LXR (EC50 = 20 nM for LXRα); FXR (EC50 = 5 μM); RORα (Ki = 132 nM); RORγ (Ki = 51nM)

In a dose- and time-dependent way, T0901317 (5-50 μM; 72 hours) effectively reduces the cell growth of human ovarian cancer cell lines, CaOV3, SKOV3, and A2780 [5]. Cell cycle arrest at the G1-S checkpoint is indicated by T0901317 (10 μM; 24-72 hours) which decreases the percentage of cells in S phase and increases the percentage of cells in G0/G1 phase. Time-dependently, the proportion of cells in the G0/G1 phase rises [5]. Twenty-four hours at 10–40 μM, T0901317 significantly increases early apoptosis [5]. After 48 hours, T0901317 (5–40 μM; 48 hours) increases the expression of the proteins p21 and p27 in a dose-dependent manner [5].

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CDCA activated the chimeric receptor with the expected EC50 in the range of 40 μM; surprisingly however, we also noted that T0901317 activated FXR with an EC50 of ∼5 μM. Although the FXR potency of T0901317 is considerably less than that described for LXR (∼50 nM) it is approximately 10-fold more potent than the natural FXR ligand CDCA. To determine if T0901317 was acting as a direct ligand for FXR we utilized a biochemical ligand sensing assay in which we assessed the ability of T0901317 to induce a conformational change in the FXRLBD sufficient to cause recruitment of coactivator proteins, SRC-1 or SRC-2. Consistent with the cell-based transfection assay, T0901317 induced FXR recruitment of either SRC-1 (Fig. 1B) or SRC-2 (Fig. 1C) with greater potency than the natural FXR ligand, CDCA. EC50 values for CDCA were 37 and 18 μM for SRC-1 and SRC-2, respectively, while the values for T0901317 were 7 and 4 μM for the two coactivators. Maximal efficacies for the two ligands were similar. The activity of a structurally unique LXR ligand, GW3965, was also assessed in these assays and found to be inactive (<10% efficacy at 10 μM) indicating that the dual LXR/FXR activity was specific for T0901317 (data not shown) [2].
To determine if T0901317 exhibited FXR agonist activity the context of a natural FXR target gene, we examined the ability of this compound to induce expression of either the bile salt export protein (BSEP) or the short heterodimer partner (SHP) in Huh7 cells. As illustrated in Figs. 2A and B, CDCA induces the expression of both BSEP and SHP mRNA in a dose-dependent manner. Consistent with our previous data, T0901317 also increases the expression of both FXR target genes in a dose-dependent manner with maximal efficacy similar to that of CDCA (Figs. 2C and D). Thus, our data demonstrate that the high affinity LXR ligand T0901317 also exhibits FXR agonist activity albeit at a lower potency [2]. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) inhibited transactivation activity of RORα and RORγ but not RORβ. T0901317 was found to directly bind to RORα and RORγ with high affinity (Ki = 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed RORα/γ-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by RORα at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine RORα/γ function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders.[3]

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T0901317 treatment resulted in a significant (P <0.001) inhibition of cell proliferation in a time- and dose-dependent manner in CaOV3, SKOV3 and A2780 cells. Western blot analysis demonstrated an induction of p21 and p27 with a concominant reduction in phospho-RB protein levels. Cell cycle analysis demonstrated a significant (P <0.001) arrest in the G1 cell cycle phase. Significant induction of Caspase-3 and BAX gene expression occurred with treatment. Induction of apoptosis was confirmed by significant (P < 0.001) elevation of caspase activity on FACS analysis, caspase-glo assay, BAX protein induction and decreased caspase 3 precursor protein expression on Western blot analysis. LXR α/β knockdown experiments did not reverse the anti-proliferative and cytotoxic effects of T0901317.
Conclusions: The LXR agonist, T0901317, significantly suppresses cell proliferation and induces programmed cell death in a dose- and time-dependent manner. Our results indicate that T0901317 induces its anti-proliferative and cytotoxic effects via an LXR-independent mechanism [4].

T0901317 (10 mg/kg/day; oral; 12 weeks) slows the rate at which atherosclerosis advances [5]. T0901317 (ip; 50 mg/kg; twice weekly for 7 days) prevents insulin resistance and obesity in male C57BL/6 mice fed a high-fat diet [6].

