Endogenous Metabolite; Microbial Metabolite

Taurocholic acid (24 hours, 100 μM) In PBMC taken from HBeAg-positive CHB patients, salt can lower the percentage of CD3+CD8+ T and NK cells [2]. IFN-α-stimulated cytokines and cytotoxic granule levels (IFN-γ, TNF-α, granzyme B) in CD3+CD8+ T and NK cells are decreased by taurocholic acid (100 μM, 24 h) sodium [2].

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Taurocholic acid (TCA) inhibits the immunoregulatory activity of IFN-α in vitro [2]
Given that IFN-α is an important immunomodulator33 and that our results indicate that Taurocholic acid (TCA) suppresses both the response to IFN-α therapy in CHB patients and the effector functions of CD3+CD8+ T and NK cells in vitro and in vivo (Figs. 2–5), we postulated that TCA inhibits IFN-α function by inhibiting its immunoregulatory activity. To test this hypothesis, due to the lack of appropriate and convenient animal models of HBeAg-positive CHB,34 we first stimulated freshly isolated PBMCs from HBeAg-positive CHB patients with IFN-α or TCA plus IFN-α for 24 h. We then performed intracellular staining of IFN-γ, TNF-α, granzyme B, and perforin in CD3+CD8+ T and NK cells and found that CD3+CD8+ T and NK cells stimulated with IFN-α produced higher levels of cytokines and cytotoxic granules than control cells (Fig. 6), which was consistent with previous work.35,36 Moreover, CD3+CD8+ T and NK cells that were stimulated with TCA plus IFN-α produced lower cytokine and cytotoxic granule levels than those that were stimulated with IFN-α alone (Fig. 6). Overall, these findings indicate that Taurocholic acid (TCA) inhibits the immunomodulatory effects of IFN-α in vitro.

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In vitro, Taurocholic acid (TCA) stimulated increased VEGF-A secretion by cholangiocytes, which was blocked by wortmannin and stimulated cholangiocyte proliferation that was blocked by VEGFR-2 kinase inhibitor.[3]

In C57BL/6 mice (tail vein injection of rAAV8-1.3HBV), taurocholic acid (oral gavage, 100 mg/kg, 2 weeks) salt can increase HBV replication by decreasing the percentage of NK and CD3+CD8+ T cells [2]. By upregulating VEGF-A expression, taurocholic acid sodium (1% in diet, 1 week) protects cholangiocyte injury caused by hepatic artery ligation (HAL) [3].

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Our present study has shown that lipopolysaccharide (LPS) and cyclosporin A (CsA) could increase or decrease the gene and protein expressions of TNF-α and IL-1β respectively. Taurocholic acid (TCA) (0.25g/kg, 0.125g/kg) could recover the suppressed expressions of TNF-α and IL-1β and increase the ratio of CD4(+)/CD8(+). In vitro, TCA (15μg/mL) could inhibit the increased production of TNF-α and IL-1β; TCA (0.15μg/mL-15μg/mL) could inhibit the increased gene expressions of IL-1β and TNF-α. TCA (0.15μg/mL) could recover the suppressed expressions of TNF-α and IL-1β. Conclusion: The function of immunoregulation of Taurocholic acid (TCA) may be accomplished through modulating the gene and protein expressions of TNF-α and IL-1β and elevating CD4(+)/CD8(+) T-cell ratio. [1]

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Taurocholic acid (TCA) impairs the effector functions of CD3+CD8+ T and NK cells in vivo [2]
To determine whether TCA suppresses the effector functions of CD3+CD8+ T and NK cells in vivo, we gavaged C57BL/6 mice with 100-mg/kg TCA daily or a control diet for 2 weeks after tail vein injection with rAAV8-1.3HBV for 6 weeks (Fig. 5A). The serum level of TCA was significantly elevated after gavage (Fig. S6). We found that treatment with TCA significantly reduced the percentage of NK and CD3+CD8+ T cells (Fig. 5B). In addition, CD8+ T and NK cells from C57BL/6 mice treated with TCA produced lower levels of cytokines and cytotoxic granules than those from mice given a control diet (Fig. 5C, D). Importantly, compared to those given the control diet, mice treated with TCA had higher serum HBsAg, HBeAg, and HBV DNA levels (Fig. 5E). These findings indicate that TCA promotes HBV replication by decreasing the percentage and impairing the effector functions of CD3+CD8+ T and NK cells in vivo.

