Target: Tubulin (β‑tubulin, colchicine‑binding site) – Fenbendazole binds to free tubulin and mildly inhibits polymerization; Competitive colchicine binding assay suggests binding at the colchicine site (Fig. S1) [1].
Target: Hypoxia‑inducible factor (HIF) pathway – Fenbendazole stabilizes HIF‑1α protein and induces HIF‑dependent gene expression (e.g., p21) via free tubulin binding, not via mitochondrial inhibition [2].

When applied to human non-small cell lung cancer (NSCLC) tumor cell lines (H460 and A549) that have wild-type p53, fenbendazole (1 uM; 24 hours) dramatically slows cell growth [1]. Perfect p53 protein levels are raised and cell engraftment is induced by fenbendazole (1 uM; 24 hours) [1]. Human NSCLC cells undergo the mitotic phase of the cell life cycle when exposed to 1 uM of fenbendazole for 24 hours [1]. Azole (1 uM; 24 hours) can partially alter the A549 cells' microtubule network [1].

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In Vitro: Fenbendazole (1 μM, 24 h) partially alters the microtubule network in A549 human NSCLC cells as shown by immunofluorescence (α‑tubulin staining), causing loss of intactness of the microtubule cage around the nucleus, but less pronounced than colchicine (50 ng/ml) which causes complete depolymerization [1].
In an in vitro tubulin polymerization assay using purified bovine tubulin (1.8 mg/ml), Fenbendazole (10 μM) caused mild inhibition of polymerization measured by turbidity at 340 nm, whereas colchicine (100 nM) caused stronger inhibition [1].
Fenbendazole (1 μM, 24 h) modestly decreased polymerized tubulin fraction in A549 cells (soluble/polymerized tubulin assay) compared to control, while nocodazole and colchicine nearly abolished polymerized tubulin. Acetylated tubulin levels were not altered by Fenbendazole, in contrast to nocodazole, colchicine and vincristine which markedly reduced acetylation [1].
Fenbendazole (1 μM) induced early elevation of cyclin B1/CDK1 levels (8 h vs 16 h in control) and increased phospho‑histone H3 (Ser10) at 12‑24 h, indicating mitotic arrest. Flow cytometry showed time‑dependent increase in sub‑G1 apoptotic cells (∼30% at 32 h) [1].
Fenbendazole (1 μM) significantly reduced cell growth in H460 and A549 NSCLC cells (MTT assay). Tumor cell lines with wild‑type p53 were more sensitive than p53 mutant/null cells. FZ induced p53 target genes (p21, MDM2) and increased nuclear p53 accumulation. In p53‑null H1299 cells transfected with WT p53, FZ enhanced apoptosis [1].
Fenbendazole (1 μM, 4 h) inhibited glucose uptake in H460 and A549 cells (2‑NBDG fluorescence assay) and reduced lactate levels in culture supernatants. It decreased mRNA expression of GLUT‑4 and hexokinase II (HKII) and increased p53‑regulated metabolic genes (TIGAR, SCO2, GLS2, proline oxidase) [1].
Fenbendazole (1 μM, 24 h) reduced hexokinase II enzymatic activity in cell lysates (spectrophotometric assay). In vitro, purified yeast HKII activity was inhibited by increasing doses of Fenbendazole (0‑100 μM) [1].
Fenbendazole (1 μM, 24 h) increased mitochondrial p53 translocation (GFP‑p53 transfection and Western blot of mitochondrial fractions), accompanied by mitochondrial membrane depolarization (JC‑1 staining) [1].
In primary mouse cortical neurons, Fenbendazole (1 μM, 8 h) increased p21 mRNA (real‑time PCR) and at 1 μM (20 h) increased p21 protein (Western blot). It also stabilized HIF‑1α protein in HT22 cells (4 h) and increased ODD‑Luc reporter activity (a surrogate for HIF‑1α stability) in a concentration‑dependent manner (0.5‑2 μM) [2].
Fenbendazole (0.25‑1 μM) protected embryonic cortical neurons from homocysteic acid (HCA)‑induced oxidative stress in a dose‑dependent manner (MTT assay). Neuroprotection was abrogated by pretreatment with Taxol (0.1 μM), which reduces free tubulin levels. At neuroprotective concentrations (≤1 μM), Fenbendazole did not disrupt neuronal morphology (β‑tubulin III immunostaining) [2].
Fenbendazole (10 μM) had no effect on isolated mouse liver mitochondrial respiration rates (State 4 and State 3), membrane potential (Safranine O), or ROS emission (Amplex Red), indicating that its HIF‑stabilizing effect is not due to direct mitochondrial dysfunction [2].
Fenbendazole did not inhibit fumarase activity in vitro (unlike mebendazole), as measured by continuous optical density assay at 240 nm [2].

