Endogenous Metabolite; Na+/taurocholate cotransporting polypeptide (NTCP)

Cholic acid competitively bound NTCP on HepG2 cells and significantly inhibited the uptake of CA-LPs-DOX·HCl, which indicated that CA-LPs-DOX·HCl are also uptaken via NTCP-mediated endocytosis pathway[1].

Cholic Acid Feeding Leads to Increased CYP2D6 Expression in CYP2D6-Humanized Mice. Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme, but the factors governing transcriptional regulation of its expression remain poorly understood. Based on previous reports of small heterodimer partner (SHP) playing an important role as a transcriptional repressor of CYP2D6 expression, here we investigated how a known upstream regulator of SHP expression, namely cholestasis triggered by cholic acid (CA) feeding in mice, can lead to altered CYP2D6 expression. To this end, CYP2D6-humanized (Tg-CYP2D6) mice were fed with a CA-supplemented or control diet for 14 days, and hepatic expression of multiple genes was examined. Unexpectedly, CA feeding led to insignificant changes in SHP mRNA but also to significant (2.8-fold) decreases in SHP protein levels. In silico analysis of the SHP gene regulatory region revealed a putative binding site for a microRNA, miR-142-3p. Results from luciferase reporter assays suggest that miR-142-3p targets the SHP gene. Hepatic expression of miR-142-3p was significantly increased in CA-fed mice (∼5-fold), suggesting a potential role of miR-142-3p in the regulation of SHP expression in cholestasis. The decreased SHP protein levels were accompanied by increased expression and activity of CYP2D6 in the liver of CA-fed mice. These results suggest potential roles of differential hepatic levels of bile acids in the transcriptional regulation of CYP2D6 expression[2].

Endocytosis pathway activity using uptake inhibitors[1]
HepG2 cells were pretreated with the inhibitors NaN3 (1 mg/mL), genistein (50 μg/mL), MβCD (10 mM), nystatin (50 μg/mL), chlorpromazine (10 μg/mL), and cholic acid (1 mg/mL) for 30 min. After removing the inhibitors, the cells were incubated with CA-LPs for 2 h, and the cellular uptake of LPs was determined as described in the “In vitro cellular uptake assays” section.

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HepG2 cell uptake after transport across the Caco-2 cell monolayer[1]
Caco-2 cells were seeded in the AP chamber of Transwell plates, and HepG2 cells were seeded in a glass-bottom culture dish. The AP chambers of the Transwell plates were placed on the glass-bottom culture dish. The cells in the AP chamber were washed three times with serum-free medium, and the Caco-2 cells were incubated with pure DOX·HCl or CA-LPs-DOX·HCl (containing DOX·HCl at 5 μg/mL) in serum-free medium at 37°C for 2 h. After incubation, the HepG2 cells in the glass-bottom culture dish were washed three times with PBS at 4°C, trypsinized, washed three times with PBS again, and then suspended in 0.4 mL of PBS. The mean concentrations of DOX·HCl in the cells were measured by flow cytometry.

Tg-CYP2D6 mice were previously described (Corchero et al., 2001). Adult male mice (8 weeks of age and weighing 20–25 g) were used for the experiments. Mice were fed with normal chow or 1% (w/w) CA/Cholic acid-supplemented diet. After feeding for 14 days, mice were sacrificed, and blood and liver tissue samples were collected. As a cholestasis model, Tg-CYP2D6 mice were fed a cholic acid (CA)–supplemented diet for over 1 week. The treatment was known to increase bile acid pool size by 2-fold and to replace ∼90% of bile acids with CA, recapitulating the features of cholestatic conditions in humans[2].