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In BDL rats with HAL, chronic feeding of Taurocholic acid (TCA) prevented HAL-induced loss of bile ducts and HAL-induced decreased cholangiocyte secretion. Taurocholic acid (TCA) also prevented HAL-inhibited VEGF-A and VEGFR-2 expression in liver sections and HAL-induced circulating VEGF-A levels, which were blocked by wortmannin administration.[3]

Preparation of splenic lymphocytes supernatants and total RNA [1]
Splenic lymphocytes were suspended in RPMI-1640 medium supplemented with 3 mM l-glutamine, 10 mM hepes buffer, 100 U/mL penicillin and streptomycin and 10% FBS at a concentration of 1 × 106 cells/mL which was added to six-well culture plate (2 mL/well) with LPS (final concentration 10 μg/mL) or CsA (final concentration 0.01 μg/mL). The cells were randomly divided into 6 groups: control group (normal mice lymphocytes), LPS/CsA group (cells with LPS/CsA only); the remaining 4 groups were treated with different concentrations of Taurocholic acid (TCA) (0.015 μg/mL, 0.15 μg/mL, 1.5 μg/mL, and 15 μg/mL). After incubation for 48 h, lymphocyte supernatants and total RNA were prepared in corresponding methods.

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In vitro cell culture and stimulation [2]
Freshly isolated human PBMCs were cultured in 96-well plates at 37 °C in a 5% CO2 incubator. Cells were incubated in medium alone or with IFN-α (1000 U/ml) or IFN-α (1000 U/ml) plus Taurocholic acid (TCA) (100 μM) for 24 h. Subsequently, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 5 h, and then detection of the intracellular staining of IFN-γ, TNF-α, granzyme B, and perforin was performed by gating for NK and CD8+ T cells by flow cytometry.

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Evaluation of the Role of VEGF-A Secretion in Taurocholate-Mediated NRIC Proliferation [3]
After trypsinization, NRIC were seeded into 96-well plates (10,000 cells/well) in a final volume of 200 μl of medium. NRIC were stimulated in vitro for 48 hours with taurocholic acid (20 μM) in the absence or presence of 1-hour preincubation with wortmannin (100 nM) or VEGFR-2 Kinase Inhibitor I (100 nM). The proliferation of NRIC was evaluated by the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Absorbance was measured at 490 nm on a microplate spectrophotometer. Data were expressed as the fold change of treated cells as compared to vehicle-treated controls. To support the concept that the supernatant of NRIC stimulates cholangiocyte proliferation to different extents (depending on the amount of VEGF present in the supernatant of these cells), we treated NRIC for 24 hours at 37°C with the supernatant of NRIC obtained after 24 hours of incubation with BSA or 20 μM Taurocholic acid (TCA) (containing higher levels of VEGF-A compared to BSA-treated supernatant) in the absence or presence of 1-hour pre-incubation with wortmannin (100 nM) before measuring cell growth by immunoblots for PCNA.

Animal/Disease Models: C57BL/6 mice[2]
Doses: 100-mg/kg
Route of Administration: po (oral gavage), for 2 weeks after tail vein injection with rAAV8-1.3HBV for 6 weeks
Experimental Results: decreased the percentage of NK and CD3+CD8+ T cells . Increases serum HBsAg, HBeAg, and HBV DNA levels.

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Taurocholic acid (TCA) dissociated and depurated [1]
Fresh bovine and/or sheep galls were collected from a slaughterhouse. The bile was deproteinated using alcohol after filtered by filter paper, and then it was condensed using rotary evaporator after depigmented by activated carbon. Crude bile acids were obtained after salting out, extracting and dewatering. Taurocholic acid (TCA) was dissociated and depurated from crude bile acid by chromatography techniques and the purity was detected by high performance liquid chromatography. Its purity was > 98.7%.
Kunming mice (half male and half female), weight 20 ± 2 g, were obtained from the experimental center, Inner Mongolia University. All animals were maintained at a controlled temperature (22 ± 2 °C), and a regular light/dark cycle (7:00 am–7:00 pm, light) and all animals had free access to food and water. The animals were divided into 7 groups of 8 each (Table 1). All animals were treated orally by administration of intra-gastric gavage (i.g.) once daily and sacrificed after 7 days of treatment. Peripheral blood, serum and spleen were prepared for flow cytometry, ELISA and mRNA extraction respectively.

