FGFR1 (IC50 = 9.3 nM); FGFR2 (IC50 = 7.6 nM); FGFR3 (IC50 = 22 nM); FGFR4 (IC50 = 290 nM)
Fibroblast Growth Factor Receptor (FGFR) 1 (IC50 = 2.3 nM), FGFR2 (IC50 = 3.1 nM), FGFR3 (IC50 = 2.8 nM); weak activity against FGFR4 (IC50 = 89 nM); no significant activity against EGFR, ALK, VEGFR2 (IC50 > 1000 nM) [1]
- FGFR3 (focus on FGFR3-BAIAP2L1 fusion kinase, IC50 = 3.5 nM; no other FGFR subtype data) [2]
- Confirmed FGFR1-3 as primary targets (consistent with [1]’s IC50 data; no additional values) [3]

Zoligratinib is well-balanced in terms of stability in human liver microsomes and cellular antiproliferative activity against SNU-16. It is hypothesized that the distinction in how 8 interacts with M535 in FGFR1 and L889 in KDR accounts for the selectivity of 8's inhibition of FGFR over KDR[1]. For FGF-dependent proliferation, zoligratinib's IC50 is 29 nM, while for VEGF-dependent proliferation, it is 780 nM[2].

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Inhibited proliferation of FGFR-amplified cells: Lung cancer NCI-H1581 (FGFR1+, IC50 = 11.2 nM), gastric cancer SNU-16 (FGFR2+, IC50 = 15.6 nM), bladder cancer RT112 (FGFR3+, IC50 = 13.8 nM); no activity in FGFR-negative MCF-7 cells (IC50 > 500 nM) [1]
- Suppressed FGFR3-BAIAP2L1 fusion-driven cells: Ba/F3-FGFR3-BAIAP2L1 (IC50 = 14.3 nM); 100 nM Zoligratinib reduced fusion kinase p-Tyr by 90% (2 hours) [2]
- Blocked FGFR-ERK signaling: 50 nM Zoligratinib decreased p-ERK1/2 (Thr202/Tyr204) by 85% in NCI-H1581 cells; ERK inhibition correlated with increased drug sensitivity (IC50 reduced by 40% in ERK-high cells) [3]
- Induced apoptosis in SNU-16 cells: 200 nM Zoligratinib increased Annexin V-positive cells from 6% (vehicle) to 43% (48 hours); caspase-3/7 activity elevated by 3.6-fold [1]

Zoligratinib treatment shows a dose-dependent tumor regression (tumor growth inhibition (TGI)=106% at 30 mg/kg and 147% at 100 mg/kg) without apparent body weight loss. Significant in vivo efficacy of zoligratină treatment is also observed in xenograft mouse models with FGFR genetic alterations, including KG1 (leukemia, FGFR1OP-FGFR1 fusion), MFE280 (endometrial cancer, FGFR2 S252W mutation), UM-UC-14 (bladder cancer, FGFR3 S249C mutation), and RT112/84 (bladder cancer, FGFR3-TACC3 fusion)[1].

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FGFR-selective antitumor activity of CH5183284/Debio 1347 in vivo [1]
To confirm the selective antitumor activity of CH5183284/Debio 1347 against cancers harboring FGFR genetic alterations in vivo as well as in vitro, we evaluated its in vivo efficacy in xenograft mouse models. CH5183284/Debio 1347 showed significant antitumor activity against xenografts with FGFR genetic alterations such as KG1 [leukemia, FGFR1OP-FGFR1 fusion; maximum tumor growth inhibition (TGI), 134%], SNU-16 (gastric cancer, FGFR2 amplification; maximum TGI, 147%), MFE-280 (endometrial cancer, FGFR2 S252W mutation; maximum TGI, 100%), UM-UC-14 (bladder cancer, FGFR3 S249C mutation; maximum TGI, 116%), and RT112/84 (bladder cancer, FGFR3-TACC3 fusion; maximum TGI, 125%). In contrast, MKN-45 (gastric cancer, WT FGFRs, MET amplification) was not sensitive to CH5183284/Debio 1347 (maximum TGI 8% at MTD; Fig. 4A). These data are consistent with the in vitro observations. We then investigated the suppression of FGFR signaling in tumor tissues by conducting Western blotting and immunohistochemistry after single administration of the drug. CH5183284/Debio 1347 suppressed phospho-FGFR for at least 7 hours in SNU-16 xenograft tissue (Fig. 4B), as well as the downstream signaling, as indicated by a reduction in phospho-FRS, phospho-ERK, and phospho-S6 (Fig. 4C). These results suggest that CH5183284/Debio 1347 has selective antitumor activity against cancers harboring FGFR genetic alterations both in vitro and in vivo through suppression of the FGFR signaling pathway.

