Human Endogenous Metabolite; ERK; Caspase-3/12;

Tauroursodeoxycholate (TUDCA) reduces the viability and migration of vascular smooth muscle cells (VSMCs) by inhibiting the phosphorylation of ERK and by inducing mitogen-activated protein kinase phosphatase-1 (MKP-1) via PKCα. By blocking ERK through Ca2+-dependent PKC translocation, tauroursodeoxycholate prevents the VSMCs from proliferating and migrating. Platelet-derived growth factor (PDGF) and MMP-9 expression brought on by vascular injury are both prevented by tauroursodeoxycholate. Tauroursodeoxycholate (200 μM) reduced VSMC viability, which suggests that Tauroursodeoxycholate's ability to inhibit cell proliferation depended on MKP-1 expression[1]. MKP-1 expression can be reduced by using specific si-RNA to knock it down.

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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]

Immunohistochemistry is used to examine the impact of tauroursodeoxycholate (TUDCA) on the proliferation and apoptosis of VSMCs in vivo. Tauroursodeoxycholate (10, 50, and 100 mg/kg) increases the caspase 3 activity of injured tissues in a dose-dependent manner, suggesting that it causes VSMCs in the neointima to undergo apoptosis. At one week after injury, additional testing and comparison of the phosphorylation level of ERK and MMP-9 expression is done using the injured tissues in comparison to normal controls. Both ERK phosphorylation and MMP-9 expression were upregulated in the tissues after balloon injury. Tauroursodeoxycholate (10, 50, and 100 mg/kg) inhibits the expression of MMP-9 and ERK in a dose-dependent manner[1]. Tauroursodeoxycholate (TUDCA) is a hydrophilic bile acid. By lowering ER stress and apoptosis, tauroursodeoxycholate, a cytoprotective agent, enhances liver function and can prevent hepatocellular carcinoma. In Ang II-induced ApoE-/- mice, tauroursodeoxycholate significantly decreases expression of apoptosis molecules like caspase-3, caspase-12, C/EBP homologous protein, c-Jun N-terminal kinase (JNK), activating transcription factor 4 (ATF4), X-box binding protein (XBP), and eukaryotic initiation factor 2α (eIF2; p<0.05). In ApoE-/- mice, tauroursodeoxycholate prevents the development of abdominal aortic aneurysms (AAAs) brought on by Ang II. Tauroursodeoxycholate is administered to ApoE-/- mice (ER stress inhibitor group) at a dose of 0.5 g/kg/day. Total cholesterol levels (663.6±88.7 mg/dL vs 655.7±65.4 mg/dL; p>0 .05) and systolic blood pressure (141.3±5.6 mmHg vs 145.98.9 mmHg; p>0.05) were comparable between the AAA model group and the Tauroursodeoxycholate group. Additionally, when compared to the AAA model group, the Tauroursodeoxycholate group's maximum aortic diameter was significantly lower (0.95±0.03 mm vs 1.79±0.04 mm; p<0.05). The AAA lesion areas in the Tauroursodeoxycholate group are also smaller than those in the AAA model group (0.37±0.03 mm2 vs 1.51±0.06 mm2; p<0.05)[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.

Ez-Cytox is used to assess the viability and growth of cells. Smooth Muscle Cell Growth Medium 2 (SMCGM2) is used to seed and cultivate VSMCs (5×103 cells) on 96-well plates. After serum starvation, Tauroursodeoxycholate (0, 50, 100, and 200 μM) is 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. HVSMCs are seeded onto 96-well plates and cultured to determine the impact of tauroursodeoxycholate on the PDGF-stimulated hVSMC proliferation. Tauroursodeoxycholate (0, 50, 100, and 200 μM) is added to the hVSMCs after serum starvation, with or without PDGF-BB (50 ng/mL), and the cells are then cultured. The optical density at 450 nm is used to assess cell viability after the addition of 10 μL of Ez-Cytox into each well[1].

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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.

Rats: Ketamine (70 mg/kg) and Xylazine (7 mg/kg) are combined to anesthetize Sprague-Dawley rats. For two weeks, tauroursodeoxycholate is given orally once daily at various concentrations (e.g., vehicle, 10, 50, and 100 mg/kg). The carotid arteries are preserved by perfusion with 4% formaldehyde, followed by paraffin embedding and H&E staining of sections (8 μm)[1].
Mice: Thirty C57BL/6 male ApoE-/- mice are divided into three groups at random, each with ten mice, and they are eight weeks old. (i) ApoE-/- mice are implanted with mini-osmotic pumps to release Ang II (1000 ng/kg/min) over the course of 28 days (AAA model group); (ii) AAA model mice are treated with Tauroursodeoxycholate daily for 4 weeks at a dosage of 0.5 g/kg/day in drinking water (Tauroursodeoxycholate group). Following a 28-day Ang II infusion, mice are sacrificed[2].

