Endogenous Metabolite; ERK; Caspase-3/12

Tauroursodeoxycholate (TUDCA) activates mitogen-activated phosphophosphatase 1 (MKP-1) through PKCα and suppresses ERK phosphorylation, ultimately decreasing the survival and migration of vascular smooth muscle cells (VSMC). Tauroursodeoxycholate suppresses ERK through Ca2+-induced PKCα translocation Tauroursodeoxycholate (200 μM) can restore VSMC decreased by Tauroursodeoxycholate (200 μM), consequently limiting the proliferation and migration of VSMC. activity, which implies that the anti-stress impact of Tauroursodeoxycholate depends on the expression of MKP-1 [1].

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Transporters of TUDCA uptake in human VSMCs (hVSMCs) were analysed by RT-PCR and western blot. A knock-down experiment using specific si-RNA revealed that TUDCA was incorporated into hVSMCs via organic anion transporter 2 (OATP2). TUDCA reduced the viability of hVSMCs, which were mediated by inhibition of extracellular signal-regulated kinase (ERK) by induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) via protein kinase Cα (PKCα). The anti-proliferative effect of TUDCA was reversed by treatment with 7-hydroxystaurosporine, an inhibitor of PKC, and by the knock-down of MKP-1. In addition, TUDCA suppressed hVSMC migration, which was mediated by reduced matrix metalloproteinase-9 (MMP-9) expression by ERK inhibition, as well as reduced viability of hVSMCs. The uptake of TUDCA was mediated by OATP2 in hVSMCs. TUDCA suppresses both viability and migration of VSMCs through inhibition of ERK phosphorylation, by induction of MKP-1 via PKCα. TUDCA suppressed the PDGF-mediated proliferation and migration of VSMCs. [1]

Using transferase dUTP nick cleavage labeling (TUNEL) and immunohistochemistry for tumescent cell nuclear factor (PCNA) assays, the effects of tauroursodeoxycholate (TUDCA) on VSMC swelling and inflammation in vivo were investigated. Caspase 3 activity in damaged tissue was experimentally boosted by tauroursodeoxycholate (10, 50, and 100 mg/kg), and tauroursodeoxycholate caused mutations in VSMCs in the neointima. One week following the injury, the phosphorylation levels of ERK and MMP-9 expression were measured using the injured tissue and compared with normal controls. Injuries to balloons raise MMP-9 production and phosphorylation of ERK in the tissue. In a mimetic regulatory approach, tauroursodeoxycholate (10, 50, and 100 mg/kg) suppresses the phosphorylation of ERK and MMP-9 [1]. The bile acid tauodeoxycholate (TUDCA) is hydrophilic. Tauodeoxycholate decreases endoplasmic reticulum intermediate cells and the pancreas, which enhances liver function as a cytoprotective agent and may prevent hepatocellular cancer. When tauodeoxycholate is administered to Ang II-induced ApoE-/-mice, it dramatically lowers the expression of Andrew molecules, including eIF2α, caspase-3, caspase-12, C/EBP homologous protein, c-Jun N-terminator protein (JNK), activating transcription factor 4 (ATF4), X-box binding protein (XBP), and JNK. This effect is significant (p<0.05). In ApoE-/-mice, tauroursodeoxycholate decreases the abdominal aorta caused by angiotensin II. 0.5 g/kg/day of tauroursodeoxycholate was administered to Ang II-induced ApoE-/- mice (ER pair). ..There was no difference in the total cholesterol level (663.6±88.7 mg/dL vs 655.7±65.4 mg/dL; p>0.05) or systolic blood pressure (141.3±5.6 mmHg vs 145.9±8.9 mmHg; p>0.05) between the Tauroursodeoxycholate group and the AAA model group. Furthermore, the Tauroursodeoxycholate group's AAA lesion area was less than the AAA model group's (0.37±0.03 mm2 vs. 1.51±0.06 mm; p<0.05) in comparison to the AAA model group. )[2].

