Apoptosis inducer; anthelmintic

Researchers found that the mechanism by which oxbendazole inhibited the proliferation of pig trophoblast (pTr) and porcine luminal epithelial (pLE) cells was intracellular cell signaling. When 200 nM dosages of oxbendazole were applied to both cell types, the phosphorylation of ERK1/2, P90RSK, and S6 decreased while the expression of phosphorylated JNK, AKT, and P70S6K increased [1].

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Oxibendazole (OBZ) inhibits growth of 22Rv1 and PC-3 cells.[2]
The ability of OBZ to inhibit the growth of 22Rv1 and PC-3 cells was determined by counting cell number. OBZ markedly inhibited the cell viability of 22Rv1 and PC-3 cells in a dose-dependent manner (Fig. 1A). As little as 0.12 µM of OBZ was observed to significantly inhibit the growth of the 22Rv1 and PC-3 cells, respectively (both P<0.05). The 22Rv1 cells were more sensitive to OBZ treatment, with a half-maximal inhibitory concentration (IC50) value of 0.25 µM, compared with 0.64 µM in PC-3 cells. OBZ inhibited the cell viability of 22Rv1 and PC-3 cells in a time-dependent manner (Fig. 1B and C). These results demonstrated that OBZ inhibited the growth of PCa cells in vitro with varied efficiency.

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Oxibendazole (OBZ) causes apoptosis of 22Rv1 and PC-3 cells. [2]
The apoptosis-inducing capability of OBZ in 22Rv1 and PC-3 cells was evaluated by Annexin V-FITC and PI double staining. Provided that the IC50 value was 0.25 µM in 22Rv1 cells, 0.25 µM OBZ was used to treat 22Rv1 and PC-3 cells for 48 h. A notable increase in the number of apoptotic cells was observed in the OBZ-treated group compared with DMSO-treated cells (the negative control) (Fig. 2A and B). The apoptotic rate of 22Rv1 cells was 1.41% in DMSO-treated cells and 9.45% in OBZ-treated cells. The apoptotic rate in PC-3 cells was 0.92 and 4.58% in DMSO- and OBZ-treated cells, respectively (Fig. 2C). These results indicated that treatment with OBZ resulted in an increased apoptotic rate in PCa cells, and the apoptosis-inducing capability of OBZ was more marked in 22Rv1 compared with PC-3 cells.

When given oxbendazole (25 mg/kg/day) in nude mice, the average size of 22Rv1 tumors was 47.96% lower than in control animals. Oxbendazole treatment triggers an upregulation of microRNA -204 (miR-204) expression [2].

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OBZ inhibits 22Rv1 tumor growth in nude mice. The antitumor effect of OBZ was next evaluated in vivo. First, 22Rv1 cells were injected into the right flank of nude mice. Approximately 10 days after injection of the cells, the tumor sizes were measurable. On day 10, the mice were treated with OBZ (25 mg/kg) by intragastric gavage. The treatment was administered once a day for 14 days. The control group of mice was treated in the same way, but OBX was substituted with corn oil. OBZ significantly repressed tumor growth in a time-dependent manner, with a significant difference identified at 20 days after cancer cell inoculation (P<0.05; Fig. 3A). The mean tumor volume of the OBZ-treated group was 0.63 cm3, whereas in the control group it was 1.20 cm3; OBZ inhibited growth of the tumor by ~47.96%. Additionally, the mean body weight of the tumor-bearing mice was 24.06±1.28 and 23.10±3.39 g in the OBZ-treated and control groups, respectively. However, this difference was not statistically significant, demonstrating that OBZ did not exert a significant general toxic effect in vivo, consistent with the results of a previous study.[2]
Oxibendazole might lead to early pregnancy failure in pigs.[1]

Cell proliferation assays[2]
Cells were seeded in 96-well plates at a cell density of 1×104 cells per well in 100 µl RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C with an atmosphere of 5% CO2 overnight. 22Rv1 and PC-3 cells were treated with 0.12, 0.25, 0.50, 1.00, 2.00 and 3.00 µM Oxibendazole (OBZ) for 48 h, or with 0.25 and 1.00 µM Oxibendazole (OBZ) for 96 h. In order to assess the role of miR-204 in mediating the effect of Oxibendazole (OBZ), 22Rv1 and PC-3 cells were transfected with the miR-204 or miR-204 inhibitor, followed by treatment with 1 µM OBZ or dimethyl sulfoxide (DMSO; as a control) for 48 h. Cells were trypsinized and live cell numbers were counted in four areas under an inverted microscope (magnification, ×40) using a hemocytometer and the trypan blue exclusion assay.

