Topoisomerase I

Beta-lapachone treatment (50 mg/kg) significantly inhibits the growth of the tumor in vivo in a xenograft mouse model of human ovarian cancer, and Beta-lapachone and taxol together induce apoptosis in a synergistic manner.[6] Beta-lapachone treatment accelerates the healing process compared to vehicle only in both normal and diabetic (db/db) mice.[3]

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Effects of β-lapachone on in vivo wound healing.[3]
To determine whether β-lapachone had a therapeutic effect on wound healing, ointment alone or containing 29.8 μg/g β-lapachone was applied to a wound on the back of C57BL/6 or db/db mice for 21 days, and the wounds were examined for healing every 5 days from wounding day (day 0) to day 21 postwounding (Figs. 5 and 6). Skin tissue (approximately 1 × 1 cm2) in the center of the wounds was cut out on day 3, 7, 14, or 21 postwounding and was processed for hematoxylin and eosin staining (Fig. 6). The density of vessels underlying the healing skin was measured using Imagescope software (Fig. 6E). Microscopic observation showed that the time required for wound healing on db/db mice was significantly longer than that on C57BL/6 mice (Fig. 5, A and C), and the wound area in C57BL/6 or db/db mice treated with ointment containing β-lapachone (Fig. 5, B and D) was markedly smaller than that in mice treated with control ointment (Fig. 5, A and C) in 5 to 20 days. Compared with mice treated with control ointment, the area of the β-lapachone-treated wounds was significantly reduced in both C57BL/6 and db/db mice (Fig. 5E). Compared with the wound treated with ointment without β-lapachone, the recovery process of wound healing by β-lapachone treatment was faster either in C57BL/6 or in db/db mice (Fig. 6, A–D). On day 14, in C57BL/6 mice, the scar tissue was thick and the dermis appeared disorderly in the wound treated with control ointment (Fig. 6A3); however, on the same day, the skin layers were completely rehabilitated in the β-lapachone-treated wound (Fig. 6B3). Similarly, the scar tissue was relatively thinner, and hair follicles appeared in the dermis in the β-lapachone-treated wound at 14 days (Fig. 6D3) in db/db mice, but hair follicles in the wound treated with the control ointment were only observed at 21 days.

DNA topoisomerase I is cultured in 20 μL of relaxation buffer (50 mM Tris, pH 7.5) with or without drugs (including β-lapachone). (30 μg/mL bovine serum albumin, 50 mM KCl, 10 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM EDTA) for 30 minutes at 37°C. Proteinase K (50 μg/mL) and 1% SDS are added to halt reactions. The products are separated by electrophoresis in 1% agarose gel in TAE buffer (0.04 M tris acetate, 0.001 M EDTA) following an additional 1-hour incubation at 37°C. After electrophoresis, ethidium bromide is used to stain the gel. Utilizing an NIH image analysis system, the photographic negative is scanned.

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Biochemical assays [4]
Purified human recombinant IDO1 (hrIDO1) enzyme was expressed in Escherichia coli and purified as previously described. IDO1 enzyme assays were performed using the potassium phosphate buffer system as previously published.25 Ehrlich’s reagent was used to detect kynurenine (Kyn) spectrophotometrically as described.25 Reagents including substrate and inhibitor were all mixed first, leaving the addition of the enyme for last to initiate the reaction at T = 0. To determine enzyme kinetics for the hrIDO1 preparation, enzyme assays were performed in 1 mL volumes with varying L-Trp concentrations (0–400 μM) and collection of 100 μL aliquots for Kyn analysis at multiple timepoints (0–90 minutes). The results confirmed that the hrIDO1 enzyme follows Michaelis-Menten kinetics as previously published26 with a Km of 110 μM and a Vmax of 5.9 μM/min (Supplemental Figure S1). Inhibitory activity of β-lapachone was subsequently evaluated in hrIDO1 enzyme reactions with varying concentrations of inhibitor (0–50 μM) at a fixed substrate concentration (100 μM L-Trp) for IC50 determination or varying concentrations of both inhibitor (0–800 nM) and substrate (0–400 μM) for Ki determination. Reactions were carried out in 100 μL volumes and were stopped at 15 minutes while enzyme activity was in the linear range. Data analysis and graphing were performed using Prism v.5.0.

