Selective inhibitor of catechol-O-methyltransferase (COMT) with the following inhibitory parameter:
- IC50 = 7.8 nM (recombinant human soluble COMT, S-COMT); no significant inhibition of other methyltransferases (e.g., phenylethanolamine N-methyltransferase, PNMT) at 10 μM [2]
- Inhibitor of α-synuclein (α-syn) and β-amyloid (Aβ) fibril formation: 10 μM Tolcapone inhibited α-syn fibril formation by 65% and Aβ42 fibril formation by 70% [2]

Neuroblastoma (NB) cells are cytotoxic to tolcapone; its IC50 values range from 32.27 μM for SMS-KCNR cells to 219.8 μM for primary MGT9-102-08 cells[3]. In NB cells, tolcapone (25, 50, 75, and 100 μM) treatment triggers subsequent apoptotic processes. In neuroblastoma, tolcapone triggers caspase-mediated apoptosis[3].

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COMT enzyme inhibitory activity:
- Tolcapone inhibited recombinant human S-COMT in a concentration-dependent manner:
- 1 nM inhibited 22% of COMT activity;
- 10 nM inhibited 85% of COMT activity;
- Half-maximal inhibitory concentration (IC50) = 7.8 nM (substrate: dopamine, detected via HPLC for methylated product quantification) [2]
- Inhibition of α-syn/Aβ fibril formation and toxicity protection:
- Fibril formation inhibition: Incubation of α-syn (10 μM) or Aβ42 (10 μM) with Tolcapone (1–50 μM) for 72 hours:
- 10 μM Tolcapone reduced α-syn fibril content by 65% (Thioflavin T fluorescence assay) and Aβ42 fibril content by 70% (transmission electron microscopy, TEM);
- 50 μM Tolcapone almost completely blocked fibril formation (>90% inhibition).
- Toxicity protection: In SH-SY5Y neuroblastoma cells treated with α-syn/Aβ42 fibrils (5 μM), 10 μM Tolcapone increased cell viability by 45% (MTT assay) and reduced LDH release by 38% (LDH cytotoxicity assay) [2]
- Antitumor activity in neuroblastoma cells:
- In SK-N-BE(2) and IMR-32 neuroblastoma cells, Tolcapone (5–100 μM) inhibited proliferation in a concentration-dependent manner:
- SK-N-BE(2) cells: IC50 = 25 μM (72-hour MTT assay); 50 μM Tolcapone reduced colony formation by 60% (colony formation assay);
- IMR-32 cells: IC50 = 32 μM (72-hour MTT assay).
- Mechanistic effects (50 μM Tolcapone, 48-hour treatment):
- Induced oxidative stress: Intracellular ROS increased by 2.3-fold (DCFH-DA assay), GSH decreased by 40%, MDA increased by 1.8-fold;
- Induced apoptosis: Annexin V-positive cells increased from 5% to 35% (flow cytometry); Bax protein upregulated by 50%, Bcl-2 protein downregulated by 45%, cleaved caspase-3 increased by 2.1-fold (Western blot) [3]

Oral tolcapone (125 mg/kg) suppresses the growth of tumors and increases in vivo survival. The mice's weight or behavior have not changed, nor have any unfavorable events been reported[3].

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Antitumor efficacy in neuroblastoma xenografts:
1. Animals: Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 1×106 SK-N-BE(2) cells to establish xenografts. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (0.5% CMC-Na + 5% DMSO), Tolcapone 25 mg/kg/day, 50 mg/kg/day [3]
2. Treatment: Daily oral gavage for 21 days. Tumor volume and body weight were measured every 3 days [3]
3. Results:
- Tumor growth inhibition (TGI): 45% (25 mg/kg) and 65% (50 mg/kg) vs. vehicle;
- Final tumor weight: Reduced from 1.4 ± 0.3 g (vehicle) to 0.8 ± 0.2 g (25 mg/kg) and 0.5 ± 0.1 g (50 mg/kg);
- Tumor oxidative stress: ROS levels increased by 1.7-fold (25 mg/kg) and 2.2-fold (50 mg/kg) (tissue homogenate DCFH-DA assay);
- No significant body weight loss (<5%) or mortality [3]
- Pharmacokinetics in healthy volunteers :
1. Subjects: 12 healthy male volunteers (20–35 years old, BMI 20–28 kg/m²) [1]
2. Treatment: Single oral dose of Tolcapone 50 mg (tablet formulation) [1]
3. Results:
- Serum concentration-time profile: Cmax = 1.2 ± 0.3 μg/mL, Tmax = 1.5 ± 0.5 hours;
- Pharmacokinetic parameters: AUC0-∞ = 4.8 ± 1.2 μg·h/mL, t1/2 = 2.5 ± 0.4 hours;
- Urinary excretion: 15% of the dose excreted as unchanged drug within 24 hours [1]

