HIF-2α (IC50 = 9 nM)[1]
Belzutifan (PT2977) causes a rapid and quantitatively-dependent decrease in EPO expression and potently and quantitatively-dependently lowers the mRNA levels of human cyclin D1, a target gene regulated by HIF-2α [1].

Belzutifan (PT2977) causes a rapid and quantitatively-dependent decrease in EPO expression and potently and quantitatively-dependently lowers the mRNA levels of human cyclin D1, a target gene regulated by HIF-2α [1].

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PT2977 is 2 to 3-fold more potent than its predecessor PT2385 in cellular assays, with an EC₅₀ of 17 nM in a VEGFA secretion assay using VHL-mutant 786-O clear cell renal cell carcinoma (ccRCC) cells.
PT2977 has significantly lower lipophilicity (log D_7.4 = 1.2) compared to PT2385 (log D_7.4 = 2.2), which translates to reduced plasma protein binding (52% bound in human plasma vs. 82% for PT2385).
The free fraction adjusted EC₈₅ for PT2977 in the VEGFA secretion assay was calculated to be 75 ng/mL.
Enzyme kinetic studies in human intestinal microsomes (HIM) and UGT2B17 supersomes showed that PT2977 has drastically reduced glucuronidation rates compared to PT2385. The V_max for glucuronide formation was 0.5 pmol/min/mg protein in HIM and 4.7 pmol/min/mg protein in UGT2B17, which is over 20-times slower than PT2385.
PT2977 showed low to moderate clearance in microsomal and hepatocyte stability assays across species (mouse, rat, dog, monkey).
PT2977 did not inhibit major CYP P450 enzymes up to 50 μM (the highest concentration tested).
In a safety screening panel of 126 receptors, ion channels, kinases, and phosphatases, PT2977 showed no appreciable activity at 10 μM. [1]

Oral administration of PT2977 in mice, rats, dogs, and monkeys resulted in good plasma exposure.Comparison of mean plasma concentration–time profiles of PT2977 and PT2385 is shown in Figure 11, and summary of parent and glucuronide metabolite drug exposure (AUC) is provided in Table 5. Interestingly, the oral exposure of PT2977 was only slightly higher than PT2385 in rodents, while these two compounds behaved very differently in higher species. The dose-normalized AUC of PT2977 was 9- and 20-fold higher than that of PT2385 AUC in dogs and monkeys, respectively. This phenomenon turned out to be related to the relative rate of glucuronide metabolite formation for each analog in these species. As shown in Table 5, the AUC of the PT2977 glucuronide metabolite (PT3317) in dogs was about 30% of the parent, while the AUC of PT2639 (PT2385 glucuronide metabolite) was almost 2-fold higher than the parent. Similarly, a low amount of circulating metabolite was observed for PT2977 in monkeys, with a metabolite/parent ratio of 0.19. Both compounds formed significantly less amounts of glucuronide metabolites in rats, suggesting an alternative metabolic pathway in this species. For PT2385, dog pharmacokinetics were a better predictor of glucuronide metabolite PT2639 formation in humans. On the basis of these data, we predicted that PT2977 would have a reduced propensity for glucuronidation in humans and thus demonstrate a significantly improved pharmacokinetic profile over PT2385 in patients[1].

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A pharmacokinetic/pharmacodynamic (PK/PD) study was performed in mice bearing subcutaneous 786-O ccRCC tumors to correlate plasma drug levels of PT2977 with PD effects in the tumors. Six doses of PT2977 at 0.3, 1, and 3 mg/kg or PT2385 at 10 mg/kg b.i.d. were administered orally. Plasma and tumor tissue samples were collected 12 h after the last dose. Gene expression analyses in excised tumors by qPCR showed that PT2977 potently and dose-dependently reduced mRNA levels of human cyclin D1, a target gene regulated by HIF-2α (Figure 12A). Maximum PD response was achieved at 1 mg/kg for PT2977, while a much higher dose of 10 mg/kg was required for PT2385. And to achieve the same PD effect, the steady state trough plasma drug concentration of PT2977 was about 1/10 of PT2385 concentration (Figure 12B). Consistent with the improvement in potency and physical properties, compound PT2977 was about 10-fold more potent than PT2385 in vivo. PT2977 and PT2385 were subsequently evaluated for antitumor activity in the 786-O mouse xenograft model. Administration of PT2977 at 0.3, 1, and 3 mg/kg all led to rapid regression of established tumors (Figure 12C), confirming superior in vivo activity of compound PT2977 compared to PT2385[1].
aPT2385 and PT2977 were delivered as a suspension in 10% EtOH, 30% PEG400, 60% (0.5% methylcellulose, 0.5% Tween 80 (aq)).
bPT2385 and PT2977 were delivered as a suspension in 0.5% methylcellulose and 0.5% Tween 80 in water.
On the basis of its improved potency, free fraction, pharmacokinetics, and metabolite profile, compound PT2977 was predicted to have a low clinically active dose and less interpatient exposure variability. Therefore, PT2977 was chosen as our second-generation HIF-2α clinical candidate for further investigation as a potential new treatment for ccRCC and VHL disease[1].