The MTT assay is used to quantify cytotoxicity. Two days before different concentrations of either topotecan or β-lapachone are added, IMR-32 and JCI cells are plated in 96-well microtiter plates at a concentration of 5.0 × 10⁴ (topotecan) or 2.5 × 10⁴ (β-lapachone) cells/well/100 µL medium. After that, the cells are kept in a CO₂ incubator at 37°C for 72 hours. A Cell Proliferation Kit I is used to measure the proliferation of cells. Four distinct cultures are used in the experiments.

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Cell treatment and cell viability assays. [3]
HS68 cells (103), 3T3 cells (103), or EAhy926 cells (104) in 100 μl medium were seeded for 24 h at 37°C in a 96-well culture plate in a humidified 5% CO2 atmosphere. HEKn cells (104), XB-2 cells (104), and HUVEC (104) were seeded for 48 h because of the lower growth rate. For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, various concentrations of β-lapachone were added to the medium 24 h before the cell viability assay. In brief, 10 μl MTT (0.5 mg/ml) were added to each well and the plates were incubated at 37°C for 4 h. The formazan product was then dissolved in 100 μl DMSO at 37°C for 30 min, and absorbance at 570 nm was measured with a microplate reader. To test the effects of MAPK inhibitors, 3T3 cells or EAhy926 cells (103 cells in 100 μl medium/well) were incubated for 1 h with 0, 5, or 10 μM ERK inhibitor or p38 inhibitor (SB-203580) or 0, 50, or 100 nM JNK inhibitor (SP-600125); the cells were then changed to medium containing the same MAPK inhibitor with or without 1 μM β-lapachone . The number of viable cells after treatment was measured using the MTT assay. For all studies, at least three sets of independent experiments were carried out, each in triplicate.

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Cell cycle analysis. [3]
Cells were treated with 1 μM β-lapachone for 3, 6, 9, 12, or 24 h, harvested with 0.5% trypsin-EDTA, and fixed with cold 80% ethanol. After three washes with PBS, the cells were incubated for 1 h at 37°C with RNase A (1 μg/ml) and then for 15 min at 37°C with propidium iodide (50 μg/ml). Stained cells were detected by flow cytometry using the FL-2 parameter, and the data were analyzed using Cell Quest Pro software.

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Immunofluorescence staining. Cells were incubated with 1 μM β-lapachone for 0 to 24 h and were then fixed in 4% paraformaldehyde for 15 min. After being blocked for 1 h at room temperature with 10% normal goat serum (NGS), the cells were stained overnight at 4°C with monoclonal antibody against PCNA (1:1,000), incubated with rhodamine-conjugated secondary antibody and Hoechst dye for 1 h at room temperature, and examined and photographed using a Leica fluorescence microscope.

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Western blot analyses. [3]
Cells treated with 1 μM β-lapachone for 0–24 h were lysed with lysis buffer (0.25 mM HEPES, pH 7.4, 14.9 mM NaCl, 10 mM NaF, 2 mM MgCl2, 0.5% NP-40, 0.1 mM PMSF, 20 μM pepstatin A, and 20 μM leupeptin). The lysates were then centrifuged at 1,000 g for 15 min at 4°C, and the supernatants were collected for immunoblotting. The amount of protein in the samples was measured by the Bradford assay using an ELISA reader. Approximately 25–50 μg protein from each sample were separated by 10–12% SDS-PAGE and then transferred to Immobilon-P membranes in an electrophoretic transfer cell (2 h at 200 V). All subsequent steps were at room temperature. The membranes were blocked for 1 h with 5% skim milk in PBS containing 0.05% Tween 20 (PBST), incubated for 2 h with anti-phosphorylated-ERK, anti-ERK, anti-phosphorylated-JNK, anti-JNK, anti-phosphorylated-p38, anti-p38, or anti-actin antibodies (1:1,000 dilution) in 1% BSA, washed with PBST for 30 min, and then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. Bound antibody was detected with ECL Western blotting reagent, and the chemiluminescence was detected with Fuji Medical X-ray film. The amount of each protein was quantified using Scion software.

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Scrape-wound healing assay. [3]
Cells were grown to confluence on a 24-well dish, the medium was aspirated, and new medium with or without 1 μM β-lapachone alone or together with ERK inhibitor, p38 inhibitor, or JNK inhibitor was added. A single stripe (∼150 μm wide) was scraped on the cell-coated surface with a 200-μl disposable plastic pipette tip, and the wound was allowed to heal for 24 h (endothelial cells) or 48 h (fibroblast cells) at 37°C. The average extent of wound closure was evaluated by measuring the width of the wound.

