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Niclosamide (Clonitralide, Fenasal, BAY-2353, NSC-178296, WR-46234) is a potent and orally bioavailable chlorinated salicylanilide analog with anthelmintic and potential antineoplastic activity. It can inhibit DNA replication and inhibit STAT with IC50 of 0.7 μM in a cell-free assay. It is an oral antihelminthic drug used to treat tapeworm infection for about 50 years. Niclosamide is also used as a molluscicide for water treatment in schistosomiasis control programs. Recently, several groups have independently discovered that niclosamide is also active against cancer cells by targeting multiple signaling pathways (NF-κB, Wnt/β-catenin, Notch, ROS, mTORC1, and Stat3).
Physicochemical Properties
| Molecular Formula | C13H8CL2N2O4 | |
| Molecular Weight | 327.12 | |
| Exact Mass | 325.986 | |
| Elemental Analysis | C, 47.73; H, 2.47; Cl, 21.68; N, 8.56; O, 19.56 | |
| CAS # | 50-65-7 | |
| Related CAS # |
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| PubChem CID | 4477 | |
| Appearance |
PALE, YELLOW CRYSTALS ALMOST COLORLESS CRYSTALS A cream-colored or yellowish-white powder Bright yellow crystalline solid |
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| Density | 1.6±0.1 g/cm3 | |
| Boiling Point | 424.5±45.0 °C at 760 mmHg | |
| Melting Point | 225-230ºC | |
| Flash Point | 210.5±28.7 °C | |
| Vapour Pressure | 0.0±1.0 mmHg at 25°C | |
| Index of Refraction | 1.709 | |
| LogP | 5.41 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 21 | |
| Complexity | 404 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | ClC1C([H])=C(C([H])=C([H])C=1N([H])C(C1C([H])=C(C([H])=C([H])C=1O[H])Cl)=O)[N+](=O)[O-] |
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| InChi Key | RJMUSRYZPJIFPJ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C13H8Cl2N2O4/c14-7-1-4-12(18)9(5-7)13(19)16-11-3-2-8(17(20)21)6-10(11)15/h1-6,18H,(H,16,19) | |
| Chemical Name | 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | antihelminthic; STAT3 (IC50 = 0.25 μM in HeLa cells); ROS; NF-κB; mTORC1; Wnt/β-catenin; Notch; |
| ln Vitro | In BD140A, SW-13, and NCI-H295R cells, niclosamide (0.6 nM–46 μM) therapy reduces the proliferation of adrenocortical carcinoma cells [3]. In HeLa cells, niclosamide administration (0.05–5 μM, 24 h) suppresses STAT3-mediated luciferase reporter activity [4]. In Vero E6 cells, treatment with niclosamide (10 μM) suppresses viral replication [5]. In SNB-19 cells, nicolosamide (GMP) (maximum 2 μM, 24 h) suppresses Zika virus infection [6]. Particularly in the early phases of osteoclastogenesis, nicolosamide (GMP) (1.5 μM, 5 d) suppresses the transdifferentiation of macrophages to precursor osteoclast bodies triggered by factor-κB ligand (RANKL) [7]. |
| ln Vivo |
The oral gavage form of niclosamide (100 mg/kg, 200 mg/kg; once weekly; 8 weeks) suppresses the formation of adrenocortical carcinoma tumors in vivo [3]. Niclosamide inhibits ACC tumor growth in vivo[3] To confirm our in vitro observations, the effect of niclosamide treatment was evaluated in ACC xenografts. Niclosamide treatments, at both doses (100 mg/kg and 200 mg/kg), were well tolerated, with no observed toxicity or side effects in the mice. There were no significant weight differences among the groups (Fig. 5). Four weeks after treatment, mice treated with niclosamide at 100 mg/kg and 200 gm/kg showed a 60% and 80% inhibition in tumor growth, respectively, as compared to the vehicle control group (P < 0.01 for both groups) (Fig. 5). The same treatment schedule was maintained for 8 weeks, at which time, more than 90% tumor growth inhibition was observed for the two treated groups, as compared to the control group. |
| Enzyme Assay |
