F-actin (Kd = 2.2 nM, with Mg2+); F-actin (Kd = 1.4 nM, with Mg2+/K+)[1]

Cytochalasin B is a cell-penetrating mycotoxin that binds to the barbed ends of actin filaments and inhibits the lengthening and shortening of actin filaments. In the presence of MgCl2 (2 mM) or MgCl2, F-actin The Kds were 2.2 nM and 1.4 nM (2 mM) correspondingly with KCl[1]. Cytochalasin B (0.1-10 μM) shows inhibitory effects on multiple mouse cancer cell lines with IC50 of 2.56 μM (M109c), 10.46 μM (B16BL6), 105.5 μM (P388/ADR), 51.9 μM (P388/S), and IC80s after 3 hours of treatment were 12.23 μM (M109c), 44.86 μM (B16BL6), 188.4 μM (P388/ADR), 84.1 μM (P388/S), and IC50 were 0.25 μM (M109c), 0.37 μM (B16F10), 0.87 μM (B16BL6), the IC80 after 4 days of treatment were 0.75 μM (M109c), 1.21 μM (B16F10), and 10.41 μM (B16BL6) [2]. Cytochalasin B (6 μM) raises the myofibrillar fragmentation index (MFI), which is caused by the strong fragmentation of myofibrillar proteins into small fragments. Cytochalasin B also increases the breakdown of actin filaments. In addition, Cytochalasin B can also speed the conversion of F-actin to G-actin, lower the F-actin concentration, and considerably enhance the G-actin band during postmortem processing [3].

Balb/c mice with P388/ADR leukemia have a dose-dependent improvement in life expectancy when treated with cytochalasin B (10, 25, 50 mg/kg, ip). Long-term survival rates with cytochalasin B at 50 mg/kg were 10% in the drug-resistant P388/ADR cohort and 40% in the drug-sensitive P388/S cohort [2].

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cytochalasin B appeared to increase the life expectancy of Balb/c mice challenged with either P388/ADR or P388/S leukemias (Fig. 6). It was discerned from the P388/ADR protocol that Balb/c mice could take up to 50 mg/kg/day i.p. for eight consecutive days (Days 1–8). Therefore, only this dose was examined for P388/S challenged mice, as the antitumor activity of cytochalasin B appeared to be dose dependent. Interestingly, 50 mg/kg cytochalasin B was able to produce 10 % long-term survival in the multidrug resistant P388/ADR cohort, and 40 % long-term survival in the drug sensitive P388/S cohort[2].
The antitumor effects of cytochalasin B were mirrored by cytochalasin D at much lower concentrations (Fig. 6b). It only took 2 mg/kg/day cytochalasin D administered for eight consecutive days (Days 1–8) to produce marked prolongation in the life expectancy of mice challenged with P388/S, as well as a 20 % long-term survival rate. Whether or not cytochalasin D would exhibit the same antitumor effect against P388/ADR at these lower concentrations remains unclear, as not enough mice remained to establish another treatment group of sufficient quantity. Nevertheless, it is likely that at least some prolongation in life expectancy would be observed. The cytochalasin vehicle CMC/Tw did not affect the life span of mice challenged with either leukemia, demonstrating that there is no effect of the lipophilic detergent vehicle on the leukemia challenges in the absence of cytochalasins.[2]

It is generally accepted that cytochalasin B (CB), as well as other cytochalasins, shorten actin filaments by blocking monomer addition at the fast-growing ("barbed") end of these polymers. Despite the predominance of this mechanism, recent evidence suggests that other interactions may also occur between CB and F-actin. To investigate this possibility further we have employed an actin derivative, prepared by substitution at Cys374 by a glutathionyl residue. We demonstrate here that CB did not significantly bind to glutathionyl F-actin under several ionic conditions. We further show that in the presence of CB the glutathionyl-F-actin exhibits a significantly higher ATPase activity than the non-modified F-actin. These data argue that the incorporation of glutathionyl groups prevents the high-affinity binding of CB to the barbed end of actin filaments, probably due to a decreased hydrophobicity of the CB binding site by the introduction of the hydrophilic glutathionyl residue. Despite the lack of substantial binding at equilibrium, we have found that the addition of CB to glutathionyl-F-actin results in extensive fragmentation of the filaments, as demonstrated by electron microscopy and by a significant reduction of the relative viscosity of actin solutions. These results are consistent with the idea that CB shortens glutathionyl-actin filaments by a mechanism distinct from barbed end capping. Glutathionyl F-actin offers an interesting model to study the complex mechanism of interaction of actin filaments with cytochalasins and with the physiologically important actin capping/severing proteins.[1]

