LXRβ/liver X receptor β

In transactivation experiments, XL041 (BMS-852927) demonstrates 20% LXRα and 88% LXRβ activity in comparison to the full pan agonist. With an EC50 of 9 nM and 26% activity in an in vitro human whole blood endogenous target gene activation assay (WBA), XL041 is a very powerful drug. Comparable binding affinities of BMS-852927 for LXRα and LXRβ (19 and 12 nM, respectively) have been reported [1].

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When the fluorine R3 substitution was combined with the 2,6-diCl A-ring (15/BMS-852927) very potent hWBA activity was observed with an EC50 value of 9 nM (26% efficacy). Although 15/BMS-852927 has similar LXRα and LXRβ binding Ki values (19 and 12 nM, respectively), in agonist assays the compound achieved 88% efficacy toward LXRβ and only 20% efficacy toward LXRα compared to a full pan-agonist. When tested in antagonist mode, 15 was a potent LXRα antagonist with an IC50 value of 69 nM (83% inhibition); whereas no antagonism was observed in LXRβ assays up to 10,000 nM (Supporting Information). Further SAR investigation with a 2-Cl,6-F R1 substitution pattern provided 16 with a hWBA potency of 41 nM (33%). Introduction of an R3 chlorine atom in 17 caused a decrease in efficacy in all four agonist assays with a potent hWBA EC50 value of 5 nM just above the limit of assay detection (16%). Analogue 18 with hydrogen at R2 had in vitro activity consistent with the gem-dimethyl analogue 12. The monomethyl analogue 19 was identified as a metabolite of 15, and upon synthesis the profile showed it to be a potent, partial LXR agonist as well [2].

At effective dosages, XL041 (BMS-852927) exhibits very positive characteristics in cynomolgus monkeys and mice. After giving XL041 to C57BL/6J mice for seven days, the system's cholesterol efflux was effectively stimulated in a dosage-dependent manner. The highest efflux rate was observed in the group given a dose of 3 mg/kg/day, which was 70% higher than the vehicle. Mice that were LDLR knockout (KO) had similar outcomes. In a further 12-week trial, XL041 prevented atherosclerosis from progressing in mice lacking the LDLR gene. Significantly, the dose-response for increasing macrophage reverse cholesterol transfer (RCT) (0.03-3 mg/kg/day) and suppressing atherosclerosis (0.1-3 mg/kg/day) was comparable. is a key underlying process by which LXR agonists impact the illness [1].

In vitro Assay Methods: [2]
Ligand Binding Assays. Baculovirus encoding human RXRα and human LXRα or human LXRβ were used to co-infect Sf9 cells. Infected cell lysates were prepared and supernatants containing soluble RXRα-LXR or RXRα-LXR heterodimers were used in scintillation proximity ligand binding assays in which compounds were tested for the ability to compete for binding with 50 nM 3H-24,25- epoxycholesterol (NEN Life Science Products / Perkin Elmer). 1 The determined Ki represents the average of at least two independent dose response experiments. The binding affinity for each compound was be determined by non-linear regression analysis using the one site competition formula to determine the IC50 where: Y = Bottom + (Top - Bottom)/(1+10X-logIC50). The Ki was then calculated: Ki = IC50/(1 + [Concentration of Ligand]/Kd of Ligand). For this assay, typically the Concentration of Ligand = 50 nM and the Kd of ligand for the receptor was 200 nM as determined by saturation binding.

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Transient Transfection Reporter Assays. [2]
LXR & LXR LXRE assays (Isotype-specific). For agonist assays CV-1 cells were co-transfected with pCXM-hLXRα or pCMX-hLXRβ, and LXREx1-tk-luc plasmids in bulk and replated at a density of 8,000 cells/well in 384-well plates containing 5 µL media with 0.5% DMSO or the test agonist. 2 LXR antagonist assays were carried out the same way but in presence of 60 nM of the agonist Pan Agonist A. 100% inhibition was calculated based on values obtained in the absence of agonist. The first two columns of each plate contained Pan Agonist A, to determine the 0% inhibition level. Cells were incubated for 18-20 hours, lysed and assayed for luciferase activity using a Northstar HTS Workstation (Applied BioSystems). The dose response curves are generated from a 10 point curve with concentrations differing by ½ log units. Each point represented the average of 4 wells of data from a 32 384 well plate. The data from this assay wass fitted to the following equation, from which the EC50 value may be solved: Y = Bottom + (Top-Bottom)/(1+10((logEC50-X)*HillSlope)). The EC50 is defined as the concentration at which an agonist elicits a response that is half way between the Top (maximum) and Bottom (baseline) values. The EC50 values represented are the averages of at least 2 independent experiments. The determination of the relative efficacy or % efficacy for an agonist was by comparison to the maximum response achieved by Pan Agonist A (Supporting Information Figure 1), which was measured individually in each dose response experiment.