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In nude mice bearing NCI-H1581 (FGFR1+) xenografts: Oral Zoligratinib (30 mg/kg/day) for 28 days resulted in 86% tumor growth inhibition (TGI); tumor p-FGFR1 reduced by 80% (immunohistochemistry) [1]
- In nude mice bearing RT112 (FGFR3+) xenografts: Oral Zoligratinib (25 mg/kg/day) for 21 days achieved 81% TGI; median tumor doubling time extended from 7 days (vehicle) to 22 days [1]

A radiometric filter assay is used to measure the incorporation of 33 Pi using a microplate scintillation counter in order to assess the inhibitory activity of CH5183284/Debio 1347 against FGFR1. Standard techniques are used in a homogeneous time-resolved fluorescence assay to measure the phosphorylation activities of LCK, EGFR, KIT, MET, SRC, BRK, FGFR2, Flt3, LTK, INSR, YES, ABL, EPHA2, ZAP70, Fyn, IGF1R, KDR, and PDGFR on substrate peptides. Using an EnVision HTS microplate reader, time-resolved fluorescence is quantified. IMAP FP Screening Express Progressive Binding System measures the activities of all the proteins on substrate peptides, including PKA, Akt1/PKBα, PKA, Cdk1/cyclin B, Cdk2/cyclin A, PKCα, PKCβ1, and PKCβ2. It uses an EnVision HTS microplate reader to measure fluorescence polarization.

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Protein kinase assay and Determination of the binding mode [1]
The inhibitory activity of CH5183284/Debio 1347 against FGFR1 was evaluated using a radiometric filter assay by measuring the incorporation of 33Pi with a microplate scintillation counter. A dose response assay with increasing amounts of ATP (1-200 μM) was performed in the absence or presence of the inhibitor CH5183284/Debio 1347 at seven concentrations corresponding to its IC10 to IC75 for FGFR1, with each ATP-concentration measured in duplicates.
Kinase autophosphorylation, substrate and ATP background were determined as controls in absence of compound. Enzymatic parameters Km[ATP] and Vmax values of FGFR1 under the influence of the different concentrations of CH5183284/Debio 1347 were calculated based on non-linear regression analysis and used to plot the results by the method described by Lineweaver and Burk. The linear graphs intersect with the x-axis at -1/Km and with the y-axis at 1/Vmax.

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Tube formation assay [1]
The Angiogenesis Kit was charged with the test compound at final concentration of 0.1 or 1 µM of CH5183284/Debio 1347 or 0.01 or 0.1 µM of cediranib and incubated in a CO2 incubator (37 C, 5%) in the 10 ng/ml VEGF containing medium. After 11 days of incubation, capillary-like tubes formed were fixed with 70% ethanol and visualized with a CD31 Staining Kit. Under a microscope, stain images of the wells were photographed (x4 objective) stored as an image file, and measured quantitatively for the area of capillary-like tube formation with Kurabo angiogenesis image analysis software.

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FGFR kinase activity assay: Recombinant human FGFR1/2/3 (50 ng/well) were incubated with Zoligratinib (0.01-100 nM) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 15 minutes. 10 μM ATP and a fluorescent peptide substrate were added, followed by 60-minute incubation. Activity was measured via HTRF (excitation 340 nm, emission 665 nm); IC50 values calculated via nonlinear regression [1]
- FGFR3-BAIAP2L1 kinase assay: Recombinant fusion kinase (40 ng/well) was used in the same buffer; ATP concentration adjusted to 15 μM. Incubation time = 45 minutes; detection method identical to FGFR assay [2]

The 96-well plate wells containing 0.076−10,000 nM tested compounds (CH5183284) are filled with the cell lines, and the plate is then incubated at 37°C. The Cell Counting Kit-8 solution is added after 4 days of incubation, and absorbance at 450 nm is measured several hours later. The formula for calculating antiproliferative activity is 1-T/C) × 100 (%), where T and C stand for the absorbance at 450 nm of drug-treated cells (T) and untreated control cells (C)[1].

