Natural product; CRISPR/Cas9; HSV-1; Arf-GEFs

After 15 or 40 hours of treatment with brefeldin A (BFA), the endoplasmic reticulum (ER) swells significantly and moves to the periphery of normal kidney (NRK) cells. Actin and MT cytoskeleton are significantly disrupted by prolonged Brefeldin A therapy [1]. Brefeldin A and ADPR conjugate mediates the ADP-ribosylation of BARS. When created with cells obtained from CD38+ HeLa cells treated with BFA, bars demonstrate BAC binding [3]. Brefeldin A reduces MDA-MB-231 colony formation in 3D and 2D cultures, promotes identity-independent cell death in MDA-MB-231 breast cancer cells, and blocks MDA-MB migration and MMP 9 (matrix metal Peptidase 9) activity-231[2].

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Cancer stem cells (CSCs) are a subset of cancer cells in tumors or established cancer cell lines that can initiate and sustain the growth of tumors in vivo. Cancer stem cells can be enriched in serum-free, suspended cultures that allow the formation of tumorspheres over several days to weeks. Brefeldin A (BFA) is a mycotoxin that induces endoplasmic reticulum (ER) stress in eukaryotic cells. We found that BFA, at sub-microgram per milliliter concentrations, preferentially induced cell death in MDA-MB-231 suspension cultures (EC50: 0.016 µg/mL) compared to adhesion cultures. BFA also effectively inhibited clonogenic activity and the migration and matrix metalloproteinases-9 (MMP-9) activity of MDA-MB-231 cells. Western blotting analysis indicated that the effects of BFA may be mediated by the down-regulation of breast CSC marker CD44 and anti-apoptotic proteins Bcl-2 and Mcl-1, as well as the reversal of epithelial-mesenchymal transition. Furthermore, BFA also displayed selective cytotoxicity toward suspended MDA-MB-468 cells, and suppressed tumorsphere formation in T47D and MDA-MB-453 cells, suggesting that BFA may be effective against breast cancer cells of various phenotypes.[2]

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Glycoprotein D (gD-1) is an essential virion envelope component of herpes simplex virus type 1 (HSV-1) normally transported to the plasma membrane of the infected cells. In the present study, the intracellular transport of gD-1 was inhibited in cultured HSV-1 infected human fibroblasts by Brefeldin A (BFA) 1 microgram/ml medium added for 12 h after virus adsorption. Immunofluorescence light- and confocal microscopy revealed abolished transport of gD-1 to the plasma membrane, juxtanuclear accumulation of gD-1, and a disorderly arrangement of the tubulin fibres. Withdrawal of BFA influence for more than 60 min resulted in incomplete transport but increasing accumulation of gD-1 in the plasma membrane and in Golgi-like areas close to the nuclei. The tubulin pattern was almost normalized 6 h after removal of BFA. The egress of infectious HSV-1 particles released 9 h post-BFA treatment was not fully reestablished. The results indicate that BFA effects were not completely reversible and caused a sort of cytotoxic influence involving the structure of tubulin.[7]

In vivo antitumor efficacy of M-BFA (BFA encapsulated in mixed nanomicelles based on TPGS and F127 copolymers) [8]
Encouraged by the outstanding in vitro cytotoxicity and high tumor accumulation in vivo of M-BFA, antitumor efficacy of M-BFA was further investigated using HepG2 tumor-bearing xenograft model. The mice were divided into three groups and given the following formulations intravenously every day for 14 days: PBS, M-BFA 5 mg/kg and M-BFA 10 mg/kg. As shown in Fig. 7A, C, M-BFA 10 mg/kg group displayed the potent antitumor effect and dramatically delayed tumor progression, whereas mice treated with M-BFA 5 mg/kg showed no obvious inhibition. The tumor growth inhibition rate (TGI %) value in the M-BFA 10 mg/kg group was about 42.08% ± 3.29%, which was 2-fold higher than that of the M-BFA 5 mg/kg group. All of three groups caused minimal animal weight loss during the entire experiment (Fig. 7B), indicating low toxicity. Hematoxylin and eosin staining (H&E) analysis revealed that M-BFA exhibited extensive tumor necrosis (Fig. 7D). As shown in Fig. 7D, after the administration of M-BFA, large-scale tumor cells showed sheet necrosis. The necrosis foci were appeared pink, even part of the necrotic tumor tissues were dissolved, forming a cavity (red arrow). In the necrotic foci, more neutrophils infiltrating (green arrow) was detected. Moreover, the tumor cells had large nucleocytoplasmic ratio, even a few cells appeared mitosis (yellow arrow)

