Bile acid derivative; anticancer; Microbial Metabolite; Endogenous Metabolite

Glycocholic acid (GC) exacerbates epirubicin-induced cell death and amplifies the cytotoxicity of epirubicin. It also greatly increases the intracellular accumulation of epirubicin in Caco-2 cells and the absorption of epirubicin in the rat small intestine. Human intestinal MDR1, MDR-related protein (MRP) 1 and MRP2 mRNA expression levels were significantly decreased by glycocholic acid and epirubicin, which also down-regulated the MDR1 promoter region, inhibited Bcl-2 mRNA expression, induced Bax mRNA expression, and significantly increased the Bax-to-Bcl-2 ratio as well as the mRNA levels of p53, caspase-9, and -3. Glycocholic acid and anticancer medications work together to regulate MDR via pathways connected to apoptosis, P-gp and MRP regulation, and other processes [1].

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Cancer-cell resistance to chemotherapy limits the efficacy of cancer treatment. The primary mechanisms of multidrug resistance (MDR) are "pump" and "non-pump" resistance. We evaluated the effects and mechanisms of Glycocholic acid (GC), a bile acid, on inhibiting pump and non-pump resistance, and increasing the chemosensitivity of epirubicin in human colon adenocarcinoma Caco-2 cells and rat intestine. GC increased the cytotoxicity of epirubicin, significantly increased the intracellular accumulation of epirubicin in Caco-2 cells and the absorption of epirubicin in rat small intestine, and intensified epirubicin-induced apoptosis. GC and epirubicin significantly reduced mRNA expression levels of human intestinal MDR1, MDR-associated protein (MRP)1, and MRP2; downregulated the MDR1 promoter region; suppressed the mRNA expression of Bcl-2; induced the mRNA expression of Bax; and significantly increased the Bax-to-Bcl-2 ratio and the mRNA levels of p53, caspase-9 and -3. This suggests that GC- and epirubicin-induced apoptosis was mediated through the mitochondrial pathway. We conclude that simultaneous suppression of pump and non-pump resistance dramatically increased the chemosensitivity of epirubicin. A combination of anticancer drugs with GC can control MDR via a mechanism that involves modulating P-gp and MRPs as well as regulating apoptosis-related pathways. [1]

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Combined Glycocholic acid/GC and epirubicin treatment significantly increased the cytotoxicity of epirubicin [1]
We used the MTT dye reduction method to assay cell-growth inhibition. Fig. 1A shows the results of the viability assay after treatment of Caco-2 cells with 0, 100, 250, 300, 400, and 500 μM of GC after 0, 24, 48, and 72-h of incubation. The effect of GC on cell viability % as measured by MTT assay was time dependent with the most profound effects seen after 72 h. After incubation with 250 μM of GC, the viability was 80 ± 3% after 72-h incubation, whereas it was 96 ± 4% and 90 ± 4% after 24-h and 48-h incubation, respectively. We fixed the period of incubation at 72 h, which was consistent with the studies of Kosmider et al. (2004) and Budman et al. (2007). They found that the most profound effect of growth inhibition and apoptosis induction was after 72-h of incubation. Because our aim was to develop GC as an adjuvant to intensify the potency of epirubicin, we chose 250 μM of GC ( Semiquantitative RT-PCR of P-gp, MRP1, MRP2, Bcl-2, Bax, and caspases [1]
To analyze the effect of Glycocholic acidGC and epirubicin on pump and non-pump resistance-related proteins, the mRNA expression levels of MDR1, MRP1, MRP2, Bax, Bcl-2, caspase-3, caspase-8, caspase-9, and p53 were evaluated using RT-PCR. GC and epirubicin, used together or separately for 72 h, significantly down regulated the corresponding mRNA levels of MDR1, MRP1, MRP2, and Bcl-2 (P < 0.05) (Figs. 2A and B). The same treatment for 72 h, however, significantly upregulated the corresponding mRNA levels of Bax, caspase-3, caspase-9, and p53 (P < 0.05). Combined GC and epirubicin treatment resulted in significantly (P < 0.05) greater down- and upregulation of MDR1, MRP1, MRP2, and Bax expression than did treatment with GC or epirubicin alone. GC and epirubicin, individually or combined, significantly increased the Bax-to-Bcl-2 ratio (Fig. 2C), but did not significantly affect caspase-8 expression in Caco-2 cells (Fig. 2B). GC combined with epirubicin did not increase the mRNA levels of p53, caspase-9, and caspase-3 more than epirubicin alone did (P > 0.05).