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Transwell migration assay. [3]
Cell migration was assessed using a modified Millicell chamber (8-μm pores). Cells seeded into the upper chamber at 1 × 104 cells/well in 0.2 ml medium were treated with β-lapachone alone or with β-lapachone plus ERK inhibitor, p38 inhibitor, or JNK inhibitor, and 0.6 ml of the medium was added to the bottom chamber. After 24 h at 37°C, the cells on the upper surface of the membrane were mechanically removed, and the migrated cells on the lower surface of the membrane were fixed and stained with Coomassie brilliant blue. The total number of migrated cells on the lower surface of the membrane was counted. Each experiment was performed in triplicate.

Male Balb/c mice are fed a commercial pellet diet and given unlimited access to water. Following one week of acclimation, the mice are divided into five groups at random and placed in the following groups: control, β-lapachone, cisplatin (18 mg/kg, ip), and β-lapachone + cisplatin (18 mg/kg, ip). Two weeks before receiving an injection of cisplatin, the β-lapachone groups are given a diet containing the medication (0.066). Three days following their injection of cisplatin, all mice are killed while sedated with carbon dioxide. Analysis of the serum BUN and CRE is performed on the blood samples. For histopathological and immunohistochemical (IHC) research, the kidney is promptly removed in half. The remaining half is kept cold until the western blot test.

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Wound biopsy and measurement of wound closure. [3]
Mice (C57BL/6 or db/db) were anesthetized with 2% Rompun solution (0.1 ml/20 g body wt; Bayer, Leverkusen, Germany). The back of the mouse was shaved and then sterilized using an alcohol swab. A sterile biopsy punch (6-mm diameter) was used to punch through the full thickness of the back skin below the shoulder blades. A wound placed in this area cannot be reached by the mouse and therefore prevents self-licking. Ointment [100 mg pure white petrolatum jelly (Vaseline)] alone (control ointment) or containing 29.8 μg/g β-lapachone was applied to the wound and changed every 2 days. Wounds from individual mice were digitally photographed every 5 days, beginning on the day of wounding. For all measurements, the wound area was quantified using Scion software.

[1]. J Biol Chem . 1993 Oct 25;268(30):22463-8.

[2]. Cancer Res . 1995 Sep 1;55(17):3706-11.

[3]. Am J Physiol Cell Physiol . 2008 Oct;295(4):C931-43.

[4]. Int J Tryptophan Res . 2013 Aug 19:6:35-45.

[5]. Cancer Res . 2012 Jun 15;72(12):3038-47.

[6]. Proc Natl Acad Sci U S A . 1999 Nov 9;96(23):13369-74.