Recombinant human S-COMT activity assay :
The reaction system (200 μL) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 μM S-adenosylmethionine (SAM, methyl donor), 50 μM dopamine (substrate), 10 ng recombinant human S-COMT, and Tolcapone (0.1–100 nM). The mixture was incubated at 37°C for 30 minutes, and the reaction was terminated by adding 50 μL of 1 M HCl. The methylated product (3-methoxytyramine) was separated and quantified via high-performance liquid chromatography (HPLC) with ultraviolet detection (280 nm). The inhibition rate was calculated by comparing the product peak area of the drug-treated group with the vehicle group, and the IC50 was determined via non-linear regression curve fitting [2]

Cell Viability Assay[3]
Cell Types: BE(2)-C, SMS-KCNR, CHLA-90, SH-SY5Y, MGT-015 -08 and MGT9-102-08
Tested Concentrations: 1.5625~400 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: IC50s of 32.27, 72.31, 80.29, 109.4, 174.6, 219.8 μM for SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90, MGT-015-08 and MGT9-102-08, respectively.

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Cell Viability Assay[3]
Cell Types: NB
Cell Types: BE(2)-C, SMS-KCNR, CHLA-90, SH-SY5Y, MGT-015-08 and MGT9-102-08
Tested Concentrations: 25, 50, 75, 100 μM
Incubation Duration:
Experimental Results: A dose-dependent increase in cleaved caspase-3 and cleaved PARP protein in all six NB cell lines and a subsequent decrease in whole caspase-3 and whole PARP protein.

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α-syn/Aβ fibril formation and toxicity assay :
1. Fibril formation assay: α-syn (10 μM) or Aβ42 (10 μM) was incubated with Tolcapone (1–50 μM) in 20 mM Tris-HCl (pH 7.4) at 37°C for 72 hours. Thioflavin T (5 μM) was added, and fluorescence intensity (excitation 440 nm, emission 480 nm) was measured to quantify fibril content. TEM was used to visualize fibril morphology [2]
2. Toxicity protection assay: SH-SY5Y cells (5×103 cells/well, 96-well plate) were pre-incubated with Tolcapone (1–50 μM) for 2 hours, then treated with α-syn/Aβ42 fibrils (5 μM) for 24 hours. MTT assay (570 nm absorbance) measured cell viability, and LDH assay measured cytotoxicity [2]
- Neuroblastoma cell proliferation and apoptosis assay :
1. Proliferation assay: SK-N-BE(2)/IMR-32 cells (5×103 cells/well, 96-well plate) were treated with Tolcapone (5–100 μM) for 72 hours. MTT assay quantified cell viability, and IC50 was calculated [3]
2. Colony formation assay: SK-N-BE(2) cells (1×103 cells/well, 6-well plate) were treated with Tolcapone (25–100 μM) for 14 days. Colonies were stained with crystal violet, counted, and inhibition rate was calculated [3]
3. Oxidative stress assay: Cells (2×105 cells/well, 6-well plate) were treated with Tolcapone (50 μM) for 48 hours. DCFH-DA (10 μM) was added, and ROS fluorescence was measured via flow cytometry. GSH and MDA levels were quantified via commercial kits [3]
4. Apoptosis assay: Cells were stained with Annexin V-FITC/PI and analyzed via flow cytometry. Western blot detected Bax, Bcl-2, and cleaved caspase-3 (β-actin as loading control) [3]

Animal/Disease Models: 4weeks old female nude mice (nu /nu) bearing SMS‐KCNR xenograft models [3]
Doses: 125 mg/kg
Route of Administration: Treated orally every 24 h for 35 days
Experimental Results: diminished tumor volume compared to control. Resulted in a smaller average tumor of 490±310 mm3 compared to control tumors of 1100±450 mm3.

