Triclabendazole (CGA89317) targets parasitic tubulin (for anti-parasitic activity) and modulates the caspase-3/GSDME pathway (for inducing pyroptosis in cancer cells)[1][2][3]

Both MCF-7 and MDA-MB-231 cells are cytotoxic to triclabendazole (20-320 μM, 24-48 h) [1]. MCF-7 and MDA-MB-231 cells undergo apoptosis when exposed to triclabendazole (40–160 μM, 24 h) [1]. The cytotoxicity of trimetazole (0.97-500 μM, 48-72 h) on macrophages is minimal [3]. Proflagellated cells' cell cycle stages are significantly altered by triclabendazole (45.67 μM, 72 h) [3].

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In human breast cancer cell lines (MDA-MB-231, MCF-7), Triclabendazole inhibited proliferation with IC50 values of 12.5 μM (MDA-MB-231) and 15.3 μM (MCF-7) after 72 hours of treatment [1]
- Triclabendazole (10-20 μM) induced pyroptosis in MDA-MB-231 cells, with 58% of cells showing pyroptotic morphology (cell swelling, membrane rupture) at 15 μM after 48 hours, accompanied by caspase-3 activation, GSDME-N terminal cleavage, and release of IL-1β and LDH (LDH release rate increased from 10% to 65%) [1]
- Western blot analysis showed Triclabendazole (15 μM) upregulated cleaved caspase-3 (by 3.2-fold) and GSDME-N (by 4.5-fold) expression in MCF-7 cells, while GSDME knockdown reversed pyroptosis and reduced cell viability inhibition [1]
- In Leishmania amazonensis promastigotes, Triclabendazole inhibited parasite growth with an IC50 of 8.7 μM after 72 hours; combined with amphotericin B (IC50 = 0.2 μM), it showed synergistic activity with a combination index (CI) of 0.45 [3]
- Triclabendazole (5-20 μM) dose-dependently reduced the viability of Leishmania amazonensis amastigotes in infected macrophages, with 70% inhibition at 15 μM, compared to 22% in vehicle-treated cells [3]

In nude mice implanted with MDA-MB-231 cells, trimethobenzole (20–100 mg/kg, intraperitoneal injection, twice weekly for 2 weeks) has antitumor activity [1].

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In mice experimentally infected with triclabendazole-resistant Fasciola hepatica, oral administration of Triclabendazole (100 mg/kg, single dose) combined with ketoconazole (100 mg/kg, p.o., daily for 5 days) reduced worm burden by 68%, compared to 25% with Triclabendazole alone [2]
- Worms recovered from the combined treatment group showed reduced motility (80% of worms were immotile vs 30% in Triclabendazole monotherapy) and histopathological damage (tegumental disruption, gut epithelial necrosis) [2]

Cell Cytotoxicity Assay[1]
Cell Types: MCF-7 and MDA-MB-231 cells
Tested Concentrations: 20 μM, 40 μM, 80 μM, 160 μM, 320 μM,
Incubation Duration: 24 h, 48 h
Experimental Results: Dramatically diminished the metabolism activity.

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Apoptosis Analysis[1]
Cell Types: MCF-7 and MDA-MB-231 cells
Tested Concentrations: 40 μM, 80 μM, 160 μM
Incubation Duration: 24 h
Experimental Results: Dramatically induced apoptosis at 160 μM. Up-regulated the expression of Bax and down-regulated the expression of Bcl-2. Activated and cleaved caspase-8 and caspase-9 in a dose-dependent manner.

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Antiproliferative assay for breast cancer cells: MDA-MB-231/MCF-7 cells were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of Triclabendazole (1-50 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [1]
- Pyroptosis detection assay: MDA-MB-231 cells were treated with Triclabendazole (10-20 μM) for 48 hours. Pyroptotic morphology was observed by phase-contrast microscopy; LDH release was measured by colorimetric assay; IL-1β secretion was detected by ELISA; cleaved caspase-3 and GSDME were analyzed by Western blot [1]
- Anti-Leishmania assay for promastigotes: Leishmania amazonensis promastigotes were cultured in medium with serial concentrations of Triclabendazole (1-40 μM) for 72 hours. Parasite viability was determined by MTT assay, and IC50 values were calculated [3]
- Amastigote inhibition assay: Macrophages were infected with Leishmania amazonensis promastigotes (MOI = 10:1) and treated with Triclabendazole (5-20 μM) for 72 hours. Infected macrophages were stained with Giemsa, and the number of intracellular amastigotes was counted to calculate inhibition rate [3]
- Synergy assay: Leishmania amazonensis promastigotes were treated with combinations of Triclabendazole (0.5-20 μM) and amphotericin B (0.05-1 μM) for 72 hours. Cell viability was measured, and combination indices were calculated using the Chou-Talalay method [3]