Previous inquiries into the effects of Brefeldin A (BFA) have largely concentrated on dynamics of ER-Golgi membrane traffic, predominantly after relatively short treatments with the drug. We have now analyzed the effects of long BFA treatment on overall cell morphology, behavior of resident and cycling Golgi proteins, and microtubular and actin cytoskeletons organization. Prolonged (15 h or 40 h) treatment of normal rat kidney (NRK) cells with BFA caused dramatic swelling of the Endoplasmic Reticulum (ER) and shifted its localization to the periphery of the cells. The Golgi complex was disassembled and Golgi proteins redistributed and persisted in partially distinct compartments. Prolonged BFA treatment resulted in marked disruption of the MT and actin cytoskeleton. Peripheral MT were absent and tubulin staining was concentrated in short astral MT emanating from the microtubule organizing center (MTOC). Actin stress fibers were largely absent and actin staining was concentrated within a perinuclear area. Within this region, actin localization overlapped that of the membrane transport factor p115. BFA effects on Golgi structure and on MT and actin organization showed the same threshold -- all could be partially reversed after 30 min and 15 h BFA treatment but were irreversible after 40h incubation with the drug. The observed effects were not induced by signaling pathways involved in apoptotic phenomena or in ER stress response pathways. These results suggest that BFA inhibits the activity of key molecules that regulate MT and actin cytoskeleton dynamics. The findings can be used as the basis for elucidating the molecular mechanism of BFA action on the cytoskeleton.[1]

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ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.[4]

Brefeldin A (BFA) is a mycotoxin that induces endoplasmic reticulum (ER) stress in eukaryotic cells. We found that BFA, at sub-microgram per milliliter concentrations, preferentially induced cell death in MDA-MB-231 suspension cultures (EC50: 0.016 µg/mL) compared to adhesion cultures. BFA also effectively inhibited clonogenic activity and the migration and matrix metalloproteinases-9 (MMP-9) activity of MDA-MB-231 cells. Western blotting analysis indicated that the effects of BFA may be mediated by the down-regulation of breast CSC marker CD44 and anti-apoptotic proteins Bcl-2 and Mcl-1, as well as the reversal of epithelial-mesenchymal transition. Furthermore, BFA also displayed selective cytotoxicity toward suspended MDA-MB-468 cells, and suppressed tumorsphere formation in T47D and MDA-MB-453 cells, suggesting that BFA may be effective against breast cancer cells of various phenotypes[2].

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2D Clonogenic Assay [2]
After pretreatment with 0–50 μg/mL Brefeldin A (BFA) for 24 h, the cells were reseeded at a density of 1 × 103 cells per well in the 6-well plate and cultured for additional 12 days, with medium changed every 3 days. The colonies were then fixed for 15 min with methanol-acetic acid (3:1) and stained with crystal violet (1%) for 30 min at room temperature.

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Wound-Healing Motility Assay [2]
Scratch wounds were created using a p10 micropipette tip in six-well plates with overnight confluent cultures. After cell debris were washed three times with phosphate-buffered saline (PBS), cells were replenished with complete medium containing 0–50 μg/mL Brefeldin A (BFA). Images of wound healing were captured by phase-contrast microscopy at indicated times after wounding.

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Gelatin Zymography [2]
Supernatants were collected from cell cultures treated with 0–50 μg/mL Brefeldin A (BFA) for 24 h, filtered through a 0.22 μm filter, and concentrated 50 X by using Centricon spin columns with 10 kD cutoff. Concentrated supernatants were resolved on non-reducing SDS-PAGE using 10% polyacrylamide gels containing 0.1% SDS and 1 mg/mL gelatin. After electrophoresis, the gels were washed three times with 50 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl, 5 mM CaC12, 5 μM ZnCl, 0.02% NaN3, 0.25% Triton X-100 at room temperature for 30 min each time, and then the gels were incubated in the same buffer without Triton X-100 at 37 °C for 20 h. Coomassie Brilliant Blue R-250 staining was used to reveal gelatin-clear zones created by MMPs.