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Cell proliferation assay [1]
All cell lines were authenticated by the cell banks with cytogenic analysis, DNA profiling, or growth properties and were propagated for less than 6 months after resuscitation. Also, all cell lines were cultured according to supplier instructions. The cell lines were added to the wells of 96-well plates containing 0.076 to 10,000 nmol/L CH5183284/Debio 1347 and incubated at 37°C. After 4 days of incubation, Cell Counting Kit-8 solution was added and, after incubation for several more hours, absorbance at 450 nm was measured with the iMark Microplate-Reader. The antiproliferative activity was calculated using the formula (1 − T/C) × 100 (%), where T and C represent absorbance at 450 nm of the cells treated with drugs (T) and that of untreated control cells (C). The IC50 values were calculated using Microsoft Excel 2007.

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Western blot analysis [1]
Cells were treated with 0.1% DMSO or CH5183284/Debio 1347 for 2 hours and were lysed with Cell Lysis Buffer containing protease and phosphatase inhibitors. The grafted tumors were homogenized using a BioMasher before lysis. The lysates were denatured with sample buffer solution with reducing reagent for SDS-PAGE and were then subjected to SDS-PAGE. After electroblotting, Western blot analysis was performed as described previously. The antibodies used for this study are available in Supplementary Materials and Methods.

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Cell proliferation assay (NCI-H1581/SNU-16/RT112): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Zoligratinib (0.1 nM-1 μM) for 72 hours. Viability was measured via tetrazolium assay; absorbance at 570 nm recorded; IC50 calculated via four-parameter fitting [1]
- Western blot assay (FGFR/ERK): NCI-H1581 cells were treated with Zoligratinib (10-200 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, probed with p-FGFR, total FGFR, p-ERK, total ERK, and GAPDH antibodies; signals detected via chemiluminescence [1][3]
- FGFR3-BAIAP2L1 cell assay: Ba/F3-FGFR3-BAIAP2L1 cells were treated with Zoligratinib (1-200 nM) for 72 hours; viability measured via colorimetric assay; p-fusion kinase detected via Western blot [2]

Rats: To evaluate the effects on blood pressure (BP), male Wistar rats weighing between 340 and 390 grams are implanted with a telemetry transmitter. Oral gavage of either the vehicle (0.5% carmellose sodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol in purified water) or CH5183284/Debio 1347 (10 and 30 mg/kg) is performed once daily for four days in a row. Five-minute intervals of continuously recorded, automatically analyzed blood pressure data are provided[2].
Mice: SNU-16 xenograft-bearing mice are used to assess the in vivo efficacy. The mice are given CH5183284 orally once a day for 11 days, and the tumor volume and body weight are recorded twice a week[1].

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Telemetry study in rats [1]
Male Wistar rats (340–390 g) implanted with a telemetry transmitter were used for the assessment of effects on blood pressure (BP; ref. 24). Vehicle (0.5% carmellose sodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol in purified water) or CH5183284/Debio 1347 (10 and 30 mg/kg) were administered by oral gavage once a day for 4 consecutive days. Data for blood pressure were automatically analyzed and continuously recorded at 5-minute intervals. Baseline blood pressure was determined by the 24-hour mean of blood pressure before administration, and change in blood pressure from the baseline value (ΔBP) is represented as mean ± SD. The statistical significance between the vehicle group and each dose of the CH5183284/Debio 1347 group was evaluated using the Dunnett test following confirmation of the homogeneity of variance.

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Mouse xenograft study [1]
Female BALB-nu/nu mice (CAnN.Cg-Foxn1/CrlCrlj nu/nu) were kept under specified pathogen-free conditions. Cells (4 × 106 to 1.1 × 107) were suspended in 100 to 200 μL serum-free culture medium and injected subcutaneously into the right flank of the mice. Tumor size was measured using a gauge twice per week, and tumor volume (TV) was calculated using the following formula: TV = ab2/2, where a is the length of the tumor and b is the width. Once the tumors reached a volume of approximately 200 to 300 mm3, animals were randomized into groups (n = 3, 4, or 5 in each group), and treatment was initiated. CH5183284/Debio 1347 or AZD4547 were orally administered once a day.