Absorption, Distribution and Excretion
After ingestion, bile acids are first absorbed in the small intestine and then transported via the bloodstream to the liver for further metabolism. The metabolic pathway of orally administered bile acids is the same as that of endogenous bile acids. Bile acids are absorbed along the gastrointestinal tract via passive diffusion. After absorption, bile acids enter the bile acid pool in the body and undergo enterohepatic circulation primarily in their conjugated form. In the liver, bile acids are conjugated with glycine or taurine by bile acid-CoA synthase and bile acid-CoA:amino acid N-acetyltransferase. Conjugated bile acids are actively secreted into bile primarily via the bile acid efflux pump (BSEP) and then released into the small intestine along with other bile components. Conjugated bile acids are mainly reabsorbed in the ileum via apical sodium-dependent bile acid transporters and then returned to the liver via transporters including sodium-taurocholate cotransport polypeptides and organic anion transporters to enter the next enterohepatic circulation. Any conjugated bile acids not absorbed by the ileum enter the colon, where they undergo deconjugation and 7-dehydroxylation reactions under the action of bacteria, producing bile acids and deoxycholic acids. These substances may be reabsorbed in the colon or excreted in feces. Healthy individuals compensate for the loss of bile acids by synthesizing bile acids de novo from cholesterol, thus maintaining normal levels in the bile acid pool. Rat excretion studies show that bile acids (CA) are almost entirely excreted in feces as metabolites. Only small amounts of unconjugated bile acids are found in rat feces. Urinary excretion of bile acids is minimal; mice fed a 1% CA diet excreted 2000 times more bile acids in feces than in urine. Studies have shown that bile acids in the guinea pig small intestine are primarily absorbed distally (ileum) rather than proximally. For more complete data on the absorption, distribution, and excretion of the nine bile acids, please visit the HSDB records page.

