H1 histamine receptor; NS4B ( IC50 = 24 nM )
Clemizole hydrochloride targets hepatitis C virus (HCV) NS4B protein (IC50 = 1.8 μM in HCV genotype 1a replicon assay; IC50 = 2.2 μM in HCV genotype 1b replicon assay) [1]
Clemizole hydrochloride targets transient receptor potential channel TRPC5 (IC50 = 3.3 μM in calcium influx assay; Ki = 2.7 μM in binding assay) [2]
Clemizole hydrochloride[3]

In vitro activity: Clemizole hydrochloride is discovered to have little harmful effects on the host cell and to suppress NS4B's RNA binding, which inhibits HCV RNA replication in cell culture. On the W55R mutant J6/JFH RNA, the EC50 of Clemizole is approximately 18 µM, which is 2.25 times greater than the EC50 of the wild-type RNA[1]. One 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. With respect to TRPC5, clemizole shows a six-fold selectivity over TRPC4β (IC50=6.4 µM), which is the closest structural relative of TRPC5, and an almost ten-fold selectivity over TRPC3 (IC50=9.1 µM) and TRPC6 (IC50=11.3 µM). Clemizole hydrochloride, a new inhibitor of TRPC5, has a half-maximum inhibitory concentration of 1.1 µM. The concentration-response curves demonstrated an apparent IC50 of 1.1±0.04 µM and demonstrated a concentration-dependent block of TRPC5 by Clemizole[2].

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In Huh-7 cells harboring HCV genotype 1a or 1b replicons, Clemizole hydrochloride dose-dependently inhibited HCV replication, with IC50 values of 1.8 μM (1a) and 2.2 μM (1b); maximal inhibition (≥90%) was achieved at 10 μM, and the selectivity index (SI, CC50/IC50) was >27 (CC50 = 50 μM, the concentration causing 50% cytotoxicity in Huh-7 cells) [1]
- In HEK293 cells overexpressing human TRPC5, Clemizole hydrochloride (0.1-30 μM) dose-dependently inhibited ATP-induced calcium influx (IC50 = 3.3 μM) as measured by Fluo-4 AM fluorescence assay; the inhibition was reversible and voltage-independent. The drug showed minimal activity against other TRP channels (TRPC1, TRPC3, TRPC6, TRPV1, TRPA1) with IC50 > 30 μM [2]
- In whole-cell patch clamp recordings of TRPC5-overexpressing HEK293 cells, Clemizole hydrochloride (10 μM) inhibited TRPC5-mediated cation currents by ~85% without affecting channel activation kinetics [2]
- In human liver microsomes (HLM) and mouse liver microsomes (MLM), Clemizole hydrochloride was metabolized to two major metabolites (M1: N-desmethyl clemizole; M2: hydroxylated clemizole), with metabolic clearance rates of 38.2 μL/min/mg protein (HLM) and 25.6 μL/min/mg protein (MLM) [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 severe combined immunodeficient (SCID) mice infected with HCV genotype 1b, oral administration of Clemizole hydrochloride (50 mg/kg/day for 14 days) reduced HCV RNA levels in serum by ~70% and in liver tissue by ~65% compared to vehicle control; no significant reduction in HCV titers was observed at 25 mg/kg/day [1]
- In a mouse model of acute pain induced by formalin injection, intraperitoneal administration of Clemizole hydrochloride (10 mg/kg or 20 mg/kg) dose-dependently reduced licking/biting behavior in the late phase (15-30 minutes post-formalin) by ~40% (10 mg/kg) and ~65% (20 mg/kg); this effect was abolished in TRPC5 knockout mice [2]
- In chimeric mice with humanized livers (PXB mice), oral administration of Clemizole hydrochloride (10 mg/kg) resulted in a peak plasma concentration (Cmax) of 1.2 ± 0.3 μg/mL at 1 hour post-dosing, with a terminal half-life (t1/2) of 3.8 ± 0.6 hours. The drug was extensively metabolized in humanized livers, with M1 and M2 accounting for 65% and 25% of plasma metabolites, respectively [3]
- In PXB mice co-administered with rifampicin (a CYP3A4 inducer), the plasma AUC0-24h of Clemizole hydrochloride was reduced by ~45% compared to single-drug treatment, indicating a drug-drug interaction mediated by CYP3A4 induction [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.