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We studied the dose and exposure-response relationships of BMS-852927 for lipid, plasma CETP, and blood mRNA endpoints and compared them to the full pan LXR agonist T0901317. In 14-day oral dosing studies, BMS-852927 was much less potent than T0901317 in elevating plasma TG and LDL-C, with a ≥40-fold rightward shift in the plasma exposure response curves (Figure 1A). The plasma CETP mass exposure-response curve was similarly right shifted (Figure 1A). In contrast, the two compounds had similar potencies for the induction of blood ABCG1 mRNA (Figure 1A), a surrogate measure of RCT stimulation. While blood ABCA1 mRNA was also induced by both compounds, it was not used in these therapeutic window analyses due to an artifactual induction of this gene from oral gavaging the animals, evident in the effect seen in vehicle-treated animals in Figure S1A, available online. Importantly, maximal blood ABCG1 induction by BMS-852927 (16-fold) occurred at an exposure that had no effect on plasma TG or LDL-C (4.9-fold over the BMS-852927 WBA EC50). BMS-852927 treatment also caused an exposure-dependent increase in HDL-C, reaching a maximal 38% above baseline (Figure 1A). The effect of the compound on all of the above endpoints was at or near steady state by day 4 of the 14-day treatment (Figures S1A–S1F). The effect of BMS-852927 and another LXR agonist, BMS-779788, on the induction of hepatic lipogenic genes was also evaluated as part of cynomolgus monkey toxicology studies. T0901317 was not tested; however, BMS-779788 has approximately 2-fold greater LXRα activity than BMS-852927 (38% versus 20%), with greater overall agonist activity (50% versus 26%) (Kick et al., 2015), and, therefore, was an informative comparator. BMS-852927 was much less active than BMS-779788 in the induction of the lipogenic genes SREBP1c, FAS, and SCD1, resulting in only 2- to 3-fold induction with liver exposures of 36-fold over its WBA EC50 (Figure 1B). By comparison, BMS-779788 induced the same genes 5- to 21-fold at exposures of 12-fold over its EC50. Consistent with this, BMS-852927 treatment had no effect on liver TG up to 3 mg/kg/day for 7 days as measured by magnetic resonance spectroscopy (MRS), while BMS-779788 caused a dose-dependent increase up to 75% above baseline (Figure 1C). [5]

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The effect of activation of liver X receptor by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)ethyl]phenyl] benzenesulfonamide (T0901317) on high fat diet (HFD)-induced obesity and insulin resistance was examined in C57BL/6 mice. When on HFD continuously for 10 weeks, C57BL/6 mice became obese with an average body weight of 42 g, insulin resistant, and glucose intolerant. Twice weekly intraperitoneal injections of T0901317 at 50 mg/kg in animals on the same diet completely blocked obesity development, obesity-associated insulin resistance, and glucose intolerance. Quantitative real-time PCR analysis showed that T0901317-treated animals had significantly higher mRNA levels of genes involved in energy metabolism, including Ucp-1, Pgc1a, Pgc1b, Cpt1a, Cpt1b, Acadm, Acadl, Aox, and Ehhadh. Transcription activation of Cyp7a1, Srebp-1c, Fas, Scd-1, and Acc-1 genes was also seen in T0901317-treated animals. T0901317 treatment induced reversible aggregation of lipids in the liver. These results suggest that liver X receptor could be a potential target for prevention of obesity and obesity-associated insulin resistance [6].

Radioligand Receptor Binding Assay [3]
Forty-five or 90 ng of purified GST-RORα or GST-RORγ was incubated with various concentrations of [ 3H]25-hydroxycholesterol in assay buffer (50 mM HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mM NaCl, and 5 mM MgCl2) to determine the Kd value. Nonspecific binding was defined in the absence of protein and excess of nonradioactive 25-hydroxycholesterol and was shown to be identical. The assays were terminated by rapid filtration through presoaked Whatman GF/B filters (0.5% polyethylenimine in phosphate-buffered saline) in Multiscreen plates and were washed (3 × 0.1 ml) with ice-cold assay buffer. The radioligand binding results were analyzed using Prism software. For the competition assay, various concentrations of T0901317 were incubated with receptor in the presence of 3 nM [ 3H]25-hydroxycholesterol.

