Antihelminthic; STAT3 (IC50 = 0.25 μM in HeLa cells); ROS; NF-κB; mTORC1; Wnt/β-catenin; Notch;
Niclosamide olamine targets multiple signaling pathways. It is a potent inhibitor of STAT3 with an IC50 of 0.25 μM. The compound also reversibly inhibits mTORC1 signaling, leading to stimulation of autophagy. Niclosamide olamine suppresses PI3K-mTORC1 signaling, blocking fibroblast proliferation. It acts as a mitochondrial uncoupler, disrupting mitochondrial metabolism in parasites. The compound has anticancer bioactivity and inhibits DNA replication in Vero E6 cells. Its ability to modulate STAT3, mTORC1, and mitochondrial function underlies its diverse biological activities.

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 vitro, niclosamide olamine has demonstrated potent inhibition of STAT3 with an IC50 of 0.25 μM in HeLa cells. The compound stimulates autophagy by reversibly inhibiting mTORC1 signaling. It suppresses PI3K-mTORC1 signaling to block fibroblast proliferation. Niclosamide olamine inhibits DNA replication in Vero E6 cells. It acts as a mild mitochondrial uncoupler and has anticancer bioactivity. The compound improves insulin levels, body weight, and muscle wasting, inhibits artery constriction, and activates non-canonical autophagy to enhance fibroblast apoptosis sensitivity. Its in vitro activities make it a compound of interest for cancer, metabolic, and infectious disease research.

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].
In vivo, niclosamide olamine has been studied for its effects on various disease models. The compound improves insulin levels, body weight, and muscle wasting in animal models. It inhibits artery constriction and has renal protective effects in diabetic and adriamycin-induced non-diabetic nephropathy models. Its STAT3 inhibition and mTORC1 modulation contribute to its in vivo effects. The compound's anthelmintic activity has been demonstrated in parasite infection models. Its safety and efficacy have been evaluated in various preclinical studies. The compound's oral bioavailability supports its use in in vivo studies.

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]
For in vitro biochemical assays, niclosamide olamine is evaluated for its effects on STAT3 and mTORC1 signaling. STAT3 inhibition is assessed using STAT3 phosphorylation assays or reporter gene assays, with an IC50 of 0.25 μM determined in HeLa cells. mTORC1 inhibition is assessed by measuring phosphorylation of downstream targets such as S6K and 4EBP1. Mitochondrial uncoupling is assessed by measuring oxygen consumption or mitochondrial membrane potential. PI3K-mTORC1 signaling inhibition is assessed by Western blotting. DNA replication inhibition is assessed by measuring incorporation of radiolabeled thymidine. These cell-free and cell-based assays help characterize the compound's multiple mechanisms of action.

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.
In vitro cellular assays for niclosamide olamine are performed using various cell lines including HeLa cells, Vero E6 cells, and cancer cell lines. Cells are cultured in standard media and treated with niclosamide olamine at various concentrations. STAT3 phosphorylation is assessed by Western blotting using phospho-specific antibodies. mTORC1 signaling is assessed by measuring phosphorylation of S6K and 4EBP1. Autophagy is assessed by measuring LC3-II conversion and p62 levels. Cell proliferation is assessed using MTT or BrdU incorporation assays. DNA replication is measured by thymidine incorporation. Fibroblast proliferation is assessed using cell counting or proliferation assays. These cellular assays help validate the compound's inhibitory effects on multiple signaling pathways.

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 animal experiments with niclosamide olamine are conducted to study its effects on cancer, metabolic diseases, and infections. Tumor xenograft models are used to study anticancer activity. Diabetic and adriamycin-induced nephropathy models are used to study renal protective effects. Parasite infection models are used to study anthelmintic activity. Niclosamide olamine is administered orally due to its oral bioavailability. Efficacy endpoints include tumor growth inhibition, improvement in metabolic parameters, renal function improvement, and parasite load reduction. Tissue samples are analyzed for STAT3 phosphorylation, mTORC1 signaling, and markers of autophagy. The compound's safety and tolerability are monitored through body weight, clinical signs, and clinical chemistry.

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.

Pharmacokinetic properties of niclosamide olamine have been characterized to support its development. The compound is orally active, indicating good absorption following oral administration. It is metabolized in the liver and eliminated primarily in feces. The compound has a relatively short half-life. Its olamine salt form has improved water solubility compared to the free acid. Detailed PK parameters such as half-life, Cmax, Tmax, AUC, volume of distribution, and clearance are available in the pharmacological literature. The compound's pharmacokinetic properties support its use in oral formulations for anthelmintic and anticancer research.

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
The toxicological profile of niclosamide olamine has been characterized through its use as an anthelmintic. The compound has an excellent safety profile for its approved indications. Common adverse effects are generally mild and may include gastrointestinal disturbances. The compound is well-tolerated at therapeutic doses. Comprehensive toxicity studies including acute, subchronic, and chronic toxicity assessments have been conducted. The compound's safety in pregnancy and lactation has been evaluated. It is intended for research use and clinical applications require appropriate medical supervision. The compound should be used with caution in patients with liver disease. Researchers should follow standard laboratory safety practices when handling niclosamide olamine.

[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.
Niclosamide olamine is a valuable research tool for studying STAT3 signaling, mTORC1 function, and mitochondrial metabolism. Its potent STAT3 inhibition (IC50 = 0.25 μM) makes it useful for investigating the role of STAT3 in cancer, inflammation, and other diseases. The compound's ability to modulate mTORC1 and induce autophagy provides opportunities for studying autophagy regulation and developing autophagy-based therapies. Its mitochondrial uncoupling activity makes it relevant for studying mitochondrial function and metabolism. The compound's anticancer bioactivity and renal protective effects make it useful for studying these therapeutic applications. Niclosamide olamine is also relevant for studying anthelmintic mechanisms and parasite metabolism.

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.)