Antihelminthic; STAT3 (IC50 = 0.25 μM in HeLa cells); ROS; NF-κB; mTORC1; Wnt/β-catenin; Notch;

Treatment with niclosamide monohydrate (0.6 nM-46 µM) reduces adrenocortical cancer cell proliferation in BD140A, SW-13 and NCI-H295R cells [3]. Niclosamide monohydrate (0.05-5 μM, 24 hours) treatment suppresses STAT3-mediated luciferase reporter activity in HeLa cells [4]. Treatment with niclosamide monohydrate (10 μM) reduces viral replication in Vero E6 cells [5].

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].

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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.

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].

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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 viability assay [4]
Cell Types: Hela Cell
Tested Concentrations: 0.05-5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibits STAT3-mediated luciferase reporter gene activity, IC50 is 0.25 μM.

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Cell viability assay [5]
Cell Types: Vero E6 Cell
Tested Concentrations: 10 μM
Incubation Duration: 2 days
Experimental Results: Inhibition of the synthesis of SARS-CoV viral antigen in Vero E6 cells.

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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.

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NCI-H295R and SW-13 cells, which form multicellular aggregates (MCA) or spheroids, were plated in Ultra Low Cluster 24-well plates 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.

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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.

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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.

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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/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-week
Experimental Results: Tumor growth was inhibited by 60%-80% compared to the control group.

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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.

[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.

Nicoloxamide is a secondary amide formed by the condensation of the carboxyl group of 5-chlorosalicylic acid and the amino group of 2-chloro-4-nitroaniline. It is an oral anthelmintic approved for the treatment of tapeworm infections. It possesses a variety of pharmacological effects, including pisicant, molluscicide, antiparasitic agent, anticoronavirus agent, anthelmintic, apoptosis inducer, and STAT3 inhibitor. It belongs to the monochlorobenzene, salicylaniline, C-nitro compounds, secondary amides, and benzamides. Its structure is related to 5-chlorosalicylic acid. Nicoloxamide is an anthelmintic used to treat tapeworm infections. Worms (nematodes) are multicellular organisms that can infect large populations and cause a variety of diseases. Over 1 billion people are infected with intestinal nematodes, and millions more are infected with filarial worms, flukes, and tapeworms. These parasites pose an even greater threat to livestock. Nicolamide was once marketed in the United States under the brand name Nilocide, but Bayer voluntarily withdrew it from the market in 1996. Nicolamide is a highly bioavailable, orally administered chlorosalicylic acid aniline drug with anthelmintic and potential antitumor activities. After oral administration, nicotinic acid specifically induces the degradation of the androgen receptor (AR) variant V7 (AR-V7) via a proteasome-mediated pathway. This downregulates AR variant expression, inhibits AR-V7-mediated transcriptional activity, and reduces AR-V7 binding to the prostate-specific antigen (PSA) gene promoter. Nicolamide also blocks AR-V7-mediated STAT3 phosphorylation and activation. This inhibits AR/STAT3-mediated signaling and prevents the expression of STAT3 target genes. In summary, this may inhibit the growth of AR-V7-overexpressing cancer cells. The AR-V7 variant, encoded by sequential splicing of AR exons 1/2/3/CE3, is upregulated in multiple cancer cell types and is associated with cancer progression and resistance to AR-targeted therapies. Nicotinamide is used to treat most tapeworm infections. Worms (nematodes) are multicellular organisms that can infect large numbers of humans and cause a variety of diseases. Over one billion people are infected with intestinal nematodes, and millions more with filarial worms, flukes, and tapeworms. They pose an even greater threat to livestock. This is an effective anthelmintic against most tapeworms.

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Drug Indications
For the treatment of tapeworm and intestinal fluke infections: Taenia saginata (beef tapeworm), Taenia solium (pork tapeworm), Diphyllobothrium latum (broad tapeworm), Fasciolopsis buski (fasciolopsis buski). Nicotinamide can also be used as a molluscicide to control schistosomiasis.

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Therapeutic Uses Nicotinamide is used only in free base form, primarily as a tapeworm killer, and secondarily as a fluke killer. This drug is a chewable tablet, each containing 500 mg of nicosulfanilamide. It is highly effective against tapeworm infections caused by Taenia solium, Taenia suis, and C. broadenum; however, it is more difficult to treat tapeworms such as Hymenolepis minimus, Hymenolepis dwarfusus, and Dipylidium canis. For infections caused by Taenia solium, Taenia suis, and C. broadenum, the recommended single oral dose is: 2 grams for adults, 1.5 grams for children weighing over 34 kg, and 1.0 gram for children weighing 11-34 kg. Other tapeworm infections may require repeated treatment, for example: 2 grams once daily for adults for 7 days; 1.5 grams once daily for children weighing over 34 kg, followed by 1 gram daily for 6 days; and 1 gram once daily for children weighing 11-34 kg, followed by 500 mg daily for 6 days. The safety of this drug in children under 2 years of age has not been established. Because niclosamide is effective only against intestinal tapeworms, it is ineffective against cysticercosis. As a trematode, niclosamide is primarily effective against intestinal trematodes (such as Clonorchis brucei).

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Pharmacodynamics
Nicolosamide is an anthelmintic used to treat tapeworm infections. Its mechanism of action likely involves uncoupling the electron transport chain from ATP synthase. Disruption of this key metabolic pathway prevents the production of adenosine triphosphate (ATP), an essential molecule for providing energy for metabolism.