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AlphaScreen [3]
The assays were performed in triplicate in white opaque 384-well plates. The final volume was 20 μl for the generation of compound dose-response curves (0.5–7.5 μM). All dilutions were made in assay buffer (100 mM NaCl, 25 mM HEPES, and 0.1% bovine serum albumin, pH 7.4). The final DMSO concentration was 0.25%. A mix of 12 μl of His-RORα-LBD (75 nM), beads (30 μg/ml of each), and 4 μl of increasing concentrations of compound (0.02–8 μM) was added to the wells, and the plates were sealed and incubated for 1 h at room temperature in the dark. After this preincubation step, 4 μl of Biotin-RIP140B (25 nM) was added, the plates were sealed and further incubated for 2 h at room temperature in the dark. The plates were read on a PerkinElmer Envision 2104, and data were analyzed using Prism software.

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Caspase-3 and -7 assay [4]
Vybrant FAM Caspase-3 and -7 Assay Kit V35118, was used to quantitatively determine the percentage of cells actively undergoing apoptosis according to the manufacturer's instructions. Briefly, ovarian carcinoma cells were seeded overnight in 6 wells plates at a density of 2 × 105 per well. Cells were then treated for 24 h with T0901317 (10 μM) or 0.1% DMSO as negative control. Cells were then trypsinized and collected and 1 × 105 cells per sample were stained with 10 μl of FLICA reagent and 7-AAD and incubated at 37°C in 5% CO2 for one hour. Cells were then washed with 1× wash buffer, centrifuged at 1500 RPM for 5 minutes. The supernatant was discarded, 400 μL of 1× wash buffer was added and samples were analyzed by flow cytometry according to manufacturer's recommendations.

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Caspase-3/7 activation assay [4]
Caspase-3/7 activation assays were performed using a Caspase-Glo™ 3/7 assay kit according to the manufacturer's instructions. Briefly, ovarian carcinoma cells were seeded in 96-well plates at a density of 1 × 104 cells/well. After 24 h, cells were treated with different concentrations of T0901317 (5, 10, 20, 40 and 50 μM) or 0.1% DMSO as negative control. Caspase-Glo 3/7 reagent (100 μl) was then added to each well including medium alone, untreated control cells or cells treated with T0901317 for 6 h. The plate was then incubated at room temperature for 1 h and the luminescence of each sample was measured with a Veritas Microplate Luminometer.

Cell Proliferation Assay[5]
Cell Types: A2780, CaOV3 and SKOV3 ovarian cancer cell lines
Tested Concentrations: 5, 10, 20, 40 or 50 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibited cellular proliferation in all cell lines in a dose-dependent and time-dependent manner.

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Cell Cycle Analysis[5]
Cell Types: A2780, CaOV3 and SKOV3 cells
Tested Concentrations: 10 μM
Incubation Duration: 24, 48 or 72 hrs (hours)
Experimental Results: diminished the percentage of cells in S phase and increased the percentage of cells in the G0/G1 phase.

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Apoptosis Analysis[5]
Cell Types: CaOV3 cells
Tested Concentrations: 10 to 40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Resulted in a significant increase of cells in early apoptosis.

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Western Blot Analysis[5]
Cell Types: CaOV3 cells
Tested Concentrations: 5 to 40 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Resulted in an increase of p21 and p27 protein expression in a dose-dependent manner.

Animal/Disease Models: 8- to 10weeks old LDL receptor null mice[5]
Doses: 10 mg/kg
Route of Administration: po (oral gavage) daily; for 12 weeks
Experimental Results: Inhibited the progression of atherosclerosis.

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Cynomolgus Monkey Studies [5]
In a PD study, animals were randomized into six treatment groups (n = 3/group) and dosed once daily with vehicle, 10 mg/kg/day T0901317, and 0.1, 0.3, 1, or 3 mg/kg/day BMS-852927 for 14 days. Blood RNA and plasma lipids were determined at baseline and days 1, 4, 7, and 14 of dosing for the PD study, and on days 1 and 7 for the liver TG MRS study (see below). In a cynomolgus monkey liver mRNA study conducted as part of a larger toxicology study, animals were randomized into four treatment groups (n = 5/group) and dosed daily for 28 days with vehicle, and 0.3, 3, or 30 mg/kg/day BMS-852927. A similar study was conducted with BMS-779788 in which animals were treated for 14 days with vehicle and 1, 10, or 30 mg/kg/day BMS-779788. Liver samples from both studies were taken at 24 hr after the final dose for compound concentration and mRNA determinations. All blood and liver mRNAs were quantitated as described in detail in the RNA preparation and analysis section of the Supplemental Experimental Procedures.