PK study [8]
Female SD rats (5 per group) were dosed intravenously with M-BFA BFA encapsulated in mixed nanomicelles based on TPGS and F127 copolymers) in 10% solutol HS-15% and 90% saline (v/v) at dose level of 520 mg/kg. Blood samples were collected from all of the animals at predose and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h postdose into tubes containing heparin sodium and 200 mM DDPV. Plasma was separated from the blood by centrifugation at 6800 rpm for 6 min at 4 °C and stored at − 80 °C until analysis. Method development and biological samples analysis were performed by Triple Quad 5500 LC-MS/MS with verapamil as an internal standard (Table S2). PK parameters derived from concentration–time profiles containing T1/2, Cmax, AUC(0−t), AUC(0-∞) were calculated using Phoenix WinNonlin 7.0 by the Study Director.

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Animal treatment and tumor inhibition in vivo [8]
Female BALB/c mice (20 ± 2 g, 5–6 weeks) randomly divided into three groups (5 per group). HepG2 cells (1 × 107/mouse) were implanted subcutaneously into the under back area of mice to establish HepG2 tumor model. The treated mice were checked daily to investigate the size changes of tumors after implanted the HepG2 cells. When the average tumor volume reached around 100 mm3 (volume = (tumor length) × (tumor width)2/2), all mice were ready for the subsequent studies. The HepG2 tumor-bearing nude mice were administrated with M-BFA every day for 14 days. PBS solution was used as the control group. The dosage of BFA in other groups was 5 mg/kg and 10 mg/kg body weight. The tumor size of each mouse was measured every 2 days. Tumor volume (V) was determined by the following equation: V = L × W2/2, where L and W are length and width of the tumor, respectively. The mice were anesthetized with diethyl ether at the end of experiment. The excised organs and tumor tissues were washed with cold PBS (pH 7.4) and were weighed and photographed.

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In vivo fluorescence imaging of tumor [8]
The biodistribution of M-BFA was observed by in vivo imaging. Amphiphilic ICG (Fig. S11) was dissolved in water (1 mg/mL), and then directly added in M-BFA solution (80 μg/mL). ICG molecules entered the nanomicelles via hydrophobic interaction. The mice were intravenously injected with 100 μL free ICG (100 μg/mL) and ICG-loaded M-BFA (containing 100 μg/mL ICG). The mice were anesthetized and imaged using a 808 nm excitation laser at predetermined time by the IVIS Spectrum image system. After 48 h, the mice were sacrificed and various organs were collected to image the fluorescence distribution.

In vivo pharmacokinetic study [8]
The initial characterization of Brefeldin A (BFA) plasma pharmacokinetics (PK) revealed that BFA decreased quite rapidly in an apparent biexponential manner in the mouse. The biological half-life (T1/2) of BFA in the body was 0.17 h [53]. On the basis of these, PK properties of M-BFA were evaluated in Sprague-Dawley (SD) rats. After intravenous administration dosing at 520 mg/kg (equivalent to BFA 20 mg/kg), the concentrations of M-BFA in plasma were analyzed. The results showed that M-BFA demonstrated moderate PK profile, the T1/2 was approximately 0.35 h (vs 0.17 h of BFA) and the maximum plasma concentration (Cmax) was 4065.68 ng/mL. It achieved a sufficient plasma exposure in rats, with an area under the concentration–time curve (AUC0−t) value of 3153.75 h*ng/mL.

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Biodistribution of M-BFA in tumor-bearing mice [8]
Given that Brefeldin A (BFA) does not have autofluorescence, to analyze the in vivo biodistribution and tumor-targeting efficacy of M-BFA, the indocyanine green (ICG)-loaded M-BFA was employed and injected to HepG2 tumor-bearing mice for optical imaging analysis. Fluorescence imaging was carried out at 0, 1, 2, 4, 8, 24 and 48 h after injection. As shown in Fig. 6A, after 8 h, ICG fluorescence was observed in tumor site in ICG-loaded M-BFA group. After 24 h, there was a sharp contrast between the accumulation of ICG-loaded M-BFA and free ICG in tumor tissue. The intensity of fluorescence increased to the strongest at this time point and remained strong after injected 48 h. In contrast, no obvious fluorescence was found in the tumor site in free ICG group. We further observed that the fluorescence mainly distributed in the liver in the initial stage. After injected 48 h, the fluorescence disappeared in the whole mice body.

mouse LD50 intraperitoneal 250 mg/kg Japanese Journal of Antibiotics., 34(51), 1981 [PMID:7241806]

[1]. Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons. Eur J Cell Biol. 1999 Jan;78(1):1-14.

