Lithocholic acid analog

Isoallolithocholic acid (3β-Hydroxy-5α-cholanic acid) (20 μM) reduces Th17 cell differentiation by approximately 50% without affecting RORγt expression [1]. The regulatory effect of isoallolithocholic acid on Tregs is cell type specific, and it has no effect on T cell differentiation into Th1 or Th2 cells [1]. The upregulation of FoxP3 expression in response to isoallolithocholic acid necessitates mitoROS[1].

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Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and Isoallolithocholic acid/isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. [1]

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Lastly, we investigated if in vitro treatment of T cells with Isoallolithocholic acid/isoalloLCA produced Tregs competent to exert suppressive function in vivo. The same number of FoxP3+ T cells (CD45.2), sorted from T cell cultures with low or high TGF-β concentrations (TGF-β-lo/-hi Tregs) in the absence or presence of isoalloLCA, were adoptively transferred into Rag1 KO mice that had also received CD45RBhi naïve CD4+ T cells (CD45.1) (Extended Data Fig. 9a, b). Mice that received CD45RBhi or CD45RBhi and TGF-β-lo Tregs developed significant weight loss and shortened colon phenotypes, both of which are indicators of colitis-associated symptoms (Extended Data Fig. 9c–f). In contrast, adoptive transfer of isoalloLCA-treated Tregs protected mice from developing colitis-associated symptoms to the same degree as those transferred with TGF-β-hi Tregs (Extended Data Fig. 9c–f). Tregs treated with isoalloLCA were more stable in terms of FoxP3 expression, compared to TGF-β-lo Tregs treated with DMSO, analyzed eight weeks post transfer (Extended Data Fig. 9g–j). In addition, mice receiving isoalloLCA-treated Tregs had reduced numbers of CD45.1+ T effector cells (Extended Data Fig. 9k). Therefore, isoalloLCA likely promotes stability of Treg cells and enhances their function following adoptive transfer in vivo, leading to decreased proliferation of T effector cells. [1]

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The modulatory effects of 3-oxoLCA on Th17 cells and Isoallolithocholic acid/isoalloLCA on Tregs were cell-type specific, as neither compound affected T cell differentiation into Th1 or Th2 cells assessed by the expression of the cytokines IFN-γ and IL-4 and the transcription factors T-bet and GATA3 (Fig. 1a, b and Extended Data Fig. 3f, g). Although 3-oxoLCA did not affect Tregs (Fig. 1b and Extended Data Fig. 2e), isoalloLCA reduced Th17 cell differentiation by ~50% without affecting RORγt expression (Fig. 1a, b and Extended Data Fig. 3h). Both compounds exhibited dose-dependent effects (Extended Data Fig. 4a). While 3-oxoLCA did not affect cell proliferation, isoalloLCA addition to T cells led to reduced proliferation compared to DMSO control (Extended Data Fig. 4b). IsoalloLCA treatment did not impair cell viability (Extended Data Fig. 4c) or T cell receptor (TCR)-mediated activation, as indicated by similar expression of TCR activation markers such as CD25, CD69, Nur77 and CD44 (Extended Data Fig. 4d). TCR activation promotes Treg-enhancement by isoalloLCA, as increasing TCR activation with higher concentrations of anti-CD3 resulted in stronger effects on FoxP3 expression without affecting cell viability (Extended Data Fig. 4e, f). [1]

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IsoalloLCA/Isoallolithocholic acid promotes Treg differentiation [1]
We next sought to uncover the mechanism by which Isoallolithocholic acid/isoalloLCA exerts its enhancing effects on Tregs. LCA has a 3α-hydroxyl group as well as a cis 5β-hydrogen configuration at the A/B ring junction and can undergo isomerization, presumably via the actions of gut bacterial enzymes2, to form isoLCA (3β,5β), alloLCA (3α,5α) or isoalloLCA (3β,5α) (Fig. 3a). Among LCA isomers, isoalloLCA has the lowest log D value (2.2), comparable to previously reported log D values of chenodeoxycholic acid (CDCA, 2.2) and ursodeoxycholic acid (UDCA, 2.2) (Extended Data Table 1), suggesting isoalloLCA is less lipophilic than other isomers. IsoalloLCA, but not the other LCA isomers, enhanced FoxP3 expression, confirming that both the 3β-hydroxyl group and trans (5α-hydrogen) A/B ring configuration of isoalloLCA are required for Treg enhancement (Fig. 3b). Compared to DMSO-treated cells, isoalloLCA-treated cells inhibited T effector cell proliferation in vitro, indicating they had acquired regulatory activity (Extended Data Fig. 5a, b). T cells isolated from FoxP3-GFP reporter mice exhibited both increased FoxP3 mRNA expression (Fig. 3c) and enhanced GFP levels following isoalloLCA treatment (Extended Data Fig. 5c). Thus, isoalloLCA-induced enhanced expression of FoxP3 occurs at the FoxP3 mRNA transcriptional level.