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Mouse Studies [5]
To study effects of LXR agonists on neutrophils, C57BL/6 mice pre-acclimated to oral dosing (n = 8/group) were randomly assigned to vehicle; 0.03, 0.1, 1, or 3 mg/kg/day BMS-852927; and 0.3 or 3 mg/kg/day GW3965 and dosed orally for 3 days. Following anesthesia with isoflurane, blood was collected by retro-orbital bleeding and analyzed for neutrophil levels using an Advia hematology instrument employing peroxidase staining. In an atherosclerosis prevention study, 8- to 10-week-old LDL receptor null mice fed a western diet were orally gavaged daily with vehicle, BMS-852927 (0.1, 1, or 3 mg/kg/day), or 10 mg/kg/day T0901317 for 12 weeks. At the end of treatment, mice were euthanized and atherosclerosis was quantitated en face in Oil Red O-stained aortas by image analysis. Lesion area was expressed as percent of total aortic area.

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Animals and Animal Treatments [6]
Male C57BL/6 mice were housed under a 12-h light–dark cycle. The mice were divided into two groups (n = 5) and fed with HFD from Bio-Serv for 10 weeks. Beginning in week 1, one group of animals (treated group) was intraperitoneally (i.p.) given T0901317 solubilized in DMSO twice weekly at a dose of 50 mg/kg. The second group was given the same volume of DMSO as a control, and named as control group. The food intake and body weight of the mice in both groups were determined twice weekly. The BMI value was calculated as body weight (grams) divided by the square of the anal–nasal length (centimeters). The body composition was analyzed using EchoMRI-100 from Echo Medical Systems. Animals were sacrificed at the end of 10 weeks for histological and biochemical analysis.

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To test the reversibility of T0901317-induced lipid accumulation in the liver, the mice were divided into three groups (n = 5), including a control, T0901317 treated, and T0901317 withdrawal group. On day 1, the T0901317-treated group was put on high fat diet and started daily treatment with T0901317 (i.p., 50 mg/kg) for 7 days, while the control group was on high fat diet and treated with DMSO. Mice in the T0901317 withdrawal group were first given T0901317 (i.p., 50 mg/kg) daily for 7 days, stayed treatment free for additional 7 days, and then sacrificed.

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IPGTT and ITT During the last week of the experiment, the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed after the last T0901317 treatment. For IPGTT, mice were fasted overnight before the injection of glucose (2 g/kg, i.p.), and the blood glucose level was measured at the predetermined time points using glucose test strips and glucose meters. For ITT, the mice fasted for 4 h before the injection of insulin (Humulin, 0.5 U/kg), and the blood glucose level was measured at predetermined time points using the same method as above. The Homeostasis Model of Assessment–Insulin Resistance (HOMA-IR) value was calculated using previously established formula: HOMA-IR = [fasting insulin (nanograms per milliliter) × fasting plasma glucose (milligrams per deciliter)/405].

[1]. Role of LXRs in Control of Lipogenesis. Genes Dev. 2000 Nov 15;14(22):2831-8.

[2]. T0901317 Is a Dual LXR/FXR Agonist. Mol Genet Metab. Sep-Oct 2004;83(1-2):184-7.

[3]. The Benzenesulfoamide T0901317 [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide] Is a Novel Retinoic Acid Receptor-Related Orphan Receptor-Alpha/Gamma Inverse Agonist. Mol Pharmacol. 2010 Feb;77(2):228-36.

[4]. Anti-proliferative effect of LXR agonist T0901317 in ovarian carcinoma cells. J Ovarian Res. 2010 May 26;3:13.

[5]. Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils. Cell Metab. 2016 Aug 9;24(2):223-33.

[6]. The Liver X Receptor Agonist T0901317 Protects Mice From High Fat Diet-Induced Obesity and Insulin Resistance. AAPS J. 2013 Jan;15(1):258-66.