For 12 days, fenbendazole (1 mg; oral; once daily) can dramatically reduce the weight and size of tumors [1].
In Vivo: In female athymic nu/nu mice bearing A549 human NSCLC xenografts (tumor size 2‑3 mm), oral administration of Fenbendazole (1 mg/mouse) every second day for 12 days significantly reduced tumor volume and weight compared to vehicle control (olive oil). Tumor vascularity (hemoglobin content) was reduced, and TUNEL staining showed increased apoptosis. Immunohistochemistry revealed increased p53‑positive cells and decreased CD31‑positive endothelial cells in FZ‑treated tumors [1].
In a separate study, Fenbendazole was not tested in vivo for neuroprotection; however, mebendazole (a related benzimidazole) at 50 mg/kg oral gavage for 5 days increased ODD‑Luc activity in the brain of transgenic mice, indicating HIF stabilization [2].
Fenbendazole has been reported to have a high safety margin and low toxicity in experimental animals (referenced in [1]), but no specific in vivo efficacy data for neurological models are provided in these papers [1][2].

Cell cycle analysis [1]
Cell Types: A549 cells
Tested Concentrations: 1 uM
Incubation Duration: For 24 h
Experimental Results: Caused an early increase in cyclin B1/CDK1 levels (8 hrs (hours) compared to 16 hrs (hours) for control untreated cells). p-Histone H3 (Ser10) was found to be upregulated at 12 and 24 hrs (hours).

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Apoptosis analysis [1]
Cell Types: A549 Cell
Tested Concentrations: 1 uM
Incubation Duration: 8, 16, 24, 32, 40, 48 hrs (hours)
Experimental Results: The number of apoptotic cells increased in a time-dependent manner, along with cyclin B1 levels diminished, and approximately 30% of the cells underwent apoptosis after 32 hrs (hours).

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Western Blot Analysis[1]
Cell Types: H460 Cell
Tested Concentrations: 1 uM
Incubation Duration: 24 hrs (hours)
Experimental Results: Resulted in increased p53 protein levels in mitochondrial fractions.