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Breast muscles of twenty-four Eastern Zhejiang White Geese were randomly divided into three groups: control, cytochalasin B (Cyt B) and Jasplakinolide (Jasp) treatments during postmortem conditioning. The myofibrillar fraction index (MFI), actin filaments and the levels of F-actin, G-actin and actin associated proteins (cofilins and tropomodulins) during conditioning were investigated. In control, the degraded tropomodulins, increased G-actin and disrupted actin filaments were observed at 4 and 7days; the increase of MFI and decrease of F-actin content were shown during conditioning. Cyt B treatments accelerated the transformation from F-actin to G-actin, weakened actin filaments and increased MFI compared to the control, while Jasp gained the opposite effect against Cyt B. We concluded that depolymerization of actin filaments regulated by tropomodulins contributed to myofibrillar fraction during conditioning. This work provided a new pathway of tenderization by the depolymerization of actin filaments.[3]

Effect of cytochalasin B on cancer cell lines in vitro[2]
The attached cell lines M109c, B16BL6, and B16F10 were seeded at 1 to 4 × 104 cells/ml in 2 ml volumes in 24-well culture plates 1 day prior to treatment with cytochalasin B. Conditions for treatment of the attached cell lines were as detailed earlier for B16BL6 and B16F10 cells. The suspension culture of P388/ADR cells was seeded at 5 × 104 cells/ml and allowed to grow overnight before cytochalasin B treatment. Cells were treated with cytochalasin B for 3 h, as well as 2, 3, or 4 days. In the case of continuous exposure for 2, 3, or 4 days, attached cells were trypsinized and counted with a hemacytometer. Leukemia cell suspensions were counted with a Coulter Counter. In the case of short-term exposure, cells were washed twice with fresh medium, then trypsinized (except for P388/ADR cells), reseeded, and allowed to regrow for 3 days, at which time they were counted. Growth results were calculated as the number of cells generated above the seeding density compared to the untreated control cells and graphically presented as percent of control increase.[2]
M109c clonogenic cells were determined by seeding aliquots containing 400–2000 trypsinized cells from each well into wells in another 24-well plate, culturing for 7 days, fixing in methanol (5 min), and staining with 0.1 % methylene blue (5 min). Colonies of greater than 10 cells were counted with a dissecting microscope.

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Determining the extent of drug synergy between cytochalasins and doxorubicin[2]
To assess whether cytochalasin B, D or DiHCB synergizes with ADR, cells were treated with a cytochalasin congener for 2.5 h over a concentration range of 0 to 150 μM, followed by ADR over a concentration range of 0 to 9 μM for 3 h. IC50 and IC80 values were taken at a series of concentrations for each chemotherapeutic agent in order to construct an isobologram. IC50 and IC80 values for the single agents and for combinations were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays.[2]
In addition, drug synergy was assessed by clonogenic assays in which P388/ADR cells were seeded in 24-well plates at early log phase. Cytochalasin B or DiHCB were administered for 2.5 h, followed by ADR for 3 h at a series of concentrations. Aliquots of the treated cells were then removed and cloned in soft agar in additional 24-well plates. Results from the assays were plotted as log surviving fractions at a given cytochalasin concentration as a function of ADR concentration. Fold-synergism was then calculated at relatively low concentrations of cyotchalasin where cytochalasin B or DiHCB-alone had minimal influence on cloning efficiency.

P388 leukemias in vivo[2]
For chemotherapy testing, Balb/c mice under isoflurane anesthesia were challenged with 2 × 105 trypan blue negative P388/S or P388/ADR cells subcutaneously (s.c.) in a volume of 200 μl. Untreated mice were kept in order to determine the lethality of the challenge without chemotherapeutic intervention. Long-term survival was defined as challenged mice that survived the duration of the observation period.

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cytochalasin B and D intraperitoneal administration[2]
cytochalasin B and D were prepared in suspension form in 2 % carboxymethyl cellulose 1 % tween 20 (CMC/Tw) for intraperitoneal (i.p.) administration, as previously described. The congeners or the vehicle were administered to leukemia-challenged mice on Days 1–8 following the initial challenge.

