For the purpose of enhancing the intake of tiny molecules like glucose and nanoparticles, methyl-β-cyclodextrin is widely employed to raise the permeability of cells[4]. Cyclodextrins are a kind of cyclic oligosaccharides that have a lipophilic core chamber and a hydrophilic outside. Generally speaking, cyclodextrin molecules do not penetrate lipophilic membranes since they are rather big molecules with many hydrogen sources and acceptors. The principal application of cyclodextrins in the pharmaceutical industry has been as complexing agents to improve the aqueous solubility, bioavailability, and stability of poorly soluble medicines. Drugs' bioavailability is one of the many uses for cyclodextrins in medicinal applications[4]. By rapidly removing cholesterol from the plasma membrane, methyl-β-cyclodextrin causes PEL cells to undergo caspase-dependent death. All PEL cell lines are inhibited in their development by methyl-β-cyclodextrin in a way that is dose dependent. Every cell line has an IC50 of 3.33–4.23 mM[5]. Among the several agents that deplete cholesterol from cells, methyl-β-cyclodextrin—a highly soluble cyclic heptasaccharide with a β glucopyranose unit—has been found to be the most efficient[5].

Methyl-β-cyclodextrin effectively suppresses PEL cell growth and invasion in a PEL xenograft mice model without causing any evident side effects. Mice treated with methyl-β-cyclodextrin seem healthy, whereas those not treated have enlarged abdomens. The mice treated with methyl-β-cyclodextrin had significantly lower body weights than the control group. Mice treated with methyl-β-cyclodextrin had a much smaller volume of ascites than mice not treated with it[4]. Cyclodextrins have been found in studies on humans and animals to be useful in enhancing the distribution of nearly any kind of medication formulation. Around 30 distinct pharmaceutical products with drug/cyclodextrin complexes are available on the market at the moment[6] throughout the world.

Tetrazolium dye methylthiotetrazole (MTT) assay[5]
The antiproliferative activities of M-β-CyD against PEL cell lines were measured by the methylthiotetrazole (MTT) method. Briefly, 2 × 104 cells were incubated in triplicate in a 96-well microculture plate in the presence of different concentrations of M-β-CyD (0–10 mM) in a final volume of 0.1 ml for 24 h at 37 °C. Subsequently, MTT (0.5 mg/ml final concentration) was added to each well. After 3 h of additional incubation, 100 μl of a 0.04 N HCl was added to dissolve the crystals. Absorption values at 570 nm were determined with an automatic enzyme-linked immunosorbent assay (ELISA) plate reader. Values were normalized to untreated (control) samples.

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Cell viability assay[5]
Cell viability was examined by the propidium iodide (PI) exclusion method as described previously. Briefly, BCBL-1 cells (2 × 105 cells/ml) were cultured in the presence or absence of M-β-CyD for 1–6 h in 6-well culture plates. After being incubated, cells were stained with PI (final concentration; 2 μg/ml) and cell viability was analyzed by LSR II flow cytometry.

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PEL cells are incubated in triplicate in a 96-well microculture plate in the presence of different concentrations of methyl-β-cyclodextrin (0-10 mM) in a final volume of 0.1 mL for 24 h at 37°C. Subsequently, MTT (0.5 mg/mL final concentration) is added to each well. After 3 h of additional incubation, 100 μL of a 0.04 N HCl is added to dissolve the crystals. Absorption values at 570 nm are determined[1].

NOD/Rag-2/Jak3-double deficient (NRJ) mice were housed and monitored in our animal research facility according to institutional guidelines. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at Kumamoto University. Eight- to ten-week-old female NRJ mice were intraperitoneally inoculated with 7 × 106 BCBL-1 cells suspended in 200 μl PBS. The mice were then treated with intraperitoneal injections of PBS or M-β-CyD (500 mg/kg per day). Tumor burdens were evaluated by measuring body weights and ascites.[5]

[1]. Methyl-β-cyclodextrin, an actin depolymerizer augments the antiproliferative potential of microtubule-targeting agents. Sci Rep. 2019 May 21;9(1):7638.

[2]. Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell. 1999;10(4):961-974.

[3]. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat Cell Biol. 2019 Aug;21(8):991-1002.

[4]. Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J Biol Chem. 2002 Dec 20;277(51):49631-7.

[5]. The antitumor effects of methyl-β-cyclodextrin against primary effusion lymphoma via the depletion of cholesterol from lipid rafts. Biochem Biophys Res Commun. 2014 Dec 12;455(3-4):285-9.

[6]. Cyclodextrins in delivery systems: Applications. J Pharm Bioallied Sci. 2010 Apr;2(2):72-9.

