Vehicle for drug delivery

Treatment of cells with HP-β-CD activates the transcription factor EB, a fundamental regulator of lysosomal activity and autophagy, and promotes autophagic clearance [1]. HP-β-CD therapy lowered intracellular cholesterol and effectively suppressed leukemic cell proliferation through G2/M cell cycle arrest and death. After 72 hours of exposure, the IC50 values of HP-β-CD were in the range of 3.86-10.09 mM. HP-β-CD also displayed anticancer effects on CML cells bearing the T315I BCR-ABL mutation (conferring resistance to most ABL tyrosine kinase inhibitors) and hypoxia-adapted CML cells with leukemic stem cell features. In addition, the colony-forming ability of human primary AML and CML cells is reduced by HP-β-CD [2].

Because cells generated from patients with lysosomal storage illnesses have reduced activity of the lysosomal autophagy system, HP-β-CD treatment increases transcription factor EB-mediated clearance of proteolipid aggregates and accumulates [1]. Leukemia mice models can have a much higher survival rate when HP-β-CD is injected intraperitoneally. Mice given HP-β-CD systemically did not exhibit any overt negative effects [2].

Effects of HP-β-CyD on in vitro cell growth[2]
Cell viability was assessed using a trypan blue dye exclusion method and cell proliferation was evaluated using a modified methyl-thiazol-diphenyl- tetrazolium (MTT) assay with SF reagent as described previously. Cells, including human primary hepatocytes were seeded in flat-bottomed 96-well plates at a density of 1×104 cells in 100 μL medium per well, and incubated with HP-β-CyD at various concentrations for 72 hours. The mean of three replicates was calculated for each concentration.

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Western blot analysis[2]
Whole cell lysates of leukemic cells treated with or without HP-β-CyD were prepared from cells using lysis buffer, as reported previously, with minor modification. Protein was separated using a 10% NuPAGE electrophoresis system, transferred to a nitrocellulose membrane, blocked with 5% bovine serum albumin at room temperature for 1 hour, and incubated with primary antibodies at 4°C overnight. Antibodies against Akt, phosphorylated-Akt (Thr308 or Ser473), phospholyrated-Erk1/2 (Thr202/Thr204), phospholyrated-Stat5, Lyn, Stat5, Erk1/2, Actin, and phospholyrated-Lyn were used as primary antibodies. Horseradish peroxidase-coupled immunoglobulin IgG was used as the secondary antibody. An enhanced chemiluminescence kit was used for detection. The results are representative of at least two independent experiments. Intensity of the immunoblot signals after background subtraction was quantified using ImageJ software.

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Cell-cycle analysis[2]
Cell-cycle analyses of human leukemic cell lines were performed as described previously. In brief, 1×106 cells were treated with the indicated concentration of HP-β-CyD. Twelve or twenty-four hours after HP-β-CyD treatment, cells were collected and fixed with 70% ethanol. Cells were then incubated with 0.1% Triton X-100 and 0.5% RNase A at room temparature for 30 minutes and stained with 50 μg/mL propidium iodide. Cellular DNA content was analyzed by flow cytometry, and cell-cycle profiles were determined using a FACS Caliber flow cytometer with CellQuest software. Data are the mean ± SD of three independent experiments.

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Apoptosis assays[2]
Apoptosis assay was performed by staining cells with 7-amino-actinomycin D (7-AAD) and annexin V, according to the manufacturer’s instructions. Cells were cultured in 6-well plate at a density of 4×105 cells, and incubated with various concentrations of HP-β-CyD for 12 or 24 hours. Then, cells were stained with 7-amino-actinomycin D (7-AAD) and Annexin V-APC, and analyzed using a FACSAriaII system with Diva software.

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Hematopoietic colony-forming assays[2]
HP-β-CyD toxicity in normal hematopoietic progenitors was investigated using a standard methylcellulose culture assay as described previously. A total of 2×104 mononuclear cells from the BM of 10-week-old C57BL/6N mice were exposed to 0, 5, 15, or 25 mM HP-β-CyD in 1 mL MethoCult M3434 . After 8 days of culture, the number of colonies was counted using an inverted microscope. Data represent the mean number of colonies ± SD (n = 3). Clinical samples were obtained with informed consent. Mononuclear cells from leukemia patients were cultured in semi-solid medium containing recombinant cytokines.

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Cholesterol assays[2]
Leukemic cells (3×106) were incubated with 5 or 10 mM HP-β-CyD in HBSS (pH 7.4) at 37°C for 1, 2, or 3 hours. Cell culture supernatants were recovered by centrifugation (3,000 rpm, 5 min). The concentration of total cholesterol in the supernatants was determined using a Cholesterol E-test Wako. Data are the mean ± SD of three experiments. Cellular lipids were extracted with methanol:chloroform (1:2), and total cholesterol and free cholesterol were determined enzymatically. The amount of esterified cholesterol was calculated by subtracting free cholesterol from total cholesterol. Cellular protein concentration was determined by BCA assay. Data are the mean ± SD of three experiments. For filipin staining, cells were incubated with β-CyDs (10 mM) for 1 hour. Thereafter, cellular cholesterol was detected using a Cholesterol Cell-Based Detection Assay Kit.