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Combined Glycocholic acidGC and epirubicin treatment significantly decreased the luciferase activity of the hMDR1 promoter region [1]
To investigate the potential effect of GC and epirubicin on the transcriptional regulation of hMDR1, 159 bp (residues −120 to +39) of the hMDR1 promoter was cloned upstream of the firefly luciferase reporter gene in the pGL3-basic vector. These 159 bp of hMDR1 DNA elements consist of the proximal promoter region that encodes AP-1, CAAT, the GC box, and the Y-box necessary for efficient transcription of hMDR1. For normalization, a Renilla luciferase reporter gene of pRL-TK vector was co-transfected. The luminescent activity of the hMDR1 promoter-pGL3 and pRL-TK vector was subsequently measured using a dual luciferase assay system. The effects of GC, epirubicin, and GC combined with epirubicin on the activity of the hMDR1 regulatory promoter region of 159 bp elements in Caco-2 cells were then compared. GC and GC combined with epirubicin for 72 h significantly suppressed hMDR1 promoter activity (P < 0.05; Fig. 3). The positive control, rifampicin, significantly increased the luciferase activity (P < 0.001), which indicated that rifampicin had significantly induced hMDR1 promoter activity. GC combined with epirubicin inhibited hMDR1 promoter-related luciferase activity significantly more than did GC or epirubicin alone (P < 0.05). In a negative control experiment, the activity levels of the hMDR1 promoter-deficient pGL3-basic luciferase reporter vectors were not affected by GC, epirubicin, or GC combined with epirubicin, which suggested that there was no nonspecific direct interaction between the individual testing agents and the luciferase reporter vectors.

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GC/Glycocholic acid significantly increased the intracellular accumulation of epirubicin in Caco-2 cells [1]
We used flow cytometry to determine whether combined GC and epirubicin treatment of Caco-2 cells increased the retention of epirubicin. In addition, the functional involvement of MDR-related proteins such as P-gp in the efflux of epirubicin was verified by the addition of verapamil. Verapamil, a calcium channel blocker, is a typical P-gp substrate and one of the most studied MDR modulators (Germann, 1996, Lo and Huang, 2000). Studies have suggested that verapamil competes with other substrates for binding to P-gp and thus reverses MDR (Gottesman and Pastan, 1993). Pretreatment with 25 μM of Ver (Ver25), 250 μM of Ver (Ver250), or 250 μM of GC (GC250) for 72 h significantly increased the intracellular accumulation of epirubicin at 180 min in Caco-2 cells (P < 0.05; Fig. 4). GC 250 exhibited significantly higher retention of epirubicin than Ver25 did (P < 0.05; Fig. 4). But GC 250 demonstrated significantly lower retention of epirubicin than Ver250 did (P < 0.05; Fig. 4). These results suggest that inhibiting P-gp and MRPs by using GC combined with epirubicin may account for the increase in the intracellular uptake of epirubicin in Caco-2 cells.

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Combined Glycocholic acid/GC and epirubicin treatment significantly induced chromatin condensation [1]
We used fluorescence microscopy and fluorescent DNA-binding dye AO staining to evaluate whether the cytotoxic effect of GC and epirubicin was related to their effect on apoptosis induction in Caco-2 cells. Viable cells had a uniform bright green nucleus (Fig. 5). Apoptotic cells displayed bright green areas of condensed or fragmented chromatin in the nucleus. No obvious morphological changes were seen in the control group (Fig. 5A). However, Caco-2 cells exposed to GC, epirubicin, or GC combined with epirubicin for 72 h showed condensed chromatin in the nucleus and appearance of apoptotic bodies (Fig. 5B–D, white arrows). Based on Fig. 5B–D, it was found that more bright spots were observed in cells exposed to GC combined with Epi compared to Epi or GC alone (P < 0.05 in both cases), which is consistent with our other data.

