NS4B ( IC50 = 24 nM ); H1 histamine receptor

Clemizole hydrochloride has an incredibly short plasma half-life (measured at 0.15 hours); in C57BL/6J mice, it biotransforms very quickly into a variety of lesser metabolites as well as a glucuronide (M14) and a dealkylated metabolite (M12)[3].

Clemizole has an IC50 of 24±1 nM for RNA binding by NS4B and an EC50 of 8 µM for viral replication. In the low micromolar range (IC50 = 1.0-1.3 µM), clemizole effectively inhibits TRPC5 currents and Ca(2+) entry.

Huh7.5 cells are kept in DMEM with 10% FBS, 1% L-glutamine, 1% Penicillin, 1% Streptomycin, and 1% non-essential amino acids added. Following treatment with 0.05% trypsin-0.02% EDTA and seeding at a 1:5 dilution, cell lines are passaged twice a week. Trypsinization and centrifugation at 700 g for five minutes are used to gather subconfluent Huh7.5 cells. After three ice-cold RNase-free PBS washes, the cells are resuspended in PBS at a density of 1.5 x 10⁷ cells/mL. Using the T7 MEGAscript kit, XbaI linearized DNA templates are transcriptionally generated to create wild-type or mutant FL-J6/JFH-5′C19Rluc2AUbi RNA for electroporation. This is followed by purification (RNA transcription and fluorescent labeling). In a 2-mm-gap cuvette (BTX), we combined 5 µg of RNA with 400 µL of washed Huh7.5 cells. We then pulsed (0.82 kV, five 99 µs pulses) using a BTX-830 electroporator. Pulsed cells are diluted into 10 mL of growth medium that has been preheated after a 10-minute recovery period at 25°C. Six-well plates are seeded with a common stock of cells from multiple electroporations (5×10⁵ cells per well). Following a 24-hour period, the medium is changed, and the cells are cultured with successive dilutions of the different inhibitory substances (such as Clemizole hydrochloride) found in the screen. Analysis is done on 17 of the 18 identified compounds that are commercially available. Regarding water-soluble compounds, untreated cells are employed as a negative control. Cells that have not been treated are cultured in the presence of equivalent concentrations of the solvent as a negative control for compounds (like Clemizole hydrochloride) that have been solubilized in DMSO. The medium is swapped out every day. Both a luciferase assay and a viability assay based on Alamar Blue are performed on the cells 72 hours after treatment. Cells are incubated for three hours at 37°C with 10% Alamar Blue reagent following a 72-hour treatment period. The FLEXstation II 384 is then used to scan the plates and detect fluorescence. The normalization of the signal with respect to untreated samples or samples grown in the presence of DMSO depends on the solvent used to dissolve the inhibitory compound (e.g., Clemizole hydrochloride), water, or DMSO[1].

[1]. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol. 2008 Sep;26(9):1019-27.

[2]. Clemizole hydrochloride is a novel and potent inhibitor of transient receptor potential channel TRPC5. Mol Pharmacol. 2014 Nov;86(5):514-21.

[3]. Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction. J Pharmacol Exp Ther. 2013 Feb;344(2):388-96.

Clemizole is a member of the class of benzimidazoles that is 1H-benzimidazole substituted by a pyrrolidin-1-ylmethyl and a 4-chlorobenzyl groups at positions 2 and 1 respectively. It has a role as a histamine antagonist. It is a member of pyrrolidines, a member of benzimidazoles and a member of monochlorobenzenes. It is a conjugate base of a clemizole(1+). It derives from a hydride of a 1H-benzimidazole.

C19H20CLN3

325.8352

325.134

C, 70.04; H, 6.19; Cl, 10.88; N, 12.90

442-52-4

Clemizole hydrochloride; 1163-36-6; Clemizole-d4; 6011-39-8 (penicillin); 17162-20-8 (sulfate)

2782

Solid powder

1.3±0.1 g/cm3

506.1±40.0 °C at 760 mmHg

259.9±27.3 °C

0.0±1.3 mmHg at 25°C

1.656

5.1

0

2

4

23

377

0

ClC1=CC=C(C=C1)CN2C(CN3CCCC3)=NC4=CC=CC=C24

CJXAEXPPLWQRFR-UHFFFAOYSA-N

InChI=1S/C19H20ClN3/c20-16-9-7-15(8-10-16)13-23-18-6-2-1-5-17(18)21-19(23)14-22-11-3-4-12-22/h1-2,5-10H,3-4,11-14H2

1-[(4-chlorophenyl)methyl]-2-(pyrrolidin-1-ylmethyl)benzimidazole

Clemizole; AL 20; AL20; AL-20; P 48; Reactrol

2934.99.03.00

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)

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.)