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Metabolism/Metabolites
This study investigated the mechanism and sequence of cholesterol side-chain hydroxylation during bile acid synthesis in ex vivo perfused rabbit liver, and compared the importance of 26- and 25-hydroxylation in rabbit bile acid biosynthesis. [G-(3)H]cholestylic acid was observed to be generated when the liver was perfused with 5β-[G-(3)H]cholestyrannosyl-3α,7α-diol, 5β-[G-(3)H]cholestyrannosyl-3α,7α-12α-triol, and 5β-[G-(3)H]cholestyrannosyl-3α,7α,26-triol. [G-(3)H]chenodeoxycholic acid was not detected in bile. These results suggest that the potential precursor of chenodeoxycholic acid undergoes hydroxylation at the 12α position before or after cholesterol side-chain hydroxylation. Furthermore, no other intermediates (tetrahydroxy or pentahydroxycholestyrannosyl alcohols) were found in bile when the liver was perfused with these compounds. When rabbit liver was perfused with 5β-[24-(14)C]cholestan-3α,7α,25-triol, bile acid precursors were detected in the bile. 5β-[24-(14)C]cholestan-3α,7α,25-triol is hydroxylated at the 12α position in the liver to generate the corresponding 5β-cholestan-3α,7α,12α,25-tetraol. The tetraol is further metabolized into a series of pentanols (5β-cholestan-3α,7α,12α,22,25-pentanol; 5β-cholestan-3α,7α,12α,23,25-pentanol; 5β-cholestan-3α,7α,12α,24,25-pentanol; and 5β-cholestan-3α,7α,12α,25,26-pentanol). The main bile acids obtained from 5β-cholestan-3α,7α,25-triol perfusion are bile acids. Experiments show that in rabbit liver, cholesterol side chains can undergo 12α-hydroxylation after hydroxylation at either the C-25 position (5β-cholestan-3α,7α,25-triol) or the C-26 position (5β-cholestan-3α,7α-26-triol). Clearly, rabbits can synthesize bile acids via either the classical 26-hydroxylation pathway or via a 25-hydroxylation intermediate. In classical bile acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage to generate 5β-cholestan-3α,7α,12α-triol. The side chain reaction of this triol can then proceed via either the mitochondrial 27-hydroxylation pathway or the microsomal 25-hydroxylation pathway. We developed highly specific and precise detection methods for determining the activities of key enzymes in two metabolic pathways, including 5β-cholestan-3α,7α,12α-triol 25- and 27-hydroxylases and 5β-cholestan-3α,7α,12α,25-tetraol 23R-, 24R-, 24S-, and 27-hydroxylases. Extracts from mitochondrial or microsomal incubation mixtures were purified using a single-use silica column, derivatized to trimethylsilyl ether, and then quantitatively analyzed using high-resolution selected ion monitoring gas chromatography-mass spectrometry. Compared to adding the substrate to acetone, adding the substrate to 2-hydroxypropyl-β-cyclodextrin increased mitochondrial triol 27-hydroxylase activity by 132%, but decreased the activity of enzymes in the microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5β-cholestane-3α, 7α, 12α, 25-tetraol 23R-, 24R-, 24S-, and 27-hydroxylases) by 13% to 60% (using human liver as a model). The enzyme activities of these two pathways are typically 2 to 4 times higher in mouse and rabbit livers compared to human livers. In all species, microsomal triol 25-hydroxylase activity is 4 to 11 times higher than mitochondrial triol 27-hydroxylase activity, but under our experimental conditions, the activity of tetraol 24S-hydroxylase is similar to that of triol 27-hydroxylase. We investigated the regulation of these two pathways in rabbit liver after interfering with bile acid synthesis. Cholesterol feeding upregulated the activity of enzymes involved in the 25-(64%; -142%) and 27-(77%) hydroxylation pathways, while bile drainage upregulated only the activity of enzymes in the 25-hydroxylation pathway (178%; -371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways of bile acid biosynthesis are more active in mouse and rabbit livers than in human livers, and are regulated differently in rabbit livers. Deoxycholic acid is the major metabolite of bile acids. Patients with 3α-hydroxysteroid dehydrogenase deficiency (3α-HSD) and Δ4-3-oxoreceptor deficiency (Δ4-3-oxoR), as well as subjects with normal bile acid metabolism, showed predominantly bile acids and deoxycholic acid in serum and bile after bile acid treatment, while chenodeoxycholic acid and its metabolites appeared to be reduced. Therefore, patients receiving bile acid treatment are exposed to higher-than-normal levels of deoxycholic acid, although the specific values of these concentrations are not yet clear. In single-dose and repeated-dose studies, deoxycholic acid exhibited lethal effects, gastrointestinal toxicity, and hepatotoxicity at approximately half the dose required for bile acids to produce the same effect. Therefore, deoxycholic acid is considered to be more toxic than bile acids and may indeed be a contributing factor to the toxicity of some bile acids. Bacterial mutagenicity data for deoxycholic acid are unclear, but in vitro micronucleus assays indicate its genotoxicity. Furthermore, this study used a comet assay to investigate the genotoxicity of bile acids (primarily chenodeoxycholic acid and deoxycholic acid) on human colon cells and HT 29 colon tumor cells. Results showed that both bile acids induced significant dose-dependent genotoxic effects in both cell types, with deoxycholic acid exhibiting stronger genotoxicity. Cell viability was above 75%. A comet assay modified with nuclease III indicated that DNA damage may be mediated by the production of reactive oxygen species, but the addition of antioxidants mitigated this damage to some extent. Short-term carcinogenicity studies showed that deoxycholic acid, like bile acids, has carcinogenic properties. In rat liver, deoxycholic acid (75–150 mg/kg) exhibited pro-cancer activity, manifested by a significant increase in the number of α-glutamyl transferase-positive (α-GT+) liver lesions, a phenomenon not observed in the control group treated only with the carcinogen diethylnitrosamine (DEN). Deoxycholic acid (20 mg/kg) enhanced the formation and growth of azomethane (AOM)-induced aberrant crypt lesions in the rat colon. In a parallel study, deoxycholic acid did not significantly induce the formation of aberrant crypt lesions in the absence of AOM. However, one study concluded that deoxycholic acid may act not only as a pro-cancer agent but also as an initiator of a multi-stage carcinogenic process. Known metabolites of bile acids include bile acid glucuronide.