Protein Kinase profiling assay (Table S1): Assay for 22 different proteins kinases was carried out by a CRO. All of the protein kinases were expressed either in Sf9 insect cells or in E.coli as recombinant GST-fusion proteins or His-tagged proteins. Protein kinases were purified by affinity chromatography using either GSH-agarose or Ni_NTH-agarose. A radiometric protein kinase assay was used for measuring the kinase activity of the 22 protein kinases. Briefly, for each protein kinase, 50 μl reaction cocktail containing 60 mM HEPES-NaOH, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 0.02 0.2 0 10 20 30 40 50 60 70 80 90 100 110 Drug Conc.(μM) Relative colony number (% of control) IC50 : 0.1μM S9 μg/ml PEG20000, 1 μM [γ-33P]-ATP(appox.6×1005cpm), test compound, adequate amount of enzyme and its substrate. The PKC-alpha assay additionally contained 1 mM Cacl2, 4 mM EDTA, 5 μg/ml phosphatidylserine and 1 μg/ml 1, 2-Dioleyl-glycerol). The reaction cocktails were incubated at 37o C for 60 minutes and stopped with 50 μl 2% (v/v) H3PO4. Incorporation of 33Pi was determined with a microplate scintillation counter. The activities and the IC50 values were calculated using Quattro Workflow V2.28[4]. In summary, niclosamide, an FDA-approved anthelmintic drug, was identified as a new small-molecule inhibitor of the STAT3 signaling pathway. This drug potently inhibited the activation, nuclear translocation, and transactivation of STAT3 but had no obvious effects on the closely related STAT1 and STAT5 proteins, the upstream JAK1, JAK2, and Src kinases, or other receptor tyrosine kinases. Furthermore, niclosamide inhibited the transcription of STAT3 target genes and induced cell growth inhibition, apoptosis, and cell cycle arrest of cancer cells with constitutively active STAT3. Although niclosamide does not have an ideal pharmarcokinetic profile (i.e., poor oral bioavailability) in humans as an anticestodal drug, it represents a new potent lead compound with salicylic amide scaffold for development of STAT3 pathway inhibitors as new molecularly targeted anticancer drugs. The further structural optimization and extensive mechanism study on niclosamide are undergoing and will be reported in due course.[4] |
| Cell Assay |
Cellular proliferation assay[3] 3 × 103 and 6 × 103 cells were plated in 96-well plates depending on the cell line. 100 µL of fresh culture medium containing the drug or vehicle was added. Cell count was determined using the CyQuant kit, according to the manufacturer’s instructions, and cell number was measured using a SpectraMax M5 microplate reader (ex485/em538). Assays were performed in quadruplicate and the experiments were repeated three times. NCI-H295R and SW-13 cells, which form multicellular aggregates (MCA) or spheroids, were plated in Ultra Low Cluster 24-well plates (Costar, Corning, NY) at 1 × 105 cells/0.5 mL or 6 × 104 cells/0.5 mL depending on the cell line. Spheroids were allowed to develop for one or two weeks at 37°C in 5% CO2, and media was exchanged twice a week. Spheroids were treated with niclosamide or the vehicle at varying concentrations, and imaged weekly. Caspase 3/7 activity assay[3] Cells were plated in 96-well plates and treated with niclosamide or the vehicle. Caspase 3/7 activity was measured using the Caspase-Glo 3/7 assay, according to manufacturer’s instructions. Cell cycle analysis[3] Cells plated in six-well plates were treated with niclosamide or the vehicle. At 48 hours, cells were fixed for 30 minutes in 70% ethanol at 4°C, and stained with 50 µg/mL of propidium iodide containing 100 mg/mL of ribonuclease A. Flow cytometry was performed on a Canto I flow cytometer using CellQuest software. Data was generated for at least 20,000 events per sample and analyzed using Modfit software. Cellular migration assays[3] NCI-H295R and SW-13 cells were