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Establishment of a recombinant adeno-associated virus type 8 (rAAV8)-mediated HBV replication mouse model [2]
rAAV8 carrying the 1.3-mer wild-type HBV genome (rAAV8-1.3HBV) was used to establish an immunocompetent mouse model for chronic HBV infection.27 A total of 5 × 1010 viral genomes/200 μl virus were injected into the tail vein of each C57BL/6 mouse. The mice were bled every other week to monitor the HBsAg, HBeAg, and HBV DNA levels. After 6 weeks, mice were fed by oral gavage for 2 weeks with either 100-mg/kg Taurocholic acid (TCA) daily or a control diet. Following this, the mice were sacrificed. Male Fischer 344 rats (150 to 175 gm) were kept in a temperature-controlled environment (22°C) with a 12-hour light-dark cycle and fed ad libitum rat chow. The studies were performed in: (i) BDL (for isolation of cells) or bile duct incannulated (BDI, for bile collection) rats that (immediately after BDL or BDI) were fed bile acid control diet or 1% taurocholic acid diet (which represents an approximate dose of 275 μmol/day) for 1 week; (ii) rats that (immediately after BDL or BDI + HAL) were fed bile acid control diet or 1% taurocholic acid diet; and (iii) rats that (immediately after BDL or BDI + HAL) were fed 1% Taurocholic acid (TCA) for 1 week in the presence of daily injections of 0.9% NaCl or wortmannin (0.7 mg/kg body weight). The groups of animals used in the study are summarized in Table 1. Since we have previously shown that daily injections of wortmannin or DMSO (in which wortmannin is dissolved) to BDL or BDI rats do not affect cholangiocyte apoptosis, proliferation and functional activity, these groups of animals were not included in the study. BDL, BDI and HAL were performed as described. Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg body weight, IP).[3]

Absorption, Distribution and Excretion
Bile acids undergo bidirectional transport within the proximal tubules of mammals via carrier-mediated processes. After secretion into the bile ducts, most (95%) of bile acids are reabsorbed in the intestine (primarily the terminal ileum), returned to the liver, and then secreted back into the bile (enterohepatic circulation). This study investigated the distribution kinetics of [(3)H]taurocholic acid ([(3)H]TC) in the livers of normally perfused and cholestatic rats using a multi-indicator dilution technique and several physiologically based pharmacokinetic models. Serum biochemical levels, [(3)H]TC efflux profiles, and bile recovery rates were determined in three experimental groups: (i) control group; (ii) 17α-ethinylestradiol (EE) treatment group (low dose); and (iii) EE treatment group (high dose). EE treatment induced cholestasis in a dose-dependent manner. A hepatobiliary TC transport model capable of identifying capillary mixing, active cellular uptake, and active efflux into bile and plasma more accurately describes the distribution of [(3)H]TC in normal and cholestatic livers than other pharmacokinetic models. Compared to normal livers, patients with moderate and severe cholestasis showed approximately 5-fold and 18-fold reductions in bile elimination rate constants, respectively, and 1.7-fold and 2.7-fold increases in hepatocyte-to-plasma efflux rate constants, respectively, and 1.8-fold and 2.8-fold reductions in [(3)H]TC bile recovery rates, respectively. The pharmacokinetic parameters of [(3)H]TC predicted based on liver pathophysiology (e.g., serum bilirubin levels and bile excretion of [(3)H]TC) correlated well with the observed parameters. In conclusion, these results indicate that the pharmacokinetic changes of taurocholic acid in the liver of cholestatic rats are closely related to cholestasis-related liver pathophysiological changes. It has been reported that adjuvant-induced inflammation affects hepatic drug metabolism. This study used taurocholic acid as a model drug to further investigate the effect of inflammation on hepatic drug transport. The hepatic distribution dynamics of [(3)H]taurocholic acid in perfused normal and adjuvant-treated rat livers were studied using a multi-indicator dilution method, and the data were analyzed using a previously reported hepatobiliary taurocholic acid transport model. In addition, real-time RT-PCR was performed to determine the mRNA expression levels of bile acid transporters in normal and diseased livers. Compared with the control group, the adjuvant-treated rats showed impaired taurocholic acid uptake and bile excretion, as evidenced by decreased inflow rate constant k(in) (0.65 ± 0.09 vs. 2.12 ± 0.30) and elimination rate constant k(be) (0.09 ± 0.02 vs. 0.17 ± 0.04), while significantly increased effluent rate constant k(out) (0.07 ± 0.02 vs. 0.02 ± 0.01). The mRNA expression of bile acid transporters in the liver of adjuvant-treated rats was altered. Compared with normal rats (0.93 ± 0.05, n = 6), the liver taurocholic acid extraction rate in adjuvant-treated rats (0.86 ± 0.05, n = 6) was significantly reduced. The liver extraction rate was strongly correlated with changes in liver ATP content (r(2) = 0.90). In conclusion, systemic inflammation significantly affects liver ATP content/production and the activity of related transporters, leading to impaired transporter-mediated solute transport and pharmacokinetics. For more complete data on the absorption, distribution, and excretion of taurocholic acids (6 in total), please visit the HSDB record page.

