RORα (Ki = 172); RORγ (Ki = 111)

By reducing the expression of the IL-17A gene and the synthesis of IL-17A protein, SR1001 prevents the proliferation of TH17 cells. In a dose-dependent manner, SR1001 diminishes the coactivator TRAP220 NR box 2 peptide's responsiveness to RORγ (IC50 value ≈ 117 nM). Moreover, SR1001 suppresses the expression of cytokines when added to secretions or human TH17 cells [1].

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SR1001 is a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. [1]

Using the T1317 scaffold as a lead compound we developed a derivative, SR1001 (Fig. 1a and Supplementary Fig. 1) that was devoid of all LXR activity yet retained its ability to suppress the activity of RORα and RORγ. We found that SR1001 repressed both GAL4-RORα and GAL4-RORγ transcriptional activity in a dose dependent manner (Fig. 1b), but demonstrated no effect on LXRα activity (Fig. 1b). We assessed the specificity of SR1001 in a panel of all 48 human nuclear receptors in a cell-based cotransfection assay and did not observe activity on receptors other than RORα or RORγ (data not shown). We examined the direct binding of SR1001 to RORα and RORγ using competitive radioligand binding assays. SR1001 dose dependently displaced [3H]25-hydroxycholesterol binding to RORα and RORγ (Ki = 172 and 111 nM, respectively) (Fig. 1c) but demonstrated no binding to RORβ (data not shown). [1]

We examined whether SR1001 would affect RORα- and RORγ-dependent regulation of an Il17 promoter-driven luciferase reporter. HEK293 cells were transfected with the Il17 reporter and either full-length RORα or RORγ and treated with SR1001 or vehicle. As illustrated in Fig. 1d, SR1001 dose-dependently suppressed the Il17 promoter driven activity by each of the receptors. Since SR1001 bound RORα and RORγ, resulting in suppression of each receptors’ transcriptional activity, we expected that SR1001 would inhibit coactivator binding to the receptors. SR1001 reduced the interaction of a coactivator TRAP220 NR box 2 peptide with RORγ in a dose dependent manner (Fig. 1e) (IC50 value ~117 nM). Collectively, these data demonstrate that SR1001 function as an inverse agonist ligand of RORα/RORγ. [1]

Next, we determined whether SR1001 affected endogenous Il17a gene expression. The EL4 murine tumor cell line constitutively expresses RORα (Rora), RORγt (Rorc), and IL-17A (Il17a). EL4 cells were treated with either control siRNA or a mixture of RORα/γ siRNA followed by treatment with either vehicle or SR1001. Reduction in the expression of RORα and RORγt significantly reduced the expression of IL-17A mRNA as measured by quantitative PCR (Fig. 2a). More importantly, treatment of cells with SR1001 suppressed Il17a mRNA expression whereas treatment of RORα/γ depleted cells displayed a significantly blunted response indicating that SR1001 suppression of Il17a mRNA expression is RORα/RORγ dependent (Fig. 2a). Furthermore, SR1001 suppressed the expression of the RORα and RORγ target gene G6Pase in HepG2 cells, a human hepatocellular carcinoma cell line, providing further proof that the effect of SR1001 is mediated by RORα and RORγ. [1]

We hypothesized that SR1001 would inhibit binding of the coactivator SRC2 to either RORα or RORγ when these receptors are occupying the Il17 promoter. We performed a sequential chromatin immunoprecipitation assay (ChIP-reChIP) assessing the relative amount of SRC2 associated with either RORα or RORγ resident at the Il17 promoter in EL4 cells. SR1001 suppressd the ability of SRC2 to bind to RORα and RORγ at the Il17 promoter and increased the recruitment of the corepressor NCoR (Fig. 2b, lanes 3 and 4 and Fig. 2c, lanes 3 and 4). Thus, SR1001 suppresses Il17a expression by directly inhibiting coactivator binding and promoting the recruitment of corepressors to RORα and RORγ. [1]

