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
Following ingestion, absorption of cholic acid will first be by the small intestine, and is then transported to the liver by the blood for further processing.
Orally administered cholic acid is subject to the same metabolic pathway as endogenous cholic acid. Cholic acid is absorbed by passive diffusion along the length of the gastrointestinal tract. Once absorbed, cholic acid enters into the body's bile acid pool and undergoes enterohepatic circulation mainly in conjugated forms. In the liver, cholic acid is conjugated with glycine or taurine by bile acid-CoA synthetase and bile acid-CoA: amino acid N-acetyltransferase. Conjugated cholic acid is actively secreted into bile mainly by the Bile Salt Efflux Pump (BSEP), and then released into the small intestine, along with other components of bile. Conjugated cholic acid is mostly re-absorbed in the ileum mainly by the apical-sodium-dependent-bile acid transporter, passed back to the liver by transporters including sodium-taurocholate cotransporting polypeptide and organic anion transport protein and enters another cycle of enterohepatic circulation. Any conjugated cholic acid not absorbed in the ileum passes into the colon where deconjugation and 7-dehydroxylation are mediated by bacteria to form cholic acid and deoxycholic acid which may be re-absorbed in the colon or excreted in the feces. The loss of cholic acid is compensated by de-novo synthesis of cholic acids from cholesterol to maintain the bile acid pool in healthy subjects.
Excretion studies in rat showed that cholic acid (CA) is almost exclusively excreted in the feces in the form of metabolite. Only minor amounts of CA were found in the unconjugated form in rat feces. Urinary excretion of bile acids is minimal and in mice fed a 1% CA diet, the excretion of bile acids was 2000-fold higher in feces than in urine
Cholic acid was shown to be mainly absorbed in the distal (ileal) rather than proximal segments of the small intestine in the guinea pig.
For more Absorption, Distribution and Excretion (Complete) data for CHOLIC ACID (9 total), please visit the HSDB record page.

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Metabolism / Metabolites
The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-(3)H]cholic acid was observed when the liver was perfused with 5beta-[G-(3)H]cholestane-3alpha, 7alpha-diol, 5beta-[G-(3)H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-(3)H]cholestane-3alpha, 7alpha, 26-triol. No [G-(3)H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-(14)C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-(14)C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.
In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.
Deoxycholic acid is the main metabolite of cholic acid. Patients with 3alpha-HSD deficiency and delta4-3-oxoR deficiency and subjects with a normal bile acid metabolism have shown that upon treatment with cholic acid, serum and bile predominantly contain cholic acid and deoxycholic acid, while chenodeoxycholic acid and its metabolites appear to be reduced. Under cholic acid treatment, patients are therefore exposed to higher than normal deoxycholic acid concentrations, although the exact quantifications of these concentrations have not been described. In single- and repeat-dose studies, deoxycholic acid showed lethal effects, gastrointestinal and hepatic toxicities at approximately half the doses needed for cholic acid to produce the same effects. It is therefore considered that deoxycholic acid is more toxic than cholic acid and may in fact be the causative agent of some of cholic acid's toxicity. Mutagenicity data from bacterial test for deoxycholic acid is ambiguous but deoxycholic acid was genotoxic in an in vitro micronucleus assay. Additionally, ... the genotoxic potential of BA (focusing on chenodeoxycholic acid and deoxycholic acid) on human colonocytes and colon tumor cells HT 29 by a comet assay /was investigated/. In both cell types a clear dose-dependent genotoxic effect induced by the two bile acids was observed, with deoxycholic acid being more genotoxic. Viability of cells appeared to be greater than 75%. Use of a nuclease III modified comet assay suggested that the DNA damage could be mediated by reactive oxygen species production but was somewhat protected by inclusion of anti-oxidants. Short term carcinogenicity studies suggest that deoxycholic acid like cholic acid has carcinogenicity promoting properties. In rat liver, deoxycholic acid (75-150 mg/kg) exerted promoting activity as evidenced by significantly increased values of alpha-glutamyl transpeptidase-positive (alpha-GT+) liver foci compared with the corresponding controls given the carcinogen, diethylnitrosamine (DEN) alone. Deoxycholic acid (20 mg/kg) enhanced the development and growth of azoxymethane (AOM)-induced aberrant crypt foci in rat colons. In a parallel study, deoxycholic acid in the absence of AOM did not significantly induce aberrant crypt foci. However, /a study/ concluded that deoxycholic acid may act not only as promoters but also initiators of the multistage process of carcinogenesis.
Cholic acid has known human metabolites that include Cholic acid glucuronide.