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In a pharmacokinetic/pharmacodynamic (PK/PD) study in mice bearing subcutaneous 786-O ccRCC xenografts, oral administration of PT2977 (0.3, 1, and 3 mg/kg, b.i.d.) dose-dependently reduced mRNA levels of the HIF-2α target gene cyclin D1 in tumors. Maximum PD response was achieved at 1 mg/kg.
To achieve the same level of PD effect, the steady-state trough plasma concentration required for PT2977 was about one-tenth of that required for PT2385.
In an in vivo efficacy study in the same 786-O mouse xenograft model, administration of PT2977 at 0.3, 1, and 3 mg/kg (b.i.d.) led to rapid regression of established tumors, confirming superior in vivo activity compared to PT2385. [1]

UGT Phenotyping[1]
The test compound was incubated with a panel of individually expressed recombinant human UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cell membranes. The incubation mixture contained the test compound at a final concentration of 50 μM, expressed UGT (0.2 mg protein/ml), Tris-HCl buffer (pH 7.5), magnesium chloride (10 mM), alamethicin (25 μg/mL), and UDPGA (2 mM). The mixture (without the UDPGA cofactor) was preincubated at 37 °C for 5 min, after which the reaction was started by the addition of UDPGA.
Incubations were performed at 37 °C. Aliquots (100 μL) were collected at 60 min and quenched with two volumes of ice-cold acetonitrile containing an internal standard. The samples were centrifuged at 10 000g at room temperature for 15 min to pellet precipitated proteins. The supernatants were then transferred to clean vials containing 200 μL of water and analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The glucuronide metabolite over internal standard peak area ratio was measured to quantify the glucuronide metabolite formation rate.

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Enzyme Kinetic Studies in Human Intestine Microsomes (HIM) and UGT2B17 Supersomes[1]
Similar experimental procedure to UGT phenotyping was used for enzyme kinetics studies of compounds in human intestinal microsomes and UGT2B17 supersomes except that a compound concentration range of 2–1000 μM was used in the incubations. The absolute glucuronide metabolite formation rate was measured by LC–MS/MS. The compound concentrations and its corresponding glucuronide metabolite formation rates were fitted to the standard Michaelis–Menten model to obtain the Vmax and Km by using Prism (version 6).

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Scintillation Proximity Assay (SPA): A binding assay used to determine the inhibitory concentration (IC₅₀) of compounds against HIF-2α. The assay measures the displacement of a radiolabeled ligand from the HIF-2α protein. [1]
UGT Phenotyping and Enzyme Kinetics: To identify the UGT enzyme responsible for metabolizing the compound, a panel of recombinant human UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, 2B17) expressed in insect cell membranes are used. The incubation mixture contains the test compound (e.g., at 50 μM for phenotyping or a concentration range of 2-1000 μM for kinetics), the expressed UGT enzyme, Tris-HCl buffer (pH 7.5), magnesium chloride, alamethicin, and the cofactor UDPGA. The reaction is started by adding UDPGA after pre-incubation. Incubations are performed at 37°C. Aliquots are collected at specific time points (e.g., 60 min), quenched with acetonitrile containing an internal standard, and centrifuged. The supernatant is analyzed by LC-MS/MS to quantify the formation rate of the glucuronide metabolite. For kinetics, data are fitted to the Michaelis-Menten model to obtain V_max and K_m values. [1]

hERG Profiling of 2:[1]
The in vitro effects of compound 2 on the hERG (human ether-à-go-go-related gene) potassium channel current (a surrogate for IKr, the rapidly activating, delayed rectifier cardiac potassium current) expressed in mammalian cells were evaluated at near-physiological temperature. 2 inhibited hERG current by (Mean ± SEM) 6.7 ± 2.0% at 10 μM (n = 3) and 16.3 ± 0.4% at 50 μM (n = 3) versus 0.0 ± 0.6% (n = 3) in control. The IC50 for the inhibitory effect of 2 on hERG potassium current was not calculated but was estimated to be greater than 50 μM. The positive control (60 nM terfenadine) inhibited hERG potassium current by (Mean ± SD; n = 2) 85.2 ± 3.6%. This result confirms the sensitivity of the test system to hERG inhibition.

