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.

Beta-lapachone is a benzochromenone that is 3,4-dihydro-2H-benzo[h]chromene-5,6-dione substituted by geminal methyl groups at position 2. Isolated from Tabebuia avellanedae, it exhibits antineoplastic and anti-inflammatory activities. It has a role as an antineoplastic agent, an anti-inflammatory agent and a plant metabolite. It is a benzochromenone and a member of orthoquinones.
Lapachone has been used in trials studying the treatment of Cancer, Carcinoma, Advanced Solid Tumors, Head and Neck Neoplasms, and Carcinoma, Squamous Cell.
beta-Lapachone has been reported in Catalpa longissima, Handroanthus guayacan, and other organisms with data available.
Lapachone is a poorly soluble, ortho-naphthoquinone with potential antineoplastic and radiosensitizing activity. Beta-lapachone (b-lap) is bioactivated by NAD(P)H:quinone oxidoreductase-1 (NQO1), creating a futile oxidoreduction that generates high levels of superoxide. In turn, the highly reactive oxygen species (ROS) interact with DNA, thereby causing single-strand DNA breaks and calcium release from endoplasmic reticulum (ER) stores. Eventually, the extensive DNA damage causes hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme facilitating DNA repair, accompanied by rapid depletion of NAD+/ATP nucleotide levels. As a result, a caspase-independent and ER-stress induced mu-calpain-mediated cell death occurs in NQO1-overexpressing tumor cells. NQO1, a flavoprotein and two-electron oxidoreductase, is overexpressed in a variety of tumors.

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beta-Lapachone is a plant product that has been found to have many pharmacological effects. To date, very little is known about its biochemical target. In this study, we found that beta-lapachone inhibits the catalytic activity of topoisomerase I from calf thymus and human cells. But, unlike camptothecin, beta-lapachone does not stabilize the cleavable complex, indicating a different mechanism of action. beta-Lapachone inhibits topoisomerase I-mediated DNA cleavage induced by camptothecin. Incubation of topoisomerase I with beta-lapachone before adding DNA substrate dramatically increases this inhibition. Incubation of topoisomerase I with DNA prior to beta-lapachone makes the enzyme refractory, and treatment of DNA with beta-lapachone before topoisomerase has no effect. These results suggest a direct interaction of beta-lapachone with topoisomerase I rather than DNA substrate. beta-Lapachone does not inhibit binding of enzyme to DNA substrate. In cells, beta-lapachone itself does not induce a SDS-K(+)-precipitable complex, but it inhibits complex formation with camptothecin. We propose that the direct interaction of beta-lapachone with topoisomerase I does not affect the assembly of the enzyme-DNA complex but does inhibit the formation of cleavable complex. [1]

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beta-Lapachone and certain of its derivatives directly bind and inhibit topoisomerase I (Topo I) DNA unwinding activity and form DNA-Topo I complexes, which are not resolvable by SDS-K+ assays. We show that beta-lapachone can induce apoptosis in certain cells, such as in human promyelocytic leukemia (HL-60) and human prostate cancer (DU-145, PC-3, and LNCaP) cells, as also described by Li et al. (Cancer Res., 55: 0000-0000, 1995). Characteristic 180-200-bp oligonucleosome DNA laddering and fragmented DNA-containing apoptotic cells via flow cytometry and morphological examinations were observed in 4 h in HL-60 cells after a 4-h, > or = 0.5 microM beta-lapachone exposure. HL-60 cells treated with camptothecin or topotecan resulted in greater apoptotic DNA laddering and apoptotic cell populations than comparable equitoxic concentrations of beta-lapachone, although beta-lapachone was a more effective Topo I inhibitor. beta-Lapachone treatment (4 h, 1-5 microM) resulted in a block at G0/G1, with decreases in S and G2/M phases and increases in apoptotic cell populations over time in HL-60 and three separate human prostate cancer (DU-145, PC-3, and LNCaP) cells. Similar treatments with topotecan or camptothecin (4 h, 1-5 microM) resulted in blockage of cells in S and apoptosis. Thus, beta-lapachone causes a block in G0/G1 of the cell cycle and induces apoptosis in cells before, or at early times during, DNA synthesis. These events are p53 independent, since PC-3 and HL-60 cells are null cells, LNCaP are wild-type, and DU-145 contain mutant p53, yet all undergo apoptosis after beta-lapachone treatment. Interestingly, beta-lapachone treatment of p53 wild type-containing prostate cancer cells (i.e., LNCaP) did not result in the induction of nuclear levels of p53 protein, as did camptothecin-treated cells. Like other Topo I inhibitors, beta-lapachone may induce apoptosis by locking Topo I onto DNA, blocking replication fork movement, and inducing apoptosis in a p53-independent fashion. beta-Lapachone and its derivatives, as well as other Topo I inhibitors, have potential clinical utility alone against human leukemia and prostate cancers.[2]

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Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.[3]

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β-lapachone is a naturally occurring 1,2-naphthoquinone-based compound that has been advanced into clinical trials based on its tumor-selective cytotoxic properties. Previously, we focused on the related 1,4-naphthoquinone pharmacophore as a basic core structure for developing a series of potent indoleamine 2,3-dioxygenase 1 (IDO1) enzyme inhibitors. In this study, we identified IDO1 inhibitory activity as a previously unrecognized attribute of the clinical candidate β-lapachone. Enzyme kinetics-based analysis of β-lapachone indicated an uncompetitive mode of inhibition, while computational modeling predicted binding within the IDO1 active site consistent with other naphthoquinone derivatives. Inhibition of IDO1 has previously been shown to breach the pathogenic tolerization that constrains the immune system from being able to mount an effective anti-tumor response. Thus, the finding that β-lapachone has IDO1 inhibitory activity adds a new dimension to its potential utility as an anti-cancer agent distinct from its cytotoxic properties, and suggests that a synergistic benefit can be achieved from its combined cytotoxic and immunologic effects.[4]

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Agents, such as β-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than β-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with β-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to β-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.[5]

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Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of beta-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after beta-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. beta-Lapachone causes cell-cycle delays in late G(1) and S phase, and taxol arrests cells at G(2)/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone "collisions" at cell-cycle checkpoints.[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.)