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Combined Glycocholic acid/GC and epirubicin treatment significantly increased the sub-G1 accumulation of DNA content [1]
Caco-2 cells treated with GC alone, epirubicin alone, or both for 72 h, showed a pattern typical of DNA content that reflected sub-G1, G0/G1, S, and G2/M phases of the cell cycle (Fig. 6). The percentages of the sub-G1 phase of Caco-2 cells, which correspond to the proportions of apoptotic cells, significantly increased after all three treatments. The percentage of the sub-G1 phase of cells treated with GC plus epirubicin (46.3%; P < 0.001) was significantly higher than the percentage treated with GC alone (26.8%) or epirubicin alone (20.6%).

The effect of Glycocholic acid/GC on the absorption of epirubicin in everted gut sacs of rats [1]
Effect and mechanism(s) of modulators on intestinal absorption of drugs in MDR spectrum may differ depending on cell types, species, and physicochemical properties of substrates of efflux transporter proteins (Komarov et al., 1996, Lo, 2003). Whereas cell lines are efficient methods to study mechanistic interaction of anticancer drugs and MDR modulators at the cellular level, studies using other systems, such as ex vivo everted sac, in situ perfusion, and in vivo experiments, are necessary to assess the absorption/exsorption mechanism(s) of epirubicin in the presence and absence of Glycocholic acid/GC or vaerapamil. Caco-2 cell monolayers have the advantage of human origin, but the system is static, gives very low rates of transport, and exaggerated enhancement of the paracellular route compared with small intestine (Barthe et al., 1999). Ex vivo everted sac technique provides quantitative information on mechanisms of drug absorption from the mucosal side to the serosal side through testing the drug content in the intestinal sac, whiles P-gp and MRPs in the apical cell membrane may limit bioavailability by expelling drugs from the mucosal cells. This model maintains tissue viability, gives reliable data, and appears particularly useful for studying drug interactions with transporters, although it is a closed system (Barthe et al., 1999, van de Kerkhof et al., 2007). Permeability values for small hydrophilic molecules using the improved everted sac gives data close to those for humans, while values with Caco-2 cells are orders of magnitude lower (Barthe et al., 1999). In situ rat intestinal perfusion is a reliable technique to investigate drug absorption potential in combination with intestinal metabolism. However, this system is time-consuming and therefore not suited for screening purposes (Bohets et al., 2001). It gives no information on events at the cellular level, and absorption may be reduced by anesthesia and surgical manipulation (Barthe et al., 1999). Finally, in vivo absorption in animals can provide valuable bioavailability data; however, it is even more time- and cost-consuming, so it is not practical for screening (Barthe et al., 1999, Bohets et al., 2001). Therefore, everted sac system is an appropriate ex vivo model to study epirubicin absorption. In addition, the regional difference of drug absorption in intestinal tissues has been reported (Makhey et al., 1998, Englund et al., 2006). Therefore, effect of GC or verapamil on epirubicin absorption at separate intestinal segments, including jejunum and ileum was investigated. Epirubicin was transported from the mucosal side to the serosal side at different segments of the rats’ small intestines for 60 min (Fig. 7A and B). The epirubicin concentrations, measured in sacs pretreated with 250 μM of GC or verapamil, were significantly higher than those in the control groups in both the jejunum and the ileum (P < 0.01, n = 3 rats per group). Ileal epirubicin absorption in combination with GC or verapamil was significantly higher (P < 0.05) than that in jejunum. The enhancing effect of verapamil was higher than that of GC (P < 0.05 compared to Epi plus GC), which was consistent with the intracellular accumulation study. This implies that GC or verapamil exhibited an inhibitory effect on P-gp and/or MRPs, thus causing a decrease in epirubicin efflux, an increase in epirubicin absorption, or both, for the combined treatment.