plated in six-well plates and treated with varying concentrations of niclosamide or the vehicle for 24 hours. Cells were trypsinized and plated in transwell chambers at a density of 1 × 105 cells per 0.5 mL. The lower chamber was filled with DMEM supplemented with 10% FBS as a chemoattractant. Cells were allowed to migrate for 24 hours or 48 hours depending on the cell line, and were fixed and stained with Diff-Quik. Cells were imaged and counted in three random fields per well, and the experiments performed in triplicate. For the wound-healing assay in BD140A cells, which do not migrate in the Boyden chamber model, cells were plated in six-well plates until confluent and treated with niclosamide or the vehicle. The cells were scratched using a sterile pipette tip and photographed at various time points. |
| Animal Protocol |
Animal/Disease Models: Nu+/Nu+ mice injected with NCI-H295R cells[3] Doses: 100 mg/kg, 200 mg/kg Route of Administration: po (oral gavage); 100 mg/kg, 200 mg/kg; once a week; 8 weeks Experimental Results: demonstrated a 60%-80% inhibition in tumor growth, as compared to the control group. In vivo mouse studies[3] Animal studies were approved by the National Cancer Institute Animal Care and Use Committee. Mice were maintained according to National Institutes of Health (NIH) Animal Research Advisory Committee (ARAC) guidelines. 5 × 106 NCI-H295R cells were injected into the flank of Nu+/Nu+ mice. Tumors were allowed to grow and mice were randomized into three treatment groups (8 mice per treatment group). Mice were treated with 100 mg/kg of niclosamide, 200 mg/kg of niclosamide, or the vehicle (PEG500) everyday by oral gavage. Tumor sizes were measured in two dimensions every week with calipers and recorded. |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Niclosamide appears to be minimally absorbed from the gastrointestinal tract—neither the drug nor its metabolites have been recovered from the blood or urine. VERY LITTLE IS ABSORBED FROM THE GI TRACT ... . EXPOSING RAINBOW TROUT TO (14)C-BAYER 73 CAUSED BILE TO WATER (14)C RATIO OF 10,000:1. AFTER 24 HR OF EXPOSURE, THIN LAYER CHROMATOGRAPHY OF UNFRACTIONATED BILE FROM FISH SHOWED 1 MAJOR RADIOACTIVE PEAK. UNCHANGED BAYER 73 WAS FOUND IN BILE. Niclosamide is excreted in feces. When male and female volunteers each were treated orally with 2000 mg of radiocarbon-labeled niclosamide, between 2 and 25% of the dose was eliminated in the urine over a 4-day period; the remainder was found in the feces. Elimination of niclosamide equivalents was essentially complete after 1-2 days. Maximal niclosamide equivalents in serum ranged from 0.25 to 6.0 ppm; the variation associated with this parameter was attrributed to differential rates of absorption among the individual. For more Absorption, Distribution and Excretion (Complete) data for NICLOSAMIDE (6 total), please visit the HSDB record page. Metabolism / Metabolites ANTHELMINTIC NICLOSAMIDE...REDUCED TO CORRESPONDING AMINO DERIVATIVE BY BOTH MOUSE- & SHEEP-LIVER ENZYME PREPARATIONS & BY ENZYMES FROM CESTODES & NEMATODES. ...NICLOSAMIDE WAS NOT HYDROLYZED EITHER BY MAMMALIAN & HELMINTH ENZYME PREPN OR BY WHOLE HELMINTHS. In warm-blooded organisms, the nitro group is reduced to an amino group (5,2'-dichloro-4-aminosalicylanilide). Pregnant rats were treated orally with niclosamide at 1000 mg/kg on day 13, 19, or 20 of gestation, and rats were sacrificed at 4, 8, 16, or 24 hr posttreatment. Highest concentrations of niclosamide and 2,5'-dichloro-4'-aminosalicylanilide were detected in liver and kidney 8 hr after treatment. Niclosamide, but not its amino metabolite, was present in fetuses from rats treated on day 13, whereas both compounds were found in fetuses from rats treated on day 19 or 20. It was suggested that 19- and 20-day-old fetuses, but not 13-day-old fetuses, were able to metabolize niclosamide. Niclosamide ... is absorbed from the gastrointestinal tract, and mutagenic metabolites are excreted in the free form and as conjugated glucuronides. as in the case of other secondary amides, phase I metabolism of niclosamide may result in hydrolytic cleavage of the amide bond, giving rise to 5-chlorosalicylic acid and 2-chloro-4-nitroaniline. ... |