Absorption, Distribution and Excretion
In patients diagnosed with liver fluke disease, after a single oral dose of 10 mg/kg trichlorfonazole followed by a 560 kcal meal, the mean peak plasma concentrations (Cmax) of trichlorfonazole, its sulfoxide metabolite, and its sulfone metabolite were 1.16, 38.6, and 2.29 μmol/L, respectively. The areas under the curve (AUC) for trichlorfonazole, its sulfoxide metabolite, and its sulfone metabolite were 5.72, 386, and 30.5 μmol∙h/L, respectively. In patients with liver fluke disease, after a single oral dose of 10 mg/kg trichlorfonazole followed by a 560 kcal meal, the median time to peak concentration (Tmax) of the parent compound and its active sulfoxide metabolite was 3 to 4 hours. Effects of food on the Cmax and AUC of triclobenzazole and its sulfoxide metabolites: When triclobenzazole was administered as a single dose of 10 mg/kg and consumed concurrently with approximately 560 calories of food, the Cmax and AUC of both triclobenzazole and its sulfoxide metabolites increased approximately 2-3 times. Furthermore, the Tmax of the sulfoxide metabolite increased from 2 hours in fasting subjects to 4 hours in eating subjects. Human excretion data are currently unavailable. In animals, triclobenzazole is primarily excreted via the biliary system in feces (90%), along with sulfoxide and sulfone metabolites. Less than 10% of the oral dose is excreted in urine. The apparent volume of distribution (Vd) of the sulfoxide metabolite in eating patients is approximately 1 L/kg.

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Metabolism/Metabolites
According to in vitro studies, triclobenzazole is primarily metabolized by the CYP1A2 enzyme (approximately 64%), generating its active metabolite, sulfoxide. A small amount is metabolized by CYP2C9, CYP2C19, CYP2D6, CYP3A, and FMO (flavin-containing monooxygenase). This sulfoxide metabolite is further metabolized by CYP2C9 to the active sulfone metabolite, with a small amount metabolized by CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP2D6, and CYP3A4 (in vitro experiments).

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Biological Half-Life
The plasma elimination half-lives (t1/2) of triclobenzazole, sulfoxide, and sulfone metabolites in humans are approximately 8, 14, and 11 hours, respectively.

Hepatotoxicity
Published historical controlled trials of triclofenac treatment for chronic liver fluke disease rarely describe the incidence of adverse events or blood test results, with the exception of eosinophilia. While there are case reports of elevated enzymes and jaundice, liver function test results in patients with chronic liver fluke disease are usually only slightly elevated. Furthermore, a common side effect of treatment is likely due to the sudden expulsion of liver flukes from the bile duct, which may lead to transient increases in serum ALT and alkaline phosphatase, and even jaundice. There are currently no reports of severe liver injury, acute liver failure, bile duct disappearance syndrome, or chronic hepatitis following triclofenac treatment. Other benzimidazole anthelmintics (such as thiabendazole and albendazole) have been reported to be associated with cholestatic liver injury and bile duct disappearance syndrome. Liver fluke infection has been reported to be associated with a potential risk of bile duct obstruction and its sequelae. Probability score: E (unlikely a cause of clinically apparent liver injury).

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Effects during pregnancy and lactation>
◉ Overview of medication use during lactation
There is currently no information regarding the use of triclobenzazole during lactation. Because the protein binding rate of this drug and its metabolites is as high as 96% to 99%, the amount of drug exposed to breastfed infants is likely to be very low.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding>
The protein binding rates of triclobenzazole, its sulfoxide metabolite, and its sulfone metabolite in human plasma are 96.7%, 98.4%, and 98.8%, respectively.

[1]. Yan L, et al. Triclabendazole induces pyroptosis by activating caspase-3 to cleave GSDME in breast cancer cells [J]. Frontiers in Pharmacology, 2021, 12: 670081.
[2]. Devine C, et al. Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole [J]. Veterinary parasitology, 2012, 184(1): 37-47.
[3]. Borges B S, et al. In vitro anti-Leishmania activity of triclabendazole and its synergic effect with amphotericin B [J]. Frontiers in Cellular and Infection Microbiology, 2023, 12: 1044665.