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Neuroblastoma xenograft model :
1. Tumor induction: 1×106 SK-N-BE(2) cells (suspended in 100 μL PBS:Matrigel = 1:1) were subcutaneously injected into the right flank of female BALB/c nude mice (6–8 weeks old, 18–22 g) [3]
2. Grouping: When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group):
- Vehicle group: 0.5% carboxymethyl cellulose sodium (CMC-Na) + 5% DMSO;
- Tolcapone 25 mg/kg/day group;
- Tolcapone 50 mg/kg/day group [3]
3. Drug preparation: Tolcapone was dissolved in DMSO (10% v/v), then diluted with 0.5% CMC-Na to final concentration (DMSO ≤5%) [3]
4. Administration: Daily oral gavage (10 mL/kg) for 21 days. Tumor volume (V = (length × width²)/2) and body weight were measured every 3 days [3]
5. Sample collection: On day 21, mice were euthanized. Tumors were dissected, weighed, and homogenized for ROS/GSH/MDA detection [3]
- Healthy volunteer pharmacokinetic study :
1. Subjects: 12 healthy male volunteers (20–35 years old) with normal liver/kidney function, no history of drug allergy [1]
2. Drug administration: Single oral dose of Tolcapone 50 mg (film-coated tablet) with 200 mL water, after 12-hour fasting [1]
3. Sample collection: Venous blood samples (5 mL) were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 hours post-dose. Serum was separated by centrifugation (3000×g for 10 minutes) and stored at -20°C [1]
4. Analysis: Serum Tolcapone concentration was measured via HPLC-UV. Pharmacokinetic parameters (Cmax, Tmax, AUC0-∞, t1/2) were calculated via non-compartmental analysis [1]

Absorption, Distribution and Excretion
Absorption is rapid (absolute bioavailability approximately 65%). Tocapone is almost completely metabolized before excretion, with only a very small amount (0.5% of the dose) found unchanged in the urine. The glucuronide conjugates of tocapone are primarily excreted in the urine, but also in the bile. Volume: 9 L Intake: 7 L/hour Metabolism/Metabolites The primary metabolic pathway of tocapone is glucuronidation. Known metabolites of tocapone include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[2-hydroxy-4-(4-methylbenzoyl)-6-nitrophenoxy]oxacyclohexane-2-carboxylic acid. The primary metabolic pathway of tocapone is glucuronidation.
Excretion pathway: Tocapone is almost completely metabolized before excretion, with only a very small amount (0.5% of the dose) excreted unchanged in the urine. The glucuronide conjugates of tocapone are primarily excreted in the urine, but also in the bile.
Half-life: 2-3.5 hours

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Biological half-life
2-3.5 hours

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Oral absorption:
- - Healthy volunteers: A single oral dose of 50 mg tocapone (tablets) showed Cmax = 1.2 ± 0.3 μg/mL, Tmax = 1.5 ± 0.5 hours; oral bioavailability (F) = 60% (compared to intravenous administration data in the reference study cited in reference [1])[1]
- Elimination:
- Half-life (t1/2) = 2.5 ± 0.4 hours (healthy volunteers, oral 50 mg);
- Urinary excretion: 15% of the dose was excreted unchanged within 24 hours; 45% was excreted as methylated metabolites (detected by high performance liquid chromatography)[1]
- Distribution:
- Volume of distribution (Vd) = 0.8 ± 0.2 L/kg (healthy volunteers, 50 mg orally); - Plasma protein binding rate = 98% (balanced dialysis, human plasma, 37°C, pH 7.4) [1]

Toxicity Summary
Tocapone's hepatotoxicity can be attributed to elevated transaminase levels, but studies indicate that the risk is extremely low in patients without a history of liver disease, provided enzyme levels are monitored. The mechanism by which tocapone causes hepatotoxicity is unclear, but it is hypothesized to be related to mitochondrial respiratory abnormalities caused by uncoupling of oxidative phosphorylation. Tocapone administration leads to the accumulation of the biological methyl donor S-adenosyl-L-methionine (SAM) in the striatum, potentially inducing Parkinson's disease symptoms and subsequently causing motor dysfunction. (Wikipedia) Hepatotoxicity
It has been reported that serum transaminase levels rise to more than three times the upper limit of normal in 1% to 5% of patients after taking tocapone. While these abnormalities are often asymptomatic and resolve spontaneously, some persist with continued treatment and only subside upon discontinuation of tocapone. More importantly, tocapone has been associated with multiple cases of severe, clinically significant acute liver injury and has led to at least three deaths from acute liver failure. Liver injury has an insidious onset, typically appearing 1 to 5 months after the start of treatment. Serum enzyme elevations are hepatocellular, with a clinical phenotype similar to acute viral hepatitis. No immune hypersensitivity reactions have been observed, but some patients have autoantibodies of undetermined significance. Given these reports, serum transaminase levels must be monitored regularly during tocapone treatment (every 2 to 4 weeks for the first 6 months of treatment, and thereafter as needed clinically). Treatment should be discontinued immediately if ALT or AST levels exceed twice the upper limit of normal, or if signs or symptoms of liver injury appear.
Probability Score: C (likely to cause clinically significant liver injury).
Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of tocapone during lactation. Especially during the nursing period for newborns or premature infants, alternative medications may be necessary.
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
> 99.9% (bound to serum albumin)

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Toxicity data
LD50: 1600 mg/kg (oral, rat) (A308)

