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
Transported by carrier-mediated processes bidirectionally across mammalian proximal tubule.
After secretion into the biliary tract, bile acids are largely (95%) reabsobed in the intestine (mainly in the terminal ileum), returned to the liver, and then again secreted in bile (enterohepatic circulation).
The disposition kinetics of [(3)H]taurocholate ([(3)H]TC) in perfused normal and cholestatic rat livers were studied using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. The serum biochemistry levels, the outflow profiles and biliary recovery of [(3)H]TC were measured in three experimental groups: (i) control; (ii) 17 alpha-ethynylestradiol (EE)-treated (low dose); and (iii) EE-treated (high dose) rats. EE treatment caused cholestasis in a dose-dependent manner. A hepatobiliary TC transport model, which recognizes capillary mixing, active cellular uptake, and active efflux into bile and plasma described the disposition of [(3)H]TC in the normal and cholestatic livers better than the other pharmacokinetic models. An estimated five- and 18-fold decrease in biliary elimination rate constant, 1.7- and 2.7-fold increase in hepatocyte to plasma efflux rate constant, and 1.8- and 2.8-fold decrease in [(3)H]TC biliary recovery ratio was found in moderate and severe cholestasis, respectively, relative to normal. There were good correlations between the predicted and observed pharmacokinetic parameters of [(3)H]TC based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of [(3)H]TC). In conclusion, these results show that altered hepatic /taurocholate/ pharmacokinetics in cholestatic rat livers can be correlated with the relevant changes in liver pathophysiology in cholestasis.
It has been reported that the adjuvant-induced inflammation could affect drug metabolism in liver. /The authors/ further investigated the effect of inflammation on drug transport in liver using taurocholate as a model drug. The hepatic disposition kinetics of [(3)H]taurocholate in perfused normal and adjuvant-treated rat livers were investigated by the multiple indicator dilution technique and data were analyzed by a previously reported hepatobiliary taurocholate transport model. Real-time RT-PCR was also performed to determine the mRNA expression of liver bile salt transporters in normal and diseased livers. The uptake and biliary excretion of taurocholate were impaired in the adjuvant-treated rats as shown by decreased influx 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) compared with control rat group, whereas the efflux rate constant k(out) was greatly increased (0.07 +/- 0.02 vs. 0.02 +/- 0.01). The changes of mRNA expression of liver bile salt transporters were found in adjuvant-treated rats. Hepatic taurocholate extraction ratio in adjuvant-treated rats (0.86 +/- 0.05, n = 6) was significantly reduced compared with 0.93 +/- 0.05 (n = 6) in normal rats. Hepatic extraction was well correlated with altered hepatic ATP content (r(2) = 0.90). In conclusion, systemic inflammation greatly affects hepatic ATP content/production and associated transporter activities and causes an impairment of transporter-mediated solute trafficking and pharmacokinetics.
For more Absorption, Distribution and Excretion (Complete) data for TAUROCHOLIC ACID (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
Taurocholic acid has known human metabolites that include 2-[[(4R)-4-[(3R,5R,7R,10S,12S,13R)-7,12-Dihydroxy-10,13-dimethyl-3-sulfooxy-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.

Interactions
Sitosterol & taurocholate given together to rats inhibited cholesterol 7alpha-hydroxylase activity.
Chickens receiving taurocholate iv did not show active tubular excretion; however, it inhibited tubular excretioN of phenolsulfonphthaleiN & of n-methylnicotinamide.
In the anesthetized rat, the low incidence of erosions with indomethacin was markedly increased by concurrent gastric perfusion with acid saline & taurocholate.
When a combination of aspirin & taurocholic acid was introduced to 8 subjects the mean electrical potential difference also fell significantly from 38.6 1.8 mv to 17.9 1.8 mv, but mean duration of this change (27 min) was significantly longer than found after individual admin.
For more Interactions (Complete) data for TAUROCHOLIC ACID (14 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mice ip 620 mg/kg
LD50 Rat ip 450 mg/kg

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rabbit LDLo intravenous 110 mg/kg SENSE ORGANS AND SPECIAL SENSES: OTHER: EYE; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; LUNGS, THORAX, 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. It contains a taurocholate.
The product of conjugation of cholic acid with taurine. Its sodium salt is the chief ingredient of the bile of carnivorous animals. It acts as a detergent to solubilize fats for absorption and is itself absorbed. It is used as a cholagogue and cholerectic.