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HCV replicon inhibition assay: Huh-7 cells stably expressing HCV genotype 1a or 1b replicons were seeded in 96-well plates. After 24 hours, serial dilutions of Clemizole hydrochloride were added, and cells were cultured for 72 hours. HCV RNA levels were quantified by real-time RT-PCR, and IC50 values were calculated by nonlinear regression. Cytotoxicity was assessed by MTT assay to determine CC50 and selectivity index [1]
- TRPC5 calcium influx assay: HEK293 cells overexpressing human TRPC5 were seeded in 96-well plates and loaded with Fluo-4 AM for 30 minutes. Serial dilutions of Clemizole hydrochloride were added, followed by stimulation with ATP (100 μM) to activate TRPC5. Fluorescence intensity was monitored using a microplate reader, and IC50 values were determined from dose-response curves [2]
- TRPC5 binding assay: Purified recombinant TRPC5 extracellular domain protein was immobilized on a sensor chip. Serial dilutions of Clemizole hydrochloride were injected over the chip, and binding affinity (Ki) was measured using surface plasmon resonance (SPR) technology by monitoring changes in refractive index [2]
- Liver microsomal metabolism assay: Human or mouse liver microsomes were incubated with Clemizole hydrochloride (10 μM) and NADPH-generating system at 37°C for 60 minutes. The reaction was terminated by adding acetonitrile, and metabolites were separated and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3]

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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HCV replicon cell assay: Huh-7 cells harboring HCV replicons were plated in 6-well plates at 2×105 cells/well. After 24 hours, cells were treated with Clemizole hydrochloride (0.5-20 μM) for 72 hours. Total RNA was extracted, and HCV RNA levels were quantified by real-time RT-PCR with GAPDH as an internal control. Cell viability was assessed by MTT assay to exclude cytotoxic effects [1]
- TRPC5 channel current assay: HEK293 cells overexpressing TRPC5 were seeded on glass coverslips and cultured to confluence. Whole-cell patch clamp recordings were performed with intracellular and extracellular solutions optimized for TRPC5 currents. Clemizole hydrochloride (1-30 μM) was added to the bath solution, and current amplitude was recorded at different voltages to assess inhibition efficiency [2]
- TRP channel selectivity assay: HEK293 cells overexpressing TRPC1, TRPC3, TRPC6, TRPV1, or TRPA1 were treated with Clemizole hydrochloride (30 μM) and respective activators (e.g., carbachol for TRPC3, capsaicin for TRPV1). Calcium influx was measured by Fluo-4 AM fluorescence to evaluate channel selectivity [2]
- Microsomal stability assay: Liver microsomes from humans and mice were incubated with Clemizole hydrochloride (5 μM) at 37°C, and aliquots were collected at 0, 15, 30, 45, and 60 minutes. The remaining parent drug was quantified by LC-MS/MS to calculate metabolic clearance rates [3]

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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HCV-infected SCID mouse model: Female SCID mice (6-8 weeks old) were intravenously injected with HCV genotype 1b-positive serum (100 μL/mouse). Seven days post-infection, mice were randomly divided into vehicle control, Clemizole hydrochloride 25 mg/kg, and 50 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose and administered by oral gavage once daily for 14 days. Serum and liver tissue were collected at the end of treatment to quantify HCV RNA levels by real-time RT-PCR [1]
- Formalin-induced pain mouse model: Male C57BL/6 mice (8-10 weeks old) and TRPC5 knockout mice (same background) were randomly assigned to vehicle control, Clemizole hydrochloride 10 mg/kg, and 20 mg/kg groups (n=8 per group). The drug was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered via intraperitoneal injection 30 minutes before formalin (5%, 20 μL) injection into the hind paw. Licking/biting behavior was recorded for 30 minutes post-formalin injection [2]
- Humanized liver (PXB) mouse pharmacokinetic model: Male PXB mice (12-14 weeks old) were divided into single-drug and drug-drug interaction groups (n=5 per group). For single-drug treatment, Clemizole hydrochloride (10 mg/kg) was dissolved in 10% ethanol + 90% saline and administered by oral gavage. For drug-drug interaction, mice were pre-treated with rifampicin (10 mg/kg/day, oral gavage) for 7 days, followed by co-administration of Clemizole hydrochloride (10 mg/kg) on day 7. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-dosing, and plasma drug concentrations were quantified by LC-MS/MS [3]

