events were observed. There was no significant correlation between the frequency of adverse events and the administered dose. Commonly reported adverse events included nausea and headache, though these were deemed unlikely to be directly related to the drug, as their onset and duration did not correlate with the pharmacokinetic profile. The main potential adverse effects anticipated from preclinical toxicology studies were elevated liver enzyme levels and inflammatory reactions at the injection site; however, these were not observed, even in the highest dose groups. These data indicate that THDCA/TDCA is well-tolerated and has a favorable safety profile within this dose range.

[1]. Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat. Hepatology. 1998 Feb;27(2):520-5.

[2]. Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line. Biochim Biophys Acta. 2002 Jan 30;1580(1):31-9.

Tauride-deoxycholic acid (TDCA) is a conjugate of bile acid and taurine, and it is the conjugated acid of tauride-deoxycholic acid (1-). This study used HepG2 cell culture (a suitable human hepatocyte model) to compare the cytotoxic and cytoprotective effects of two hydrophilic bile acids, TUDCA and THDCA/TAUdeoxycholic acid. Low concentrations of both bile acids showed no cytotoxicity, but with increasing concentration and exposure time, a trend towards enzyme release into the culture medium gradually emerged, with THDCA releasing more than TUDCA. In mixed experiments with a fixed DCA concentration and increasing either TUDCA or THDCA concentration, the difference in the effects of the two bile acids was more significant, with THDCA+DCA consistently inducing higher enzyme release than TUDCA+DCA. The mixed experiments showed that equimolar doses of the hydrophobic bile acid DCA alone were far more toxic than THDCA or a mixture of TUDCA and DCA (as shown in Figure 2). The difference in behavior between THDCA and TUDCA may be related to their physicochemical properties. THDCA/taurine deoxycholic acid has a retention time on high performance liquid chromatography (HPLC) very close to that of TUDCA, thus exhibiting hydrophilic bile acid characteristics; on the other hand, its critical micelle concentration (CMC) is similar to that of hydrophobic bile acids. Given that the cytotoxicity of bile acids is related to their relative hydrophobicity, the effect of THDCA on HepG2 cells reflects the balance between its hydrophilicity and hydrophobicity. Hydrophobic bile acids can induce changes in the permeability of large monolayer vesicle membranes at concentrations below their critical micelle concentration (CMC), while hydrophilic bile acids can only be detected at concentrations far above physiological levels. Our data on bile acid-induced hemolysis also support this view, suggesting that even highly hydrophilic bile acids can cause cell membrane damage at high concentrations. Therefore, under our study conditions, low concentrations of THDCA showed no cytotoxicity to HepG2 cells, but at higher concentrations, it exhibited moderate cell lysis, an effect not observed in the more hydrophilic TUDCA. [2]

C26H45NO6S

499.70

499.297

2958-04-5

38411-85-7

119046

White to off-white solid powder

1.216g/cm3

1.552

4.869

4

6

7

34

858

10

C[C@H](CCC(=O)NCCS(=O)(=O)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2C[C@@H]([C@H]4[C@@]3(CC[C@H](C4)O)C)O)C

HMXPOCDLAFAFNT-BHYUGXBJSA-N

InChI=1S/C26H45NO6S/c1-16(4-7-24(30)27-12-13-34(31,32)33)19-5-6-20-18-15-23(29)22-14-17(28)8-10-26(22,3)21(18)9-11-25(19,20)2/h16-23,28-29H,4-15H2,1-3H3,(H,27,30)(H,31,32,33)/t16-,17-,18+,19-,20+,21+,22+,23+,25-,26-/m1/s1

2-[[(4R)-4-[(3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-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]pentanoyl]amino]ethanesulfonic acid

Taurohyodeoxycholic acid; 2958-04-5; Taurine Hyodeoxycholate; 2-[[(4R)-4-[(3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-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]pentanoyl]amino]ethanesulfonic acid; D6V086EMME; CHEMBL270516; THDCA; TAUROHYODEOXYCHOLIC ACID SODIUM SALT;

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)

DMSO :~100 mg/mL (~200.12 mM)
H2O :~25 mg/mL (~50.03 mM; ultrasonic and warming and adjust pH to 12 with NaOH and heat to 60°C)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.16 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 20.8 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 2: ≥ 2.08 mg/mL (4.16 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 20.8 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 3: ≥ 2.08 mg/mL (4.16 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (50.03 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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