Toxicity Summary
IDENTIFICATION AND USE: Cholic acid is used in biochemical research, as a pharmaceutical intermediate, and as an emulsifying agent in foods (up to 0.1%). It is also a medication used for the treatment of bile acid synthesis disorders due to single enzyme defects and for the adjunctive treatment of peroxisomal disorders including Zellweger spectrum disorders in patients who exhibit manifestations of liver disease, steatorrhea or complications from decreased fat soluble vitamin absorption. HUMAN EXPOSURE AND TOXICITY: Cholic acid is a primary bile acid. Primary bile acids are biosynthesized in the liver and are key constituents of normal bile. When formulated and used as a treatment for patients with bile acid synthesis disorders the major toxic effect is on liver function. The dose of cholic acid to be administered to the patients is intended to restore a concentration that is equivalent to that physiologically present in humans. Therefore any perceived genotoxic risk from cholic acid and deoxycholic acid to the patients would be equivalent to that of a normal healthy adult that produces these bile acids intrinsically. Overall, cholic acid showed nonsignificant mutagenic activity in a battery of genotoxicity tests performed in vitro. ANIMAL STUDIES: Bile acids are known to regulate bile acid synthesis and transport by the farnesoid X receptor in the liver (FXR-SHP) and intestine (FXR-Fgf15). Mice lacking the farnesoid X receptor (FXR) involved in the maintenance of hepatic bile acid levels are highly sensitive to cholic acid-induced liver toxicity. Serum aspartate aminotransferase (AST) activity was elevated 15.7-fold after feeding a 0.25% cholic acid diet for five days, whereas only slight increases in serum AST (1.7- and 2.5-fold) were observed in wild-type mice fed 0.25 and 1% cholic acid diet, respectively. Primary bile acids were studied as possible colon tumor promoters or inhibitors in a rat model of chemically induced colon cancer. Cholic acid feeding increased the number of animals with tumors, the number of tumors per animal, and the number of tumors per tumor-bearing animal. Tumor enhancement was attributed to deoxycholic acid, the bacterial metabolite of cholic acid. Administration of cholic acid (1.0% of the diet) to male rats for 3 days resulted in increased numbers of DNA synthesizing epithelial cells per colonic crypt column as compared to those found in either control or 0.2% cholic acid-fed rats. The mutagenicity of bile acids was detected by a fluctuation test using Salmonella typhimurium TA100 and TA98 as tester strains. Cholic acid and deoxycholic acid were mutagenic in this test. The mutagenicity of the bile acids on a molar basis was roughly one-fourth that of methyl methanesulfonate, a moderately potent mutagen. 0.5% cholic acid ingested by pregnant hamsters, caused ductal/ductular proliferation and hepatobiliary inflammatory damage in a different degree of intensity in adult animals and mild intensity in the young; and also the number of the young was reduced in the litter. The ingestion of these bile acids by hamsters, during gestational period caused different degrees of toxicity on maternal and neonatal hepatobiliary systems. In rat developmental studies there was apparent pathological change of fetal rats brain in cholic acid-treated groups, the neuronal degeneration and the mitochondria swelling was mainly found in low cholic acid group, the neuronal necrosis and the mitochondria decrease was mainly found in high cholic acid group.

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Hepatotoxicity
In small, open label trials, cholic acid was found to improve fat soluble vitamin absorption and to ameliorate many of the clinical features of the bile acid synthetic defects including improvement in serum aminotransferase levels, decrease in bilirubin and jaundice and improvement in general health and growth. In some instances, higher doses of cholic acid were associated with elevations in serum aminotransferase levels. These abnormalities, however, were mild, transient and rapidly reversed with lowering the daily dose. There have been no reports of clinically apparent liver injury with jaundice attributed to cholic acid therapy given in standard doses.
Likelihood score: D[HD] (possible cause of liver injury but only when given in high doses).