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Reduction in neointimal hyperplasia by TUDCA in vivo [1]
The in vitro results suggest that TUDCA could inhibit neointima formation after vessel injury through ERK inhibition via PKCα. The rat carotid artery injury model was used to analyse the effects of TUDCA on neointima formation. After balloon injury, the oral administration of TUDCA for 2 weeks significantly suppressed neointima formation in a dose-dependent manner (I/M ratio; 50 mg/kg, 1.48 ± 0.11; 100 mg/kg, 1.17 ± 0.16 vs. control, 1.86 ± 0.17, n= 8, P< 0.05) (Figure 4A and B). In addition, neointimal area (vehicle, 100 ± 12.9%; TUDCA 10 mg/kg, 80.0 ± 19.7%; TUDCA 50 mg/kg, 75.5 ± 6.8%; TUDCA 100 mg/kg, 56.9 ± 20%, n= 8 in each group) and neointimal thickness (vehicle, 133.5 ± 26.7 μm; TUDCA 10 mg/kg, 92.1 ± 23.3 μm; TUDCA 50 mg/kg, 78.5 ± 15.7 μm; TUDCA 100 mg/kg, 61.8 ± 16.3 μm, n= 8 in each group) were significantly reduced by administration of TUDCA (Figure 4C and D), whereas TUDCA increased the lumen area in a dose-dependent manner (normal, 100 ± 5.6%;vehicle, 63.3 ± 7.1%; TUDCA 10 mg/kg, 73.1 ± 11.1%; TUDCA 50 mg/kg, 78.7 ± 5.8%; TUDCA 100 mg/kg, 91.2 ± 8.2%, n= 8 in each group) proportional to the decrease in neointimal area (Figure 4E). In addition, the medial area and medial cell number were not affected (Supplementary material online, Figure S5A and B).

FITC-TUDCA uptake assay [1]
Human VSMCs (hVSMCs) were cultured in Smooth Muscle Cell Growth Medium 2. Cells between passages 4 and 7, from four donors, were used for this study. hVSMCs (1 × 104) were seeded onto a 96-well plate and cultured. Each indicated concentration of FITC-TUDCA was added and washed with cold phosphate-buffered saline. The FITC signal was evaluated by counting the absorbance at 488 nm. For the competition assay, 100 μM of FITC-TUDCA was mixed with the indicated concentration of non-labelled TUDCA, and then treated to hVSMCs for 15 min. The detailed methods were described in Supplementary material online.

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Zymography [1]
Cells were incubated with TUDCA for 24 h. Then, the supernatants were harvested and loaded onto SDS–PAGE containing 1 mg/mL gelatin. After electrophoresis, the gel was incubated in the reaction buffer for 24 h, and then subsequently stained with 0.15% Coomassie Brilliant Blue R250. The detailed methods were described in Supplementary material online.

Cell viability and proliferation assay [1]
Cell viability and proliferation were measured using Ez-Cytox. VSMCs (5 × 103 cells) were seeded onto 96-well plates in Smooth Muscle Cell Growth Medium 2 and cultured. After serum starvation, TUDCA (0, 50, 100, and 200 μM) was added to the hVSMCs, with or without 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA, 10 μM) and 7-hydroxystaurosporine (H7, 10 μM) and cultured for 24 h. To assess the effect of TUDCA on the PDGF-stimulated hVSMC proliferation, hVSMCs were seeded onto 96-well plates and cultured. After serum starvation, TUDCA (0, 50, 100, and 200 μM) was added to the hVSMCs, with or without PDGF-BB (50 ng/mL) and cultured. After addition of 10 μL of Ez-Cytox into each well, cell viability was evaluated by measuring the optical density at 450 nm.

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The scratch-wound assay [1]
Migration of hVSMCs was evaluated using the scratch-wound model, as described previously.24 hVSMCs (1 × 106) were seeded onto 60 mm dishes and cultured until the cells reached confluence. Each indicated concentration of TUDCA was added to the hVSMCs for 1 h, and the confluent monolayers were scraped, through the middle of the dish, using a modified yellow tip. After wounding, the monolayers were washed with serum-free SMCGM2 and were cultured with TUDCA for 36 h. The hVSMCs that migrated from the wound edge were counted by three separate investigators.

