NS4B ( IC50 = 24 nM ); H1 histamine receptor
Hepatitis C Virus (HCV) NS5A protein (KD = 0.8 μM in binding assay; IC50 = 1.2 μM for HCV replicon inhibition) [1]
Transient Receptor Potential Canonical 5 (TRPC5) (IC50 = 37 nM for TRPC5-mediated Ca²⁺ influx; Ki = 29 nM for TRPC5 binding via ITC) [2]
Cytochrome P450 3A4 (CYP3A4) (inhibits activity with IC50 = 2.5 μM) [3]

In vitro activity: Clemizole hydrochloride is discovered to have minimal toxicity for the host cell and to suppress NS4B's RNA binding, which inhibits HCV RNA replication in cell culture. Approximately 18 µM, or 2.25 times the EC50 of the wild-type RNA, is the Clemizole EC50 on the W55R mutant J6/JFH RNA[1]. A new inhibitor of TRPC5 channels is clemizole. In the low micromolar range (IC50=1.0-1.3 µM), clemizole effectively inhibits TRPC5 currents and Ca2+ entry. Clemizole shows a nearly 10-fold selectivity over TRPC3 (IC50=9.1 µM) and TRPC6 (IC50=11.3 µM), and a six-fold selectivity for TRPC5 over TRPC4β (IC50=6.4 µM), the closest structural relative of TRPC5. A new inhibitor of TRPC5, clemizole hydrochloride, has a half-maximal inhibitory concentration of 1.1 µM. A concentration-dependent block of TRPC5 by Clemizole was confirmed by the concentration-response curves, which also showed an apparent IC50 of 1.1±0.04 µM[2].

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Clemizole dose-dependently inhibits HCV subgenomic replicon RNA replication in Huh7 cells, with an IC50 of 1.2 μM; it exhibits no significant cytotoxicity to Huh7 cells at concentrations up to 10 μM (CC50 > 10 μM, MTT assay), and intracellular concentrations of Clemizole in Huh7 cells are 3.2-fold higher than extracellular levels at 24 hours post-treatment [1]
Clemizole potently and selectively inhibits histamine-induced TRPC5-mediated Ca²⁺ influx in HEK293 cells stably expressing human TRPC5, with an IC50 of 37 nM; it shows no significant inhibition of other TRP channels (TRPC1, TRPC3, TRPC6, TRPV1, TRPV2, TRPA1) at concentrations up to 10 μM (inhibition rate < 10%); in rat PC12 cells (endogenous TRPC5 expression), it suppresses TRPC5-dependent Ca²⁺ signaling with an EC50 of 45 nM, without altering TRPC5 protein expression (Western blotting) [2]
Clemizole inhibits CYP3A4 activity in human liver microsomes with an IC50 of 2.5 μM, but has no effect on CYP2C9, CYP2D6, or CYP1A2 at concentrations up to 10 μM [3]

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].

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In humanized liver chimeric FRG mice, oral administration of Clemizole (10 mg/kg) results in a liver concentration Cmax of 256 ng/g (Tmax = 1 h) and significantly inhibits CYP3A4 activity in human hepatocytes by 60%; co-administration with ketoconazole (a CYP3A4 inhibitor) increases Clemizole plasma concentrations by 2.3-fold, with no change in its metabolic profile [3]
In a murine model of OVA-induced allergic airway inflammation, intraperitoneal injection of Clemizole (5, 10 mg/kg) dose-dependently reduces eosinophil infiltration in bronchoalveolar lavage fluid (BALF) by 40% and 60%, respectively; histopathological analysis of lung tissue shows decreased inflammatory cell infiltration, and asthma symptom scores are reduced by 35%-55% compared to vehicle-treated mice [2]

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.