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Interactions
Aluminum-based antacids have been shown to adsorb bile acids in vitro and can reduce the bioavailability of Cholbam. Take Cholbam at least 1 hour before or 4 to 6 hours (or at as great an interval as possible) after an aluminum-based antacid.
Bile acid binding resins such as cholestyramine, colestipol, or colesevelam adsorb and reduce bile acid absorption and may reduce the efficacy of Cholbam. Take Cholbam at least 1 hour before or 4 to 6 hours (or at as great an interval as possible) after a bile acid binding resin.
Phenobarbital treatment increases the transport capacity (Vmax) for bile salt in the ileum of the rat. ... Rats treated with phenobarbital for 10 days had a 4-fold smaller cholic acid pool compared to control rats. Additionally, the daily production of cholic acid was decreased in phenobarbital treated rats compared to control rats. Phenobarbital has an antagonistic effect to the desired action of cholic acid in patients and may potentially endanger the established metabolic control. Therefore use of Phenobarbital in patients treated with cholic acid has been contraindicated.
Avoid concomitant use of inhibitors of the bile salt efflux pump (BSEP) such as cyclosporine /with Cholbam/. Concomitant medications that inhibit canalicular membrane bile acid transporters such as the BSEP may exacerbate accumulation of conjugated bile salts in the liver and result in clinical symptoms. If concomitant use is deemed necessary, monitoring of serum transaminases and bilirubin is recommended.
For more Interactions (Complete) data for CHOLIC ACID (8 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mouse ip 330 mg/kg
LD50 Mouse oral 4950 mg/kg
LD50 Mouse iv 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
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Cholic acid is included in the database.
Cholbam is indicated for the treatment of bile acid synthesis disorders due to single enzyme defects (SEDs). /Included in US product label/
Cholbam is indicated for adjunctive treatment of peroxisomal disorders (PDs) including Zellweger spectrum disorders in patients who exhibit manifestations of liver disease, steatorrhea or complications from decreased fat soluble vitamin absorption. /Included in US product label/
The safety and effectiveness of Cholbam on extrahepatic manifestations of bile acid synthesis disorders due to single enzyme defects (SEDs) or peroxisomal disorders (PDs) including Zellweger spectrum disorders have not been established.
For more Therapeutic Uses (Complete) data for CHOLIC ACID (7 total), please visit the HSDB record page.

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Drug Warnings
The safety and effectiveness of Cholbam has been established in pediatric patients 3 weeks of age and older for the treatment of bile acid synthesis disorders due to single enzyme defects (SEDs), and for adjunctive treatment of patients with peroxisomal disorders (PDs) including Zellweger spectrum disorders who exhibit manifestations of liver disease, steatorrhea or complications from decreased fat soluble vitamin absorption.
Monitor liver function and discontinue Cholbam in patients who develop worsening of liver function while on treatment. Concurrent elevations of serum gamma glutamyltransferase (GGT), alanine aminotransferase (ALT) may indicate Cholbam overdose. Discontinue treatment with Cholbam at any time if there are clinical or laboratory indicators of worsening liver function or cholestasis.
The developmental and health benefits of breastfeeding should be considered along with the mother's clinical need for Cholbam and any potential adverse effects on the breastfed infant from Cholbam or from the underlying maternal condition.
No studies in pregnant women or animal reproduction studies have been conducted with Cholbam. Limited published case reports discuss pregnancies in women taking cholic acid for 3beta-HSD deficiency resulting in healthy infants. These reports may not adequately inform the presence or absence of drug-associated risk with the use of Cholbam during pregnancy. The background risk of major birth defects and miscarriage for the indicated population is unknown. However, the background risk in the U.S. general population of major birth defects is 2-4% and of miscarriage is 15-20% of clinically recognized pregnancies.
For more Drug Warnings (Complete) data for CHOLIC ACID (6 total), please visit the HSDB record 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.)