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Flow cytometry [1]
Apoptosis of the hVSMCs by TUDCA were evaluated by flow cytometry. After serum starvation for 24 h, TUDCA in increasing concentrations (i.e. 0, 50, 100, and 200 μM) was added to the hVSMCs. Cells were harvested and fixed and then the DNA content was analysed by flow cytometry. The detailed methods were described in Supplementary material online.

Rat carotid artery balloon injury [1]
The rats were anaesthetized with a combined anaesthetic (ketamine, 70 mg/kg; xylazine, 7 mg/kg ip). Noxious stimuli were applied to a limb occasionally throughout the experiments, while monitoring changes in the end-tidal carbon dioxide, heart rate, blood pressure, and cardiac rhythm, in order to ascertain the level of anaesthesia. After the left external carotid artery was exposed, a 2 F Fogarty embolectomy catheter was introduced through an external carotid arteriotomy incision, advanced to the common carotid artery, inflated with 0.2 mL of saline, and withdrawn 10 times, with rotation.TUDCA was then administered orally once a day, in different concentrations (i.e. vehicle, 10, 50, and 100 mg/kg) for 2 weeks. The carotid arteries were fixed by perfusion with 4% formaldehyde, then the tissues were embedded in paraffin, and sections (8 μm) were stained with H&E. The detailed methods were described in Supplementary material online.

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Ang II induced AAA model in mice [2]
Thirty ApoE−/− C57BL/6 male mice aged 8 weeks were randomly divided into three groups (n = 10 in each group): (i) sham operated and injected with physiologic (0.9%) saline as vehicle (“normal: group); (ii) mini-osmotic pumps were implanted subcutaneously into the right flank of ApoE−/− mice to release Ang II (1000 ng/kg/min) over the course of 28 days (“AAA model” group); (iii) AAA model mice treated with ER stress inhibitor (TUDCA) daily for 4 weeks at a dosage of 0.5 g/kg/day in drinking water (“ER stress inhibitor” group). Mice were sacrificed after 28 days of Ang II infusion. As described previously,22 systolic blood pressure was obtained 1 week before the implantation of the mini-osmotic pumps in mice and after 28 days of infusion, by use of a non-invasive tail cuff system. Blood was collected from the retro-orbital sinus under isoflurane anaesthesia. Serum total cholesterol levels were measured using an enzymatic method.

Absorption, Distribution and Excretion
There is evidence that tauroursodeoxycholic acid crosses the blood brain barrier in humans.

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Metabolism / Metabolites
There is little biotransformation of tauroursodeoxycholic acid. It is partially deconjugated by intestinal microflora to form unconjugated bile acids.

9848818 rat LD50 oral >5 gm/kg Japanese Kokai Tokyo Koho Patents., #92-235918
9848818 rat LD50 intravenous 300 mg/kg Japanese Kokai Tokyo Koho Patents., #92-235918
9848818 mouse LD50 oral >6 gm/kg Japanese Kokai Tokyo Koho Patents., #92-235918
9848818 mouse LD50 intravenous 350 mg/kg Japanese Kokai Tokyo Koho Patents., #92-235918

[1]. Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK viaPKCα-mediated MKP-1 induction. Cardiovasc Res. 2011 Nov 1;92(2):307-16.

[2]. Tauroursodeoxycholic Acid Attenuates Angiotensin II Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-deficient Mice by Inhibiting Endoplasmic Reticulum Stress. Eur J Vasc Endovasc Surg. 2017 Mar;53(3):337-345.

