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
WHOLE BODY RESIDUES WERE LOW IN INVERTEBRATES EXPOSED TO (14)C-BAYLUSCIDE (1 UG/L). WITHIN 48 HR, INVERTEBRATES (DAPHNIDS, SOWBUGS, SCUDS, GLASS SHRIMP, CRAYFISH, DAMSELFLY LARVAE, AND MIDGE LARVAE) REACHED PLATEAUS WHICH WERE 4 TO 87 TIMES THAT OF THE CONCN TO WHICH THEY WERE EXPOSED. A 50% REDUCTION IN THESE RESIDUES OCCURRED WITHIN 24 HR AFTER THE ORGANISMS WERE TRANSFERRED TO FRESH WATER.
RESIDUES OF CLONITRALIDE WERE RAPIDLY ACCUMULATED BY FISH EXPOSED TO THE LAMPRICIDE. MUSCLE RESIDUE LEVELS INCREASED TO NEAR THE TREATMENT CONCN DURING EXPOSURE. AFTER 10 DAYS OF WITHDRAWAL, RESIDUES IN PLASMA, BILE, & MUSCLE OF RAINBOW TROUT & COHO SALMON DECREASED TO LESS THAN 1% OF THEIR RESPECTIVE PEAK CONCN. IN ALL SPECIES TESTED, RESIDUES IN MUSCLE DROPPED BELOW THE LIMIT OF DETECTION (0.01 UG/G) WITHIN 3-14 DAYS. AFTER AN INITIAL INCREASE DURING EARLY WITHDRAWAL, BILE RESIDUES DECLINED STEADILY BUT HAD NOT DROPPED BELOW INITIAL LEVELS IN CHANNEL CATFISH IN 14 DAYS, OR BELOW DETECTABLE LEVELS (0.01 UG/ML) IN COHO SALMON AFTER 28 DAYS OF WITHDRAWAL.
RAINBOW TROUT EXPOSED TO CLONITRALIDE IN THE WATER QUICKLY BEGAN TO EXCRETE CONJUGATED & FREE CLONITRALIDE IN THE URINE, WITH THE LARGEST AMOUNT BEING EXCRETED DURING THE 12 HR EXPOSURE. THE TROUT CONTINUED TO EXCRETE CLONITRALIDE 60 HR AFTER EXPOSURE. FIFTEEN TIMES AS MUCH CONJUGATED AS FREE CLONITRALIDE WAS EXCRETED. AMONG FISH GIVEN IP INJECTIONS, 25% OF THE INJECTED DOSE WAS EXTRACTED IN THE URINE, & 20% RECOVERY IN THE BILE. CLONITRALIDE EXPOSURE HAD NO EFFECT ON THE URINE OUTPUT OR RENAL EXCRETION OF SODIUM, POTASSIUM, CALCIUM, OR CHLORIDE IONS.

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Metabolism / Metabolites
GLUCURONIDE CONJUGATION HAS BEEN EXHIBITED IN RAINBOW TROUT EXPOSED TO CLONITRALIDE.

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.

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Interactions
THE CHEMICALS 3-TRIFLUOROMETHYL-4-NITROPHENOL OR A COMBINATION OF 3-TRIFLUOROMETHYL-4-NITROPHENOL & CLONITRALIDE CAUSE SOME MORTALITIES OF OLIGOCHAETA & HIRUDINEA, IMMATURE FORMS OF EPHEMEROPTERA, & CERTAIN TRICHOPTERA, SIMULIIDAE, & AMPHIBIA. THE COMBINATION OF 3-TRIFLUOROMETHYL-4-NITROPHENOL & CLONITRALIDE MAY AFFECT SOME PELECYPODA & GASTROPODS, BUT ITS OVERALL EFFECTS ON INVERTEBRATES ARE PROBABLY LESS THAN THOSE OF 3-TRIFLUOROMETHYL-4-NITROPHENOL ALONE. GRANULAR CLONITRALIDE IS LIKELY TO INDUCE MORTALITIES AMONG OLIGOCHAETES, MICROCRUSTACEANS, CHIRONOMIDS, & PELECYPODS. NO EVIDENCE EXISTS THAT THE LAMPRICIDES HAVE CAUSED THE CATASTROPHIC DECLINE OR DISAPPEARANCE OF ANY SPECIES. THE OVERALL IMPACT OF CHEMICAL CONTROL OF SEA LAMPREYS ON AQUATIC COMMUNITIES HAS BEEN MINOR COMPARED WITH THE BENEFITS DERIVED.

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Non-Human Toxicity Values
LD50 Rat ip 250 mg salt/kg
LD50 Rat oral >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.

Clonitralid is a yellow solid. Insoluble in water. (NTP, 1992)
An antihelmintic that is active against most tapeworms. (From Martindale, The Extra Pharmacopoeia, 30th ed, p48)

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Mechanism of Action
/Bayluscide/ ... is an effective inhibitor of oxidative phosphorylation, but ... 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.)