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Flow cytometry[2]
Apoptosis was determined using a double-staining Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit. The 22Rv1 and PC-3 cells were treated with DMSO control or 0.25 µM Oxibendazole (OBZ). After 48 h, the cells were collected, washed in phosphate-buffered saline (PBS) and suspended in binding buffer. The cells were then stained using annexin V and propidium iodide (PI) (5 µl). Following incubation for 15 min at room temperature in the dark, the cells were diluted and analyzed using a flow cytometer. When green fluorescence (FITC) was plotted against red fluorescence (PI), the cell populations could be detected in a dot-plot that indicated the following conditions: Viable cells (FITC−/PI−), early apoptotic cells (FITC+/PI−) and late apoptotic cells (FITC+/PI+). The data were reported as the percentage of early apoptotic cells (FITC+/PI−) and late apoptotic cells (FITC+/PI+). Oxibendazole significantly reduced the viability of porcine trophectoderm and uterine luminal epithelial cells[1]
We analyzed the effects of oxibendazole on the proliferation of porcine trophectoderm (pTr) cells and porcine uterine luminal epithelial (pLE) cells by using BrdU reagents to assess their cell viability. When different doses of oxibendazole were compared, a 200 nM dose of oxibendazole was found to have led to greatly decreased proliferation of pTr and pLE cells (Fig. 1A and B). Specifically, 45.7% of pTr cells and 46.5% of pLE cells were found to be viable at 200 nM of oxibendazole, which was...

Xenograft tumor development in nude mice[2]
At the exponential growth stage, 22Rv1 cells were harvested, washed and suspended in PBS. A trypan blue exclusion assay was performed to ensure cell viability (>99%) prior to inoculation. The cells were counted and 2×106 cells suspended in 0.1 ml PBS were subcutaneously injected into the right flank of each mouse. At 10 days after tumor cell inoculation, each mouse in the Oxibendazole (OBZ)-treated group was provided with 25 mg/kg homogeneous suspension of Oxibendazole (OBZ) by intragastric gavage. The treatment was administered once a day for 14 days; mice in the control group was provided with the same amount of corn oil. Tumor size was measured in two dimensions every other day. Tumor volume (measured in cm3) was calculated using the following formula: Tumor volume=axb2×0.5 (a, length; b, width).

Metabolism / Metabolites
Hepatic

mouse LDLo oral 32 gm/kg American Journal of Veterinary Research., 38(809), 1977 [PMID:560153]

[1]. Oxibendazole induces apoptotic cell death in proliferating porcine trophectoderm and uterine luminal epithelial cells via mitochondria-mediated calcium disruption and breakdown of mitochondrial membrane potential. Comp Biochem Physiol C Toxicol Pharmacol. 2019 Jun;220:9-19.

[2]. Oxibendazole inhibits prostate cancer cell growth. Oncol Lett. 2018 Feb;15(2):2218-2226.

N-(6-propoxy-1H-benzimidazol-2-yl)carbamic acid methyl ester is a member of benzimidazoles and a carbamate ester.
Oxibendazole is a polymerase inhibitor in phase III trials for the treatment of helminth intestinal infections.
See also: Diethylcarbamazine Citrate; Oxibendazole (component of).

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Drug Indication
Investigated for use/treatment in infectious and parasitic disease (unspecified) and pediatric indications.

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Mechanism of Action
Oxibendazole causes degenerative alterations in the tegument and intestinal cells of the worm by binding to the colchicine-sensitive site of tubulin, thus inhibiting its polymerization or assembly into microtubules. The loss of the cytoplasmic microtubules leads to impaired uptake of glucose by the larval and adult stages of the susceptible parasites, and depletes their glycogen stores. Degenerative changes in the endoplasmic reticulum, the mitochondria of the germinal layer, and the subsequent release of lysosomes result in decreased production of adenosine triphosphate (ATP), which is the energy required for the survival of the helminth. Due to diminished energy production, the parasite is immobilized and eventually dies.

C12H15N3O3

249.2658

249.111

C, 57.82; H, 6.07; N, 16.86; O, 19.26

20559-55-1

4622

White to off-white solid powder

1.3±0.1 g/cm3

459ºC

230-231°C

1.635

2.5

2

4

5

18

288

0

RAOCRURYZCVHMG-UHFFFAOYSA-N

InChI=1S/C12H15N3O3/c1-3-6-18-8-4-5-9-10(7-8)14-11(13-9)15-12(16)17-2/h4-5,7H,3,6H2,1-2H3,(H2,13,14,15,16)

methyl N-(6-propoxy-1H-benzimidazol-2-yl)carbamate

Oxibendazole; 20559-55-1; Loditac; Filaribits Plus; Anthelcide EQ; Oxibendazolo; Oxibendazolum; SK&F 30310;

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 : ~5 mg/mL (~20.06 mM)

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.)