[2]. Brefeldin A reduces anchorage-independent survival, cancer stem cell potential and migration of MDA-MB-231 human breast cancer cells. Molecules. 2014 Oct 29;19(11):17464-77.

[3]. Erythroleukemia cells acquire an alternative mitophagy capability. Sci Rep. 2016 Apr 19;6:24641.

[4]. Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS. Proc Natl Acad Sci U S A. 2013 Jun 11;110(24):9794-9.

[5]. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015 Feb 5;16(2):142-7.

[6]. Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting. J Virol. 2003 Mar;77(5):3204-16.

[7]. A time-related study of Brefeldin A effects in HSV-1 infected cultured human fibroblasts. APMIS. 1995;103(7-8):530-539. doi:10.1111/j.1699-0463.1995.tb01402.x.

[8]. Brefeldin A delivery nanomicelles in hepatocellular carcinoma therapy: Characterization, cytotoxic evaluation in vitro, and antitumor efficiency in vivo. Pharmacol Res . 2021 Oct:172:105800.

Brefeldin A is a metabolite from Penicillium brefeldianum that exhibits a wide range of antibiotic activity. It has a role as a Penicillium metabolite.
A metabolite from Penicillium brefeldianum that exhibits a wide range of antibiotic activity.
brefeldin A has been reported in Penicillium camemberti, Penicillium brefeldianum, and other organisms with data available.
A fungal metabolite which is a macrocyclic lactone exhibiting a wide range of antibiotic activity.

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Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor Brefeldin A (BFA) or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.[3]

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Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor Brefeldin A (BFA) or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.[6]

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Hepatocellular carcinoma (HCC) is one of the major cancers with high mortality rate. Traditional drugs used in clinic are usually limited by the drug resistance and side effect and novel agents are still needed. Macrolide brefeldin A (BFA) is a well-known lead compound in cancer chemotherapy, however, with poor solubility and instability. In this study, to overcome these disadvantages, BFA was encapsulated in mixed nanomicelles based on TPGS and F127 copolymers (M-BFA). M-BFA was conferred high solubility, colloidal stability, and capability of sustained release of intact BFA. In vitro, M-BFA markedly inhibited the proliferation, induced G0/G1 phase arrest, and caspase-dependent apoptosis in human liver carcinoma HepG2 cells. Moreover, M-BFA also induced autophagic cell death via Akt/mTOR and ERK pathways. In HepG2 tumor-bearing xenograft mice, indocyanine green (ICG) as a fluorescent probe loaded in M-BFA distributed to the tumor tissue rapidly, prolonged the blood circulation, and improved the tumor accumulation capacity. More importantly, M-BFA (10 mg/kg) dramatically delayed the tumor progression and induced extensive necrosis of the tumor tissues. Taken together, the present work suggests that M-BFA has promising potential in HCC therapy.[8]

C16H24O4

280.36

280.167

C, 68.55; H, 8.63; O, 22.83

20350-15-6

5287620

Typically exists as White to off-white solids at room temperature

1.1±0.1 g/cm3

492.7±45.0 °C at 760 mmHg

200-205ºC

180.8±22.2 °C

0.0±2.8 mmHg at 25°C

1.513

1.61

2

4

0

20

388

5

O([H])[C@]1([H])C([H])([H])[C@]2([H])C([H])=C([H])C([H])([H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])OC(C([H])=C([H])C([H])([C@@]2([H])C1([H])[H])O[H])=O |c:10,t:31|

KQNZDYYTLMIZCT-KQPMLPITSA-N

InChI=1S/C16H24O4/c1-11-5-3-2-4-6-12-9-13(17)10-14(12)15(18)7-8-16(19)20-11/h4,6-8,11-15,17-18H,2-3,5,9-10H2,1H3/b6-4+,8-7+/t11-,12+,13-,14+,15+/m0/s1

(1S,2E,7S,10E,12R,13R,15S)-12,15-Dihydroxy-7-methyl-8-oxabicyclo[11.3.0]hexadeca-2,10-dien-9-one

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 (8.92 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 (8.92 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 (8.92 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: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 6: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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.)