Cell growth inhibition assay [1]
Cell viability was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Caco-2 cells were trypsinized, disaggregated through a pipette, and counted with a hemacytometer. The cells were seeded in 96-well plates (104 cells/well), allowed to attach overnight, and then treated with different concentrations of epirubicin with or without Glycocholic acid/GC. After 0, 24, 48, or 72-h incubation, 20 μl of MTT reagent at the concentration of 5 mg/ml was added to each well and the cells were incubated for an additional 4 h. The supernatant from each well was then carefully removed, and 100 μl of dimethylsulfoxide (DMSO) was added to each well and thoroughly mixed. The plate was read for optical density at 545 nm, using an MRX microplate reader. The absorbance value (OD545) was transformed to the cell number in each well of the 96-well plates. Cell viability (%) was calculated by dividing the number of cells incubated with epirubicin and with GC by the number of cells incubated with tissue culture medium only (control). The cell-growth inhibition potency of epirubicin and the combination of epirubicin and GC are expressed as IC50 values, defined as the concentration of a drug necessary to inhibit the growth of cells by 50%. Data are the means ± S.D. of four experiments (Pakunlu et al., 2006).

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Semiquantitative RT-PCR of P-gp, MRP1, MRP2, Bcl-2, Bax, caspases, and p53 [1]
Caco-2 cells were pretreated for 72 h with 250 μM of Glycocholic acid/GC with or without 10 μg/ml of epirubicin. RNA was isolated from the cells using a kit according to the manufacturer's instructions. RNA yield and purity were assessed using spectrophotometric analysis. Total RNA (1 μg) from each sample was subjected to RT-PCR with templates, dNTPs, AMV reverse transcriptase, Tfl DNA polymerase, and the corresponding 5′- and 3′-primers of MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, and p53 (Table 1) in 50 μl of the total reaction volume. The PCR cycles for these proteins are given in Table 2. Expression levels of MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, and p53 mRNA were evaluated by measuring RT-PCR products after amplification. An aliquot of each reaction mixture was analyzed using electrophoresis on a 2% agarose gel, and amplified DNA was visualized using a DNA gel stain staining (Chearwae et al., 2004). The gels were digitally photographed and scanned using a gel documentation system and commercial software. Gene expression of MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, and p53 was calculated as the ratio of mean band density of analyzed RT-PCR product to that of the internal standard (GAPDH). The expression values of different mRNAs treated with epirubicin or GC, or both, were compared to the expression values of the control (treated with medium only). The expression values of different mRNAs treated with epirubicin combined with GC were also compared with the expression values of different mRNAs treated with GC or epirubicin alone (Chearwae et al., 2004, Pakunlu et al., 2004). Means ± S.D. from four independent measurements are shown.

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Transfection and dual luciferase activity assay [1]
Cells were plated at a density of 2 × 105 per well in six-well plates and allowed to attach overnight. We mixed 2 μg/well of the hMDR1 promoter-pGL3 firefly luciferase reporter gene constructs and 0.2 μg/well of the pRL-TK Renilla luciferase reporter gene (Promega) with 6 μl of lipofectin reagent, and then incubated the cells at 25 °C for 15 min and then at 37 °C for 15 h. Next, the cells were treated with 25 μM of rifampicin (a positive control), 10 μg/mL of epirubicin, 250 μM of GC, or a combination of epirubicin and Glycocholic acid/GC for 72 h. After the incubation, luciferase reporter gene activity was evaluated with a dual luciferase reporter assay system. Briefly, the cells were washed twice with cold PBS and then lysed in 300 μl of reporter lysis buffer. After they had been incubated at 37 °C for 15 min, the lysates were mixed in a vortex blender for 15 s and centrifuged at 4 °C for 30 s. The luciferase reaction was then initiated by auto-injecting 100 μl of a reagent with luciferin (Luciferase Assay Reagent II) to 20 μl of lysate supernatants; the resulting luminescence was measured using a luminometer (Chen et al., 2004). After we quantified the firefly luminescence, we quenched the reaction and then initiated a Renilla luciferase reaction by simultaneously adding 100 μl of reagent to the same tube. After completing a background correction (for activity in untreated control cells), we calculated the results as the level of hMDR1 promoter-pGL3 activity divided by pRL-TK activity. The total cellular protein concentration was determined using a protein assay.Data are the means ± S.D. of four independent experiments.