In B6 mice, Isoallolithocholic acid (0.03% in diet; 7 days) increases Treg cells[1].

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Bile acids modulate T cell activities in vivo [1]
Researchers examined whether 3-oxoLCA and Isoallolithocholic acid/isoalloLCA influence Th17 and Treg cell differentiation in vivo using the mouse model. Segmented filamentous bacteria (SFB), a murine commensal, is known to induce Th17 cell differentiation in the small intestine of B6 mice41. C57BL/6NTac mice from Taconic Biosciences (Tac) have abundant Th17 cells in their small intestine owing to the presence of SFB. In contrast, C57BL/6J mice from Jackson Laboratories (Jax), which lack SFB, have few intestinal Th17 cells. To determine whether 3-oxoLCA suppresses Th17 cell differentiation in vivo, we gavaged Jax-B6 mice with an SFB-containing fecal slurry and fed these animals either a control diet or 0.3% (w/w) 3-oxoLCA-containing chow for one week (Fig. 4a). The resulting average concentration of this metabolite in cecal contents was 24 picomol/mg of wet mass (approximately equivalent to μM) (Extended Data Fig. 7a, b). This concentration was sufficient to suppress Th17 differentiation in vitro (Fig. 1c). Indeed, 3-oxoLCA treatment significantly reduced the percentage of ileal Th17 cells (Fig. 4b). When we quantified the average levels of 3-oxoLCA in the stool of human patients with ulcerative colitis or in the ceca of conventionally housed mice, we observed a mean concentration of 23 or 1.0 picomol/mg, respectively (Extended Data Fig. 7c, d). SFB colonization levels were comparable between control and 3-oxoLCA-treated groups, suggesting that the change in Th17 cell percentage was not due to a decrease in SFB colonization (Extended Data Fig. 7e). In addition, Tac-B6 mice with pre-existing SFB had reduced levels of Th17 cell percentages when fed with 3-oxoLCA compared to those fed with vehicle (Extended Data Fig. 7f–h). 3-oxoLCA treatment did not affect Treg percentages (Extended Data Fig. 7i). Even under gut inflammatory conditions induced by anti-CD3 injection, known to produce a robust Th17 cell response18,42, mice treated with 1%, but not with 0.3%, 3-oxoLCA had reduced Th17 cell levels (Extended Data Fig. 7j–l).

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To examine the effects of Isoallolithocholic acid/isoalloLCA on Tregs in vivo, we fed SFB-colonized B6 mice a control diet or a diet containing 0.03% (w/w) isoalloLCA. IsoalloLCA alone was insufficient to enhance Treg percentages both at steady state (Extended Data Fig. 7m) and following anti-CD3 treatment (Extended Data Fig. 7n). We noted that 3-oxoLCA further enhanced Treg differentiation induced by isoalloLCA in vitro (Extended Data Fig. 7o, p). In line with this observation, a mixture of 0.3% (w/w) 3-oxoLCA and 0.03% (w/w) isoalloLCA significantly enhanced the Treg population in mice treated with anti-CD3 compared to control diet (Fig. 4c, d). Consistent with the mechanism in vitro, this treatment led to increased mitoROS production among CD4+ T cells in the ileal lamina propria (Extended Data Fig. 7q). Importantly, the 3-oxoLCA/isoalloLCA-induced enhanced FoxP3 expression in vivo was also dependent on the CNS3 enhancer because ΔCNS3, unlike WT, cells no longer responded to this treatment in a mixed bone marrow experiment (Fig. 4e, f). Feeding both isoalloLCA and 3-oxoLCA in chow resulted in an average concentration of 47 picomol/mg isoalloLCA in cecal contents (Extended Data Fig. 7b). This concentration was sufficient to enhance Treg differentiation in vitro (Fig. 1c). The mean concentration of isoalloLCA in the stool of human ulcerative colitis patients was 2 picomol/mg and ranged from 0 – 17 picomol/mg (Extended Data Fig. 7c). These values are within an order of magnitude of the concentrations observed in mice fed 0.03% isoalloLCA and 0.3% 3-oxoLCA, suggesting that the in vivo levels of isoalloLCA achieved are physiologically relevant [1].

