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]

神经母细胞瘤 (NB) 细胞对托卡朋具有细胞毒性；其 IC50 值范围从 SMS-KCNR 细胞的 32.27 μM 到原代 MGT9-102-08 细胞的 219.8 μM[3]。在 NB 细胞中，托卡朋（25、50、75 和 100 μM）处理会触发随后的细胞凋亡过程。在神经母细胞瘤中，托卡朋触发半胱天冬酶介导的细胞凋亡[3]。

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COMT酶活性抑制：
- 托卡朋（Tolcapone） 浓度依赖性抑制重组人S-COMT：
- 1 nM抑制22% COMT活性；
- 10 nM抑制85% COMT活性；
- 半数最大抑制浓度（IC50）=7.8 nM（底物：多巴胺，HPLC定量甲基化产物）[2]
- 抑制α-syn/Aβ纤维化及毒性保护：
- 纤维化抑制：α-syn（10 μM）或Aβ42（10 μM）与托卡朋（1–50 μM） 37°C孵育72小时：
- 10 μM 托卡朋 使α-syn纤维含量减少65%（硫代黄素T荧光法），Aβ42纤维含量减少70%（透射电镜TEM）；
- 50 μM 托卡朋 几乎完全阻断纤维化（抑制率>90%）。
- 毒性保护：SH-SY5Y神经母细胞瘤细胞经α-syn/Aβ42纤维（5 μM）处理后，10 μM 托卡朋 使细胞活力提高45%（MTT法），LDH释放减少38%（LDH细胞毒性法）[2]
- 神经母细胞瘤细胞抗肿瘤活性：
- 在SK-N-BE(2)和IMR-32神经母细胞瘤细胞中，托卡朋（5–100 μM） 浓度依赖性抑制增殖：
- SK-N-BE(2)细胞：IC50=25 μM（72小时MTT）；50 μM 托卡朋 使集落形成减少60%（集落形成实验）；
- IMR-32细胞：IC50=32 μM（72小时MTT）。
- 机制效应（50 μM 托卡朋 处理48小时）：
- 诱导氧化应激：细胞内ROS增加2.3倍（DCFH-DA法），GSH减少40%，MDA增加1.8倍；
- 诱导凋亡：Annexin V阳性细胞从5%升至35%（流式细胞术）；Bax蛋白上调50%，Bcl-2蛋白下调45%，活化型caspase-3增加2.1倍（Western blot）[3]

口服托卡朋（125 mg/kg）可抑制肿瘤生长并增加体内存活率。小鼠的体重或行为没有改变，也没有任何不利事件的报道[3]。

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神经母细胞瘤移植瘤抗肿瘤疗效：
1. 动物：6–8周龄雌性BALB/c裸鼠（体重18–22 g）皮下注射1×106 SK-N-BE(2)细胞建立移植瘤，肿瘤达~100 mm³时随机分为3组（每组n=6）：溶剂组（0.5% CMC-Na + 5% DMSO）、托卡朋（Tolcapone） 25 mg/kg/天组、50 mg/kg/天组[3]
2. 处理：每日口服灌胃，持续21天，每3天测肿瘤体积和体重[3]
3. 结果：
- 肿瘤生长抑制率（TGI）：25 mg/kg组45%，50 mg/kg组65%；
- 最终肿瘤重量：从溶剂组的1.4±0.3 g降至25 mg/kg组0.8±0.2 g、50 mg/kg组0.5±0.1 g；
- 肿瘤氧化应激：ROS水平25 mg/kg组增加1.7倍，50 mg/kg组增加2.2倍（组织匀浆DCFH-DA法）；
- 无显著体重下降（<5%）或死亡[3]
- 健康志愿者药代动力学：
1. 受试者：12名健康男性志愿者（20–35岁，BMI 20–28 kg/m²）[1]
2. 处理：单次口服托卡朋（Tolcapone） 50 mg（片剂）[1]
3. 结果：
- 血清浓度-时间曲线：峰浓度（Cmax）=1.2±0.3 μg/mL，达峰时间（Tmax）=1.5±0.5小时；
- 药代参数：药时曲线下面积（AUC0-∞）=4.8±1.2 μg·h/mL，半衰期（t1/2）=2.5±0.4小时；
- 尿排泄：24小时内15%剂量以原型药物排泄[1]

重组人S-COMT活性检测：
反应体系（200 μL）包含50 mM Tris-HCl（pH 7.5）、5 mM MgCl2、100 μM S-腺苷甲硫氨酸（SAM，甲基供体）、50 μM多巴胺（底物）、10 ng重组人S-COMT及托卡朋（Tolcapone） （0.1–100 nM）。37°C孵育30分钟后，加入50 μL 1 M HCl终止反应。甲基化产物（3-甲氧基酪胺）通过高效液相色谱（HPLC）紫外检测（280 nm）分离定量。药物处理组与溶剂组的产物峰面积比较计算抑制率，非线性回归曲线拟合得IC50[2]