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Cell Assay (cancer cells): Human NSCLC A549, H460, and H1299 cells were cultured in DMEM with 10% FBS. For immunofluorescence, cells on coverslips were treated with Fenbendazole (1 μM) or colchicine (50 ng/ml) for 24 h, permeabilized, fixed, stained with anti‑α‑tubulin primary and FITC‑secondary antibodies, and nuclei counterstained with propidium iodide [1].
Tubulin polymerization assay: Purified bovine tubulin (1.8 mg/ml) in PEM buffer (80 mM PIPES, 1 mM EGTA, 0.5 mM MgCl₂, 1 mM GTP) was incubated with DMSO, Fenbendazole (10 μM), or colchicine (100 nM). Polymerization was monitored by absorbance at 340 nm over 30 min [1].
Soluble/polymerized tubulin fractionation: Cells treated for 24 h were lysed in microtubule stabilization buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl₂, 0.1 mM EDTA, 4 M glycerol, 0.1% NP‑40, 1 mM GTP, 1 mM PMSF, protease inhibitors), centrifuged at 100,000 g for 30 min to separate soluble (supernatant) and polymerized (pellet) fractions. Western blot with anti‑α‑tubulin and β‑actin [1].
Cell cycle and apoptosis: Synchronized A549 cells treated with 1 μM Fenbendazole for indicated times, fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis also assessed by annexin V/PI staining [1].
MTT viability assay: Cells in 96‑well plates treated with compounds for 24‑48 h, then MTT added, formazan dissolved in DMSO, absorbance at 570 nm [1].
Western blot: Cells lysed in NP‑40 buffer, proteins separated by SDS‑PAGE, transferred to PVDF, probed with antibodies against cyclin B1, CDK1, pH3(Ser10), p53, p21, MDM2, Ac‑α‑tubulin, β‑actin, COX IV, etc. [1].
Glucose uptake: Cells treated with 1 μM Fenbendazole for 4 h, then incubated with 100 μM 2‑NBDG for 1 h, washed, and fluorescence measured (ex/em 485/528 nm) [1].
Lactate and glucose oxidation assays: Culture supernatants assayed using commercial kits (glucose oxidase/peroxidase and lactate assay) [1].
Hexokinase activity: Cell lysates or purified yeast HKII incubated with reaction mix (8.3 mM glycylglycine, 17 mM ATP, 0.0011% cresol red, 14 mM MgCl₂, 27 mM glucose), decrease in A₅₆₀ measured [1].
Mitochondrial isolation and p53 translocation: Cells treated for 24 h, mitochondria isolated using commercial kit, fractions analyzed by Western blot [1].
Cell Assay (neurons): Primary cortical neurons from E15 CD‑1 mouse embryos cultured in MEM with 10% FBS/5% horse serum. For neuroprotection, neurons co‑treated with HCA (5 mM) and Fenbendazole (0.125‑2 μM) for 16 h, viability assessed by MTT or live/dead staining (calcein AM/ethidium homodimer‑1). For Taxol pretreatment, 0.1 μM Taxol added 30 min before HCA and FZ [2].
HT22 HRE‑Luc and ODD‑Luc reporter assays: HT22 cells stably transfected with HRE‑luciferase or ODD‑luciferase were treated with compounds for 4‑24 h, lysed, and luciferase activity measured and normalized to protein [2].
Immunocytochemistry (neurons): Cells fixed with 4% paraformaldehyde, stained with anti‑β‑tubulin III antibody and secondary antibody, visualized by fluorescence microscopy [2].
Mitochondrial assays: Non‑synaptic mouse brain mitochondria isolated by Percoll density gradient. Respiration measured with Oxytherm oximeter (State 4 and State 3 with ADP). Membrane potential measured by Safranine O fluorescence (ex/em 495/586 nm). ROS emission measured by Amplex Red (ex/em 555/581 nm) with HRP and SOD [2].
Fumarase activity: Mitochondrial lysates incubated with malate (0.05‑0.4 mM) and varying benzimidazole concentrations, fumarate production monitored at A₂₄₀ [2].

Animal/Disease Models: Female athymic nu/nu (nude) mice xenografted with A549 cells[1]: 1 mg/mouse
Route of Administration: Oral; every other day for 12 days
Experimental Results: Result in significant reduction in tumor size and weight . The resulting decrease in hemoglobin content in the tumor means a decrease in the tumor's vascular supply.

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Animal Protocol (xenograft): Female athymic nu/nu mice (6 weeks old) were acclimated 3 days, then 5 × 10⁶ A549 cells in 100 μl PBS were injected subcutaneously into the right flank. When tumors reached 2‑3 mm in diameter (∼4 weeks), mice were orally gavaged with Fenbendazole (1 mg/mouse) dissolved in olive oil, every second day for 12 days. Control mice received olive oil only. Tumor volumes measured with digital calipers (volume = width² × length/2). At endpoint, tumors excised, weighed, and processed for hemoglobin measurement (spectrophotometry at A₅₉₀), TUNEL staining, p53 and CD31 immunohistochemistry. All procedures approved by Institutional Animal Ethics Committee [1].
Animal Protocol (HIF reporter): ODD‑Luc transgenic mice (ADU FB.129S6‑Gt(ROSA)26Sortm2(HIF1A/luc)Kael/J) were treated with mebendazole (50 mg/kg) or vehicle (PBS:sesame oil 1:1) once daily by oral gavage for 5 days. Six hours after last dose, mice received D‑luciferin (75 mg/kg i.p.) and were anesthetized with isoflurane, then imaged for bioluminescence using IVIS‑100 system. Luciferase signal from head region (ROI) quantified. (No Fenbendazole in vivo data in this study) [2].