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Measuring the intracellular accumulation of epirubicin in Caco-2 cells using flow cytometry [1]
We measured intracellular epirubicin fluorescence as previously described (Lo and Huang, 2000, Lo, 2003). Cells (104 per well) were seeded into 24-well plates to permit confluence. The cells were then rinsed twice with PBS, pretreated with 250 μM of Glycocholic acid/GC, and incubated at 37 °C for 72 h. The experiment was then done by adding 10 μg/ml of epirubicin to the culture medium. After they had been incubated at 37 °C for 3 h, the cells were washed twice with ice-cold PBS, trypsinized, centrifuged, and then resuspended in PBS. Flow cytometric analysis was then done using a flow cytometer equipped with an argon ion laser and operated at 488 nm and 15 mW. Red epirubicin fluorescence was measured through a 585/42 nm band pass filter. Data acquisition and analysis were done using commercial software (Lysis II; Becton Dickinson). Forward- and side-scatter signals were collected using linear scales, and fluorescence signals were collected on a logarithmic scale. At least 10,000 cells were analyzed in each sample. Within each experiment, determinations were done in quadruplicate.

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Chromatin condensation detection using a fluorescence microscope [1]
After we had treated the cells with medium alone, Glycocholic acid/GC, epirubicin, or GC plus epirubicin, we used centrifugation (200 g for 1 min) to collect 1 × 106 cells. The pellets were resuspended in 100 μl of PBS and 10 μl of 100 mg/ml acridine orange, and then visualized under an inverted microscope equipped with a fluorescence image capture device controlled with an Image-Pro Plus software. The characteristics of fragmented nuclei and condensed chromatin were observed and compared with those of the control (Kosmider et al., 2004).

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Sub-G1 accumulation of DNA content detection using a flow cytometer [1]
After we had treated cells with medium alone, 10 μg/ml of epirubicin, 250 μM of Glycocholic acid/GC, or a combination of epirubicin and GC for 24 h, we used centrifugation (1200 rpm for 5 min) to collect 105 cells. The pellets were resuspended in 200 μl of PBS, fixed with 800 μl of ice-cold absolute ethanol, and maintained at −20 °C for 30 min. We used centrifugation (1200 rpm for 5 min) to collect cells, washed them twice with ice-cold PBS, resuspended them in 50 μg/ml of propidium iodide in PBS, and then incubated them for 40 min at 25 °C in the dark. We analyzed the samples using a flow cytometer. Data acquisition and analysis were done using commercial software. Fluorescence signals were collected on a logarithmic scale. Each experiment was repeated between four and six times (Barcia et al., 2003, Wang et al., 2006, Yamanaka et al., 2006).

Everted sacs of rat jejunum and ileum [1]
Male Sprague–Dawley rats bred and housed in the animal center were used. The animal-use protocols were in accord with nationally approved guidelines. Everted sacs of rat jejunum and ileum were prepared using a method previously described (Lo and Huang, 2000, Lo, 2003). Rats weighing about 300 g were deprived of food for 1 day and given only double-distilled water until they were killed with carbon dioxide. The jejunum and distal ileum of the rat intestines (approximately 25 cm each) as well as the underlying mesenterium were removed. The segments were washed with iced saline before they were mounted in Tyrode's solution. The sacs were everted, filled with 3 ml of Tyrode's solution, and ligated at both ends. One of the two ends was ligated with a needle for the following sampling. The sacs were then incubated for 60 min with 50 ml of Tyrode's solution with or without 250 μM of Glycocholic acid/GC or verapamil. The solution was bubbled with air and maintained at 37 °C throughout the experiment. At 0 min, 100 μg/ml of epirubicin was added in the mucosal side. In each study, 200 μl of the solution inside the sacs was taken every 10 min for 60 min, and was replaced with fresh Tyrode's solution to keep the volume of the serosal solution constant. Each experiment was done in triplicate. The concentration of epirubicin in each sample was determined using high-performance liquid chromatography (HPLC) as described below.

[1]. Inhibit multidrug resistance and induce apoptosis by using glycocholic acid and epirubicin. Eur J Pharm Sci. 2008 Sep 2;35(1-2):52-67.