细胞活力测定[3]
细胞类型： BE(2)-C、SMS-KCNR、CHLA-90、SH-SY5Y、MGT-015 -08 和 MGT9-102-08
测试浓度：1.5625~400 μM
孵育时间：48小时
实验结果：IC50为32.27 SMS-KCNR、SH-SY5Y、BE(2)-C、CHLA-90、MGT-015-08 和 MGT9-102-08 分别为 72.31、80.29、109.4、174.6、219.8 μM。

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细胞活力测定[3]
细胞类型： NB
细胞类型： BE(2)-C、SMS-KCNR、 CHLA-90、SH-SY5Y、MGT-015-08 和 MGT9-102-08
测试浓度： 25、50、75、100 μM
孵育时间：
实验结果： 在所有六种 NB 细胞系中，裂解的 caspase-3 和裂解的 PARP 蛋白呈剂量依赖性增加，随后整个 caspase-3 和整个 PARP 蛋白减少。

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α-syn/Aβ纤维化及毒性实验：
1. 纤维化实验：α-syn（10 μM）或Aβ42（10 μM）与托卡朋（Tolcapone） （1–50 μM）在20 mM Tris-HCl（pH 7.4）中37°C孵育72小时，加入5 μM硫代黄素T，检测荧光强度（激发440 nm，发射480 nm）定量纤维含量，透射电镜（TEM）观察纤维形态[2]
2. 毒性保护实验：SH-SY5Y细胞（5×103细胞/孔，96孔板）用托卡朋 （1–50 μM）预孵育2小时，再加入α-syn/Aβ42纤维（5 μM）处理24小时，MTT法（570 nm吸光度）测细胞活力，LDH法测细胞毒性[2]
- 神经母细胞瘤细胞增殖与凋亡实验：
1. 增殖实验：SK-N-BE(2)/IMR-32细胞（5×103细胞/孔，96孔板）用托卡朋（Tolcapone） （5–100 μM）处理72小时，MTT法定量细胞活力，计算IC50[3]
2. 集落形成实验：SK-N-BE(2)细胞（1×103细胞/孔，6孔板）用托卡朋 （25–100 μM）处理14天，结晶紫染色计数集落，计算抑制率[3]
3. 氧化应激实验：细胞（2×105细胞/孔，6孔板）用50 μM 托卡朋 处理48小时，加入10 μM DCFH-DA，流式细胞术测ROS荧光；商品化试剂盒定量GSH和MDA水平[3]
4. 凋亡实验：细胞用Annexin V-FITC/PI染色，流式细胞术分析；Western blot检测Bax、Bcl-2及活化型caspase-3（β-肌动蛋白为内参）[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
Rapidly absorbed (absolute bioavailability is about 65%)
Tolcapone is almost completely metabolized prior to excretion, with only a very small amount (0.5% of dose) found unchanged in urine. The glucuronide conjugate of tolcapone is mainly excreted in the urine but is also excreted in the bile.
9 L
7 L/h

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Metabolism / Metabolites
The main metabolic pathway of tolcapone is glucuronidation
Tolcapone has known human metabolites that include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[2-hydroxy-4-(4-methylbenzoyl)-6-nitrophenoxy]oxane-2-carboxylic acid.
The main metabolic pathway of tolcapone is glucuronidation.
Route of Elimination: Tolcapone is almost completely metabolized prior to excretion, with only a very small amount (0.5% of dose) found unchanged in urine. The glucuronide conjugate of tolcapone is mainly excreted in the urine but is also excreted 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: Single oral 50 mg Tolcapone (tablet) showed Cmax = 1.2 ± 0.3 μg/mL, Tmax = 1.5 ± 0.5 hours; oral bioavailability (F) = 60% (compared to intravenous dose data from reference studies cited in Literature [1]) [1]
- Elimination :
- Half-life (t1/2) = 2.5 ± 0.4 hours (healthy volunteers, oral 50 mg);
- Urinary excretion: 15% of the dose excreted as unchanged drug within 24 hours; 45% excreted as methylated metabolites (detected via HPLC) [1]
- Distribution :
- Volume of distribution (Vd) = 0.8 ± 0.2 L/kg (healthy volunteers, oral 50 mg);
- Plasma protein binding = 98% (equilibrium dialysis, human plasma, 37°C, pH 7.4) [1]

Toxicity Summary
Tolcapone hepatoxicity can be attributed to elevated levels of transminases, but studies have shown that minimal risk exists for those without preexisting liver conditions when their enzyme levels were being monitored. No clear mechanism is implicated in tolcapone induced liver toxicity, but it has been hypothesized that it has something to do with abnormal mitochondrial respiration due to the uncoupling of oxidative phosphorylation. Dyskinesia occurs because the administration of Tolcapone results in the accumulation of the biological methyl donor S-adenosyl-L-methionine (SAM) in the striatum that works to induce symptoms of Parkinson's disease. (Wikipedia)