Cyclodextrins (CDs) are a family of cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity. CD molecules are relatively large with a number of hydrogen donors and acceptors and, thus in general, they do not permeate lipophilic membranes. In the pharmaceutical industry, CDs have mainly been used as complexing agents to increase aqueous solubility of poorly soluble drugs and to increase their bioavailability and stability. CDs are used in pharmaceutical applications for numerous purposes, including improving the bioavailability of drugs. Current CD-based therapeutics is described and possible future applications are discussed. CD-containing polymers are reviewed and their use in drug delivery is presented. Of specific interest is the use of CD-containing polymers to provide unique capabilities for the delivery of nucleic acids. Studies in both humans and animals have shown that CDs can be used to improve drug delivery from almost any type of drug formulation. Currently, there are approximately 30 different pharmaceutical products worldwide containing drug/CD complexes in the market.[6]

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Primary effusion lymphoma (PEL) is a subtype of aggressive and chemotherapy-resistant non-Hodgkin lymphoma that occurs predominantly in patients with advanced AIDS. In this study, we examined the antitumor activity of methyl-β-cyclodextrin (M-β-CyD) in vitro and in vivo. M-β-CyD quickly induced caspase-dependent apoptosis in PEL cells via cholesterol depletion from the plasma membrane. In a PEL xenograft mouse model, M-β-CyD significantly inhibited the growth and invasion of PEL cells without apparent adverse effects. These results strongly suggest that M-β-CyD has the potential to be an effective antitumor agent against PEL.[5]

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Methyl-β-cyclodextrin (MCD), an established pharmacological excipient, depolymerizes the actin cytoskeleton. In this work, we investigated the effect of MCD-mediated actin depolymerization on various cellular phenotypes including traction force, cell stiffness, focal adhesions, and intracellular drug accumulation. In addition to a reduction in the contractile cellular traction, MCD acutely inhibits the maturation of focal adhesions. Alteration of contractile forces and focal adhesions affects the trypsin-mediated detachment kinetics of cells. Moreover, MCD-mediated actin depolymerization increases the intracellular accumulation of microtubule-targeting agents (MTAs) by ~50% with respect to the untreated cells. As MCD treatment enhances the intracellular concentration of drugs, we hypothesized that the MCD-sensitized cancer cells could be effectively killed by low doses of MTAs. Our results in cervical, breast, hepatocellular, prostate cancer and multidrug-resistant breast cancer cells confirmed the above hypothesis. Further, the combined use of MCD and MTAs synergistically inhibits the proliferation of tumor cells. These results indicate the potential use of MCD in combination with MTAs for cancer chemotherapy and suggest that targeting both actin and microtubules simultaneously may be useful for cancer therapy. Importantly, the results provide significant insight into the crosstalk between actin and microtubules in regulating the traction force and dynamics of cell deadhesion.[1]

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The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.[2]

C54H94O35

1303.3032

1302.557

128446-36-6

51051622

White to off-white solid powder

1.4±0.1 g/cm3

1206.9±65.0 °C at 760 mmHg

180-182ºC

683.7±34.3 °C

0.0±0.6 mmHg at 25°C

1.567

6.95

9

35

19

89

2050

35

CO[C@H]1[C@@H]([C@H]2[C@H](O[C@@H]1O[C@@H]3[C@H](O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]4[C@H](O[C@@H]([C@H]([C@@H]4OC)OC)O[C@@H]5[C@H](O[C@@H]([C@H]([C@@H]5OC)OC)O[C@@H]6[C@H](O[C@@H]([C@H]([C@@H]6OC)OC)O[C@@H]7[C@H](O[C@@H]([C@H]([C@@H]7OC)OC)O[C@@H]8[C@H](O[C@H](O2)[C@H]([C@@H]8O)OC)CO)CO)CO)CO)CO)CO)CO)O

YZOUYRAONFXZSI-SBHWVFSVSA-N

InChI=1S/C54H94O35/c1-64-36-28(63)30-21(14-56)76-48(36)83-29-20(13-55)77-49(37(65-2)27(29)62)85-31-22(15-57)79-51(44(72-9)38(31)66-3)87-33-24(17-59)81-53(46(74-11)40(33)68-5)89-35-26(19-61)82-54(47(75-12)42(35)70-7)88-34-25(18-60)80-52(45(73-10)41(34)69-6)86-32-23(16-58)78-50(84-30)43(71-8)39(32)67-4/h20-63H,13-19H2,1-12H3/t20-,21-,22-,23-,24-,25-,26-,27-,28-,29-,30-,31-,32-,33-,34-,35-,36+,37+,38-,39-,40-,41-,42-,43+,44+,45+,46+,47+,48-,49-,50-,51-,52-,53-,54-/m1/s1

(1S,3R,5R,6R,8R,10R,11R,13R,15R,16R,18R,20R,21R,23R,25R,26R,28R,30R,31S,33R,35R,36R,37S,38R,39S,40R,41S,42R,43S,44R,45S,46R,47S,48R,49S)-5,10,15,20,25,30,35-heptakis(hydroxymethyl)-37,39,40,41,42,43,44,45,46,47,48,49-dodecamethoxy-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontane-36,38-diol

128446-36-6; Methyl-b-cyclodextrin; Methyl-; A-cyclodextrin; MFCD00074980;

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 : ≥ 100 mg/mL
H2O : ≥ 50 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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