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]acetic acid;hydrate is a bile acid glycine conjugate.

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Glycocholic acid is a bile acid glycine conjugate having cholic acid as the bile acid component. It has a role as a human metabolite. It is functionally related to a cholic acid and a glycochenodeoxycholic acid. It is a conjugate acid of a glycocholate.
The glycine conjugate of cholic acid. It acts as a detergent to solubilize fats for absorption and is itself absorbed.
Glycocholic acid has been reported in Homo sapiens and Caenorhabditis elegans with data available.
The glycine conjugate of CHOLIC ACID. It acts as a detergent to solubilize fats for absorption and is itself absorbed.

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Our previous study (Lo and Huang, 2000) suggested that sodium deoxycholate, a bile salt, increased the transepithelial transport of epirubicin, an amphiphilic compound, via paracellular and transcellular routes. Sodium deoxycholate affects the transcellular route by creating membrane perturbation, which results from its interaction with membrane lipids and proteins. This bile salt also affects the paracellular route by opening tight junctions (Sakai et al., 1997). In addition, inhibiting P-gp function using multidrug-resistance reversing agents that act via substrate competition, ATP-depletion, or membrane perturbation may antagonize MDR (Kvackajova-Kisucka et al., 2001; Lo et al., 2003). Amphiphilic bile salts such as sodium deoxycholate usually have a membrane perturbation property that changes the fluidity of Caco-2 cell membranes and rat colon epithelium, and thus modulates the efflux function of membrane-spanning proteins such as P-gp and MRPs (Sawada et al., 1991, Sakai et al., 1997). We could not rule out the possibility that GC has a similar membrane perturbing effect and thus the potential for increasing absorption and decreasing epirubicin efflux. We further hypothesize that the inhibitory effect of GC on pump-related resistance contributes, at least in part, to inhibiting P-gp and MRPs, as shown in our RT-PCR and MDR1 promoter activity studies. Furthermore, in the present study, GC combined with epirubicin induced caspases-3 and caspases-9 through the mitochondrial pathway. Pro-apoptotic stimuli induced by anticancer drugs may need a mitochondrion-dependent process involving permeabilization of the outer mitochondrial membrane and the release of mitochondrial proteins normally located in the intermembrane space (Petit et al., 1997, Minko et al., 2005). Based on the possible membrane perturbation characteristics of GC, we hypothesize that GC incorporates into mitochondrial membranes, changes the fluidity of mitochondrial membranes, and modulates the essential processes, such as the release of cytochrome c and proapoptotic proteins, and thus induces the mitochondrial pathway in apoptosis. However, the detailed mechanism requires further investigation.[1]

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Given the inhibiting effects of GC combined with epirubicin on antiapoptotic- and MDR-related proteins, we hypothesize that there is a connection between pump and non-pump resistance. Simultaneously inhibiting pump and non-pump resistance with GC combined with epirubicin may be a novel strategy for reversing MDR. A combination of anticancer drugs with GC can control MDR via a mechanism that involves modulating P-gp and MRPs as well as regulating apoptosis-related pathways.

C26H43NO6.H2O

483.638

483.32

1192657-83-2

475-31-0; 863-57-0

24856164

White to off-white solid powder

3.34

6

7

6

34

759

11

C[C@H](CCC(=O)NCC(=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.O

WDKPRHOCWKLQPK-ZUHYDKSRSA-N

InChI=1S/C26H43NO6.H2O/c1-14(4-7-22(31)27-13-23(32)33)17-5-6-18-24-19(12-21(30)26(17,18)3)25(2)9-8-16(28)10-15(25)11-20(24)29;/h14-21,24,28-30H,4-13H2,1-3H3,(H,27,31)(H,32,33);1H2/t14-,15+,16-,17-,18+,19+,20-,21+,24+,25+,26-;/m1./s1

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]acetic acid;hydrate

Glycocholic acid hydrate; 1192657-83-2; Glycocholic acid hydrate synthetic; 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]acetic acid;hydrate; MLS001304001; MFCD06408004; Glycocholic acid (hydrate); SCHEMBL60638;

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)

DMSO: 250 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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