| Toxicity/Toxicokinetics |
Toxicity Summary Niclosamide works by killing tapeworms on contact. Adult worms (but not ova) are rapidly killed, presumably due to uncoupling of oxidative phosphorylation or stimulation of ATPase activity. The killed worms are then passed in the stool or sometimes destroyed in the intestine. Niclosamide may work as a molluscicide by binding to and damaging DNA. Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Niclosamide is not marketed in the United States. No information is available on the clinical use of niclosamide during breastfeeding. Because niclosamide is not orally absorbed it is unlikely to adversely affect the breastfed infant. No special precautions are necessary. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Interactions IT WAS CONCLUDED THAT CARBARYL POTENTIATED TOXICITY OF BAYER 73 TO RAINBOW TROUT BY INCREASING ITS UPTAKE FROM WATER, POSSIBLY BY EFFECTS ON GILL BLOOD FLOW OR PERMEABILITY. Niclosamide can be admin in conjunction with several other drugs ... The effectiveness of niclosamide for tapeworms of mice is enhanced when it is given in conjunction with cucurbitine or procaine. ... To study the in utero developmental toxicity of these cmpd, pregnant female Wistar Albino rats received 1/50 LD50 of ametryne and niclosamide either individually or in combination on gestational days 5-15 before they were /sacrificed/ on day 20. The results showed neither mortality nor clinical no ... adverse effects in the treated dams throughout the study period. Also, no overt preimplantation losses were noted. ... Corrected maternal wt gain was significantly reduced under the influence of ametryne treatment. Gravid uterine weight and litter size were statistically reduced in dams treated with ametryne or a combination of ametryne and niclosamide. The percentage of postimplantation deaths was significantly raised under the influence of ametryne treatment (21.15%) or the combined treatment (25.45%) compared with 6.48% in untreated rats and 15.6% in niclosamide treated dams. ... Fetal morphological abnormality examination showed a pronounced incr in incidence of malformed fetuses (litters) up to 5.8% (29.4%), 6.48% (33.3%), and 7.9% (33.3%) in ametryne, niclosamide and combination treated groups, respectively, compared with 1.6% (11.1%) in the control group. The most common deformities were skeletal abnormalities in all groups studied, while the ametryne group showed external deformities as well. The results suggest the possibility of embryo/fetotoxicity of ametryne and fetotoxicity of niclosamide, at least at higher doses. Non-Human Toxicity Values LD50 Rat intraperitoneal 250 mg salt/kg /2-hydroxyethylammonium salt/ |
| References |
[1]. The biology and toxicology of molluscicides, Bayluscide. Pharmacol Ther. 1982;19(2):245-95. [2]. Niclosamide: Beyond an antihelminthic drug. Cell Signal. 2018 Jan;41:89-96. [3]. Identification of Niclosamide as a Novel Anticancer Agent for Adrenocortical Carcinoma. Clin Cancer Res. 2016 Jul 15;22(14):3458-66. [4]. Identification of Niclosamide as a New Small-Molecule Inhibitor of the STAT3 Signaling Pathway. ACS Med Chem Lett. 2010 Sep 7;1(9):454-9. [5]. Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide. Antimicrob Agents Chemother. 2004 Jul;48(7):2693-6. [6]. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat Med. 2016;22(10):1101-1107. [7]. Niclosamide and its derivative DK-520 inhibit RANKL-induced osteoclastogenesis. FEBS Open Bio. 2020;10(8):1685-1697. doi:10.1002/2211-5463.12921. |