To understand how ligand mediates the transcriptional activation of RORγ, we performed comprehensive differential hydrogen-deuterium exchange mass spectrometry (HDX) analysis of the RORγ LBD in the presence and absence of SR1001. This approach provides a measure of the localized ligand-induced perturbation in the conformational ensemble of the receptor. HDX kinetics of peptic peptides derived from the RORγ LBD were measured and the average difference in percentage of incorporated deuterium between apo RORγ LBD and SR1001 bound RORγ LBD are presented in Supplementary Figure 3. A negative value represents an increase in protection to exchange (more stable, less dynamic) in that region of the LBD when bound to ligand as compared to apo whereas a positive value represents a decrease in protection to exchange (less stable, more dynamic). HDX kinetics are sensitive to hydrogen bond networks and perturbations in these networks upon ligand binding can be determined using differential HDX. The differential HDX induced by SR1001 binding to RORγ correlates with the co-crystal structure of RORγ complexed with the sterol ligand, 25-hydroxycholesterol 25-OHC (PDB:3LOL) (Supplementary Fig. 4). In the RORγ/25-OHC structure, the C25 hydroxyl tail is oriented towards helix 11 (H11) and the A ring toward H1/H2. As can be inferred from PDB:3LOL, the hydroxyl group at the C1 position of 25-OHC is hydrogen bonded to Qln-286 (H1) and the 25-hydroxyl is hydrogen bonded to His-479 (H11). The regions within the RORγ LBD that show increased protection to exchange upon binding of SR1001 include portions of H1 and H11. To highlight this, the HDX data in Supplementary Fig.3 is represented graphically by overlay onto PDB:3LOL with SR1001 docked (Fig. 2d). Consistent with the differential HDX data, docking of SR1001 to PDB:3LOL suggests a similar binding mode for SR1001 to RORγ (Supplementary Fig. 4). [1]

In order to examine the role of SR1001 in modulation of this interaction between SRC2 and RORγ, we performed differential HDX on RORγ LBD in the presence and absence of the receptor interaction domain (RID) of SRC2 (Fig. 2d & Supplementary Fig.3), which contains three NR boxes (~18kDa). Several regions of the LBD demonstrate reduced HDX kinetics in the presence of SRC2 RID, indicating an interaction between the two proteins. One region stabilized is H12, containing the AF2 domain of the receptor, which has been shown to be important for NR interaction with coactivators. Furthermore, differential HDX analysis of the RORγ:SRC2 complex in the presence and absence of SR1001 clearly demonstrates that ligand disrupts the receptors interaction with SRC2 RID (Fig. 2d). These data provide strong mechanistic insight into how inverse agonists such as SR1001 repress transcriptional output of RORγ target genes. [1]

Since RORα and RORγt activity is required for optimal TH17 cell development, we explored whether SR1001 would inhibit TH17 cell differentiation. Splenocytes were cultured under TH17 polarizing conditions (TGF-β and IL-6) with SR1001 or vehicle control for 5 days. The combination of TGF-β and IL-6 increased the mRNA expression of Il17a, Il17f, Il21, and Il22, in vehicle treated cells whereas SR1001 treated cells failed to significantly upregulate these cytokines (Fig. 3). Propidium iodide staining indicated that SR1001 was not toxic and did not induce cell death (Supplementary Fig. 5). TH17 cells and inducible T regulatory cells (iTreg) are both dependent on TGFβ for their differentiation. We evaluated whether expression of the Treg-specific transcription factor Foxp3, was affected by SR1001 treatment. Similar to vehicle control, Foxp3 mRNA expression was unaffected by SR1001 treatment suggesting that inhibition of TH17 cell differentiation by SR1001 did not drive the cells into an iTreg phenotype (Supplementary Fig. 6). Furthermore, suppression of TH17 cell development with SR1001 treatment did not drive the splenocyte cultures into any of the other T helper lineages, TH1 or TH2, as indicated by the decrease in Tbx21 (T-bet) and Gata3 mRNA expression, respectively. [1]

Finally, we explored whether SR1001 would inhibit IL-17 protein production and secretion. Splenocytes were cultured under TH17 polarizing conditions and analyzed for IL-17 expression by intracellular flow cytometry. Treatment with SR1001 inhibited the expression of IL-17 from CD4+ T cells at Day 4, 5, and 6 (Fig. 4a). Similar to splenocyte cultures, intracellular flow cytometry demonstrated that SR1001 significantly repressed IL-17 expression in purified differentiated murine CD4+ T cells (CD4+CD25− CD62LhiCD44lo) (Fig. 4b). Next we assessed the effect of SR1001 on IL-17 secretion from splenocyte cultures by ELISA. Treatment with SR1001 inhibited IL-17 secretion over a three-day time course, when SR1001 was added at either the initiation of TH17 cell differentiation (initiation) or 48 hours post initiation of differentiation (post) (Fig. 4c). SR1001 was also effective at inhibiting intracellular IL-17 expression in human peripheral blood mononuclear cells (hPBMCs) (Fig. 4d). Finally, we examined the effects of SR1001 on other T helper cell lineages. Differentiation of TH1, TH2, and iTreg cells was unaffected by SR1001 treatment as similar amounts of IFNγ, IL-4, or Foxp3, respectively, were expressed compared to vehicle controls, indicating that SR1001 specifically targets TH17 cells (Supplementary Fig. 7)[1].