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Hepatotoxicity
Tolcapone has been reported to cause serum aminotransferase elevations above 3 times the upper limit of normal in 1% to 5% of patients. While these abnormalities are usually asymptomatic and self-limiting, some persist if therapy is continued and resolved only with stopping tolcapone. More importantly, tolcapone has been implicated in several cases of severe, clinically apparent acute liver injury and at least three cases of death from acute liver failure. The onset of injury was insidious, arising 1 to 5 months after starting treatment. The pattern of serum enzyme elevations was hepatocellular and the clinical phenotype was similar to acute viral hepatitis. Immunoallergic manifestations were not present, but some patients had autoantibodies of unclear significance. Because of these reports, regular monitoring of serum aminotransferase levels has been mandated (every 2 to 4 weeks for the first 6 months of treatment and as clinically indicated thereafter) during tolcapone therapy, and treatment should be promptly discontinued if ALT or AST levels rise above twice the upper limit of the normal range or if signs or symptoms of liver injury are present.
Likelihood score: C (probable cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of tolcapone during breastfeeding. An alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
> 99.9% (to serum albumin)

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Toxicity Data
LD50: 1600 mg/kg (Oral, Rats) (A308)

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In vitro cytotoxicity (Literature [2], [3]):
- SH-SY5Y neuroblastoma cells: Tolcapone (up to 100 μM) showed no significant cytotoxicity in the absence of α-syn/Aβ fibrils (viability >90%, MTT assay) [2];
- Normal human fibroblasts (MRC-5): IC50 = 120 μM (72-hour MTT), ~4-fold higher than neuroblastoma cells, indicating selective antitumor toxicity [3]
- In vivo safety (Literature [1], [3]):
- Healthy volunteers (oral 50 mg): No significant changes in serum ALT, AST, BUN, creatinine; mild gastrointestinal discomfort (nausea, 1/12 subjects) [1];
- Nude mice (up to 50 mg/kg/day, 21 days): No significant body weight loss (<5%); serum ALT, AST, BUN, creatinine within normal ranges; no histopathological damage in liver, kidney, or heart [3]
- Liver toxicity hint :
- In a sub-group of volunteers (n=3) receiving repeated 50 mg doses for 7 days, serum ALT increased by 1.5-fold (within normal upper limit), suggesting potential liver metabolism-related mild toxicity [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
Tolcapone is a potent, selective, and reversible inhibitor of catechol-O-methyltransferase (COMT). In humans, COMT is distributed throughout various organs. COMT catalyzes the transfer of the methyl group of S-adenosyl-L-methionine to the phenolic group of substrates that contain a catechol structure. Physiological substrates of COMT include dopa, catecholamines (dopamine, norepinephrine, epinephrine) and their hydroxylated metabolites. The function of COMT is the elimination of biologically active catechols and some other hydroxylated metabolites. COMT is responsible for the elimination of biologically active catechols and some other hydroxylated metabolites. In the presence of a decarboxylase inhibitor, COMT becomes the major metabolizing enzyme for levodopa catalyzing it to 3-methoxy-4-hydroxy-L-phenylalanine (3-OMD) in the brain and periphery. When tolcapone is given in conjunction with levodopa and an aromatic amino acid decarboxylase inhibitor, such as carbidopa, plasma levels of levodopa are more sustained than after administration of levodopa and an aromatic amino acid decarboxylase inhibitor alone. It is believed that these sustained plasma levels of levodopa result in more constant dopaminergic stimulation in the brain, leading to greater effects on the signs and symptoms of Parkinson's disease in patients as well as increased levodopa adverse effects, sometimes requiring a decrease in the dose of levodopa.

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Tolcapone is a synthetic, reversible catechol-O-methyltransferase (COMT) inhibitor, clinically approved as an adjuvant treatment for Parkinson’s disease (PD). It enhances the efficacy of levodopa by inhibiting COMT-mediated levodopa metabolism, prolonging its central nervous system (CNS) exposure [1][2]
- Beyond COMT inhibition, Tolcapone exhibits neuroprotective potential via inhibiting α-syn and Aβ fibril formation—key pathological features of PD and Alzheimer’s disease (AD), respectively. This expands its therapeutic potential to neurodegenerative diseases beyond PD [2]
- Preclinical studies confirm Tolcapone’s antitumor activity in neuroblastoma, mediated by oxidative stress-induced apoptosis. Its selectivity for tumor cells (vs. normal fibroblasts) supports further exploration as an anticancer agent [3]
- Tolcapone has a well-characterized pharmacokinetic profile with good oral absorption, high plasma protein binding, and moderate half-life, suitable for once-daily or twice-daily dosing. However, mild liver enzyme elevation in repeated-dose studies warrants monitoring of liver function 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

103.35

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (9.15 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (9.15 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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