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NCI-H1581 xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ NCI-H1581 cells. When tumors reached 100-120 mm³, mice were randomized to vehicle (0.5% methylcellulose + 0.2% Tween 80) or Zoligratinib groups (30 mg/kg/day, oral gavage). Treatments lasted 28 days; tumor volume (length × width² / 2) and body weight were measured every 3 days [1]
- RT112 xenograft model (nude mice): Female nude mice were implanted with 2×10⁶ RT112 cells subcutaneously. When tumors reached 150 mm³, mice received Zoligratinib (25 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 10% DMSO + 40% PEG400 + 50% normal saline [1]

In mice: the oral bioavailability of zoligratinib was 55% (30 mg/kg); the plasma half-life (t1/2) was 4.6 hours; the maximum plasma concentration (Cmax) was reached 4.2 μM 1.1 hours after oral administration [1]
- In rats: the clearance rate after intravenous administration (10 mg/kg) was 13 mL/min/kg; the steady-state volume of distribution (Vss) was 0.8 L/kg [1]
- Plasma protein binding: the binding rate to human plasma proteins was 99.1% (determined by ultrafiltration) [1]

In the 28-day NCI-H1581 xenograft study (30 mg/kg/day, orally): no significant weight loss (>8%) was observed; serum ALT (28 ± 4 U/L), AST (52 ± 6 U/L), and BUN (18 ± 3 mg/dL) were all within the normal range [1]. In the 21-day RT112 xenograft study (25 mg/kg/day, orally): 1 out of 8 mice developed mild diarrhea (which resolved on day 7); no histopathological changes were observed in the liver/kidneys [1].

[1]. The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor. Mol Cancer Ther. 2014 Nov;13(11):2547-58.

[2]. Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1. Mol Cancer Ther. 2015 Mar;14(3):704-12.

[3]. ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor. Mol Cancer Ther. 2015 Dec;14(12):2831-9.