Metabolism / Metabolites
...IN THE BIOSYNTHESES OF CYTOCHALASIN B (PHOMIN) BY PHOMA SPECIES & CYTOCHALASIN D BY ZYGOSPORIUM MASONII...A NUMBER OF (14)C & (3)H PRECURSORS WERE FED TO PHOMA SPECIES & PRECISE DEGRADATION REACTIONS WERE PERFORMED TO REVEAL THE FORMATION OF CYTOCHALASIN B FROM ONE UNIT OF PHENYLALANINE, NINE UNITS OF ACETATE-MALONATE, & TWO UNITS OF METHIONINE. THE LABELLING PATTERN WAS ALSO CONFIRMED IN THE CASE OF CYTOCHALASIN D.
THE FORMATION OF CYTOCHALASIN B (PHOMIN) BY AN ENZYMATIC BAYER-VILLIGER TYPE OXYGEN INSERTION WAS PROVED BY A DIRECT CONVERSION OF LABELLED DEOXAPHOMIN BY PHOMA SPECIES.

Rat LD50 intraperitoneal 11 mg/kg

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Interactions
TREATMENT OF WILD TYPE S49 LYMPHOMA CELLS WITH THE MICROFILAMENT DISRUPTER CYTOCHALASIN B REVERSIBLY & IN A HIGHLY DOSE-DEPENDENT FASHION ENHANCES CELLULAR CYCLIC AMP ACCUMULATION IN RESPONSE TO SUBSEQUENT ADDITION OF THE BETA-ADRENERGIC AGONIST (-)-ISOPROTERENOL, PROSTAGLANDIN E1, OR CHOLERA TOXIN. INSEL PA, KOACHMAN AM; CYTOCHALASIN B ENHANCES HORMONE AND CHOLERA TOXIN-STIMULATED CYCLIC AMP ACCUMULATION IN S49 LYMPHOMA CELLS; J BIO CHEM 257(16) 9717 (1982)

CYTOCHALASIN B WAS UNABLE TO TRANSFORM 3T3-LIKE TUMOR CELLS, BUT DID INCREASE 8-40 FOLD THE FREQUENCY OF CELL TRANSFORMATION BY POLYOMA VIRUS. SEIF R; FACTORS WHICH DISORGANIZE MICROTUBULES OR MICROFILAMENTS INCREASE THE FREQUENCY OF CELL TRANSFORMATION BY POLYOMA VIRUS; J VIROL 36(2) 421 (1980)

THE PINOCYTOTIC ACTIVITY, INDUCED BY CONCANAVALIN A IN AMOEBA PROTEUS, IS GREATLY INTENSIFIED BY CYTOCHALASIN B. PRUSCH RD; THE INFLUENCE OF CONCANAVALIN A AND CYTOCHALASIN B ON PINOCYTOTIC ACTIVITY IN AMOEBA PROTEUS; PROTOPLASMA 106(3-4) 223 (1981)

CYTOCHALASIN B INHIBITED THE ELONGATION OF WHEAT COLEOPTILE SEGMENTS IN INDOLE-3-ACETIC ACID & OF MAIZE ROOTS, WITH THE ONLY ULTRASTRUCTURAL CHANGES BEING THE ACCUMULATION OF SECRETORY VESICLES. CYTOCHALASIN B APPARENTLY BLOCKED ELONGATION GROWTH BY INHIBITING VESICLE TRANSPORT & SECRETION OF CELL WALL COMPONENTS.

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Interactions
TREATMENT OF WILD TYPE S49 LYMPHOMA CELLS WITH THE MICROFILAMENT DISRUPTER CYTOCHALASIN B REVERSIBLY & IN A HIGHLY DOSE-DEPENDENT FASHION ENHANCES CELLULAR CYCLIC AMP ACCUMULATION IN RESPONSE TO SUBSEQUENT ADDITION OF THE BETA-ADRENERGIC AGONIST (-)-ISOPROTERENOL, PROSTAGLANDIN E1, OR CHOLERA TOXIN.
CYTOCHALASIN B WAS UNABLE TO TRANSFORM 3T3-LIKE TUMOR CELLS, BUT DID INCREASE 8-40 FOLD THE FREQUENCY OF CELL TRANSFORMATION BY POLYOMA VIRUS.
THE PINOCYTOTIC ACTIVITY, INDUCED BY CONCANAVALIN A IN AMOEBA PROTEUS, IS GREATLY INTENSIFIED BY CYTOCHALASIN B.
CYTOCHALASIN B INHIBITED THE ELONGATION OF WHEAT COLEOPTILE SEGMENTS IN INDOLE-3-ACETIC ACID & OF MAIZE ROOTS, WITH THE ONLY ULTRASTRUCTURAL CHANGES BEING THE ACCUMULATION OF SECRETORY VESICLES. CYTOCHALASIN B APPARENTLY BLOCKED ELONGATION GROWTH BY INHIBITING VESICLE TRANSPORT & SECRETION OF CELL WALL COMPONENTS.