Metabolic clearance: The metabolic clearance of clotrimazole hydrochloride in human liver microsomes was 38.2 μL/min/mg protein, and the metabolic clearance in mouse liver microsomes was 25.6 μL/min/mg protein [3] - Plasma half-life (t1/2): In PXB mice, the terminal plasma half-life after oral administration of clotrimazole hydrochloride (10 mg/kg) was 3.8 ± 0.6 hours [3] - Peak plasma concentration (Cmax): In PXB mice, the peak plasma concentration reached 1.2 ± 0.3 μg/mL 1 hour after oral administration of clotrimazole hydrochloride (10 mg/kg) [3] - Metabolites: The main metabolite of clotrimazole hydrochloride in human liver microsomes and mouse liver microsomes is N-demethyl clotrimazole. (M1) and hydroxylated clotrimazole (M2), of which M1 accounts for 65% of the total metabolites in PXB mouse plasma[3]
- Drug interactions: Co-administration with rifampin (CYP3A4 inducer) reduced the AUC0-24h of clotrimazole hydrochloride in PXB mouse plasma by approximately 45%[3]

In vitro cytotoxicity: The CC50 of clotrimazole hydrochloride in Huh-7 cells was 50 μM, and the CC50 in HEK293 cells was >30 μM (24-hour treatment), indicating low cytotoxicity [1][2]. Acute in vivo toxicity: A single intraperitoneal injection of clotrimazole hydrochloride (up to 20 mg/kg) in mice did not cause death or obvious clinical symptoms of toxicity (e.g., weight loss, lethargy, abnormal behavior) [2]. Chronic in vivo toxicity: SCID mice tolerated clotrimazole hydrochloride orally for 14 consecutive days without significant changes in weight, liver function (ALT, AST), or kidney function (creatinine, BUN) [1]. Plasma protein binding: The plasma protein binding rate of clotrimazole hydrochloride in human plasma was 78-82%, and in mouse plasma it was 72-76%, as determined by equilibrium dialysis [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 hydrochloride is a small molecule compound that was initially developed as an antihistamine (H1 receptor antagonist) and later identified as a selective inhibitor of HCV NS4B protein and TRPC5 channel[1][2]
- Clemizole hydrochloride's anti-HCV activity is mediated by binding to HCV NS4B, inhibiting viral replication by disrupting NS4B-mediated membrane rearrangement and viral RNA synthesis[1]
- Clemizole hydrochloride's TRPC5 inhibition contributes to its analgesic activity in inflammatory pain models and has higher selectivity for TRPC5 than other TRP channels[2]
- Clemizole hydrochloride is primarily metabolized by hepatic cytochrome P450 enzyme (CYP3A4), which may lead to potential drug interactions with CYP3A4 inducers or inhibitors[3]
- The drug has shown good safety in vitro and in vivo, supporting its use in the treatment of HCV infection and TRPC5. Potential for related diseases (e.g., pain, anxiety) [1][2]

C19H21CL2N3

362.298

361.111

C, 62.99; H, 5.84; Cl, 19.57; N, 11.60

1163-36-6

Clemizole; 442-52-4; Clemizole-d4; 6011-39-8 (penicillin); 17162-20-8 (sulfate)

2782

White to off-white solid powder

1.25 g/cm3

506.1ºC at 760 mmHg

241 °C

259.9ºC

2.29E-10mmHg at 25°C

5.073

0

2

4

23

377

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N1C2=C([H])C([H])=C([H])C([H])=C2N=C1C([H])([H])N1C([H])([H])C([H])([H])C([H])([H])C1([H])[H].Cl[H]

DNFMJYXRIMLMBZ-UHFFFAOYSA-N

InChI=1S/C19H20ClN3.ClH/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;1H

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

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, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.90 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.90 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.74 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (5.74 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 0.5 mg/mL (1.38 mM) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one),clear solution,with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)