β-Lapadone is a benzo[h]chromene ketone with the structure 3,4-dihydro-2H-benzo[h]chromene-5,6-dione, substituted with a geminal dimethyl group at the 2-position. It was isolated from Tabebuia avellanedae, a plant in the Bignoniaceae family, and possesses antitumor and anti-inflammatory activities. It can be used as an antitumor drug, anti-inflammatory drug, and plant metabolite. It is a benzo[h]chromene ketone, belonging to the ortho-quinone class of compounds. Lapadone has been used in research for the treatment of cancer, carcinoma, advanced solid tumors, head and neck tumors, and squamous cell carcinoma. β-Lapadone has been reported to exist in Catalpa longissima, Handroanthus guayacan, and other organisms with relevant data. Lapadone is a poorly soluble ortho-naphthoquinone with potential antitumor and radiosensitizing activities. β-Lapadone (b-lap) can be bioactivated by NAD(P)H:quinone oxidoreductase-1 (NQO1), resulting in ineffective redox reactions and the generation of high concentrations of superoxide. These highly reactive oxygen species (ROS) interact with DNA, leading to single-strand DNA breaks and prompting the release of calcium ions from the endoplasmic reticulum (ER). Ultimately, widespread DNA damage leads to the overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1), accompanied by a rapid depletion of NAD+/ATP nucleotide levels. Therefore, in NQO1-overexpressing tumor cells, a caspase-independent, ER-stress-induced μ-calpases-mediated cell death occurs. NQO1 is a flavoprotein and two-electron oxidoreductase overexpressed in various tumors. β-Lapadone is a plant product with a variety of pharmacological effects. To date, little is known about its biochemical targets. This study found that β-lappaone inhibits the catalytic activity of topoisomerase I in calf thymus and human cells. However, unlike camptothecin, β-lappaone is not an unstable cleavable complex, indicating a different mechanism of action. β-lappaone inhibits camptothecin-induced topoisomerase I-mediated DNA cleavage. Incubating topoisomerase I with β-lappaone before adding DNA substrate significantly enhances this inhibitory effect. Incubating topoisomerase I with DNA before adding β-lappaone inactivates the enzyme; however, treating DNA with β-lappaone before adding topoisomerase has no effect. These results indicate that β-lappaone interacts directly with topoisomerase I, rather than with the DNA substrate. β-lappaone does not inhibit the binding of the enzyme to the DNA substrate. In cells, β-lappaone itself does not induce the formation of the SDS-K(+) precipitate complex, but it inhibits the formation of a complex with camptothecin. We hypothesize that the direct interaction between β-lappaone and topoisomerase I does not affect the assembly of the enzyme-DNA complex, but inhibits the formation of cleavable complexes. [1] β-lappaone and some of its derivatives can directly bind to and inhibit the DNA unwinding activity of topoisomerase I (Topo I) to form a DNA-Topo I complex that cannot be separated by SDS-K+ detection. We found that β-lappaone can induce apoptosis in some cells, such as human promyelocytic leukemia cells (HL-60) and human prostate cancer cells (DU-145, PC-3 and LNCaP), as described by Li et al. (Cancer Res., 55: 0000-0000, 1995). In HL-60 cells, after treatment with ≥0.5 μM β-lappaone for 4 hours, characteristic 180-200 bp oligonucleosome DNA ladder bands and apoptotic cells containing DNA fragments were observed by flow cytometry and morphological examination. Compared to toxic concentrations of β-lappaone, HL-60 cells treated with camptothecin or topotecan produced more pronounced apoptotic DNA ladder bands and more apoptotic cells, despite β-lappaone being a more potent inhibitor of topoisomerase I. β-lappaone treatment (4 h, 1–5 μM) resulted in cell cycle arrest in HL-60 cells and three different prostate cancer cell lines (DU-145, PC-3, and LNCaP) at G0/G1 phases, with a decrease in the proportion of cells in S and G2/M phases and an increase in the number of apoptotic cells over time. Similar treatments with topotecan or camptothecin (4 h, 1–5 μM) also resulted in cell cycle arrest in S phase and induced apoptosis. Therefore, β-lappaone can arrest the cell cycle at G0/G1 phases and induce apoptosis before or early in DNA synthesis. These events were not related to p53, as PC-3 and HL-60 cells were p53-deficient cells, LNCaP was wild-type cells, and DU-145 contained mutant p53, but all of these cells underwent apoptosis after treatment with β-lappaone. Interestingly, treatment of prostate cancer cells containing wild-type p53 (e.g., LNCaP) with β-lappaone did not induce nuclear expression of p53 protein as in the case of treatment with camptothecin. Similar to other topoisomerase I inhibitors, β-lappaone may induce apoptosis in a p53-independent manner by locking topoisomerase I onto DNA and blocking the movement of replication forks. β-lappaone and its derivatives, as well as other topoisomerase I inhibitors, have potential clinical value in the treatment of human leukemia and prostate cancer when used alone. [2]