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Absorption
Nicolosamide appears to be minimally absorbed in the gastrointestinal tract—the drug and its metabolites are undetectable in blood or urine.
Very little gastrointestinal absorption…Gilman, AG, TW Rall, AS Nies, and P. Taylor (eds.). Pharmacological Basis of Therapeutic Drugs, Goodman and Gilman. 8th ed. New York, NY. Pergamon Press, 1990, p. 965.
After exposing rainbow trout to (14)C-Bayer 73, the ratio of bile to (14)C in water reached 10,000:1. Twenty-four hours after exposure, thin-layer chromatography analysis of the ungraded bile of the fish revealed a single major radioactive peak. Unaltered Bayer 73 was found in the bile.

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Metabolites/Metabolites
The anthelmintic nicotinamide…can be reduced to the corresponding amino derivatives by liver enzymes in mice and sheep, as well as by enzymes in tapeworms and nematodes. …Nicolicamide cannot be hydrolyzed by enzymes in mammals and worms, or by intact worms.

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Mechanism of Action
Nicolicamide works by killing tapeworms through contact. Adult worms (but not eggs) are rapidly killed, likely due to uncoupling of oxidative phosphorylation or enhanced ATPase activity. The killed worms are then excreted in feces and sometimes destroyed in the intestines. Nicoloxacin may exert its molluscicidal effect by binding to and destroying DNA.
Nicolasamide exhibits significant activity against most tapeworms infecting humans; pinworms (Enterobius (Oxyuris) vermicularis) are also susceptible. Low concentrations of nicolasamide stimulate oxygen uptake in Hymenolepis diminuta, but high concentrations inhibit respiration and block glucose uptake. The primary action of this drug is likely the inhibition of anaerobic phosphorylation of adenosine diphosphate (ADP) in the parasite's mitochondria, an energy production process dependent on carbon dioxide fixation… Gilman, AG, TW Rall, AS Nies, and P. Taylor (eds.). Pharmacological Basis of Therapeutic Drugs (8th ed.), Goodman and Gilman, Pergamon Press, New York, 1990, p. 965.
The nicotinic activity of nicotinamide is due to its inhibition of glucose uptake in tapeworms and its uncoupling of oxidative phosphorylation in tapeworm mitochondria. The resulting blockage of the Krebs cycle leads to lactic acid accumulation, which kills the tapeworms. ...Overstimulation of mitochondrial adenosine triphosphatase (ATPase) activity may be related to the tapeworm-killing effect of nicotinamide.

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Toxicity Overview
Nicoleamide's mechanism of action is contact killing of tapeworms. Adult worms (not eggs) are rapidly killed, possibly due to uncoupling of oxidative phosphorylation or stimulation of ATPase activity. The killed worms are then excreted in feces and sometimes destroyed in the intestine. Nicotinamide may exert its molluscicidal effect by binding to and damaging DNA.

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We identified 21 active compounds, all with efficacy >80% in all three cell lines. Among them, nicotinamide was more effective than known anti-adenoid cystic carcinoma (ACC) drugs and had a lower IC50 value. We then verified the inhibitory effect of nicotinamide on cell proliferation in all three ACC cell lines. Next, we investigated the mechanism by which nicotinamide inhibits ACC cell proliferation and found that it induces caspase-dependent apoptosis and G1 phase cell cycle arrest. Nicotinamide also reduced cell migration and decreased levels of epithelial-mesenchymal transition (EMT) mediators such as N-cadherin and vimentin. In addition, nicotinamide treatment led to a decrease in β-catenin expression. We also evaluated the effects of nicotinamide on energy metabolism in ACC cell lines and found that it caused mitochondrial uncoupling. Nicotinamide treatment inhibited the growth of ACC tumors in mice and no toxicity was observed. Conclusion: Our results suggest that nicotinamide exerts its anti-ACC activity by inhibiting multiple aberrant cellular pathways and cellular metabolism in ACC. Our results provide a preclinical basis for evaluating nicotinamide therapy in ACC clinical trials. [3] Inhibition of signal transduction and transcription activator 3 (STAT3) signaling pathway is considered a novel therapeutic strategy for treating constitutively activated STAT3-mediated human cancers. In this study, we report the discovery of nicotinamide (an FDA-approved anthelmintic) as a novel small molecule STAT3 signaling pathway inhibitor. The compound effectively inhibits the activation and transcriptional function of STAT3, thereby inducing cell growth inhibition, apoptosis, and cell cycle arrest in STAT3-continuously activated cancer cells. Our study provides a novel and promising lead compound with a salicylamide backbone that can be used to develop STAT3 pathway inhibitors as novel molecularly targeted anticancer drugs. [4]

C13H8N2O4CL2.H2O

345.1349

343.997

C, 45.24; H, 2.92; Cl, 20.54; N, 8.12; O, 23.18

73360-56-2

Niclosamide;50-65-7;Niclosamide olamine;1420-04-8;Niclosamide sodium;40321-86-6;Niclosamide-13C6 monohydrate;1325559-12-3;Niclosamide;50-65-7; 36466-48-5 (piperazine); 73360-56-2 (hydrate);

12296604

Typically exists as solid at room temperature

224-229ºC

4.702

3

5

2

22

404

0

O=C(NC1=CC=C([N+]([O-])=O)C=C1Cl)C2=CC(Cl)=CC=C2O.O

ZBXRPLQCPHTHLM-UHFFFAOYSA-N

InChI=1S/C13H8Cl2N2O4.H2O/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);1H2

5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide;hydrate

5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide monohydrate; Niclosamide (monohydrate); BAY2353 monohydrate; UNII-20Z25R1145; 2',5-Dichloro-4'-nitrosalicylanilide monohydrate; Niclosamide monohydrate; 20Z25R1145;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)