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In Vitro Studies of LXR Agonist Effects on Macrophage-Dependent Neutropenia Pathways [1]
Thioglycolate-elicited C57BL/6J mouse peritoneal macrophages were cultured in DMEM-FBS and incubated with LXR agonists or vehicle in serum-free DMEM for 20 hr, followed by 5 hr treatment with 20 ng/mL LPS in the continued presence of agonists or vehicle. The effect of treatment on IL-23α and Mertk mRNAs was determined as described in detail in the RNA preparation and analysis section of the Supplemental Experimental Procedures.

Cells in each flask are washed with PBS and 2 mL of Trypsin-EDTA (0.25%) is added and incubated for five minutes at 37 C, 5% CO2. The flasks are then tapped vigorously to break up cell aggregates. After the addition of 8 ml of DMEM containing 5% charcoal/dextran-treated FBS, the entire mixture is transferred to conical tubes. Cells are then centrifuged at 1000 rpm for 5 minutes. Cell pellets are resuspended to a final count of ~7 x 106 cells/ml in freezing media (DMEM containing 20% serum and 10% DMSO). The cell suspension is aliquoted into 15-ml polypropylene tubes, 5 ml per tube. Cells are slowly frozen by placing in a Styrofoam-insulated container at -80 C overnight. Vials are transferred to an Ultracold (-140 C) freezer after 24 hours for long-term storage. Vials of cryopreserved cells are thawed rapidly in a warm water bath for five minutes. Cells are pooled and diluted to 50 ml in a 50-ml conical vial. The thawed cells are centrifuged at 1500 rpm for 5 minutes to collect the cells and the supernatant discarded. Cells are then resuspended in fresh Media II (DMEM containing 5% charcoal/dextran-treated FBS, 1% Penicillin/Streptomycin, 100 M Non-essential Amino Acids, 1 mM Sodium Pyruvate, and 2 mM L-Glutamine), counted using the Guava Cell Counter, and diluted to 1.6 x 105 cells/ml in the same media. Fifty microliters of cell mixture is added to wells in columns 1-23 of white tissue-culture treated 384- well plates containing 0.25 l of test compound dissolved in 100% DMSO. Fifty microliters of Media II is added to wells in column 24. The plates are incubated at 37C (5% CO2) for 24 hours, then 5 l of Alamar Blue reagent is added to each well. Plates are then incubated an additional two hours at 37 C, 5% CO2 and then one hour at room temperature. Fluorescence is read at Ex525/Em598. After the fluorescence is measured, 25 l of luciferase substrate is added to each well. The plates are incubated for fifteen minutes at room temperature, after which the luminescence is read on a PheraStar plate reader [1].

Cynomolgus Monkey Studies [1]
All studies were performed in male animals. 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.
To explore global effects of BMS-852927 on the transcriptome of multiple tissues, three animals each were treated with vehicle or 15 mg/kg/day BMS-852927 by oral gavage for 7 days. At 5–6 hr post-final dose, animals were sedated and then euthanized. Multiple tissues, including liver, spleen, aorta, right common carotid, bone marrow, and others, were harvested for transcriptome analysis using Affymetrix arrays as described in the Supplemental Experimental Procedures.
In vivo MR spectroscopic measurements of liver TG were made using a Bruker 4.7T/40cm MRI system using Bruker Topspin software as described in detail in the Supplemental Experimental Procedures. Lipid analysis of the same region of interest in the central liver was performed on each of the primates after 7 days of oral dosing with either vehicle or BMS-852927 and compared to previously measured baseline values of the same liver region of interest.
To determine the effect of BMS-852927 on sterol excretion, feces were collected at baseline and at the end of treatment in the 14 day PD study described above. Dried samples plus added standards were solubilized in NaOH-ethanol-water, and petroleum ether extracted material was analyzed for cholesterol by ultra-performance liquid chromatography.