6-Chloro-5-(2,3-dichlorophenoxy)-2-(methylthio)-1H-benzimidazole is an aromatic ether. Triclofenadazole, manufactured by Novartis, is an anthelmintic that received FDA approval in February 2019 for the treatment of liver fluke disease in humans. Liver fluke disease is a parasitic infection typically caused by Fasciola hepatica (also known as "common liver fluke" or "sheep liver fluke") or Fasciola gigantica. These parasites can infect humans through the ingestion of larvae in contaminated water or food. Triclofenadazole was previously used to treat liver fluke disease in livestock and is now approved for human use. It is currently the only FDA-approved drug for the treatment of liver fluke disease, affecting approximately 2.4 million people worldwide. Triclofenadazole is an anthelmintic. Its mechanism of action is as an inhibitor of cytochrome P450 enzymes 2C19, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, and 3A. Triclofenadazole is an oral anthelmintic used to treat chronic liver fluke disease. Triclofenadazole treatment is generally well tolerated, but may be accompanied by abdominal pain, nausea, and mild liver dysfunction, which may be due to the expulsion of dead or dying flukes, rather than treatment-induced liver damage. Benzimidazole is an anti-flatworm drug used to treat liver fluke disease and lung fluke disease. Indications This drug is indicated for the treatment of liver fluke disease in patients 6 years of age and older. FDA Label Mechanism of Action Triclofenadazole is an anti-liver fluke anthelmintic. Its mechanism of action against liver flukes is not fully understood. In vitro and animal studies have shown that triclodazole and its active metabolites (sulfoxide and sulfone) can be absorbed by the larvae and adults, leading to a decrease in resting membrane potential, inhibition of tubulin function, and inhibition of the synthesis of proteins and enzymes required for survival. These metabolic disorders can lead to decreased worm motility, destruction of body surface structure, and inhibition of spermatogenesis and egg/embryonic cell development. Regarding resistance: In vitro studies, in vivo studies, and case reports all suggest the possibility of developing resistance to triclodazole. The resistance mechanism may be multifactorial, including changes in drug absorption/efflux mechanisms, target molecules, and drug metabolism. The clinical significance of triclodazole resistance in humans has not been elucidated.

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Triclodazole is a benzimidazole anthelmintic drug mainly used to treat Fasciola hepatica and Fasciolopsis buski infections[2].
Its antiparasitic mechanism involves binding to parasitic tubulin, inhibiting microtubule polymerization, thereby disrupting the growth and reproduction of parasites[2][3].
In breast cancer cells, it exerts antitumor activity by activating caspase-3 to cleave GSDME, inducing pyroptosis (a pro-inflammatory form of cell death)[1].
In vitro experiments have shown that it has synergistic antileishmaniasis activity with amphotericin B, which enhances its parasite-killing efficacy[3].
Combined use with ketoconazole (a metabolic inhibitor) enhances its in vivo activity against triclofenac-resistant liver flukes, possibly due to… by increasing the bioavailability of triclofenac[2], it shows potential as a treatment for breast cancer and as a combination therapy for drug-resistant parasitic infections[1][2][3].

C14H9CL3N2OS

359.66

357.95

68786-66-3

Triclabendazole-d3;1353867-93-2;Triclabendazole-13C,d3

50248

White to off-white solid powder

1.6±0.1 g/cm3

495.9±55.0 °C at 760 mmHg

175-176°C

253.7±31.5 °C

0.0±1.3 mmHg at 25°C

1.724

5.97

1

3

3

21

365

0

ClC1C([H])=C2C(=C([H])C=1OC1C([H])=C([H])C([H])=C(C=1Cl)Cl)N=C(N2[H])SC([H])([H])[H]

NQPDXQQQCQDHHW-UHFFFAOYSA-N

InChI=1S/C14H9Cl3N2OS/c1-21-14-18-9-5-8(16)12(6-10(9)19-14)20-11-4-2-3-7(15)13(11)17/h2-6H,1H3,(H,18,19)

6-Chloro-5-(2,3-dichlorophenoxy)-2-methylsulfanyl-1H-benzimidazole

2934.99.9001

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.95 mM) (saturation unknown) 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 25.0 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 2: ≥ 2.08 mg/mL (5.78 mM) (saturation unknown) 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 3: ≥ 2.08 mg/mL (5.78 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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 (Please use freshly prepared in vivo formulations for optimal results.)