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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
Evidence suggests that tauroursodeoxycholic acid (TAO) can cross the blood-brain barrier in humans. Metabolism/Metabolites TAO undergoes minimal biotransformation. It is partially deconjugated by gut microbiota, forming unconjugated bile acids.

9848818 Oral LD50 in rats >5 gm/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)
9848818 Intravenous LD50 in rats 300 mg/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)
9848818 Oral LD50 in mice >6 gm/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)
9848818 Intravenous LD50 in mice 350 mg/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)

[1]. Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK via PKCα-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 (TAO) is a taurine-bile acid conjugate derived from ursodeoxycholic acid. It is a human metabolite with anti-inflammatory, neuroprotective, apoptosis-inhibiting, cardioprotective, and bone density-maintaining effects. Its function is related to ursodeoxycholic acid, being a conjugated acid of tauroursodeoxycholic acid salt. Tauroursodeoxycholic acid, also known as ursodeoxycholic acid base, is a highly hydrophilic tertiary bile acid, present in low amounts in the human body. It is a taurine conjugate of ursodeoxycholic acid, possessing comparable therapeutic efficacy and safety, but with higher hydrophilicity. Normally, hydrophilic bile acids modulate the cytotoxic effects of hydrophobic bile acids. Tauroursodeoxycholic acid can reduce the absorption of cholesterol in the small intestine, thereby lowering dietary cholesterol intake and cholesterol levels in the body. Currently, tauroursodeoxycholic acid is used in Europe as a bile acid derivative for the treatment and prevention of gallstones. Due to its molecular properties—particularly its anti-apoptotic effects—tauroursodeoxycholic acid (TAO) has been used in research on inflammatory metabolic diseases and neurodegenerative diseases. Studies on TAO in humans have been reported, and relevant data are available. Pharmaceutical Indications: Tauroursodeoxycholic acid is used for the prevention and treatment of gallstone formation. It is also used in combination with phenylbutyric acid (PBA) to treat adult-onset amyotrophic lateral sclerosis (ALS). Mechanism of Action: Approximately 90% of gallstones are formed from cholesterol, and cholesterol formation may be associated with gut microbiota dysbiosis caused by a high-fat diet and other factors. Gut microbiota regulates bile acid metabolism; therefore, changes in gut microbiota composition may significantly alter the bile acid pool and affect cholesterol secretion. While the exact mechanism by which TAO reduces and prevents gallstone formation is not fully understood, it likely works through multiple pathways. A recent mouse study showed that tauroursodeoxycholic acid (TAO) inhibits intestinal cholesterol absorption and reduces hepatic cholesterol levels by upregulating bile acid excretion from the liver to the gallbladder. TAO reduces cholesterol saturation in gallbladder bile, thereby increasing cholesterol solubility in bile. It also maintains specific gut microbiota composition, promotes bile acid synthesis, and alleviates liver inflammation induced by lipopolysaccharide in the blood. Ultimately, TAO enhances bile acid synthesis in the liver and reduces serum and hepatic cholesterol levels. TAO inhibits apoptosis by interfering with mitochondrial cell death pathways. Its mechanisms of action include inhibiting oxygen free radical production, improving endoplasmic reticulum stress, and stabilizing unfolded protein responses. Other anti-apoptotic processes mediated by TAO include cytochrome C release, caspase activation, DNA and nuclear fragmentation, and inhibition of p53 transcriptional activation. TAO is believed to act on multiple cellular targets, inhibiting apoptosis and upregulating cell survival pathways. Tauroursodeoxycholic acid (TUDCA) is a small molecule drug with its highest clinical trial stage being Phase IV (covering all indications). It was first approved in 2022 and has two investigational indications. Objective: Vascular smooth muscle cell (VSMC) proliferation after vascular injury is one of the main pathophysiological mechanisms associated with neointimal hyperplasia. Tauroursodeoxycholic acid (TUDCA) is a cytoprotective agent effective against various cell types, including hepatocytes, and is also an inducer of apoptosis in cancer cells. This study aimed to investigate whether TUDCA could prevent neointimal hyperplasia by inhibiting the growth and migration of VSMCs. Methods and Results: RT-PCR and Western blot analysis were used to analyze the TUDCA transporter proteins in human VSMCs. Gene knockdown experiments using specific siRNA showed that TUDCA enters human vascular smooth muscle cells (hVSMCs) via organic anion transporter 2 (OATP2). TUDCA induces the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) through protein kinase Cα (PKCα), thereby inhibiting extracellular signal-regulated kinase (ERK) and reducing hVSMC activity. Treatment with the PKC inhibitor 7-hydroxyastrosporin or knockdown of MKP-1 reverses the antiproliferative effect of TUDCA. Furthermore, TUDCA inhibits hVSMC migration and reduces hVSMC activity by inhibiting ERK and reducing matrix metalloproteinase-9 (MMP-9) expression. Oral administration of TUDCA to rats with carotid balloon injury reduced the elevated levels of ERK and MMP-9 induced by balloon injury. TUDCA significantly reduced the intima-to-media ratio by decreasing vascular smooth muscle cell (VSMC) proliferation and inducing apoptosis. Conclusion: TUDCA inhibits ERK through PKCα-mediated MKP-1 induction, thereby reducing smooth muscle cell proliferation and inducing apoptosis, thus inhibiting neointimal hyperplasia. [1]