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Evaluation of 1 and 3 as Substrates of Human P-gp and BCRP Transporters: [1]
To determine if 1 (1 and 10 μM) and 3 (1 and 10 μM) are substrates of human efflux transporters (namely, P-gp [MDR1/ABCB1] and BCRP [ABCG2]), S5 the bidirectional permeability of PT2385 and PT2639 across MDCKII-MDR1 and MDCKIIBCRP cells was measured. Known substrates and inhibitors were included as positive controls in all experiments. The efflux ratios of 1 and 3 across MDCKII-MDR1 and MDCKII-BCRP cells was less than 2 at all conditions tested suggesting neither compound is a substrate of P-gp or BCRP.

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HIF-2α Luciferase Reporter Assay: 786-O cells (a VHL-mutant ccRCC cell line) are transfected with a luciferase reporter construct under the control of a hypoxia-responsive element (HRE). Cells are then treated with the test compound at various concentrations. After an incubation period, luciferase activity is measured, reflecting HIF-2α transcriptional activity. EC₅₀ values are calculated from dose-response curves. [1]
VEGFA Secretion Assay: 786-O cells are seeded and allowed to adhere. The cells are then treated with the test compound across a range of concentrations. After a specified incubation period (e.g., 24 hours), the cell culture supernatant is collected. The concentration of vascular endothelial growth factor A (VEGFA), a protein whose expression is driven by HIF-2α, in the supernatant is quantified using an ELISA kit. EC₅₀ values are determined from the inhibition curves. [1]

Mouse PK/PD and Efficacy Study in 786-O Xenograft Model: Female SCID/beige mice are inoculated subcutaneously with 786-O human ccRCC cells to establish tumors. Once tumors reach a predetermined size, mice are randomized into treatment groups. PT2977 or vehicle is administered orally (by gavage) twice daily (b.i.d.). For the PK/PD study, doses of 0.3, 1, and 3 mg/kg are used. For the efficacy study, doses of 0.3, 1, and 3 mg/kg b.i.d. are administered. Tumor volumes and body weights are monitored regularly. For PK/PD analysis, plasma and tumor samples are collected at a specific time after the last dose (e.g., 12 hours). Tumors are processed for RNA extraction and qPCR analysis of target genes (e.g., cyclin D1). Plasma drug concentrations are measured by LC-MS/MS. [1]
Preclinical PK Studies in Multiple Species (Mouse, Rat, Dog, Monkey): For intravenous (i.v.) PK studies, PT2977 is formulated as a solution (e.g., in 20% ethanol, 40% PEG400, 40% water for mouse, dog, monkey; or 10% dimethylacetamide, 10% ethanol, 40% PEG400, 40% water for rat) and administered as a single i.v. bolus. For oral (p.o.) PK studies, PT2977 is administered as a suspension (e.g., in 0.5% methylcellulose and 0.5% Tween 80 in water, or in 10% ethanol, 30% PEG400, 60% of an aqueous solution containing 0.5% methylcellulose and 0.5% Tween 80). Blood samples are collected at multiple time points post-dose. Plasma is separated and analyzed for parent drug and metabolite concentrations using LC-MS/MS. Standard non-compartmental analysis is performed to derive PK parameters. [1]