In rat models, SR1001 successfully genetically predisposes the clinical severity of autoimmunity. Cyp7b1, Rev-erbα, and Serine 1 are hepatic ROR targets whose expression is inhibited when SR1001 is applied to C57BL/6 mice[1]. The internal citrate synthase mRNA expression pattern is eliminated by the SR1001 RORα counterregulator [2].

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Given that RORα and RORγt are required for development of TH17-mediated autoimmune diseases and SR1001 inhibits the activity of both of these receptors leading to suppressed TH17 cell development in vitro, we evaluated the effects of SR1001 treatment in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), a well characterized model of TS17 cell-mediated autoimmune disease. After myelin oligodendrocyte glycoprotein (MOG35–55) immunization at day 0, mice were treated with 25 mg kg−1 of SR1001 b.i.d. i.p. for the duration of the study. As shown in Figure 4e, SR1001 treatment delayed the onset and clinical severity of EAE. Further analysis of spinal cords from mice harvested at day 18 post-immunization revealed that SR1001 repressed Il17a mRNA expression by ~60%, as well as reduced Il21, and Il22 mRNA expression (Fig. 4f). Intracellular cytokine analysis of splenocytes indicated a significant reduction in IL-17 expression and reduced total CD4+ T cells with no effect on CD8+ T cells. mRNA expression of IL-4 and IFNγ was unaffected in both spleen and spinal cords (Supplementary Fig. 8). These data are consistent with our interpretation that SR1001 suppresses EAE through its effects on TH17 cell function in vivo. Further optimization of SR1001 may yield compounds with greater activity. While RORα and RORγt expression and activity are essential for full TH17 cell development, it is important to note that RORα and RORγ have roles outside of the immune system and are critical regulators of hepatic metabolism. Administration of SR1001 to C57BL/6 mice suppressed the expression of hepatic ROR target genes, Cyp7b1, Rev-erbα, and Serpine 1 (Pai-1) suggesting that this class of compound may have metabolic effects; however, we noted no obvious toxicity in animals treated with SR1001 [1].

Radioligand binding assay. [1]
Radioligand binding assays were performed as previously described. For the competition assay, various concentrations of SR1001 were incubated with receptor in the presence of 3 nM [ 3H]-25-hydroxycholesterol. Results were analyzed using GraphPad Prism software and the Ki was determined using the Cheng-Prusoff equation.

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Alpha Screen. [1]
The ALPHA screen assays were performed as previously described. Assays were performed in triplicate in white opaque 384-well plates under green light conditions (<100 lux) at room temperature. The final assay volume was 20 μL. All dilutions were made in assay buffer (100 mM NaCl, 25 mM Hepes, 0.1% BSA, pH 7.4). The final DMSO concentration was 0.25%. A mix of 12 L of GST-ROR- LBD (10 nM), beads (12.5 μg ml-1 of each donor and acceptor), and 4 μL of increasing concentrations (210 nM – 50 M) of compound SR1001 were added to the wells, the plates were sealed and incubated for 1h. After this preincubation step, 4 μL of BiotinTRAP220-2 peptide (50 nM) was added, the plates were sealed and further incubated for 2h. The plates were read on PerkinElmer Envision 2104 and data analyzed using GraphPad Prism software.