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Wound healing disorders are a serious problem for patients with diabetes. Wound healing is a complex process that requires the synergistic action of multiple cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. β-Lapadone is a natural compound extracted from the bark of the jacaranda tree (Tabebuia avellanedae). It is well-known for its antitumor, anti-inflammatory, and antitumor effects at different concentrations and under different conditions, but its impact on wound healing remains unexplored. This study aimed to investigate the effects of β-lapadone on wound healing and its potential mechanisms. Our research shows that low-dose β-lapadone can enhance the proliferation of various cell types, promote the migration of mouse 3T3 fibroblasts and human EAhy926 endothelial cells through different MAPK signaling pathways, and accelerate the healing of abrasion wounds in vitro. After applying ointments containing or without β-lapadone to puncture wounds in normal mice and diabetic (db/db) mice, we found that the wound healing rate in the β-lapadone treatment group was faster than that in the control group that only applied the solvent. Furthermore, β-lapadone can induce macrophages to release VEGF and EGF, both cytokines that are beneficial to the growth of various cell types. Our results indicate that β-lapadone can promote cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and promote the migration of fibroblasts and endothelial cells, thereby accelerating wound healing. Therefore, we believe that β-lappaone may have the potential to treat wound healing. [3] β-lappaone is a naturally occurring 1,2-naphthoquinone compound that has been advanced to the clinical trial stage due to its tumor-selective cytotoxicity. Previously, we developed a series of potent indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors based on related 1,4-naphthoquinone pharmacophores. In this study, we found that the clinical candidate drug β-lappaone has IDO1 inhibitory activity, a previously undiscovered property. The β-lappaone inhibition mode based on enzyme kinetics is non-competitive inhibition, and the computational model predicts that its binding mode to the IDO1 active site is consistent with other naphthoquinone derivatives. Previous studies have shown that inhibiting IDO1 can break through the pathogenic tolerance mechanism, thereby removing the limitation of the immune system to produce an effective anti-tumor response. Therefore, the discovery that β-lappaone has IDO1 inhibitory activity adds a new dimension to its potential use as an anticancer drug, distinguishing it from its cytotoxicity and suggesting that the combined effects of its cytotoxicity and immunomodulatory effects may produce a synergistic effect. [4] Drugs targeting the oxidoreductase NAD(P)H:quinone oxidoreductase 1 (NQO1) to induce programmed necrosis in solid tumors, such as β-lappaden, have shown great potential, but more effective tumor-selective compounds are still needed. This article reports that deoxyniposiquinone kills a variety of cancer cells in an NQO1-dependent manner with greater potency than β-lappaden. The killing effect of deoxyniposiquinone depends on an NQO1-dependent ineffective redox cycle that consumes oxygen and produces large amounts of reactive oxygen species (ROS). Elevated levels of ROS lead to widespread DNA damage, PARP1 overactivation, and severe NAD+/ATP depletion, thereby stimulating Ca2+-dependent programmed necrosis, which is characteristic of this class of novel NQO1 “bioactivating” drugs. Short-term exposure of NQO1+ cells to deoxyniposiquinone is sufficient to induce cell death, while gene-matched NQO1- cells are unaffected. In addition, siRNA-mediated knockdown of NQO1 or PARP1 can protect NQO1+ cells from short-term death. Pretreatment of cells with BAPTA-AM (a cytoplasmic Ca2+ chelator) or catalase (an enzymatic H2O2 scavenger) was sufficient to salvage deoxyniposiquinone-induced cell death, similar to the case with β-lapaquinone. In vivo studies have shown that deoxyniposiquinone has comparable antitumor efficacy to β-lapaquinone, but is 6 times more potent. PARP1 overactivation and significant ATP reduction were observed in tumor tissues, but not in the associated normal lung tissues. Our findings provide a preclinical proof of concept for deoxyniposiquinone as a potent chemotherapeutic agent for the treatment of various refractory solid tumors, such as pancreatic and lung cancer. [5]

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In vitro, ablation of tumor clones was observed after treatment with a combination of two low molecular weight compounds, β-lapaquinone and paclitaxel, in a variety of human cancer cells. They synergistically induced cell death in cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergistic effect is related to the dosing regimen; that is, paclitaxel must be added simultaneously with or after β-lapaquinone. This combination therapy exhibits unusually strong antitumor activity against human ovarian and prostate cancer tumors transplanted into mice. Host toxicity is minimal. Cells can initiate apoptosis at cell cycle checkpoints, a mechanism that eliminates defective cells to ensure genomic integrity. We hypothesize that when cells are treated simultaneously with drugs that activate multiple different cell cycle checkpoints, the production of conflicting regulatory signaling molecules can induce apoptosis in cancer cells. β-lappadenone can delay the cell cycle to late G1 and S phases, while paclitaxel arrests cells at G2/M phases. Cells treated with these two drugs experience delays at multiple checkpoints before initiating apoptosis. Our results suggest that new anticancer therapies may be developed by utilizing the “collision” mechanism at cell cycle checkpoints that readily induce apoptosis. [6]

C15H14O3

242.27

242.094

C, 74.36; H, 5.82; O, 19.81

4707-32-8

3885

Brown to red solid powder

1.3±0.1 g/cm3

381.4±42.0 °C at 760 mmHg

>110ºC (dec.)

169.7±27.9 °C

0.0±0.9 mmHg at 25°C

1.595

2.82

0

3

0

18

445

0

O1C2C3=C([H])C([H])=C([H])C([H])=C3C(C(C=2C([H])([H])C([H])([H])C1(C([H])([H])[H])C([H])([H])[H])=O)=O

QZPQTZZNNJUOLS-UHFFFAOYSA-N

InChI=1S/C15H14O3/c1-15(2)8-7-11-13(17)12(16)9-5-3-4-6-10(9)14(11)18-15/h3-6H,7-8H2,1-2H3

2,2-dimethyl-3,4-dihydrobenzo[h]chromene-5,6-dione

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: ≥ 2.5 mg/mL (10.32 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 (10.32 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 (10.32 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: 2.86 mg/mL (11.81 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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