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Mouse Studies [1]
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.
To determine the effect of BMS-852927 on in vivo macrophage cholesterol efflux, C57Bl/6J mice (12–24 weeks of age, n = 3–4/group) were treated with vehicle or 0.03, 0.1, or 3 mg/kg/day BMS-852927 and dosed once daily for 7 days by oral gavage. Five hours post-final dose, animals were anesthetized and bovine serum albumin:[3H]-cholesterol particulate complexes were injected i.v. via the orbital plexus of non-fasted animals. Tail vein blood samples were collected from 5 to 150 min post-injection, and, following isolation by centrifugation, plasma radioactivity was determined by liquid scintillation counting. The time course of plasma radioactivity as a percentage of injected label was plotted, and the linear portion of the sterol reappearance phase following the rapid initial clearance was used to calculate initial macrophage cholesterol efflux rates using linear regression.

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Phase 1 Single, CV201-001, and Multiple, CV201-002, Ascending Dose Studies [1]
Healthy men and women 18–45 years of age with a BMI of 18–30 kg/m2 were enrolled in randomized, double-blind, and placebo-controlled SAD and MAD studies. In each dose panel, eight subjects were randomized in a 3:1 ratio to receive BMS-852927 (n = 6) or matched placebo (n = 2). In the SAD study, subjects (mean age of 24.2 years and BMI of 24.1 kg/m2) were assigned to six sequential panels to receive a single dose of BMS-852927 (0.4, 2, 8, 20, 40, and 80 mg) or placebo. There were no deaths or SAEs in the SAD study; the most frequent treatment-emergent AE was hypertriglyceridemia reported in four (7.1%) subjects, all of whom were receiving BMS-852927 and were considered related to the study drug by the Investigator. In the MAD study, subjects were assigned to five sequential dose panels to receive BMS-852927 (0.2, 1, 2.5, 5, and 15 mg) or placebo once daily for 14 days. Exclusion criteria for the MAD study included MRI contraindications such as metal implants or prostheses.

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Phase 1b Study, CV201008 [1]
Male and female patients between the ages of 18 and 75 years with primary hypercholesterolemia and with a BMI ≤40 kg/m2 were enrolled. Subjects were required to have been receiving a stable daily dose of a statin for ≥6 weeks with serum triglyceride levels at screening <400 mg/dL. Ninety-seven subjects were randomized and received study medication, 90 (92.8%) subjects completed the study, 7 (7.2%) subjects discontinued, 6 (6.2%) subjects discontinued due to AEs, and 1 (1.0%) discontinued due to other reason. Subjects received 0.25, 1, or 2.5 mg BMS-852927 or placebo once daily for 28 days and subjects remained on their pre-study statin treatment regimen throughout the study. Safety assessments were based on medical review of AE reports, the results of electrocardiograms, and the review of clinical laboratory tests, including fasting serum lipids. Refer to the Supplemental Experimental Procedures for more detail on study design and execution.
The concentration of BMS-852927 in human plasma samples was determined using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay as described in detail in the Supplemental Experimental Procedures. In vivo Methods [2]
In vivo Pharmacokinetics in Mice [2]
The pharmacokinetics were characterized in male C57BL6 mice (21.6-36 g). Mice received compound by oral gavage (fasted overnight) as a suspension in 0.75% CMC/0.1% Tween 80 in water. Blood samples (~0.2 mL) were obtained from 3 mice per time point at 0.25, 0.5, 1, 3, 6, 8 and 24 h post dose resulting in a composite pharmacokinetic profile (three blood sample were collected from each mouse). Blood samples were allowed to coagulate and centrifuged at 4 °C (1500-2000xg) to obtain serum. Serum samples were stored at -20 C until analysis by LC/MS/MS.