Murine leukemia model[2]
Two different experimental settings were used. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Saga University (Permit number: 25-028-0). First, nude mice were intravenously transplanted with 1×106 EGFP+ Ba/F3 BCR-ABLWT cells. These mice were intraperitoneally injected with 200 μL vehicle (saline) or HP-β-CyD (50 or 150 mM) for 20 consecutive days 3 days after transplantation, and survival was monitored daily. Leukemic cell engraftment was confirmed by detection of GFP-positive cells in the recipient’s BM using flow cytometry.[2]
The second experimental setting involved a human leukemia xenograft model. BV173 cells (1×106) were intravenously injected into sublethally irradiated (2 Gy) NOD/SCID mice. After 72 hours, xenotransplanted mice were intraperitoneally injected with 200 μL vehicle or HP-β-CyD (50 or 150 mM) for 5 consecutive days every week for 13 weeks, and survival was monitored daily. The percentage of human leukemic cells in BM was determined by flow cytometry after double staining with FITC-conjugated anti-human CD19 and PE/Cy7-conjugated anti-mouse CD45 antibodies. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Mice were euthanized with ether when they became moribund or unable to obtain food or water, as recommended by the institutional guidelines of Saga University. Survival data were analyzed by a log-rank nonparametric test and shown as Kaplan-Meier survival curves (n = 10 for each group).[2]

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Lung histology[2]
HP-β-CyD was administered to NOD/SCID mice for 13 weeks as described in the above section entitled “Murine leukemia model”. Age-matched mice were used as a control. Lungs were perfused with 10% buffered formalin and excised. Tissues were fixed in 10% buffered formalin and embedded in paraffin. These blocks were then sectioned and stained with hematoxylin and eosin (H&E).

[1]. 2-Hydroxypropyl-β-cyclodextrin promotes transcription factor EB-mediated activation of autophagy: implications for therapy. J Biol Chem. 2014 Apr 4;289(14):10211-22.

[2]. 2-Hydroxypropyl-β-Cyclodextrin Acts as a Novel Anticancer Agent. PLoS One. 2015 Nov 4;10(11):e0141946.

Derivative of beta-cyclodextrin that is used as an excipient for steroid drugs and as a lipid chelator.

C63H12O42

1541.54

1540.662

128446-35-5

128446-35-5 ;107745-73-3;

4363642

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

1.4±0.1 g/cm3

1521.9±60.0 °C at 760 mmHg

278ºC (dec.)

874.2±32.9 °C

0.0±0.6 mmHg at 25°C

1.545

-6.23

21

42

28

105

2010

0

CO[C@@H]1[C@H](O[R])[C@@H](O)[C@H](C)[C@@H](CO[R])O1.[C;D2]CC(O)C.[R].[7].[R=H or]

ODLHGICHYURWBS-FOSILIAISA-N

InChI=1S/C63H112O42/c1-22(64)8-85-15-29-50-36(71)43(78)57(92-29)100-51-30(16-86-9-23(2)65)94-59(45(80)38(51)73)102-53-32(18-88-11-25(4)67)96-61(47(82)40(53)75)104-55-34(20-90-13-27(6)69)98-63(49(84)42(55)77)105-56-35(21-91-14-28(7)70)97-62(48(83)41(56)76)103-54-33(19-89-12-26(5)68)95-60(46(81)39(54)74)101-52-31(17-87-10-24(3)66)93-58(99-50)44(79)37(52)72/h22-84H,8-21H2,1-7H3/t22?,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-,55-,56-,57-,58-,59-,60-,61-,62-,63-/m0/s1

(1R,3S,5S,6R,8S,10S,11R,13S,15S,16R,18S,20S,21R,23S,25S,26R,28S,30S,31R,33S,35S,36S,37S,38S,39S,40S,41S,42S,43S,44S,45S,46S,47S,48S,49S)-5,10,15,20,25,30,35-heptakis(2-hydroxypropoxymethyl)-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-36,37,38,39,40,41,42,43,44,45,46,47,48,49-tetradecol

Hydroxypropyl betadex (2-Hydroxypropyl)-β-cyclodextrinHydroxypropyl-β-cyclodextrin; HP-β-CD; (2-Hydroxypropyl)-; A-cyclodextrin; MFCD00069372; HP-??cyclodextrin; ODLHGICHYURWBS-FOSILIAISA-N; HMS3885J22; s4760;

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 : ~50 mg/mL
H2O : ~50 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (Infinity 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 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: ≥ 2.08 mg/mL (Infinity 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 3: ≥ 2.08 mg/mL (Infinity 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 4: 100 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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Solubility in Formulation 5: 200 mg/mL (Infinity mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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