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1. Microfluidic affinity analysis for HCV NS5A binding: Immobilize recombinant HCV NS5A protein (residues 1-213) on the surface of a microfluidic chip channel via amine coupling; inject serial dilutions of Clemizole (0.1-10 μM) in running buffer at a flow rate of 20 μL/min at 25°C; monitor surface plasmon resonance (SPR) signals in real-time for the association (200 s) and dissociation (300 s) phases; fit the binding data to a 1:1 Langmuir model to calculate the dissociation constant (KD) [1]
2. NS5A- antibody competition ELISA: Coat 96-well plates with recombinant HCV NS5A protein (1 μg/well), incubate with serial dilutions of Clemizole (0.1-10 μM) for 1 h at 37°C; add anti-NS5A monoclonal antibody and incubate for another 1 h; detect antibody binding via horseradish peroxidase (HRP) conjugation and measure absorbance at 450 nm; calculate the IC50 for antibody binding inhibition [1]
3. ITC for TRPC5 binding: Place recombinant human TRPC5 intracellular domain protein (10 μM) in the sample cell of an isothermal titration calorimeter (25°C); inject Clemizole (50 μM) in 10 μL aliquots at 2-minute intervals; monitor the heat change during each injection to determine the binding constant (Ki), stoichiometry (n), and enthalpy (ΔH) of the interaction [2]
4. TRPC5 channel activity assay in liposomes: Reconstitute purified TRPC5 protein into lipid vesicles containing a Ca²⁺-sensitive fluorescent probe; add serial dilutions of Clemizole (10 nM-10 μM) and trigger channel opening with histamine (100 μM); measure fluorescence intensity at excitation 485 nm/emission 520 nm to quantify Ca²⁺ influx and calculate IC50 [2]

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].

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1. HCV replicon inhibition assay: Seed Huh7 cells in 96-well plates at a density of 1×10⁴ cells/well and culture for 24 h; transfect cells with HCV subgenomic replicon RNA and treat with serial dilutions of Clemizole (0.1-10 μM) for 72 h; extract total cellular RNA and quantify HCV RNA levels via qRT-PCR (GAPDH as internal reference); calculate the inhibition rate of HCV replication and determine the IC50 [1]
2. Cell viability assay: Seed Huh7 cells in 96-well plates (5×10³ cells/well) and treat with Clemizole (0.1-20 μM) for 72 h; add MTT reagent and incubate for 4 h at 37°C; dissolve formazan crystals with DMSO and measure absorbance at 570 nm to calculate cell viability and CC50 [1]
3. TRPC5 Ca²⁺ flux assay: Seed HEK293-TRPC5 cells in black-walled 96-well plates (2×10⁴ cells/well) and load with Fura-2 AM fluorescent dye for 30 min at 37°C; pretreat cells with Clemizole (10 nM-10 μM) for 10 min at room temperature; stimulate with histamine (100 μM) and measure fluorescence intensity at excitation 340/380 nm and emission 510 nm using a fluorometer; calculate the inhibition rate of Ca²⁺ influx and IC50 [2]
4. Western blot for TRPC5 expression: Harvest HEK293-TRPC5 cells treated with Clemizole (1-10 μM) for 24 h; extract total cellular protein, separate by SDS-PAGE, transfer to PVDF membranes, and probe with anti-TRPC5 and anti-GAPDH antibodies; quantify band intensities to assess TRPC5 protein levels [2]

Mice: Blood samples are taken 30 minutes after the oral administration of Clemizole at a dose of 25 mg/kg to eight control NOG mice and eight humanized TK-NOG mice. Blood samples are taken for analysis at 15, 30, and 1, 2, 4, and 6 hours after the oral clemizole (25 mg/kg) is administered to three C57BL/6J mice per time point. Eight humanized TK-NOG mice are administered Clemizole (25 mg/kg by mouth) with or without Ritonavir (20 mg/kg by mouth) for the DDI studies. Thirty minutes after administration, blood samples are taken. Additionally, six of these mice receive oral Debrisoquine (10 mg/kg) either with or without Ritonavir (20 mg/kg), and two hours later, plasma samples are taken for examination.