Toxicity Summary
Identification and Uses: Bile acids are used in biochemical research, as pharmaceutical intermediates, and as emulsifiers in food (up to 0.1%). They are also used to treat bile acid synthesis disorders caused by a single enzyme deficiency, and as adjunctive therapy for peroxisome disorders (including Zelvig syndrome) in patients with symptoms of liver disease, steatorrhea, or complications of reduced absorption of fat-soluble vitamins. Human Exposure and Toxicity: Bile acids are primary bile acids. Primary bile acids are biosynthesized in the liver and are key components of normal bile. When formulated and used to treat patients with bile acid synthesis disorders, their primary toxic effect is on liver function. The dose of bile acids administered to patients is intended to restore concentrations comparable to physiological levels in the human body. Therefore, any potential genotoxic risk to patients from bile acids and deoxycholic acid is comparable to the risk to normal, healthy adults capable of producing these bile acids themselves. Overall, bile acids did not show significant mutagenic activity in a series of in vitro genotoxicity studies. Animal studies: Bile acids are known to regulate their synthesis and transport via farnesol X receptors in the liver (FXR-SHP) and intestine (FXR-Fgf15). Mice lacking farnesol X receptors (FXR) involved in maintaining hepatic bile acid levels are highly susceptible to bile acid-induced hepatotoxicity. After 5 days of feeding a 0.25% bile acid diet, serum aspartate aminotransferase (AST) activity increased 15.7-fold, while wild-type mice fed 0.25% and 1% bile acid diets showed only slight increases in serum AST (1.7-fold and 2.5-fold, respectively). This study used a chemically induced rat colon cancer model to explore the potential of primary bile acids as promoters or inhibitors of colon tumors. Results showed that feeding bile acids increased the number of animals with tumors, the number of tumors per animal, and the number of tumors per animal with tumors. The tumor-promoting effect was attributed to deoxycholic acid, a bacterial metabolite of bile acids. After male rats were fed bile acids (1.0% in their diet) for three consecutive days, the number of DNA-synthesizing epithelial cells in each colonic crypt column was significantly increased compared to the control group or rats fed 0.2% bile acids. The mutagenicity of bile acids was assessed using fluctuation tests with Salmonella Typhimurium strains TA100 and TA98. The results showed that both bile acids and deoxycholic acid are mutagenic. The molar mutagenicity of bile acids was approximately one-quarter that of the moderately potent mutagen methyl mesylate. Ingestion of 0.5% bile acids in pregnant hamsters resulted in varying degrees of bile duct hyperplasia and hepatobiliary inflammation in adult hamsters, with milder damage in juvenile hamsters; furthermore, litter sizes were reduced. Ingestion of these bile acids during pregnancy can cause varying degrees of toxicity to the hepatobiliary system of both mothers and newborns. In rat developmental studies, significant pathological changes were observed in the brains of fetuses treated with bile acids. Neuronal degeneration and mitochondrial swelling were mainly observed in the low-chole acid group, while neuronal necrosis and mitochondrial reduction were mainly observed in the high-chole acid group.

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Hepatotoxicity
In small open-label trials, bile acids were found to improve the absorption of fat-soluble vitamins and improve many clinical features of bile acid synthesis defects, including lower serum transaminase levels, lower bilirubin and jaundice, and improved overall health and growth. In some cases, higher doses of bile acids were associated with elevated serum transaminase levels. However, these abnormalities were mild, transient, and rapidly reversible by reducing the daily dose. There are currently no reports of clinically significant liver injury with jaundice resulting from treatment with standard doses of bile acids.
Probability score: D[HD] (Possible liver injury, but only with high doses).

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Drug Interactions
In vitro studies have shown that aluminum-based antacids can adsorb bile acids and may reduce the bioavailability of choline. When taking choline, it should be taken at least 1 hour before or 4 to 6 hours after taking an aluminum-based antacid (or as long as possible).
Bile acid binding resins, such as cholestyramine, colestipol, or colesvelam, can adsorb and reduce the absorption of bile acids and may reduce the efficacy of choline. Cholbam should be taken at least 1 hour before or 4 to 6 hours after taking a bile acid binding resin (or as long as possible).
Phenobarbital treatment increases the ileum's capacity for bile acid transport (Vmax) in rats. …Rats treated with phenobarbital for 10 days had a 4-fold reduction in bile acid pools compared to control rats. Furthermore, the daily bile acid production in rats treated with phenobarbital was also reduced compared to control rats. Phenobarbital antagonizes the intended effects of bile acids in patients and may compromise established metabolic control. Therefore, phenobarbital is contraindicated in patients receiving bile acid therapy.
Avoid concomitant use of bile acid efflux pump (BSEP) inhibitors, such as cyclosporine (used in combination with Cholbam). Concomitant use of drugs that inhibit bile acid transporters in the bile canal membrane (e.g., BSEP) may exacerbate the accumulation of conjugated bile acids in the liver and lead to clinical symptoms. If concomitant use is necessary, monitoring of serum transaminase and bilirubin levels is recommended. For more complete data on interactions of bile acids (8 in total), please visit the HSDB record page. Non-human toxicity values: Intraperitoneal LD50 in mice: 330 mg/kg; Oral LD50 in mice: 4950 mg/kg; Intravenous LD50 in mice: 350 mg/kg