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Distribution Into Mice Brain [2]
Distribution of 15 into the brain was studied following oral administration (10 mg/kg) to C57BL6 mice (N = 12). Brain and blood samples were collected at 1, 4, 8 and 24 h post dose (N = 3 animals per time point). Brain tissues were blotted dry, weighed, and homogenized (1:6 v/v) with 50% acetonitrile/water and centrifuged (10,000xg at 4°C for 10 min). Plasma samples were obtained from blood by centrifugation at 4°C (1500-2000xg). All samples were stored at -20°C until analysis by LC/MS/MS.

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Cynomolgus Monkey Single-dose Pharmacokinetics [2]
The pharmacokinetics to evaluate clearance in cynomolgus monkeys (Table 2) was performed either by i.v. dosing of a single compound (e.g. BMS-852927) or i.v. dosing at 0.2 mg/kg in a cassette of 5 compounds that have different molecular weights. The i.v. infusion was done over 10 minutes (vehicle: 10% EtOH; 50% PEG400; 40% saline) two monkeys by similar procedures described below. The pharmacokinetics were evaluated in male cynomolgus monkeys in a crossover design (SI Table 1). Following an overnight fast, 3 animals (4.5 to 5.7 kg) received drug by IV infusion (1 mg/kg over 10 min) via a femoral vein and by oral gavage (3 mg/kg), with a 1-week washout between treatments. Serial blood samples (~0.3 mL) were collected from a femoral artery pre-dose and at 0.17 (IV only), 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, 48 and 72 h post dose, and centrifuged at 4 °C (1500-2000xg) to obtain plasma. Plasma samples were stored at -20 C until analysis by LC/MS/MS.

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Cynomolgus Monkey Pharmacodynamics Studies. [2]
Male cynomolgus monkeys were obtained from the Bioculture Group and pair-housed for 3 weeks for acclimation and animals were subsequently transferred to single housing in standard non-human primate cages at the start of the study. Water and standard primate chow 37 were provided ad libitum. Food was provided daily in the amount of 120 grams. When fasting was required, food was removed at 3 PM and returned on the following day at 9 AM, 2 hours after dosing. For the 14 day PD studies, animals were randomized into treatment groups (N=3/group; 3- 6 kg) and received the following treatments at 7 am, q.d. for 14 days by oral gavage: vehicle (0.5% carboxymethyl cellulose and 2% Tween 80), 10 mg/kg/d T0901317, and 0.1, 0.3, 1, or 3 mg/kg/d BMS-852927 or 0.3, 1, 3, 10, 30 mpk mg/kg/d BMS-832878. An initial venous blood sample was obtained on the day prior to the start of dosing (day -1) for baseline RNA and lipid measurements, followed by samples taken on days 1, 4, 7, and 14 of dosing for the PD study. Blood samples for RNA and compound exposure determinations were collected at 5-6 hours post-dose, and those for plasma lipids were collected 24 hours post-dose. Plasma triglycerides were determined using a kit for triglycerides from Roche Diagnostics. The values reported are the mean. Statistical analysis was done by ANOVA, using Dunnett’s post-hoc test. The reported inductions of ABCG1 in the text were statistically significant compared to baseline levels by ANOVA, using Dunnett’s post-hoc test with p < 0.05 or better.

BMS-852927 has an oral bioavailability of 42% (mouse), 84% (monkey), and t1/2 of 12.4h (mouse), 9.9h (monkey).[2]

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Agonists 13 and 15/BMS-852927 were nominated for further study because both compounds had robust LXRβ efficacy with low LXRα agonist efficacy (<20%), which was anticipated to improve the separation of desired efficacy from TG and LDL-C effects. In addition, 15/BMS-852927 was very potent in the hWBA. Analogues 13 and 15/BMS-852927 were not active in 16 nuclear hormone receptor agonist assays (>10 μM), except PXR with EC50 values of 3 μM (85% of full agonism) and 1 μM (108% of full agonism), respectively. When dosed in mice at 10 mg/kg, the Cmax coverage was high compared to the hWBA potency (Supporting Information). Given that LXR agonists could have deleterious effects in brain, as observed with LXR-623, the brain levels were measured and found to be low with 15 having a brain to plasma ratio of <0.05. In cynomolgus monkeys, 13 and 15 displayed good bioavailability, moderate clearance rates, and 10–12 h plasma half-lives[2].