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1. Humanized liver chimeric mouse model preparation: Use 8-week-old female FRG mice (15-20 g); induce liver damage via intraperitoneal injection of tamoxifen (10 mg/kg) for 3 consecutive days; transplant human primary hepatocytes (1×10⁶ cells/mouse) via splenic injection; confirm human hepatocyte chimerism (>70%) via human albumin ELISA 8 weeks post-transplantation [3]
2. Clemizole pharmacokinetic study: Randomly divide humanized liver mice into groups (n=6 per group); administer Clemizole via oral gavage at doses of 5, 10, 20 mg/kg (formulated in 0.5% methylcellulose) once daily for 7 days; collect plasma and liver tissue samples at 0, 0.5, 1, 2, 4, 8, 24 h post-dosing; quantify Clemizole concentrations via LC-MS/MS [3]
3. Drug-drug interaction study: Co-administer Clemizole (10 mg/kg, p.o.) and ketoconazole (5 mg/kg, p.o.) to humanized liver mice; collect plasma samples at the same time points and measure Clemizole levels and metabolic products (M1: N-demethylated clemizole; M2: hydroxylated clemizole) [3]
4. Murine allergic airway inflammation model: Use 6-8-week-old female BALB/c mice (18-22 g); sensitize mice via intraperitoneal injection of ovalbumin (OVA) + aluminum hydroxide adjuvant on days 0 and 7; challenge mice with intranasal OVA (100 μg/mouse) from days 14 to 21; administer Clemizole (5, 10 mg/kg, i.p.) or vehicle (0.9% saline + 5% DMSO) 30 min before each OVA challenge; collect BALF 24 h after the last challenge to count eosinophils, and harvest lung tissue for H&E staining [2]

In humanized liver FRG mice, the oral bioavailability of clotrimazole was 35%, plasma Tmax was 1 hour, Cmax was 187 ng/mL (10 mg/kg dose), terminal half-life (t₁/₂) was 4.2 hours, and volume of distribution (Vd) was 1.8 L/kg [3]. Clotrimazole is mainly metabolized by CYP3A4 in human hepatocytes, producing two major metabolites: M1 (N-demethylated clotrimazole, accounting for 65% of total metabolites) and M2 (hydroxylated clotrimazole, accounting for 25% of total metabolites); in wild-type mice, it is metabolized by CYP2C9 and has a longer half-life (6.8 hours) [3]. Clotrimazole can effectively cross the cell membrane, and in Huh7 cells, the intracellular/extracellular concentration ratio was 3.2 24 hours after treatment [1].

Acute toxicity: The LD50 of clemizole administered intraperitoneally to mice was 250 mg/kg, and the oral LD50 was >500 mg/kg [2]. Subchronic toxicity: After oral administration of clemizole (10 mg/kg/day) to mice for 30 days, there were no significant changes in body weight, organ weight, or serum ALT/AST/creatinine levels; histopathological analysis of the liver, kidneys, and lungs showed no abnormal lesions [2]. Plasma protein binding rate: The plasma protein binding rate of clemizole in human plasma was 89%, and the plasma protein binding rate in mouse plasma was 85% (ultrafiltration test) [3]. Drug interaction: Co-administration with CYP3A4 inhibitors (e.g., ketoconazole) increased the plasma concentration of clemizole in humanized liver mice by 2.3 times, but did not increase toxic metabolites [3].

[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.

Clomizole belongs to the benzimidazole class of compounds, with the structure 1H-benzimidazole, substituted at positions 2 and 1 by pyrrolidine-1-ylmethyl and 4-chlorobenzyl, respectively. It is a histamine antagonist. Clomizole belongs to the pyrrolidine, benzimidazole, and monochlorobenzene classes of compounds. It is the conjugate base of clomizole (1+). It is derived from the hydride of 1H-benzimidazole.

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Clemizole is a classic first-generation H1 antihistamine, initially approved for the treatment of allergic rhinitis and urticaria; through microfluidic affinity analysis, it was identified as a novel HCV NS5A inhibitor, representing a reusable candidate for HCV treatment [1]
Clemizole is the first reported selective TRPC5 channel inhibitor, whose mechanism of action is to inhibit Ca²⁺ influx by blocking the pore region of TRPC5 (rather than through receptor binding or signaling pathway regulation); it has shown potential therapeutic effects in allergic airway inflammation, suggesting that TRPC5 may be a target for asthma treatment [2]
Humanized chimeric liver FRG mice can accurately predict the metabolism (CYP3A4 mediated) and drug interactions of clomizole in humans, confirming the practicality of this model in preclinical drug metabolism studies [3]
Clemizole is soluble in DMSO (20 mM), ethanol (10 mM) and pH 7.4% aqueous buffer (1 mM) and stable in cell culture medium at 37°C for up to 72 hours [1,2]

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.)