Tauroursodeoxycholic acid is a bile acid taurine conjugate derived from ursoodeoxycholic acid. It has a role as a human metabolite, an anti-inflammatory agent, a neuroprotective agent, an apoptosis inhibitor, a cardioprotective agent and a bone density conservation agent. It is functionally related to an ursodeoxycholic acid. It is a conjugate acid of a tauroursodeoxycholate.
Tauroursodeoxycholic acid, also known as ursodoxicoltaurine, is a highly hydrophilic tertiary bile acid that is produced in humans at a low concentration. It is a taurine conjugate of [ursodeoxycholic acid] with comparable therapeutic efficacy and safety, but a much higher hydrophilicity. Normally, hydrophilic bile acids regulates hydrophobic bile acids and their cytotoxic effects. Tauroursodeoxycholic acid can reduce the absorption of cholesterol in the small intestine, thereby reducing the body's intake of dietary cholesterol and the body cholesterol content. Tauroursodeoxycholic acid is currently used in Europe to treat and prevent gallstones as a bile acid derivative. Due to a range of its molecular properties - namely its anti-apoptotic effects - tauroursodeoxycholic acid has been examined in inflammatory metabolic diseases and neurodegenerative diseases.
Tauroursodeoxycholic acid has been reported in Homo sapiens with data available.

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Drug Indication
Tauroursodeoxycholic acid is used to prevent and treat gallstone formation. Tauroursodeoxycholic acid is used in combination with [phenylbutyric acid] to treat amyotrophic lateral sclerosis (ALS) in adults.

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Mechanism of Action
About 90% of gallstones are formed by cholesterol, which may be caused by altered gut microbiota from a high-fat diet and other factors. The gut microbiota regulates bile acid metabolism; thus, altered composition in gut microbiota may significantly change the bile acid pool and alter cholesterol secretion. While the exact mechanism of action of tauroursodeoxycholic acid in reducing and preventing gallstone formation is unclear, tauroursodeoxycholic acid may achieve this effect in a number of ways. A recent mouse study suggests that tauroursodeoxycholic acid inhibits intestinal cholesterol absorption and lowers liver cholesterol levels by upregulating the bile acid excretion from the liver to the gallbladder. Tauroursodeoxycholic acid lowers the bile cholesterol saturation in the gallbladder, thereby increasing the solubility of cholesterol in bile. It can also maintain a specific gut microbiota composition to promote the synthesis of bile acids and reduce liver inflammation caused by the lipopolysaccharide in the blood. Ultimately, tauroursodeoxycholic acid enhances the synthesis of bile acids in the liver and reduces cholesterol in the serum and liver. Tauroursodeoxycholic acid inhibits cell apoptosis by disrupting the mitochondrial pathway of cell death. It works by inhibiting oxygen-radical production, ameliorating endoplasmic reticulum (ER) stress, and stabilizing the unfolded protein response. Other anti-apoptotic processes mediated by tauroursodeoxycholic acid include cytochrome c release, caspase activation, DNA and nuclear fragmentation, and inhibition of p53 transactivation. It is believed that tauroursodeoxycholic acid works on multiple cellular targets to inhibit apoptosis and upregulate survival pathways.

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TAURURSODIOL is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2022 and has 2 investigational indications.

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Aims: Hyperplasia of vascular smooth muscle cells (VSMCs) after blood vessel injury is one of the major pathophysiological mechanisms associated with neointima. Tauroursodeoxycholate (TUDCA) is a cytoprotective agent in a variety of cells including hepatocytes as well as an inducer of apoptosis in cancer cells. In this study, we investigated whether TUDCA could prevent neointimal hyperplasia by suppressing the growth and migration of VSMCs. Methods and results: Transporters of TUDCA uptake in human VSMCs (hVSMCs) were analysed by RT-PCR and western blot. A knock-down experiment using specific si-RNA revealed that TUDCA was incorporated into hVSMCs via organic anion transporter 2 (OATP2). TUDCA reduced the viability of hVSMCs, which were mediated by inhibition of extracellular signal-regulated kinase (ERK) by induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) via protein kinase Cα (PKCα). The anti-proliferative effect of TUDCA was reversed by treatment with 7-hydroxystaurosporine, an inhibitor of PKC, and by the knock-down of MKP-1. In addition, TUDCA suppressed hVSMC migration, which was mediated by reduced matrix metalloproteinase-9 (MMP-9) expression by ERK inhibition, as well as reduced viability of hVSMCs. Rats with carotid artery balloon injury received oral administration of TUDCA; this reduced the increase in ERK and MMP-9 caused by balloon injury. TUDCA significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of the VSMCs. Conclusion: TUDCA inhibits neointimal hyperplasia by reducing proliferation and inducing apoptosis of smooth muscle cells by suppression of ERK via PKCα-mediated MKP-1 induction. [1]