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Researchers next asked whether the immunomodulatory roles of 3-oxoLCA and Isoallolithocholic acid/isoalloLCA are mediated through changes in the composition of the gut bacterial community. 16S rDNA sequencing with fecal samples of mice fed bile acid-containing diets revealed no significant perturbations in the gut bacterial community, compared to those on a control diet (Extended Data Fig. 8a–e). Furthermore, 3-oxoLCA treatment reduced Th17 cell induction in the colons of germ-free B6 mice infected with Citrobacter rodentium (Extended Data Fig. 8f, g). Thus, the Th17 and Treg modulatory activities of 3-oxoLCA and isoalloLCA do not likely require the presence of a community of commensal bacteria. Altogether, these data suggest that both 3-oxoLCA and isoalloLCA directly modulate Th17 and Treg cell responses in mice in vivo [1].

Metabolic Assays [1]
In vitro differentiated cells were cultured in the presence of DMSO or Isoallolithocholic acid/isoalloLCA for 48h, and washed extensively before the assay. Oxygen consumption rate (OCR) was determined using a Seahorse XF96 Extracellular Flux Analyzer following protocols recommended by the manufacturer and according to the previously published method50. Briefly, cells were seeded on XF96 microplates (150,000 cells/well) that had been pre-coated with poly-D-lysine to immobilize cells. Cells were maintained in XF medium in a non-CO2 incubator for 30 min before the assay. The Mito stress test kit was used to test OCR by sequential injection of 1 µM oligomycin, 1.5 µM FCCP and 0.5 µM rotenone/antimycin A. Data were analyzed by wave software.

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Microscale Thermophoresis Assay [1]
The binding affinity of the compounds with RORγ ligand-binding domain (LBD) was analyzed by microscale thermophoresis (MST). Purified RORγ-LBD was labeled with the Monolith NT™ Protein Labeling Kit RED. Serially-diluted compounds, with concentrations of 1 mM to 20 nM, were mixed with 55 nM labeled RORγ-LBD at room temperature and loaded into Monolith TM standard-treated capillaries. Binding was measured by monitoring the thermophoresis with 20% LED power and ‘Medium’ MST power on a Monolith NT.115 instrument with the following time setting: 5s Fluo, Before; 20s MST On; and 5s Fluo, After. Kd values were fitted using the NT Analysis software.

Adoptive Transfer Colitis [1]
CD45RBhi adoptive transfer colitis was performed as described51. Briefly, isolated CD4+CD25−CD45RBhi naïve T cells were sorted from wild-type B6 (CD45.1) mice by FACS and 0.5 million cells were adoptively transferred into each Rag1-KO recipient mouse. In addition, the same number of in vitro cultured and sort-purified CD45.2+ FoxP3-GFP+ cells was transferred into the recipient mice. Naïve CD4 T cells, isolated from CD45.2 FoxP3-IRES-GFP mice, were cultured under TGF-β-lo (0.05 ng/ml TGF-β), Isoallolithocholic acid/isoalloLCA (20 µM isoalloLCA and 0.01 ng/ml TGF-β) or TGF-β-hi (1 ng/ml TGF-β) conditions. Mice were then monitored and weighed each week. At week 8, colon tissues were harvested, and lamina propria lymphocytes were analyzed by flow cytometry. H&E staining and disease scoring were performed by the Rodent Histopathology Core at Harvard Medical School.