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In vitro cytotoxicity (references [2], [3]):
- SH-SY5Y neuroblastoma cells: Tocapone (concentration up to 100 μM) did not show significant cytotoxicity in the absence of α-synuclein/Aβ fibrils (cell viability >90%, MTT assay) [2];
- Normal human fibroblasts (MRC-5): IC50 = 120 μM (72-hour MTT), about 4 times higher than neuroblastoma cells, indicating selective antitumor toxicity [3]
- In vivo safety (references [1], [3]):
- Healthy volunteers (oral 50 mg/kg) mg): No significant changes in serum ALT, AST, BUN, and creatinine; mild gastrointestinal discomfort (nausea, 1/12 subjects) [1]; - Nude mice (up to 50 mg/kg/day, 21 days): No significant decrease in body weight (<5%); serum ALT, AST, BUN, and creatinine within the normal range; no histopathological damage to the liver, kidneys, or heart [3]; - Hepatotoxicity indication: - In a group of volunteers (n=3), serum ALT increased 1.5 times (within the upper limit of normal) after receiving a 50 mg dose for 7 consecutive days, suggesting possible mild toxicity related to liver metabolism [1]

[1]. Eur J Clin Pharmacol. 1998 May;54(3):215-9.

[2]. Entacapone and tolcapone, two catechol O-methyltransferase inhibitors, block fibril formation of alpha-synuclein and beta-amyloid and protect against amyloid-induced toxicity. J Biol Chem. 2010 May 14;285(20):14941-14954.

[3]. Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma.Cancer Med. 2017 Jun;6(6):1341-1352.

Pharmacodynamics
Tocapone is a potent, selective, and reversible inhibitor of catechol-O-methyltransferase (COMT). In the human body, COMT is distributed in various organs. COMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the phenolic hydroxyl group of a substrate containing a catechol structure. The physiological substrates of COMT include dopa, catecholamines (dopamine, norepinephrine, epinephrine), and their hydroxylated metabolites. The function of COMT is to clear bioactive catechols and other hydroxylated metabolites. In the presence of decarboxylase inhibitors, COMT becomes the major metabolic enzyme of levodopa, catalyzing its conversion to 3-methoxy-4-hydroxy-L-phenylalanine (3-OMD) in the brain and peripheral tissues. When tocapone is used in combination with levodopa and aromatic amino acid decarboxylase inhibitors (such as carbidopa), the plasma concentration of levodopa is more sustained than when levodopa and aromatic amino acid decarboxylase inhibitors are used alone. It is believed that this sustained plasma concentration of levodopa leads to more stable dopaminergic stimulation in the brain, thus producing a more significant therapeutic effect on the signs and symptoms of Parkinson's disease, but at the same time it will increase the adverse reactions of levodopa, and sometimes it is necessary to reduce the dose of levodopa.

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Tocapone is a synthetic, reversible catechol-O-methyltransferase (COMT) inhibitor that has been clinically approved as an adjunct therapy for Parkinson's disease (PD). Tocapone enhances the efficacy of levodopa by inhibiting COMT-mediated levodopa metabolism, thus prolonging the exposure time of levodopa in the central nervous system (CNS) [1][2]. In addition to inhibiting COMT, tocapone also exhibits neuroprotective effects by inhibiting the formation of α-synuclein and Aβ fibers (key pathological features of Parkinson's disease and Alzheimer's disease, respectively). This extends its therapeutic potential to other neurodegenerative diseases beyond Parkinson's disease [2]. Preclinical studies have confirmed that tocapone has antitumor activity against neuroblastoma by inducing apoptosis through oxidative stress. Its selectivity for tumor cells (rather than normal fibroblasts) supports further research on its use as an anticancer drug [3]. Tocapone has well-defined pharmacokinetic characteristics, good oral absorption, high plasma protein binding, and a moderate half-life, making it suitable for once- or twice-daily administration. However, mild elevations in liver enzymes observed in repeated-dose studies suggest that liver function should be monitored during clinical use [1].

C14H11NO5

273.24

273.063

134308-13-7

Tolcapone-d7;Tolcapone-d4;1246816-93-2

4659569

Light yellow to yellow solid powder

1.4±0.1 g/cm3

485.6±45.0 °C at 760 mmHg

126-128ºC

205.7±17.2 °C

0.0±1.3 mmHg at 25°C

1.661

4.07

2

5

2

20

372

0

MIQPIUSUKVNLNT-UHFFFAOYSA-N

InChI=1S/C14H11NO5/c1-8-2-4-9(5-3-8)13(17)10-6-11(15(19)20)14(18)12(16)7-10/h2-7,16,18H,1H3

(3,4-dihydroxy-5-nitrophenyl)-(4-methylphenyl)methanone

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 (9.15 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: ≥ 2.5 mg/mL (9.15 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)