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While 15/BMS-852927 was considered the lead compound due to exceptional hWBA potency coupled with low LXRα efficacy (Table 3), both 13 and 15/BMS-852927 were studied in cynomolgus monkeys for 14 days to investigate the ABCG1 dose response to the lipid effects compared to those of 1. The agonists showed robust induction of the RCT target gene ABCG1 in plasma at drug concentrations that were predicted by the cynomolgus monkey WBA potency (1 cynoWBA EC50 = 310 nM (100%); 13 cynoWBA EC50 = 52 nM (29%); 15 cynoWBA EC50 = 5 nM (32%)). ABCA1 had shown variable vehicle effects in multiple cynomolgus monkey studies, precluding its use as a pharmacodynamic biomarker. Both 13 and 15 had improved TG profiles compared to 1. Fourteen days of dosing 1 at 10 mg/kg (200 nM plasma concentration at 5 h) caused a 6-fold ABCG1 induction in blood cells with TGs elevated 140% over baseline values (p < 0.05, ANOVA). After 14 days, the 1 and 3 mg/kg doses of 13 afforded 4- and 10-fold ABCG1 induction in blood cells with 85 and 310 nM plasma exposures, respectively. These doses yielded TGs of 2% and 58% above baseline (not significant). Comparatively the 0.1, 0.3, and 1 mg/kg doses of 15 provided 5 h plasma exposures of 7.5, 22, and 57 nM with 4.7-, 15-, and 11-fold ABCG1 induction on day 14. The TGs were elevated nonsignificantly 20, 8, and 10% over baseline, respectively. As anticipated, 15 provided robust ABCG1 induction at very low plasma drug concentrations, with little effect on plasma TGs. A full data set from this cynomolgus monkey study 15 is reported elsewhere [2].

[1]. 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.

[2]. Discovery of Highly Potent Liver X Receptor β Agonists. ACS Med Chem Lett. 2016 Oct 23;7(12):1207-1212.

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. [1]

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Introducing a uniquely substituted phenyl sulfone into a series of biphenyl imidazole liver X receptor (LXR) agonists afforded a dramatic potency improvement for induction of ATP binding cassette transporters, ABCA1 and ABCG1, in human whole blood. The agonist series demonstrated robust LXRβ activity (>70%) with low partial LXRα agonist activity (<25%) in cell assays, providing a window between desired blood cell ABCG1 gene induction in cynomolgus monkeys and modest elevation of plasma triglycerides for agonist 15/BMS-852927. The addition of polarity to the phenyl sulfone also reduced binding to the plasma protein, human α-1-acid glycoprotein. Agonist 15/BMS-852927 was selected for clinical development based on the favorable combination of in vitro properties, excellent pharmacokinetic parameters, and a favorable lipid profile.[2]

C29H28CL2F2N2O4S

609.511431694031

608.111

C, 57.15; H, 4.63; Cl, 11.63; F, 6.23; N, 4.60; O, 10.50; S, 5.26

1256918-39-4

49787490

White to off-white solid powder

5.7

2

7

7

40

974

0

C(C1C(=CC=CC=1Cl)Cl)(C1=NC(C(O)(C)C)=CN1C1C=CC(C2C=C(F)C(CO)=C(S(=O)(=O)C)C=2)=CC=1F)(C)C

HNAJDMYOTDNOBK-UHFFFAOYSA-N

InChI=1S/C29H28Cl2F2N2O4S/c1-28(2,26-19(30)7-6-8-20(26)31)27-34-25(29(3,4)37)14-35(27)23-10-9-16(11-22(23)33)17-12-21(32)18(15-36)24(13-17)40(5,38)39/h6-14,36-37H,15H2,1-5H3

2-(2-(2-(2,6-dichlorophenyl)propan-2-yl)-1-(3,3′-difluoro-4′-(hydroxymethyl)-5′-(methylsulfonyl)biphenyl-4-yl)-1H-imidazol-4-yl)propan-2-ol

BMS-852927; BMS 852927; BMS852927; XL041; 1256918-39-4; BMS-852,927; XL-652; EXEL-04541041; H9649L8MZN; UNII-H9649L8MZN;XL-041; XL 041.

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 : ~100 mg/mL (~164.07 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.10 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 25.0 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: ≥ 2.5 mg/mL (4.10 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 25.0 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: ≥ 2.5 mg/mL (4.10 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 25.0 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.)