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Hydrogen/deuterium exchange mass spectrometry. [1]
Differential, solution phase HDX experiments were performed with a LEAP Technologies Twin HTS PAL liquid handling robot interfaced with an Orbitrap mass spectrometer. Each exchange reaction was initiated by incubating 4 L of 10M protein complex (with or without SR1001 and SRC2 RID) with 20 L of D2O protein buffer for a predetermined time (1s, 30s, 60s, 900s, and 3600s) at 25ºC. The exchange reaction was quenched by mixing with 50 L of 3 M Urea, 0.1% TFA at 1 ºC. The mixture was passed across at an in-house packed pepsin column (2mm × 2cm) at 50 l min-1 and digested peptides were captured onto a 2mm × 1cm C8 trap column and desalted (total time for digestion and desalting was 2.5 min). Peptides were then separated across a 2.1mm × 5cm C18 column (1.9μ Hypersil Gold) with linear gradient of 4%-40% CH3CN, 0.3 % formic acid, over 5 min. Protein digestion and peptide separation were performed within a thermal chamber held at 2C to reduce D/H back exchange. Mass spectrometric analyses were carried out with capillary temperature at 225 ºC and data were acquired with a measured resolving power of 65,000 at m/z 400. Three replicates were performed for each on-exchange time point.

RNA-mediated interference. [1]
EL4 cells were first electroporated with 100nM total siRNA with the GenePulserXcell Electroporator using siRNA against mouse ROR and ROR followed by reverse using 50nM siRNA according to the instructions for Dharma-FECT 1 transfection reagent and seeded onto a 12 well plate. 24 hours post transfection, cells were treated with vehicle (DMSO) or SR1001 (10M) for 24 hours. Cells were harvested and total RNA was isolated. Quantitative reverse transcriptase PCR was performed to analyze mRNA levels of mouse ROR, RORt, Gapdh, and Il17a using SYBR green technology. Primers sequences to mouse ROR, RORt, Il17a, and Gapdh and have previously been described 5,6,7 . HepG2 cells were treated similarly to EL4 cells with the following exceptions: HepG2 cells were transfected with siRNA against human ROR and ROR at 50nM according to the instructions for DharmaFECT 1 transfection reagent. Quantitative reverse transcriptase PCR was performed to analyze mRNA levels of human ROR RORA, ROR RORC, CYCLOPHILIN, and G6Pase using SYBR green technology. The primer sequences have previously been described.

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ChIP/ReChIP. [1]
EL4 cells were treated with plate bound anti-CD3 (5g ml -1 ) and soluble anti-CD28 (1g ml -1 ) for 24 h and then treated with vehicle (DMSO) or SR1001 (10 M) for another 24 h. Anti-ROR or anti-ROR anti-body was used to do the first immunoprecipitation for all of the samples. The second immunoprecipitation was performed by using anti-rabbit IgG for ROR, anti-hamster IgG for ROR, anti-RNA Pol II, anti-SRC2, or anti-NCoR (Santa Cruz). The IL-17 primers used in PCR have been previously described.

Mice.[1]
C57BL/6J mice were used for all in vitro experiments unless otherwise noted. EAE was induced in 8 week-old male wild-type C57BL/6J mice. Male DIO mice, 22 weeks of age, were purchased from Jackson Laboratories and fed a high fat diet (HFD) (60%kCal % fat) for the duration of the study.

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Induction and clinical evaluation of EAE. [1]
EAE was induced in C57BL/6 wild-type mice by s.c. injection over four sites in the flank with 200 µg per mouse MOG35–55 peptide in an emulsion with IFA supplemented with 2.25 mg ml -1 Mycobacterium tuberculosis, strain H37Ra. Pertussis toxin dissolved in PBS was injected i.p. at 200 ng pre mouse at the time of immunization (Day 0) and 48 h later. Mice were scored daily on a scale of 0–6, as described previously: 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind-limb weakness; 3, severe hind-limb weakness; 4, complete hind-limb paralysis; 5, quadriplegia or pre-moribund state; 6, death. All mice were 7–10 weeks of age when experiments were performed. The SR1001 was dissolved in DMSO at 25 mg ml -1 and the mice were treated (i.p.) with 25 mg kg -1 SR1001 (1 µl g -1 body weight of mouse) or vehicle (DMSO, 1 µl g -1 body weight of mouse) twice per day. The treatment was started 2 days before immunization and continued until the end of experiment. Where indicated in the figure legends, mice were anesthetized with halothane and transcardially perfused with PBS, and spinal cords were removed for RNA and protein isolation