TO-901317 is an LXRalpha and LXRbeta agonist.
N-(2,2,2-Trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}benzenesulfonamide has been reported in Aspergillus puniceus with data available.

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We characterize the ability of the liver X receptor (LXRalpha [NR1H3] and LXRbeta [NR1H2]) agonist, T0901317, to activate the farnesoid X receptor (FXR [NR4H4]). Although T0901317 is a much more potent activator of LXR than FXR, this ligand actually activates FXR more potently than a natural bile acid FXR ligand, chenodeoxycholic acid. Thus, the FXR activity of T0901317 must be considered when utilizing this agonist as a pharmacological tool to investigate LXR function.[2]

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Initial biological characterization of T0901317 indicated that it was specific for LXR or retained slight FXR activity relative to LXR. In the latter study, which indicated some limited FXR activity, no potency data were provided. Our results indicate that T0901317 acts as an FXR agonist with efficacy similar to a natural bile acid ligand, CDCA. The EC50 for T0901317 ranged from 4 to 7 μM within the various FXR assays, which is within a range that is significant given that 1 μM concentrations are often used as a standard in various in vitro assays assessing LXR activity. Since T0901317 has been the primary pharmacological tool for elucidating the physiological role of the LXRs, it is apparent that the concentration of this ligand must be carefully monitored so as to avoid FXR activation and conclusions that may be erroneous due to activation of both receptors.[2]

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RORs regulate a variety of physiological processes, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we demonstrate that T0901317 represents the first synthetic ligand for RORα and RORγ, and this compound is a potent inverse agonist of these two orphan nuclear receptors. This was demonstrated by competitive radioligand binding assay and cell-based assays in which T0901317 repressed RORα/γ-dependent transactivation of reporter genes driven by the ROR-responsive promoters from the G6Pase and Cyp7b1 genes. Moreover, repression of G6Pase by T0901317 was relieved after knockdown of both RORs, concluding that this compound's effects on this gluconeogenic enzyme are ROR-dependent. Finally, we show that T0901317 reduces recruitment of the p160 coactivator SRC2 by RORα at the G6Pase promoter, thus providing a mechanism for control of this important enzyme by the RORs. The pharmacology of T0901317 has been extensively studied in animal models, with the compound exhibiting acceptable pharmacokinetic properties. More importantly, the benzenesulfonamide scaffold is amenable to a modular synthetic chemistry optimization (Michael et al., 2005). Therefore, T0901317 represents a novel chemical tool to examine RORα/γ function, and our findings offer an excellent starting point for the design of potent and selective ROR ligands with potential application in the treatment of metabolic and immune disorders. [3]

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To our knowledge, this is the first study to report the anti-proliferative and pro-apoptotic activity of T0901317 on ovarian cancer cells mediated via an LXR-independent pathway. We believe that based on our results that synthetic LXR agonists warrant further studies as anti-neoplastic agents in the treatment of ovarian cancer.[4]

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The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.[5]

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C17H12F9NO3S

481.332715034485

481.039

C, 42.42; H, 2.51; F, 35.52; N, 2.91; O, 9.97; S, 6.66

293754-55-9

447912

White to off-white solid powder

1.5±0.1 g/cm3

470.5±55.0 °C at 760 mmHg

116-122° C

238.4±31.5 °C

0.0±1.2 mmHg at 25°C

1.491

4.82

1

13

5

31

684

0

S(C1C=CC=CC=1)(N(CC(F)(F)F)C1C=CC(=CC=1)C(C(F)(F)F)(C(F)(F)F)O)(=O)=O

SGIWFELWJPNFDH-UHFFFAOYSA-N

InChI=1S/C17H12F9NO3S/c18-14(19,20)10-27(31(29,30)13-4-2-1-3-5-13)12-8-6-11(7-9-12)15(28,16(21,22)23)17(24,25)26/h1-9,28H,10H2

N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide

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)

Solubility in Formulation 1: ≥ 3 mg/mL (6.23 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 30.0 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: ≥ 3 mg/mL (6.23 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 30.0 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: ≥ 3 mg/mL (6.23 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (5.19 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: ≥ 2.5 mg/mL (5.19 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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 (Please use freshly prepared in vivo formulations for optimal results.)