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In Vitro T Cell Culture [1]
Naïve CD4+ (CD62L+ CD44− CD25− CD4+) T cells were isolated from the spleens and the lymph nodes of mice of designated genotypes with FACS sorting. For certain experiments, naïve CD4+ T cells were enriched using naïve CD4+ T cell isolation kits. Naïve CD4+ T cells (40,000 cells) were cultured in a 96-well plate pre-coated with hamster IgG in T cell medium (RPMI, 10% fetal bovine serum, 25 mM glutamine, 55 µM 2-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin) supplemented with 0.25 µg/mL anti-CD3 (clone 145–2C11) and 1 µg/mL anti-CD28 (clone 37.51). For Th0 culture, T cells were cultured with the addition of 100 U/mL of IL-2. For Th1 cell differentiation, T cells were cultured with the addition of 100 U/mL of IL-2, 10 µg/mL of anti-IL-4 (clone 11B11) and 10 ng/mL of IL-12. For Th2 cell differentiation, T cells were cultured with the addition of 10 µg/mL of anti-IFNγ (clone XMG1.2) and 10 ng/mL of IL-4. For Th17 cell differentiation, T cells were cultured with the addition of 10 ng/mL of IL-6 and 0.5 ng/mL of TGF-β. For Treg culture, T cells were cultured with the addition of 100 U/mL of IL-2 and various concentrations of TGF-β. For most in vitro experiments to test the effects of Isoallolithocholic acid/isoalloLCA, no additional TGF-β was added. Bile acids, retinoic acid or mitoQ, or mitoPQ were added either at 0 or 16h time points. Compounds with low water solubility were sonicated before adding to the culture. Cells were harvested and assayed by flow cytometry on day 3. For ROS and mitochondrial membrane potential detection, cells cultured for 2 days were incubated with 5 µM of mitoSOX, 10 µM of DCFDA or 2 µM of JC-1 for 30 min and assayed with flow cytometry.

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Flow Cytometry [1]
Cells harvested from in vitro culture or in vivo mice experiments were stimulated with 50 ng/mL PMA (Phorbol 12-myristate 13-acetate) and 1 µM ionomycin in the presence of GolgiPlug for 4h to determine cytokine expression. After stimulation, cells were stained with cell surface marker antibodies and LIVE/DEAD Fixable dye, Aqua, to exclude dead cells, fixed and permeabilized with a FoxP3/Transcription factor staining kit, followed by staining with cytokine- and/or transcription factor-specific antibodies. All flow cytometry analyses were performed on an LSR II flow cytometer and data were analyzed with FlowJo software.

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Cell Proliferation Assay [1]
Naïve CD4+ T cells were labeled with 1 µM carboxyfluorescein succinimidyl ester and cultured for 3 days prior to FACS analysis.

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In Vitro Suppression Assay [1]
A total of 2.5 × 104 freshly-purified naïve CD4+CD25−CD44−CD62Lhigh T cells from CD45.1 B6 mice were labeled with 1 µM CFSE, activated with soluble anti-CD3 (1 µg/mL) and 5 × 104 APCs in 96-well round-bottom plates for 3 days in the presence of tester cells (CD45.2). The CFSE dilution of CD45.1 Tconv cells was assessed by flow cytometry.

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Mammalian Luciferase Reporter Assay [1]
Reporter assays were conducted as previously described14. Briefly, 50,000 human embryonic kidney 293 cells per well were plated in 96-well plates in antibiotic-free Dulbecco’s Modified Eagle Media (DMEM) containing 1% fetal calf serum (FCS). Cells were transfected with a DNA mixture containing 0.5 μg/mL of firefly luciferase reporter plasmid, 2.5 ng/mL of a plasmid containing Renilla luciferase, and Gal4-DNA binding domain-RORγ (0.2 μg/mL). Transfections were performed using TransIT-293 according to the manufacturer’s instruction. Bile acids or vehicle control were added 24h after transfection and luciferase activity was measured 16 h later using the dual-luciferase reporter kit.