Toxicity/Toxicokinetics: In vitro, Fenbendazole at concentrations ≥2 μM showed a gradual negative impact on basal viability of primary cortical neurons (MTT assay) [2]. In cancer cells, higher concentrations were not detailed. In vivo, oral administration of Fenbendazole (1 mg/mouse every other day for 12 days) was well tolerated with no reported overt toxicity in nude mice. The drug is cited as having a high safety margin and low toxicity in experimental animals based on previous reports (references within [1]). No LD₅₀, organ toxicity, or specific toxicokinetic data are provided [1][2].

[1]. Fenbendazole acts as a moderate microtubule destabilizing agent and causes cancer cell death by modulating multiple cellular pathways. Sci Rep. 2018 Aug 9;8(1):11926.

[2]. Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin. Antioxid Redox Signal. 2015 Jan 10;22(2):121-34.

[3]. Fenbendazole as a potential anticancer drug. Anticancer Res. 2013 Feb;33(2):355-62.

Fenbendazole belongs to the benzimidazole class of compounds, with the structure 1H-benzimidazole, substituted at positions 2 and 5 by (methoxycarbonyl)amino and phenylthioyl groups, respectively. It is a broad-spectrum anthelmintic, particularly used in veterinary medicine to treat nematode infections. Fenbendazole is an anti-nematode drug belonging to the benzimidazole, carbamate, and aryl thioether classes of compounds. It is a benzimidazole compound with broad-spectrum anthelmintic activity and can be used to treat various gastrointestinal parasitic infections, including Giardia lamblia, Ascaris lumbricoides, hookworms, whipworms, tapeworms, pinworms, Strongyloides pulmonale, paragonimiasis, and Strongyloides stercoralis. Fenbendazole is approved for use under veterinary guidance in sheep, cattle, horses, fish, dogs, cats, rabbits, and seals.
Anti-nematode benzimidazole drugs used in veterinary medicine. See also: Bacitracin methylene disalicylate; Fenbendazole (component); Fenbendazole; Ivermectin; Praziquantel (component).

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Indications
For the treatment and control of gastrointestinal nematode infections in pigs, including: Ascaris suis (adult, intestinal, and migrating larval stages); Nematodes spp. (adult stage); Trichodeda spp. (adult stage). For the treatment of gastrointestinal nematode infections in chickens, including: Ascaris suis (L5 stage and adult stage); Heterakis gallinarum (L5 stage and adult stage); Capillaria spp. (L5 stage and adult stage).

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Additional Info: Fenbendazole is a benzimidazole anthelmintic used in veterinary medicine. It has been repurposed as an anticancer agent due to its ability to interfere with microtubules, activate p53, and disrupt glucose metabolism. Unlike classical microtubule poisons (e.g., colchicine, vinca alkaloids), Fenbendazole causes only modest microtubule depolymerization and does not affect acetylated tubulin levels. It is not a substrate or inhibitor of P‑glycoprotein (P‑gp), as shown by rhodamine 123 accumulation assay with verapamil [1]. In neurons, Fenbendazole activates HIF‑1α through free tubulin binding, leading to upregulation of p21 and protection against oxidative stress. This neuroprotective effect is independent of mitochondrial inhibition and is blocked by Taxol (which reduces free tubulin) [2]. The compound has been used orally in animals and humans for parasitic infections, and its safety profile supports further investigation for cancer and neurological diseases [1][2].

299.072

43210-67-9

Fenbendazole-d3;1228182-47-5

3334

White to off-white solid powder

1.4±0.1 g/cm3

541.4±42.0 °C at 760 mmHg

233°C

281.2±27.9 °C

0.0±1.5 mmHg at 25°C

1.679

4.34

2

4

4

21

363

0

S(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C2=C(C=1[H])N([H])C(N([H])C(=O)OC([H])([H])[H])=N2

HDDSHPAODJUKPD-UHFFFAOYSA-N

InChI=1S/C15H13N3O2S/c1-20-15(19)18-14-16-12-8-7-11(9-13(12)17-14)21-10-5-3-2-4-6-10/h2-9H,1H3,(H2,16,17,18,19)

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

Hoe881vHoe-881v Fenbendazole FenbendazolPanacurFenbendazoleamine Phenbendasol Safe-quard Hoe 881v

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 : ~10 mg/mL (~33.41 mM)
H2O : ~1 mg/mL (~3.34 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (3.34 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 10.0 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 2: ≥ 1 mg/mL (3.34 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 10.0 mg/mL clear DMSO 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.)