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Metabolism/Metabolites
Known human metabolites of taurocholic acid include 2-[[(4R)-4-[(3R,5R,7R,10S,12S,13R)-7,12-dihydroxy-10,13-dimethyl-3-sulfonoxy-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecano-1H-cyclopenta[a]phenanthrene-17-yl]valeryl]amino]ethanesulfonic acid.

Interactions
Concomitant administration of sitosterol and taurocholic acid to rats inhibited cholesterol 7α-hydroxylase activity. Intravenous injection of taurocholic acid in chickens did not show active renal tubular excretion; however, it inhibited the renal tubular excretion of phenolsulfonamide and N-methylnicotinamide. In anesthetized rats, indomethacin-induced erosion incidence was low, but concomitant gastric instillation with acidic saline and taurocholic acid significantly increased the incidence of erosion. When aspirin and taurocholic acid were concomitantly administered to 8 subjects, the mean potential difference also significantly decreased from 38.6 ± 1.8 mV to 17.9 ± 1.8 mV, but the mean duration of this change (27 minutes) was significantly longer than that after single administration. For more complete data on taurocholic acid interactions (14 in total), please visit the HSDB records page.

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Non-human toxicity values
Mouse intraperitoneal LD50: 620 mg/kg
Rat intraperitoneal LD50: 450 mg/kg
Rabbit LD50 intravenous injection: 110 mg/kg Sensory organs and special senses: Other: eyes; Behavior: seizures or effects on the epileptic threshold; Lungs, pleura, or respiration: Other changes. Zeitschrift fuer die Gesamte Experimentelle Medizin., 52(779), 1926

[1]. Effects of taurocholic acid on immunoregulation in mice. Int Immunopharmacol. 2013 Feb;15(2):217-22.

[2]. Taurocholic acid inhibits the response to interferon-α therapy in patients with HBeAg-positive chronic hepatitis B by impairing CD8+ T and NK cell function. Cell Mol Immunol. 2021 Feb;18(2):461-471.

[3]. Taurocholic acid prevents biliary damage induced by hepatic artery ligation in cholestatic rats. Dig Liver Dis. 2010 Oct;42(10):709-17.

Sodium taurocholate is a bile salt containing taurocholate ions. It is a product of the combination of bile acids and taurine. Its sodium salt is a major component of bile in carnivorous animals. As a surfactant, it can dissolve fats for absorption and is also absorbed itself. It is used as a choleretic agent. Background: Currently, there is increasing interest in traditional Chinese medicine, especially in the treatment of inflammatory diseases. Taurocholic acid (TCA), as a natural animal bile acid, has medicinal value in treating various inflammatory diseases. Objective: This study aims to evaluate the effects of TCA on the secretion of cytokines (such as TNF-α and IL-1β) and the CD4(+)/CD8(+) ratio, to preliminarily understand the immunomodulatory mechanism of TCA, and to provide a reference for our subsequent research. Materials and Methods: Real-time RT-PCR and ELISA were used to detect the gene and protein expression levels of TNF-α and IL-1β in serum, spleen, and lymphocytes, respectively. The CD4(+)/CD8(+) ratio in peripheral blood and lymphocytes was detected by flow cytometry. Results: This study showed that lipopolysaccharide (LPS) and cyclosporine A (CsA) can upregulate or downregulate the gene and protein expression of TNF-α and IL-1β, respectively. TCA (0.25 g/kg, 0.125 g/kg) restored the suppressed expression of TNF-α and IL-1β and increased the CD4(+)/CD8(+) ratio. In vitro experiments showed that TCA (15 μg/mL) could inhibit the excessive production of TNF-α and IL-1β; TCA (0.15 μg/mL-15 μg/mL) could inhibit the increase in IL-1β and TNF-α gene expression. TCA (0.15 μg/mL) could restore the suppressed expression of TNF-α and IL-1β. Conclusion: The immunomodulatory function of taurocholic acid (TCA) may be achieved by regulating the gene and protein expression of TNF-α and IL-1β and increasing the CD4+/CD8+ T cell ratio. [2] Pegylated interferon-α (PegIFNα) treatment has limited efficacy in patients with chronic hepatitis B (CHB) who are positive for hepatitis B e antigen (HBeAg). However, the mechanism of its poor efficacy is unclear. This study aimed to investigate the effect of bile acids (BA), especially taurocholic acid (TCA), on the response of CHB patients to PegIFNα treatment. In this study, the serum bile acid (BA) profiles of 110 patients with chronic hepatitis B virus (HBV) infection and 20 healthy controls (HCs) were measured by mass spectrometry. The results showed that the serum BA levels of HBeAg-positive chronic hepatitis B (CHB) patients were significantly higher than those of healthy controls and other patients in the chronic HBV infection stage, especially the tricarboxylic acid cycle (TCA) level. Furthermore, serum BA levels, particularly TCA, inhibited the efficacy of pegylated interferon-α (PegIFNα) therapy in HBeAg-positive CHB patients. Mechanistically, we used flow cytometry to detect the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B, and perforin in patients with low and high BA levels to assess the effector function of immune cells. The results showed that elevated BA levels reduced the number and proportion of CD3+CD8+ T cells and natural killer (NK) cells in HBeAg-positive CHB patients, impairing their effector function. TCA, in particular, reduced the frequency of CD3+CD8+ T cells and NK cells both in vitro and in vivo, impairing their effector function, and inhibited the immunomodulatory activity of IFN-α in vitro. Therefore, our results suggest that bile acids (BAs), particularly TCA, inhibit the response of CD3+CD8+ T cells and NK cells to pegylated interferon-α (PegIFNα) treatment by impairing the effector function of HBeAg-positive chronic hepatitis B (CHB). Our findings suggest that targeting TCA may be a promising approach to restore IFN-α responsiveness during CHB treatment. [2]

