Bile acid derivative

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].

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].

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].

[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.

Taurohyodeoxycholic acid is a bile acid taurine conjugate. It is a conjugate acid of a taurohyodeoxycholate(1-).

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In this study we have compared the cytotoxic and cytoprotective effects of two hydrophilic bile acids, TUDCA and THDCA/Taurohyodeoxycholic acid, using HepG2 cell cultures, a suitable model of human hepatocytes. Low concentrations of either bile acid resulted to be not cytotoxic, however as concentrations and time of exposure increase, a trend to increased enzyme release into the medium becomes evident, being greater with THDCA as compared to TUDCA. In mixing experiments using a fixed concentration of DCA and increasing that of either TUDCA or THDCA, the effects of the two bile acids appeared to differ to a greater extent, in that enzyme release induced by THDCA plus DCA was always greater than that caused by TUDCA plus DCA. The mixing experiments show that an equimolar dose of the hydrophobic bile acid DCA alone is by far more toxic than a mixture of THDCA or TUDCA plus DCA as shown in Fig. 2. The different behavior of THDCA as compared to TUDCA might be explained on the basis of its physical–chemical characteristics. Having a retention time very close to that of TUDCA on HPLC, Taurohyodeoxycholic acid/THDCA behaves as a hydrophilic bile acid; on the other hand its CMC is similar to that of the hydrophobic bile acids. Given that cytotoxicity of the bile acids has been related to their relative hydrophobicity the effect of THDCA on HepG2 cells reflects the balance between the hydrophilic and hydrophobic properties. Hydrophobic bile salts at concentrations lower than their CMC can induce alterations in membrane permeability of large unilamellar vesicles whereas for hydrophilic bile salts such an effect can be detected only at concentrations far higher than that reached in physiological conditions. The view is supported also by our data on bile acid-induced hemolysis, which show that at high concentrations even very hydrophilic bile acids may be damaging for membranes. Thus in the conditions of our study THDCA was not cytotoxic on HepG2 cells, at low concentrations, and showed, at higher concentrations, a moderate cytolytic effect that was not observed yet with 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.)