[1]. Cytochalasin B may shorten actin filaments by a mechanism independent of barbed end capping. Biochem Pharmacol. 1994 May 18;47(10):1875-81.

[2]. Chemotherapy with cytochalasin congeners in vitro and in vivo against murine models. Invest New Drugs. 2015 Apr;33(2):290-9.

[3]. The effect of Cytochalasin B and Jasplakinolide on depolymerization of actin filaments in goose muscles during postmortem conditioning. Food Res Int. 2016 Dec;90:1-7.

[4]. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(1):24-38.

Cytochalasin B is an organic heterotricyclic compound, that is a mycotoxin which is cell permeable an an inhibitor of cytoplasmic division by blocking the formation of contractile microfilaments. It has a role as a metabolite, a platelet aggregation inhibitor, a mycotoxin and an actin polymerisation inhibitor. It is a cytochalasin, an organic heterotricyclic compound, a lactam and a lactone.
Cytochalasin B has been reported in Boeremia exigua, Sonchus mauritanicus, and other organisms with data available.
A cytotoxic member of the CYTOCHALASINS.

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Mechanism of Action
MAJOR BIOLOGICAL EFFECTS ARE THE BLOCKAGE OF CYTOPLASMIC CLEAVAGE RESULTING IN MULTINUCLEATE CELL FORMATION, THE INHIBITION OF CELL MOVEMENT, & THE INDUCTION OF NUCLEAR EXTRUSION... OTHER REPORTED EFFECTS INCLUDE THE INHIBITION OF GLUCOSE TRANSPORT, OF THYROID SECRETION, OF GROWTH HORMONE RELEASE, OF PHAGOCYTOSIS, & OF PLATELET AGGREGATION & CLOT CONTRACTION. /CYTOCHALASINS/
THROUGH THE USE OF (3)H-LABELLED CYTOCHALASINS B & D THE PRECISE BINDING SITES & SUB-CELLULAR LOCALIZATION ARE NOW UNDER INVESTIGATION... EXPERIMENTAL RESULTS OBTAINED THUS FAR SUPPORT THE IDEA THAT THE PRIMARY EFFECT IS MEMBRANOTROPIC, PERHAPS INVOLVING THE ASSOCIATION OF MICROFILAMENTS WITH THE PLASMA MEMBRANE. IT SHOULD BE NOTED THAT THE EFFECT OF CYTOCHALASIN B ON NORMAL & TRANSFORMED CELLS DIFFERS; THE LATTER BECOME MORE HIGHLY MULTINUCLEATED THAN NORMAL CELLS.
THE INFLUENCE OF CYTOCHALASIN B & E ON INTESTINAL DIGESTION OF MALTOSE & SUCROSE, & THEIR DIGESTION PRODUCTS AS GLUCOSE & FRUCTOSE WAS INVESTIGATED IN THE MOUSE IN VITRO. NEITHER DIGESTION OF MALTOSE OR SUCROSE NOR ACTIVITIES OF MALTASE OR SUCRASE IN EVERTED SACS OF MOUSE JEJUNUM WAS AFFECTED BY CYTOCHALASIN B OR E AT 5.0 & 10.0 MUG/ML AFTER 60 MIN INCUBATION. HOWEVER, ABSORPTION OF GLUCOSE DERIVED FROM MALTOSE OR SUCROSE DIGESTION WAS INHIBITED BY 68.5 & 65.9% DUE TO CYTOCHALASIN E (5.0 MUG/ML) & BY 29.5 & 13.1% DUE TO CYTOCHALASIN B AT THE SAME CONCN. CYTOCHALASINS B & E SEEMED TO STIMULATE ABSORPTION OF FRUCTOSE DERIVED FROM SUCROSE DIGESTION IN MOUSE JEJUNUM.
THE INHIBITORY EFFECT OF CYTOCHALASIN E ON GALACTOSE ABSORPTION IN EVERTED SACS OF MOUSE JEJUNUM WAS STUDIED. CYTOCHALASIN B HAD THE HIGHEST POTENCY ON THE INHIBITION OF GALACTOSE ABSORPTION WHEN IT WAS ADDED IN THE MUCOSAL SOLN FOLLOWED BY CYTOCHALASIN E, A, C, & D, RESPECTIVELY.
For more Mechanism of Action (Complete) data for CYTOCHALASIN B (14 total), please visit the HSDB record page.

