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

In BD140A, SW-13, and NCI-H295R cells, niclosamide (0.6 nM–46 μ M) therapy can stop the growth 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 virus multiplication [5].

In vivo growth of adrenocortical carcinoma tumors is inhibited by niclosamide sodium (gavage; 100 mg/kg, 200 mg/kg; once a week; 8 weeks) [3].

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[3]
Cell Types: BD140A, SW-13 and NCI-H295R cells
Tested Concentrations: 0.6 nM-46 µM
Incubation Duration:
Experimental Results: Inhibited cellular proliferation in adrenocortical carcinoma cell lines with the IC50 of 0.12 µM, 0.15 µM, and 0.53 µM in BD140A, SW-13, and NCI-H295R, respectively.

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Cell Viability Assay[4]
Cell Types: Hela cells
Tested Concentrations: 0.05-5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited STAT3-mediated luciferase reporter activity with an IC50 of 0.25 μM.

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Cell Viability Assay[5]
Cell Types: Vero E6 cells
Tested Concentrations: 10 μM
Incubation Duration: 2 days
Experimental Results: Inhibited the synthesis of viral antigens of SARS-CoV in Vero E6 cells.

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.

Absorption, Distribution and Excretion
Systemic residues in invertebrates exposed to (14) C-Berlusk (1 μg/L) were low. Within 48 hours, residues in invertebrates (water fleas, woodlice, amphipods, glass shrimp, crayfish, damselflies, and chironomid larvae) reached 4 to 87 times the exposure concentration. These residues decreased by 50% within 24 hours of organisms being transferred to freshwater. Clonidazole residues rapidly accumulated in fish exposed to the pesticide. During exposure, residue levels in muscle increased to near the treatment concentration. Ten days after discontinuation, residues in plasma, bile, and muscle of rainbow trout and coho salmon decreased to below 1% of their respective peak concentrations. In all tested species, residues in muscle decreased to below the detection limit (0.01 UG/G) within 3–14 days. After a slight increase in bile residues at the initial stage of drug withdrawal, bile residues steadily decreased, but 14 days after withdrawal, bile residues in channel catfish remained above initial levels; 28 days after withdrawal, bile residues in coho salmon also remained above detectable levels (0.01 μg/mL). Rainbow trout, upon exposure to clonidine in water, rapidly began to excrete both bound and free clonidine in their urine, with the highest excretion occurring during the 12-hour exposure period. Rainbow trout continued to excrete clonidine even 60 hours after exposure. The excretion of bound clonidine was fifteen times that of free clonidine. In fish injected intraperitoneally with clonidine, 25% of the injected dose was excreted in the urine, and 20% was recovered in the bile. Clonidine exposure had no effect on urinary excretion or renal excretion of sodium, potassium, calcium, or chloride ions.

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Metabolism/Metabolites
Glucuronide binding has been observed in rainbow trout exposed to clonidine.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Nicoloxamide is not marketed in the United States. There is currently no information regarding the clinical use of nicoloxamide during lactation. Because nicoloxamide is not absorbed orally, it is unlikely to have adverse effects on breastfed infants. No special precautions are required.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Interactions The chemical substance 3-trifluoromethyl-4-nitrophenol or the combination of 3-trifluoromethyl-4-nitrophenol and clonidine can cause death in oligochaetes and hirudinea, mayfly larvae, and certain trichomoniasis, berberididae, and amphibians. The combination of 3-trifluoromethyl-4-nitrophenol and clonidine may have effects on certain bivalves and gastropods, but its overall impact on invertebrates is likely less than that of 3-trifluoromethyl-4-nitrophenol alone. Granular clonidine may cause mortality in oligochaetes, microcrustaceans, chironomids, and bivalves. There is currently no evidence that the pesticide caused catastrophic decline or extinction in any species. The overall impact of chemical control on the aquatic community of sea lampreys is relatively small compared to the benefits gained.

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Non-human toxicity values
Rat intraperitoneal LD50: 250 mg/kg saline
Rat oral LD50: >5000 mg/kg

[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.

Niclosamide is a yellow solid, insoluble in water. (NTP, 1992)
An anthelmintic effective against most tapeworms. (From Martindale Pharmacopoeia, 30th edition, p. 48)
Mechanism of Action
/Bayluscide/...is an effective inhibitor of oxidative phosphorylation, but...it is highly selective.

C15H15CL2N3O5

388.20

385.059

C, 46.41; H, 3.89; Cl, 18.26; N, 10.82; O, 20.61

1420-04-8

Niclosamide;50-65-7;Niclosamide sodium;40321-86-6;Niclosamide monohydrate;73360-56-2;Niclosamide;50-65-7

14992

Light yellow to yellow solid powder

155-156 °C

91-93 °C(lit.)

149 °F

4.093

4

6

3

25

414

0

XYCDHXSQODHSLG-UHFFFAOYSA-N

InChI=1S/C13H8Cl2N2O4.C2H7NO/c14-7-1-4-12(18)9(5-7)13(19)16-11-3-2-8(17(20)21)6-10(11)15;3-1-2-4/h1-6,18H,(H,16,19);4H,1-3H2

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

1420-04-8; Niclosamide-olamine; Clonitralid; Niclosamide ethanolamine salt; Bayluscide; CLONITRALIDE; 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide compound with 2-aminoethanol (1:1); Niclosamide-olamine [ISO];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 125 mg/mL (322.00 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.)