[1]. Mechanism of hepatic targeting via oral administration of DSPE-PEG-Cholic acid-modified nanoliposomes. Int J Nanomedicine. 2017 Feb 28;12:1673-1684.
[2]. Cholic acid Feeding Leads to Increased CYP2D6 Expression in CYP2D6-Humanized Mice. Drug Metab Dispos. 2017 Apr;45(4):346-352.

Therapeutic Uses
ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Bile acids are listed in this database. Bile acids (Cholbam) are indicated for the treatment of bile acid synthesis disorders caused by a single enzyme deficiency (SED). Cholbam is indicated as adjunctive therapy for patients with peroxisome disorders (PDs), including Zellweger spectrum disorders, presenting with symptoms of liver disease, steatorrhea, or complications of reduced absorption of fat-soluble vitamins. The safety and efficacy of Cholbam for extrahepatic manifestations of bile acid synthesis disorders caused by single enzyme deficiency (SED) or peroxisome disorders (PDs), including Zellweger spectrum disorders, have not been established. For more complete data on the therapeutic uses of bile acids (7 in total), please visit the HSDB record page. Drug Warnings Cholbam has been demonstrated in children aged 3 weeks and older for the treatment of bile acid synthesis disorders caused by single enzyme deficiency (SED) as adjunctive therapy for patients with peroxisome disorders (PDs), including Zellweger spectrum disorders, presenting with symptoms of liver disease, steatorrhea, or complications of reduced absorption of fat-soluble vitamins.
Liver function should be monitored during treatment, and Cholbam should be discontinued if liver function deteriorates. Simultaneous elevation of serum gamma-glutamyl transferase (GGT) and alanine aminotransferase (ALT) may indicate choline overdose. Choline should be discontinued immediately if clinical or laboratory indicators of worsening liver function or cholestasis occur.
The benefits of breastfeeding to infant development and health should be weighed against the mother's clinical need for choline, and any potential adverse effects of choline or maternal disease on the breastfed infant should be considered.
There are currently no studies on the effects of choline on maternal or animal reproduction. Limited case reports have been published, mentioning women who received cholic acid treatment for 3β-HSD deficiency and subsequently delivered healthy infants. These reports may not adequately account for the drug-related risks associated with choline use during pregnancy. The background risk of choline causing serious birth defects and miscarriage in the target population is unclear. However, in the general US population, the background risk for major birth defects is 2–4%, and the background risk for miscarriage is 15–20% of clinically confirmed pregnancies. For more complete data on drug warnings (6 in total) related to bile acids, please visit the HSDB Records page.

C24H40O5

408.579

408.287

C, 70.55; H, 9.87; O, 19.58

81-25-4

81-25-4; 361-09-1; 206986-87-0; 52886-36-9; 52886-36-9; 116380-66-6; 53007-09-3

221493

White to off-white solid powder

1.2±0.1 g/cm3

583.9±50.0 °C at 760 mmHg

197-202 ºC

321.0±26.6 °C

0.0±3.7 mmHg at 25°C

1.558

2.62

4

5

4

29

637

11

O[C@@H]1C[C@@H]2C[C@@H](CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCC(=O)O)[C@]1([C@H](C2)O)C)O

BHQCQFFYRZLCQQ-XXDQCAAPSA-N

InChI=1S/C24H40O5/c1-13(4-7-21(28)29)16-5-6-17-22-18(12-20(27)24(16,17)3)23(2)9-8-15(25)10-14(23)11-19(22)26/h13-20,22,25-27H,4-12H2,1-3H3,(H,28,29)/t13?,14?,15-,16-,17+,18+,19-,20+,22+,23+,24-/m1/s1

4-((3R,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid

Cholic Acid Cholalin E-1000 Cholbam E-1000 NSC-6135 OrphacolNSC-6135 E-1000NSC-6135 ColalinCholalic acid

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)