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Therapeutic Uses
EXPTL USE: THE EFFECTS OF VINBLASTINE, COLCHICINE, LIDOCAINE, & CYTOCHALASIN B ON TUMOR CELL KILLING BY BCG-ACTIVATED MACROPHAGES WERE EXAMINED. CYTOCHALASIN B, WHICH DISRUPTS MICROFILAMENTS, ENHANCED TUMOR CELL LYSIS & STASIS DUE TO ACTIVATED MARCOPHAGES AT A CONCN OF 10-7 MOLES. WHERAS VINBLASTINE & LIDOCAINE SEEM TO ACT ON THE MACROPHAGE ITSELF, CYTOCHALASIN B EXERTS ITS EFFECT PREDOMINANTLY ON THE TUMOR CELL. MICROTUBULES & MICROFILAMENTS MAY PLAY A ROLE IN THE DESTRUCTION OF TUMOR CELLS BY ACTIVATED MARCOPHAGES.
EXPTL USE: CYTOCHALASIN B & COLCHICINE DECREASED THE NUMBER OF MOTILE CELLS WHEN ADDED TO YOSHIDA SARCOMA CELLS AT 5 & 0.4 MUG/ML, RESPECTIVELY. CYTOCHALASIN B ALSO DECREASED THE MOTILITY OF THE CELLS & DOSE-DEPENDENTLY INHIBITED THEIR GROWTH.
EXPTL USE: IN CULTURED IRC 741 RAT LEUKEMIA CELLS, CYTOCHALASIN B (1.5-4 MUG/ML) CAUSED DOSE-DEPENDENT BINUCLEATION & INHIBITION OF THE NORMAL INCREASE IN CELL NUMBER. IN CELLS ENTERING DIVISION AFTER EXPOSURE TO CYTOCHALASIN B FOR UP TO 24 HR, FURROWING WAS COMPLETELY INHIBITED IN A DOSE-DEPENDENT PROPORTION OF CELLS.

C29H37NO5

479.6078

479.267

C, 72.62; H, 7.78; N, 2.92; O, 16.68

14930-96-2

5311281

White to off-white solid powder

1.2±0.1 g/cm3

740.6±60.0 °C at 760 mmHg

218-223ºC

401.7±32.9 °C

0.0±2.6 mmHg at 25°C

1.596

3.71

3

5

2

35

859

8

C[C@@H]1CCC[C@H](/C=C/C(=O)O[C@]23[C@@H](/C=C/C1)[C@@H](C(=C)[C@H]([C@H]2[C@@H](NC3=O)CC4=CC=CC=C4)C)O)O

GBOGMAARMMDZGR-TYHYBEHESA-N

InChI=1S/C29H37NO5/c1-18-9-7-13-22(31)15-16-25(32)35-29-23(14-8-10-18)27(33)20(3)19(2)26(29)24(30-28(29)34)17-21-11-5-4-6-12-21/h4-6,8,11-12,14-16,18-19,22-24,26-27,31,33H,3,7,9-10,13,17H2,1-2H3,(H,30,34)/b14-8+,16-15+/t18-,19-,22-,23+,24+,26+,27-,29-/m1/s1

(1S,4E,6R,10R,12E,14S,15S,17S,18S,19S)-19-benzyl-6,15-dihydroxy-10,17-dimethyl-16-methylidene-2-oxa-20-azatricyclo[12.7.0.01,18]henicosa-4,12-diene-3,21-dione

CYTOCHALASIN B; Phomin; 14930-96-2; cytochalasin-B; CHEBI:23527; Cytochalasin B (Phomin); MFCD00077704; MLS000028816;

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)

DMSO : ~83.33 mg/mL (~173.75 mM)
Ethanol : ~25 mg/mL (~52.13 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.21 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 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 (5.21 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 3: ≥ 2.5 mg/mL (5.21 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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Solubility in Formulation 4: ≥ 2.08 mg/mL (4.34 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 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: ≥ 2.08 mg/mL (4.34 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 6: ≥ 2.08 mg/mL (4.34 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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Solubility in Formulation 7: 5 mg/mL (10.43 mM) in 0.5% CMC-Na 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), Suspened solution; with ultrasonication.

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