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Background: Ischemic injury caused by hepatic artery ligation (HAL) during obstructive cholestasis induced by bile duct ligation (BDL) can lead to bile duct injury, which can be prevented by administration of VEGF-A. The potential regulatory role of bile acids in the expression and secretion of VEGF and its receptors in HAL-induced bile duct ligation (BDL) is unclear. Objective: We evaluated whether taurocholic acid (TC) could prevent HAL-induced bile duct cell injury by altering the expression of VEGFR-2 and/or VEGF-A. Methods: We used rats treated with BDL, BDL+TC, BDL+HAL, BDL+HAL+TC, and BDL+HAL+wortmannin+TC to assess cholangiocellular apoptosis, proliferation, and secretion, as well as the expression of VEGF-A and VEGFR-2, using immunohistochemistry. In vitro, we evaluated the effect of TC on VEGF-A secretion in cholangiocellular cells and the dependence of TC-induced proliferation on VEGFR-2 activity. Results: In a HAL-induced bile duct ligation (BDL) rat model, long-term TC administration prevented HAL-induced bile duct loss and decreased cholangiocellular secretion. TC also prevented the expression of VEGF-A and VEGFR-2 in HAL-suppressed liver tissue and the HAL-induced increase in circulating VEGF-A levels, all of which could be blocked by wortmannin. In vitro experiments showed that TC stimulates increased VEGF-A secretion from cholangiocellular cells, while wortmannin blocked this effect; TC also stimulates cholangiocellular proliferation, while VEGFR-2 kinase inhibitors blocked this proliferation. Conclusion: TC prevents HAL-induced biliary tract injury by upregulating VEGF-A expression. [3]

C26H44NNAO7S

537.6848

537.273

C, 58.08; H, 8.25; N, 2.61; Na, 4.28; O, 20.83; S, 5.96

145-42-6

81-24-3 (free acid);145-42-6 (sodium); 81-24-3 (free acid); 345909-26-4

23666345

White to off-white solid powder

230 °C

23 ° (C=3, H2O)

3.497

4

7

7

36

897

11

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

JAJWGJBVLPIOOH-IZYKLYLVSA-M

InChI=1S/C26H45NO7S.Na/c1-15(4-7-23(31)27-10-11-35(32,33)34)18-5-6-19-24-20(14-22(30)26(18,19)3)25(2)9-8-17(28)12-16(25)13-21(24)29;/h15-22,24,28-30H,4-14H2,1-3H3,(H,27,31)(H,32,33,34);/q;+1/p-1/t15-,16+,17-,18-,19+,20+,21-,22+,24+,25+,26-;/m1./s1

sodium;2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-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

sodium taurocholate; 145-42-6; Taurocholate sodium; Taurocholate sodium salt; Monosodium N-choloyltaurinate; Monosodium taurocholate; Taurocholic acid sodium salt; Monosodium taurocholic acid;

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)

DMSO : ~250 mg/mL (~464.96 mM)
H2O : ~100 mg/mL (~185.98 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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