| Additional Infomation |
Therapeutic Uses Anticestodal Agents; Antinematodal Agents; Molluscacides THERE ARE NO CONTRAINDICATIONS TO THE USE OF NICLOSAMIDE AS A TENIACIDE. MEDICATION (VET): Niclosamide can also be used to treat tapeworm infections of lab animals /eg mice, rabbits, monkeys, or reptiles/. ... Niclosamide is admin orally in tablet form to dogs and cats ... It is usually admin to ruminants /eg cattle, sheep and goats/ ... as a drench ... Therapeutic Use Niclosamide in the free-base form only is used primarily as a cestocide and to a lesser extent as a trematocide. The drug is formulated as a chewable tablet containing 500 mg of niclosamide. It is very effective against tapeworrn infections caused by Taenia saginata, Taenia solium, and Diphyllobothrium latum; tapeworms such as Hymenolepis diminuta, Hymenolepis nana, and Dipylidium caninum are somewhat more recalcitrant. For infections of Taenia saginata, Taenia solium, and Diphyllobothrium latum, single oral dosages of 2 gm (adult), 1.5 gm (child > 34 kg), and 1.0 gm (child 11-34 kg) are recommended. 0ther tapeworms may require repeated treatment for example, 2 gm/day in single daily doses for 7 days (adults), 1.5 gm given in a single dose on the first day followed by 1 gm/day for the next 6 days (child > 34 kg), and 1 gm given in a single dose on the first day followed by 500 mg/day for next 6 days (child 11-34 kg). Safety for use in children under 2 years of age has not been established. Since niclosamide is active only against intestinal cestodes, it is not effective for treatment of cysticercosis. As a trematocide, niclosamide is active mainly against flukes such as Fasciolopsis buski in the intestines. For more Therapeutic Uses (Complete) data for NICLOSAMIDE (10 total), please visit the HSDB record page. Drug Warnings SINCE TAPEWORM INFECTIONS GENERALLY ARE NOT LIFE THREATENING, IT IS RECOMMENDED THAT TREATMENT OF PREGNANT WOMEN BE POSTPONED UNTIL AFTER DELIVERY. VET: DO NOT TREAT LACTATING ANIMALS. ... It is important to note that the lethal action of the drug against the adult worn does not extend to the ova. Thus, use of niclosamide in Taenia solium infections may expose the patient to the risk of cysticercosis, since, following digestion of the dead segments, viable ova will be liberated into the lumen of the gut. It is desirable to give an adequate purge within 3 to 4 hours after the drug has been given, to clear the bowel of all dead segments before they can be digested. The drug causes segments to disintegrate, releasing viable eggs; hence, if used against pork tapeworm, a purge should be given 1 or 2 hr after treatment. Untoward effects occur only occasionally; nausea and abdominal pain have been reported. For more Drug Warnings (Complete) data for NICLOSAMIDE (7 total), please visit the HSDB record page. Pharmacodynamics Niclosamide is an antihelminth used against tapeworm infections. It may act by the uncoupling of the electron transport chain to ATP synthase. The disturbance of this crucial metabolic pathway prevents creation of adenosine tri-phosphate (ATP), an essential molecule that supplies energy for metabolism. |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 0.5 mg/mL (1.53 mM) in 10% DMF 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 1% DMSO+30% polyethylene glycol+1% Tween 80:30 mg/mL Solubility in Formulation 3: 5 mg/mL (15.28 mM) in 20% HP-β-CD 5% Cremophor EL (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0570 mL | 15.2849 mL | 30.5698 mL | |
| 5 mM | 0.6114 mL | 3.0570 mL | 6.1140 mL | |
| 10 mM | 0.3057 mL | 1.5285 mL | 3.0570 mL |