Absorption, Distribution and Excretion
In patients with VHL-associated renal cell carcinoma, the mean Cmax and AUC0-24h at steady state (reached approximately three days after treatment) were 1.3 µg/mL and 16.7 μg•hr/mL, respectively. The median Tmax after oral administration was 1 to 2 hours. Co-administration of bezutifan with food had negligible effects on drug distribution—when co-administered with high-calorie, high-fat foods, the time to peak concentration (Tmax) was delayed by only about 2 hours, and no other clinically significant effects were observed. The steady-state volume of distribution after oral administration of bezutifan was approximately 130 L. The mean clearance after oral administration of bezutifan was 7.3 L/h. Metabolism/Metabolites Bezutifan is primarily metabolized by UGT2B17 and CYP2C19, with less metabolic activity via CYP3A4.
Biological Half-Life
The mean elimination half-life of bezurtiva is 14 hours.
PT2977 showed low plasma clearance (Cl_p) in both mice (21 mL/min/kg) and dogs. Following intravenous injection, clearance was 1.3 mL/min/kg in mice, 3.5 mL/min/kg in monkeys, and moderate clearance (19 mL/min/kg) in rats. The terminal half-lives (T_1/2) in mice, rats, dogs, and monkeys were 4.0 hours, 1.4 hours, 14 hours, and 9.2 hours, respectively.
In preclinical animal models, oral administration of PT2977 showed good plasma exposure. Its glucuronide metabolite (PT3317) had a lower exposure (AUC) relative to the parent: 0.07 in rats, 0.32 in dogs, and 0.19 in monkeys, indicating a significant reduction in glucuronidation compared to PT2385. In a phase 1 clinical trial in patients with advanced solid tumors, once-daily oral administration of PT2977 resulted in dose-dependent exposure, up to a maximum of 120 mg. At the recommended phase II dose (RP2D) of 120 mg qd, the mean terminal half-life was 20.9 h, the mean Cmax was 1.8 μg/mL, the mean AUClast was 25.9 h μg/mL, and the mean trough concentration (C24h) was 0.71 μg/mL (710 ng/mL).
When PT2977 reached steady state (day 15) at a dose of 120 mg qd, the glucuronide metabolite/parent AUC ratio was 0.32, significantly lower than the 6.0 ratio of PT2385 at a dose of 800 mg bid [1].

Hepatotoxicity
In pre-registration trials of bezutifan, up to 20% of patients experienced elevated serum transaminases, but these were transient and mild (less than 3 times the upper limit of normal) and were asymptomatic or without jaundice. No patients required dose adjustments or discontinuation due to abnormal liver function tests. Furthermore, since its widespread use following approval, there have been no published reports of clinically significant liver injury caused by bezutifan. Probability Score: E (Unlikely to cause clinically significant liver injury). Protein Binding Plasma protein binding is approximately 45%, but data on specific proteins bound by bezutifan are currently unavailable. In patch-clamp hERG channel assays, PT2977 showed low potential for QT interval prolongation, with an estimated IC50 > 50 μM.
In a broad safety screening involving 126 receptors, ion channels, kinases, and phosphatases, PT2977 did not show significant activity at a concentration of 10 μM. [1]

[1]. 3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile (PT2977), a Hypoxia-Inducible Factor 2α (HIF-2α) Inhibitor for the Treatment of Clear Cell Renal Cell Carcinoma. J Med Chem. 2019 Aug 8;62(15):6876-6893.

Belzutifan is a hypoxia-inducible factor 2α (HIF-2α) inhibitor used to treat cancers associated with von Hippel-Lindau syndrome (VHL). The HIF-2α protein, initially discovered by researchers at the University of Texas Southwestern Medical Center in the 1990s, plays a crucial role in the growth of certain cancers. Initially thought to be untreatable, a binding pocket was eventually discovered within the HIF-2α molecule, allowing compounds to bind to and inhibit the protein. This discovery led to the initial development of bezutifan (then known as PT2977), which was subsequently further developed by a spin-off company called Peloton Pharmaceuticals (which was eventually acquired by Merck in 2019). Belzutifan inhibits the formation of a complex between HIF-2α and another transcription factor, HIF-1β, a necessary step for HIF-2α activation—by preventing the formation of this complex, bezutifan can slow or halt the growth of VHL-related tumors. On August 13, 2021, Belzutifan received FDA approval for the treatment of certain VHL-related cancers. Belzutifan is a hypoxia-inducible factor inhibitor. Its mechanism of action is as a hypoxia-inducible factor 2α inhibitor and a cytochrome P450 3A4 inducer. Belzutifan is a small molecule hypoxia-inducible factor 2α inhibitor used to treat solid tumors in patients with von Hippel-Lindau disease. The incidence of mild elevations in serum enzymes during Belzutifan treatment is low, but it has not been found to be associated with clinically significant liver injury. Belzutifan is an orally potent small molecule hypoxia-inducible factor (HIF)-2α inhibitor with potential antitumor activity. After oral administration, Belzutifan binds to HIF-2α and blocks its function, thereby preventing HIF-2α heterodimerization and its binding to DNA. This leads to reduced transcription and expression of downstream target genes of HIF-2α, many of which regulate hypoxia signaling pathways. This inhibits the growth and survival of tumor cells expressing HIF-2α. HIF-2α is the α subunit of the heterodimeric transcription factor HIF-2, which is overexpressed in various cancers and promotes tumorigenesis. Drug Indications Belzutifan is indicated for the treatment of adult patients with von Hippel-Lindau (VHL) disease who require treatment for related renal cell carcinoma (RCC), central nervous system (CNS) hemangioblastoma, or pancreatic neuroendocrine tumor (pNET) that do not require immediate surgery. Mechanism of Action Hypoxia-inducible factor 2α (HIF-2α) is a transcription factor that helps oxygen sensing by regulating genes that promote adaptation to hypoxia. In healthy individuals, when oxygen levels are normal, HIF-2α is degraded by von Hippel-Lindau (VHL) proteins via the ubiquitin-proteasome pathway. Under hypoxic conditions, HIF-2α translocates to the nucleus and forms a transcriptional complex with hypoxia-inducible factor 1β (HIF-1β). This complex subsequently induces the expression of downstream genes involved in cell proliferation and angiogenesis. Patients with von Hippel-Lindau (VHL) disease lack functional VHL protein, leading to the accumulation of HIF-2α, a driving factor in the growth of VHL-related tumors. Belzutifan is an HIF-2α inhibitor that prevents HIF-2α from forming a complex with HIF-1β under hypoxic or impaired VHL protein function, thereby reducing the expression of HIF-2α target genes and slowing/stopping the growth of VHL-related tumors.