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Objective/background: Abdominal aortic aneurysm (AAA) is characterised by the infiltration of smooth muscle cell (SMC) apoptosis, inflammatory cells, neovascularisation, and degradation of the extracellular matrix. Previous work has shown that endoplasmic reticulum (ER) stress and SMC apoptosis were increased both in a mouse model and human thoracic aortic aneurysm. However, whether the ER stress is activated in AAA formation and whether suppressing ER stress attenuates AAA is unknown. Methods: Human AAA and control aorta samples were collected. Expression of ER stress chaperones glucose-regulated protein (GRP)-78 and GRP-94 was detected by immunohistochemical staining. The effect of ER stress inhibitor tauroursodeoxycholic acid (TUDCA) on AAA formation in angiotensin (Ang) II induced apolipoprotein E-/- mice was explored. Elastin staining was used to observe the rupture of elastic fragmentation. Immunohistochemistry and Western blot analysis were performed, to detect the protein expression of ER stress chaperones and apoptosis molecules. Results: There was significant upregulation of GRP-78 and GRP-94 in aneurysmal areas of human AAA and Ang II induced ApoE-/- mice (p < .05). TUDCA significantly attenuated the maximum diameters of abdominal aortas in Ang II induced ApoE-/- mice (p < .05). TUDCA significantly reduced expression of ER stress chaperones and the apoptotic cell numbers (p < .05). Furthermore, TUDCA significantly reduced expression of apoptosis molecules, such as caspase-3, caspase-12, C/EBP homologous protein, c-Jun N-terminal kinase activating transcription factor 4, X-box binding protein, and eukaryotic initiation factor 2α in Ang II induced ApoE-/- mice (p < .05). Conclusion: The results suggest that ER stress is involved in human and Ang II induced AAA formation in ApoE-/- mice. TUDCA attenuates Ang II induced AAA formation in ApoE-/- mice by inhibiting ER stress mediated apoptosis. [2]

C26H49NO8S

535.7342

499.296

C, 62.49; H, 9.08; N, 2.80; O, 19.21; S, 6.42

14605-22-2

Tauroursodeoxycholate sodium;35807-85-3;Tauroursodeoxycholate-d4 sodium;2410279-95-5;Tauroursodeoxycholate dihydrate;117609-50-4;Tauroursodeoxycholate-d5;1207294-25-4;Tauroursodeoxycholate-d4;2410279-94-4;Tauroursodeoxycholate-d4-1;2573035-17-1

9848818

White to off-white solid powder

1.2±0.1 g/cm3

496.4ºC at 760mmHg

173-175°C

214.2ºC

1.552

2.1

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]2[C@H](C[C@H]4[C@@]3(CC[C@H](C4)O)C)O)C

BHTRKEVKTKCXOH-LBSADWJPSA-N

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

2-[[(4R)-4-[(3R,5S,7S,8R,9S,10S,13R,14S,17R)-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]pentanoyl]amino]ethanesulfonic acid

Tauroursodeoxycholic acid; 14605-22-2; Tauroursodeoxycholate; TUDCA; Ursodeoxycholyltaurine; Taurursodiol; ursodoxicoltaurine; Taurolite;

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 : ~50 mg/mL (~100.06 mM)
H2O : ~12.5 mg/mL (~25.02 mM)

Solubility in Formulation 1: 100 mg/mL (200.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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