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Context: Currently, there is a dramatically growing interest in Chinese traditional medicines, especially in the therapy of inflammatory diseases. Taurocholic acid (TCA), as a kind of natural bioactive substance of animal bile acid, has medicinal applications to treat a wide range of inflammatory diseases. Objective: The study was designed to evaluate the effects of TCA on cytokine secretion, such as TNF-α and IL-1β and on the ratio of CD4(+)/CD8(+), which is beneficial for understanding the mechanism of TCA on immunoregulation preliminarily, and also will benefit our further research. Materials and methods: The gene and protein expressions of TNF-α and IL-1β were measured by real time RT-PCR and ELISA in serum, spleen and lymphocytes respectively. The ratio of CD4(+)/CD8(+) in peripheral blood and lymphocytes was measured by flow cytometry. Results: 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. 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. [2]

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egylated interferon-alpha (PegIFNα) therapy has limited effectiveness in hepatitis B e-antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. However, the mechanism underlying this failure is poorly understood. We aimed to investigate the influence of bile acids (BAs), especially Taurocholic acid (TCA), on the response to PegIFNα therapy in CHB patients. Here, we used mass spectrometry to determine serum BA profiles in 110 patients with chronic HBV infection and 20 healthy controls (HCs). We found that serum BAs, especially TCA, were significantly elevated in HBeAg-positive CHB patients compared with those in HCs and patients in other phases of chronic HBV infection. Moreover, serum BAs, particularly TCA, inhibited the response to PegIFNα therapy in HBeAg-positive CHB patients. Mechanistically, the expression levels of IFN-γ, TNF-α, granzyme B, and perforin were measured using flow cytometry to assess the effector functions of immune cells in patients with low or high BA levels. We found that BAs reduced the number and proportion and impaired the effector functions of CD3+CD8+ T cells and natural killer (NK) cells in HBeAg-positive CHB patients. TCA in particular reduced the frequency and impaired the effector functions of CD3+CD8+ T and NK cells in vitro and in vivo and inhibited the immunoregulatory activity of IFN-α in vitro. Thus, our results show that BAs, especially TCA, inhibit the response to PegIFNα therapy by impairing the effector functions of CD3+CD8+ T and NK cells in HBeAg-positive CHB patients. Our findings suggest that targeting TCA could be a promising approach for restoring IFN-α responsiveness during CHB treatment. [2]

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Background: Ischemic injury by hepatic artery ligation (HAL) during obstructive cholestasis induced by bile duct ligation (BDL) results in bile duct damage, which can be prevented by administration of VEGF-A. The potential regulation of VEGF and VEGF receptor expression and secretion by bile acids in BDL with HAL is unknown. Aims: We evaluated whether Taurocholic acid (TC) can prevent HAL-induced cholangiocyte damage via the alteration of VEGFR-2 and/or VEGF-A expression. Methods: Utilizing BDL, BDL+TC, BDL+HAL, BDL+HAL+TC, and BDL+HAL+wortmannin+TC treated rats, we evaluated cholangiocyte apoptosis, proliferation, and secretion as well VEGF-A and VEGFR-2 expression by immunohistochemistry. In vitro, we evaluated the effects of TC on cholangiocyte secretion of VEGF-A and the dependence of TC-induced proliferation on the activity of VEGFR-2. Results: In BDL rats with HAL, chronic feeding of TC prevented HAL-induced loss of bile ducts and HAL-induced decreased cholangiocyte secretion. TC 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. In vitro, TC stimulated increased VEGF-A secretion by cholangiocytes, which was blocked by wortmannin and stimulated cholangiocyte proliferation that was blocked by VEGFR-2 kinase inhibitor. Conclusion: TC prevented HAL-induced biliary damage by upregulation of 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.)