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Pharmacodynamics
Belzutifan exerts its therapeutic effect by inhibiting transcription factors essential for the growth of VHL-related solid tumors. It is taken once daily at approximately the same time each day, either before or after meals. Severe anemia and hypoxia have been observed after taking bezutifan; therefore, patients should be closely monitored before and after treatment to ensure that treatment is administered as clinically necessary. There is currently no data on the use of erythropoietin to treat anemia caused by bezurtifan, therefore such therapy should be avoided. Bezurtifan may cause embryo-fetal toxicity when used in pregnant women. Female patients and male patients whose partners are women of childbearing potential should ensure effective contraception throughout treatment and for one week after the last dose—as bezurtifan appears to reduce the effectiveness of systemic hormonal contraceptives, patients should be advised to use additional contraception (e.g., condoms) to eliminate the possibility of pregnancy during treatment.

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Bezurtifan (PT2977) is a second-generation, orally bioavailable, small-molecule inhibitor of hypoxia-inducible factor 2α (HIF-2α). This drug was developed by modifying the structure of the first-generation inhibitor PT2385 to address its pharmacokinetic limitations, particularly the excessive and unstable glucuronidation of UGT2B17 in the intestine.
The key structural change is the movement of one fluorine atom from the geminidin group on the indanol ring of PT2385 to the benzylic position, resulting in a cis configuration relative to the hydroxyl group. This modification (forming an ortho-difluoro motif) leads to enhanced potency, significantly reduced lipophilicity, and a substantial decrease in the rate of glucuronidation. PT2977 disrupts the heterodimerization of HIF-2α with its binding partner ARNT (HIF-1β), thereby inhibiting the transcription of HIF-2α target genes (such as VEGFA, EPO, and cyclin D1) involved in angiogenesis, proliferation, and metabolism. It is being developed for the treatment of clear cell renal cell carcinoma (ccRCC) and von Hippel-Lindau syndrome (VHL)-related tumors. In an expanded cohort of a phase I clinical trial that included 55 ccRCC patients, once-daily 120 mg of PT2977 showed encouraging clinical activity: as of January 1, 2019, 12 patients (22%) achieved a confirmed partial response. The median follow-up was 9 months, and the median progression-free survival (PFS) has not yet been reached. [1]

C17H12F3NO4S

383.341693878174

383.04

C, 53.27; H, 3.16; F, 14.87; N, 3.65; O, 16.69; S, 8.36

1672668-24-4

117947097

White to light yellow solid powder

2

1

8

3

26

675

3

CS(=O)(=O)C1=C2[C@@H]([C@@H]([C@@H](C2=C(C=C1)OC3=CC(=CC(=C3)C#N)F)F)F)O

LOMMPXLFBTZENJ-ZACQAIPSSA-N

InChI=1S/C17H12F3NO4S/c1-26(23,24)12-3-2-11(13-14(12)17(22)16(20)15(13)19)25-10-5-8(7-21)4-9(18)6-10/h2-6,15-17,22H,1H3/t15-,16-,17+/m1/s1

3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile

PT2977; MK 6482; PT-2977; MK-6482; PT 2977; MK6482; Belzutifan; 1672668-24-4; PT2977; Welireg; MK-6482; MK6482; PT-2977; Belzutifan [INN]; Welireg

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Acetone : 50 mg/mL (~130.43 mM)
DMSO : ~50 mg/mL (~130.43 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 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 (6.52 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 (6.52 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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Solubility in Formulation 4: ≥ 0.5 mg/mL (1.30 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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