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Mouse Experiments [2]
The livers from staggerer mice, a naturally occurring RORα mutant were purchased from Jackson laboratories. Liver was homogenized on ice. mRNA was prepared by the Trizol method. For the qPCR, cyclophilin B was the control gene. qPCR was performed using the following primers: mCycB_F: 5′-GCAAGTTCCATCGTGTCATCAAG-3′, mCycB_R: 5′-CCATAGATGCTCTTTCCTCCTG-3′, mCS_F: 5′-GGACAATTTTCCAACCAATCTGC-3′, and mCS_R: 5′-TCGGTTCATTCCCTCTGCATA-3′. For CS assays liver was homogenized on ice using CelLytic MT buffer with added protease inhibitor (1∶100 concentration). Protein quantity was determined using a Bradford assay. The assay contained 4 µg protein per well (12 µg total in the master mix, which was aliquoted into 3 wells). Enzymatic activity was determined using the CS assay kit, as described above. For circadian gene expression experiments male C57BL6 mice (8–10 weeks of age) were either maintained on a L:D (12h∶12h) cycle or on constant darkness (1 day). At circadian time (CT) 0 animals were administered a single dose of 25 mg/kg SR1001 (i.p.) and groups of animals (n = 6) were sacrificed at CT0, CT6, CT12 and CT18. Gene expression was determined by real time QPCR. Gene expression was normalized to Cyclophin b in all experiments.

[1]. Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand. Nature. 2011 Apr 28;472(7344):491-4.

[2]. Regulation of expression of citrate synthase by the retinoic acid receptor-related orphan receptor α (RORα). PLoS One. 2012;7(4):e33804.

SR 1001 is a sulfonamide.

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T-helper cells that produce interleukin-17 (T(H)17 cells) are a recently identified CD4(+) T-cell subset with characterized pathological roles in autoimmune diseases. The nuclear receptors retinoic-acid-receptor-related orphan receptors α and γt (RORα and RORγt, respectively) have indispensible roles in the development of this cell type. Here we present SR1001, a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. Finally, SR1001 effectively suppressed the clinical severity of autoimmune disease in mice. Our data demonstrate the feasibility of targeting the orphan receptors RORα and RORγt to inhibit specifically T(H)17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.[1]

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In summary, we describe a novel, first-in-class, highly selective drug targeting the orphan NRs RORα and RORγ that effectively suppresses TH17 cell differentiation and cytokine expression and reduces the severity of disease in an animal model of multiple sclerosis. Our data indicates that the targeting of TH17 cells, by blocking RORα/γ function with a synthetic ligand, is a tractable approach for potential therapeutic intervention. Current treatments for TH17-mediated autoimmune diseases, including multiple sclerosis, utilize agents that are general immunosuppressant’s and thus the side effect profile is significant. Clearly, our data demonstrates that by targeting RORα and RORγ one can specifically inhibit TH17 cells without affecting other T helper cell lineages thereby providing a more focused therapy that will not be a general immunosuppressant. Therefore, SR1001 and derivatives of this compound may represent a novel class of superior drugs to not only treat TH17-mediated autoimmune disorders, but ROR-mediated metabolic disorders as well.[1]

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The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression.[2]

C15H13F6N3O4S2

477.401841878891

477.025

C, 37.74; H, 2.74; F, 23.88; N, 8.80; O, 13.41; S, 13.43

1335106-03-0

44241473

White to yellow solid powder

1.6±0.1 g/cm3

1.557

3.15

3

13

5

30

718

0

S(C1=C(C)N=C(NC(C)=O)S1)(NC1C=CC(=CC=1)C(C(F)(F)F)(C(F)(F)F)O)(=O)=O

OZBSSKGBKHOLGA-UHFFFAOYSA-N

InChI=1S/C15H13F6N3O4S2/c1-7-11(29-12(22-7)23-8(2)25)30(27,28)24-10-5-3-9(4-6-10)13(26,14(16,17)18)15(19,20)21/h3-6,24,26H,1-2H3,(H,22,23,25)

N-[5-[[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]sulfamoyl]-4-methyl-1,3-thiazol-2-yl]acetamide

1335106-03-0; SR1001; SR 1001; SR-1001; N-[5-[[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]sulfamoyl]-4-methyl-1,3-thiazol-2-yl]acetamide; N-(5-(N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)sulfamoyl)-4-methylthiazol-2-yl)acetamide; CHEMBL3094388; OZBSSKGBKHOLGA-UHFFFAOYSA-N;

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 : ≥ 39 mg/mL (~81.69 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.36 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 (4.36 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.36 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.)