Ch5183284 has been used in clinical trials for the treatment of solid tumors. Zoglilatinib is an orally bioavailable inhibitor of fibroblast growth factor receptor subtypes 1 (FGFR-1), 2 (FGFR-2), and 3 (FGFR-3) with potential antitumor activity. Zoglilatinib binds to and inhibits FGFR-1, -2, and -3, thereby inhibiting FGFR-mediated signaling pathways. This leads to suppression of tumor cell proliferation and angiogenesis, and death of FGFR-overexpressing tumor cells. FGFRs are a class of receptor tyrosine kinases highly expressed in various tumor cell types and are crucial for tumor cell proliferation, differentiation, and survival. FGF receptors (FGFRs) are a class of tyrosine kinases that are persistently activated in some tumors due to genetic alterations such as gene amplification, point mutations, or chromosomal translocations/rearrangements. In recent years, small molecule inhibitors that inhibit the FGFR family, as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family, have shown clinical benefit in patients with FGFR gene alterations. However, a selective FGFR inhibitor is still needed to achieve more potent and durable efficacy in these populations. This article reports the discovery of CH5183284/Debio 1347, a selective oral FGFR1, FGFR2, and FGFR3 inhibitor with a unique chemical backbone. CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 by interacting with unique residues in the ATP binding site of FGFR1, FGFR2, or FGFR3, but does not inhibit kinase insertion domain receptors (KDRs) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 showed preferential antitumor activity against cancer cells with different FGFR gene alterations in 327 cancer cell lines and xenograft models. Due to its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 carrying a "gatekeeper" mutation that leads to resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is currently in clinical trials for the treatment of patients with FGFR gene alterations. [1] Recent cancer genomic analysis studies have identified many new gene alterations, including gene rearrangements encoding members of the FGFR family. However, the functions of most fusion genes are not yet clear, and their potential in targeted therapy is unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like protein 1 (BAIAP2L1). We screened four bladder cancer patients and two lung cancer patients carrying the FGFR3-BAIAP2L1 fusion gene using PCR and FISH. To investigate the oncogenic potential of this fusion gene, we constructed a Rat-2 fibroblast cell line (Rat-2_F3-B) transfected with FGFR3-BAIAP2L1. The FGFR3-BAIAP2L1 fusion protein exhibited transforming activity in Rat2 cells, and Rat-2_F3-B cells showed high tumorigenicity in mice. Rat-2_F3-B cells were sensitive to the selective FGFR inhibitor CH5183284/Debio 1347 both in vitro and in vivo, indicating that FGFR3 kinase activity is crucial for tumorigenesis. Gene characterization analysis showed that FGFR3-BAIAP2L1 can activate growth signaling pathways such as the MAPK pathway and inhibit tumor suppressor signaling pathways such as p53, RB1, and CDKN2A. We also constructed Rat-2_F3-B-ΔBAR cells expressing the FGFR3-BAIAP2L1 variant, which lacked the Bin-Amphiphysin-Rvs (BAR) dimerization domain of BAIAP2L1. Compared with Rat-2_F3-B cells, these cells exhibited reduced tumorigenicity, FGFR3 phosphorylation levels, and F3-B-ΔBAR dimerization levels. In summary, these data suggest that constitutive dimerization via the BAR domain promotes constitutive activation of the FGFR3 kinase and is crucial for its potent oncogenic activity. [2] Drugs targeting specific gene alterations have proven beneficial for cancer treatment. Given the diverse drug resistance mechanisms of cancer cells, understanding the downstream pathways of target genes and monitoring pharmacodynamic biomarkers associated with treatment efficacy is essential. We performed transcriptomic analysis to characterize the responses of different cancer cell lines to selective fibroblast growth factor receptor (FGFR) inhibitors (CH5183284/Debio 1347), mitogen-activated protein kinase (MEK) inhibitors, or phosphatidylinositol 3-kinase (PI3K) inhibitors. FGFR and MEK inhibitors produced similar expression patterns, and the extracellular signal-regulated kinase (ERK) gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with these results, CH5183284/Debio 1347 inhibited phosphorylated ERK in all tested FGFR inhibitor-sensitive cell lines. Since the mitogen-activated protein kinase (MAPK) pathway is downstream of FGFR, we screened a series of ERK-signatured cell lines to identify pharmacodynamic biomarkers for FGFR inhibitor efficacy and identified bispecific phosphatase 6 (DUSP6) as a candidate biomarker. Although MEK inhibitors can suppress the MAPK pathway, most cell lines sensitive to FGFR inhibitors are insensitive to MEK inhibitors, and we found that FGFR can effectively feedback activate multiple pathways. Therefore, we believe that the mechanism of action of FGFR inhibitors is through inhibition of the ERK signaling pathway, rather than through feedback activation of the ERK signaling pathway. Furthermore, DUSP6 may be a pharmacodynamic biomarker for the efficacy of FGFR inhibitors in FGFR-dependent cancers. [3]

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Zoligratinib is a selective ATP-competitive FGFR1-3 inhibitor designed to target solid tumors (lung, gastric, bladder cancer) driven by FGFR amplification or fusion[1]
-FGFR gene status (amplification/fusion) is a predictive biomarker of zoligratinib sensitivity; FGFR-negative tumors are unresponsive[1]
-ERK signaling activation enhances the sensitivity of FGFR+ cells to zoligratinib, supporting its use in combination with ERK inhibitors (no quantitative data available)[3]
-It inhibits the FGFR3-BAIAP2L1 fusion kinase, a potential target for FGFR3 rearrangement cancers[2]

C20H16N6O

356.38

356.139

C, 67.40; H, 4.53; N, 23.58; O, 4.49

1265229-25-1

1265229-25-1;1265231-80-8;

66555680

Off-white to yellow solid powder

3.932

3

4

3

27

573

0

O=C(C1=C([H])C2=C([H])C([H])=C([H])C([H])=C2N1[H])C1C([H])=NN(C=1N([H])[H])C1C([H])=C([H])C2=C(C=1[H])N([H])C(C([H])([H])[H])=N2

BEMNJULZEQTDJY-UHFFFAOYSA-N

InChI=1S/C20H16N6O/c1-11-23-16-7-6-13(9-17(16)24-11)26-20(21)14(10-22-26)19(27)18-8-12-4-2-3-5-15(12)25-18/h2-10,25H,21H2,1H3,(H,23,24)

[5-amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone

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: ≥ 2.08 mg/mL (5.84 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 (5.84 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 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 (5.84 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.)