Animal/Disease Models: Segmented filamentous bacteria (SFB)-colonized Jax-B6 mice[1]
Doses: 0.03% (w/w)
Route of Administration: In diet, 7 days
Experimental Results: Was insufficient to enhance Treg percentages both at steady state and following anti- CD3 treatment alone. Dramatically enhanced the Treg population in mice treated with anti-CD3 compared to control diet in combination with 0.3% (w/w) 3-oxolithocholic acid (3-oxoLCA). decreased the number of CD45.1+ T effector cells.

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In vivo bile acid analysis [1]
Stock solutions of all bile acids were prepared by dissolving the compounds in molecular biology grade DMSO. These solutions were used to establish standard curves. Glycocholic acid (GCA) or β-muricholic acid (β-MCA) was used as the internal standard for mouse and human samples, respectively. Bile acids were extracted from mouse cecal and human fecal samples and quantified by Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) as previously reported. The limits of detection of individual bile acids in tissues (in picomol/mg wet mass) are as follows: βMCA, 0.10; Isoallolithocholic acid/isoalloLCA, 0.45; isoLCA, 0.29; LCA, 0.12; alloLCA, 0.43; and 3-oxoLCA, 0.18.

[1]. Bile acid metabolites control TH17 and Treg cell differentiation. Nature. 2019 Dec;576(7785):143-148.

Isoallolithocholic acid is a bile acid.

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Certain bile acids are thought to be tissue-damaging agents that promote inflammation due to their enhanced accumulation in patients with liver diseases and their chemical properties as detergents that disrupt cellular membranes. Recent studies, however, have begun to reveal their anti-inflammatory roles, particularly in the innate immune system by suppressing NF-κB-dependent signaling pathways and by inhibiting NLRP3-dependent inflammasome activities. Our studies reveal additional anti-inflammatory roles of two LCA metabolites found in both humans and rodents that directly affect CD4+ T cells: 3-oxoLCA suppresses Th17 differentiation while isoalloLCA enhances Treg differentiation. Our data suggest that both 3-oxoLCA and isoalloLCA are present in the stool samples of human colitis patients as well as in the ceca of conventionally-housed Jax-B6 mice (Extended Data Fig. 7c, d). Importantly, both bile acids are completely absent in germ-free B6 mice (Extended Data Fig. 7d). These data suggest that gut-residing bacteria may contribute to the production of 3-oxoLCA and isoalloLCA, although we cannot rule out the possibility that host enzymes are involved. Given the significant roles of Th17 and Treg cells in a wide variety of inflammatory diseases and their close relationship with gut-residing bacteria, our study suggests the existence of novel modulatory pathways that regulate T cell function through bile acid metabolites. Future studies to elucidate the bacteria or host enzymes that generate 3-oxoLCA and isoalloLCA will provide novel means for controlling T cell function in the context of autoimmune diseases and other inflammatory conditions.[1]

C24H40O3

376.57

376.298

C, 76.55; H, 10.71; O, 12.75

2276-93-9

Lithocholic acid;434-13-9;Isolithocholic acid;1534-35-6;Isoallolithocholic acid-d2;2410277-69-7

94228

White to off-white solid powder

1.073g/cm3

511ºC at 760mmHg

276.9ºC

1.4E-12mmHg at 25°C

1.528

5.507

2

3

4

27

574

9

C[C@H](CCC(=O)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC[C@@H]4[C@@]3(CC[C@@H](C4)O)C)C

SMEROWZSTRWXGI-XBESLWPFSA-N

InChI=1S/C24H40O3/c1-15(4-9-22(26)27)19-7-8-20-18-6-5-16-14-17(25)10-12-23(16,2)21(18)11-13-24(19,20)3/h15-21,25H,4-14H2,1-3H3,(H,26,27)/t15-,16+,17+,18+,19-,20+,21+,23+,24-/m1/s1

(4R)-4-[(3S,5S,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

Isoallolithocholic acid; 2276-93-9; Cholan-24-oic acid,3-hydroxy-, (3b,5a)-; (4R)-4-[(3S,5S,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid; 3beta-Hydroxy-5alpha-cholan-24-oic Acid; Alloisolithocholic acid; Isoallolithocholate; 3beta-Hydroxy-5alpha-cholanic 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)

DMSO: 83.33 mg/mL (221.29 mM)
H2O: < 0.1 mg/mL

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.)