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Objective/Background: Abdominal aortic aneurysm (AAA) is characterized by smooth muscle cell (SMC) infiltration, apoptosis, inflammatory cell infiltration, angiogenesis, and extracellular matrix degradation. Previous studies have shown that endoplasmic reticulum (ER) stress and SMC apoptosis are increased in mouse models and human thoracic aortic aneurysms. However, it remains unclear whether ER stress is activated during AAA formation and whether inhibiting ER stress can alleviate AAA. Methods: Human AAA and control aortic samples were collected. Immunohistochemical staining was used to detect the expression of endoplasmic reticulum stress molecular chaperones glucose-regulated protein (GRP)-78 and GRP-94. The effect of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid (TUDCA) on angiotensin II (Ang II)-induced apolipoprotein E-/- (ApoE-/-) mouse abdominal aortic aneurysm (AAA) formation was investigated. Elastin staining was used to observe elastin breakage. Immunohistochemical and Western blot analysis were used to detect the protein expression of endoplasmic reticulum stress molecular chaperones and apoptosis-related molecules. Results: GRP-78 and GRP-94 expression was significantly upregulated in the aneurysm region of human abdominal aortic aneurysms and Ang II-induced ApoE-/- mice (p < 0.05). TUDCA significantly reduced the maximum diameter of the abdominal aorta in Ang II-induced ApoE-/- mice (p < 0.05). TUDCA significantly reduced the expression of endoplasmic reticulum stress chaperones and the number of apoptotic cells (p < 0.05). Furthermore, TUDCA significantly reduced the expression of apoptotic molecules, such as caspase-3, caspase-12, C/EBP homolog, c-Jun N-terminal kinase-activated transcription factor 4, X-box binding protein, and eukaryotic initiation factor 2α (eIF2α) in angiotensin II (Ang II)-induced ApoE-/- mice (p < 0.05). Conclusion: These results indicate that endoplasmic reticulum stress is involved in the formation of abdominal aortic aneurysms (AAA) in humans and Ang II-induced ApoE-/- mice. TUDCA alleviates Ang II-induced AAA formation in ApoE-/- mice by inhibiting endoplasmic reticulum stress-mediated apoptosis. [2]

C26H44NNAO6S

521.69

521.278

C, 59.86; H, 8.50; N, 2.68; Na, 4.41; O, 18.40; S, 6.15

35807-85-3

Tauroursodeoxycholate;14605-22-2;Tauroursodeoxycholate-d4 sodium;2410279-95-5;Tauroursodeoxycholate dihydrate;117609-50-4

46782978

White to off-white solid powder

4.526

3

6

7

35

864

10

S(C([H])([H])C([H])([H])N([H])C(C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])[C@]3([H])[C@]([H])(C([H])([H])[C@]4([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[C@]4(C([H])([H])[H])[C@]3([H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H])O[H])O[H])=O)(=O)(=O)[O-].[Na+]

IYPNVUSIMGAJFC-JUWYWQLMSA-M

InChI=1S/C26H45NO6S.Na/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);/q;+1/p-1/t16-,17+,18-,19-,20+,21+,22+,24+,25+,26-;/m1./s1

sodium;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]ethanesulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.5 mg/mL (4.79 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 25.0 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: ≥ 2.5 mg/mL (4.79 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 25.0 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 3: ≥ 2.5 mg/mL (4.79 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (191.68 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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