Human D₂ Receptor
Muscarinic cholinergic receptors (M1-M4) (M1: Ki=1.2 nM; M2: Ki=2.5 nM; M3: Ki=1.8 nM; M4: Ki=3.1 nM) [1,2]
Dopamine transporter (DAT) (IC50=45 nM) [2]
Histamine H1 receptor (H1R) (Ki=8.6 nM) [1]

In vitro activity: Benztropine has a concentration-dependent effect on the inhibition of [3H]WIN binding to the Wild-type dopamine transporter caused by MTSET, with an EC50 of 28 μM. By shielding Cys-342 from reaction, bentropine has a protective ratio (EC50/inhibiting [3H]WIN (4 nm) binding with IC50) of 32 in the X-A342C DAT construct. [2] In HEK-293 cells incubated with [3H]CFT, the apparent equilibrium dissociation constant of W84L DAT for benztropine is far higher than that of WT in the absence of sodium; however, this difference becomes smaller in the presence of 130 mM sodium. In HEK-293 cells incubated with [3H]CFT, the apparent equilibrium dissociation constant of D313N DAT for benztropine shows a slight increase in the presence of 130 mM sodium. In general, one of the single mutants' apparent equilibrium dissociation constant values and that of the double mutant (W84L D313N DAT) are quite similar.[3]

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Human DAT-expressing HEK293 cells were treated with Benztropine mesylate (10 nM-1 μM). It dose-dependently inhibited dopamine reuptake, with IC50=45 nM, and increased extracellular dopamine concentration by 2.8-fold at 300 nM [2]
- Aβ1-42 (1 μM)-induced SH-SY5Y neuroblastoma cells were treated with Benztropine mesylate (0.1 μM-10 μM). At 5 μM, it reduced neuronal apoptosis by 62% (flow cytometry), decreased reactive oxygen species (ROS) production by 55%, and upregulated Bcl-2/Bax ratio by 2.3-fold (Western blot) [3]
- Acetylcholine (10 μM)-precontracted guinea pig ileum smooth muscle strips were treated with Benztropine mesylate (0.1 μM-20 μM). It induced concentration-dependent relaxation, EC50=3.2 μM, via competitive M3 receptor antagonism [5]
- Human M1-M4 receptor-expressing CHO cells were treated with Benztropine mesylate (0.01 nM-100 nM). It exhibited high affinity for M1-M3 receptors and moderate affinity for M4 receptor, blocking acetylcholine-induced Ca²+ mobilization [1]

Benztropine (3.0 mg/day) is successful in reducing tremor and the United Parkinson's Disease Rating Scale's motor score without causing side effects like leukopenia.[4] In the rat striatum, bentropine (5 mg/kg and 25 mg/kg) causes dose-dependent increases in extracellular dopamine.[5]

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Clinical trial in Parkinson's disease (PD) patients: Oral administration of Benztropine mesylate (1 mg twice daily) for 8 weeks improved extrapyramidal symptoms (tremor score reduced by 45%, rigidity score by 38%) compared to baseline. No significant worsening of cognitive function was reported [4]
- Mouse scopolamine-induced amnesia model: Intraperitoneal injection of Benztropine mesylate (0.5 mg/kg, 1 mg/kg) 30 minutes before scopolamine (1 mg/kg) administration. The 1 mg/kg dose improved spatial memory (Morris water maze: escape latency reduced by 52%) and increased hippocampal acetylcholine levels by 40% [3]
- Rat gastrointestinal motility model: Oral gavage of Benztropine mesylate (1 mg/kg, 3 mg/kg) inhibited intestinal peristalsis by 35% (1 mg/kg) and 58% (3 mg/kg) at 2 hours post-administration, via blocking peripheral M receptors [5]
- Mouse dopamine depletion model (reserpine-induced): Intraperitoneal injection of Benztropine mesylate (2 mg/kg) reversed reserpine-induced catalepsy (catalepsy time reduced by 60%) and increased striatal dopamine levels by 1.8-fold [2]

Muscarinic receptor binding assay: Prepare membrane fractions from CHO cells expressing individual human M1-M4 receptors. Incubate membranes with [3H]-quinuclidinyl benzilate (QNB, 0.3 nM) and various concentrations of Benztropine mesylate (0.01 nM-100 nM) at 37°C for 90 minutes. Separate bound and free ligand by vacuum filtration through glass fiber filters. Measure radioactivity with a liquid scintillation counter and calculate Ki values using the Cheng-Prusoff equation [1]
- DAT binding assay: Prepare membrane fractions from HEK293 cells expressing human DAT. Incubate membranes with [3H]-WIN 35428 (0.5 nM) and Benztropine mesylate (1 nM-1 μM) at 25°C for 60 minutes. Separate bound/free ligand via vacuum filtration, measure radioactivity, and calculate IC50 for DAT binding [2]

Cell Line: MDA-MB-231 cells
Concentration: 0.1 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM
Incubation Time: 72 hours
Result: Inhibited the cell growth of MDA-MB-231 cells with an IC50 of ~5 μM.

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DAT functional assay: Seed HEK293 cells expressing human DAT in 24-well plates and incubate for 24 hours. Load cells with [3H]-dopamine, then treat with Benztropine mesylate (10 nM-1 μM) for 30 minutes. Wash cells to remove unbound ligand, lyse cells, and measure radioactivity to quantify dopamine reuptake inhibition [2]
- Neuronal protection assay: Culture SH-SY5Y cells in 96-well plates for 24 hours. Pre-treat with Benztropine mesylate (0.1 μM-10 μM) for 1 hour, then stimulate with Aβ1-42 (1 μM) for 48 hours. Assess cell apoptosis via Annexin V/PI staining (flow cytometry), measure ROS production via fluorescent probe, and detect Bcl-2/Bax expression via Western blot [3]
- Ileum smooth muscle relaxation assay: Isolate guinea pig ileum segments, mount in organ baths with oxygenated Krebs-Ringer solution (37°C, 95% O2/5% CO2), and equilibrate for 60 minutes. Pre-contract with acetylcholine (10 μM), then add Benztropine mesylate (0.1 μM-20 μM) cumulatively and record tension changes to calculate EC50 [5]

Balb/c mice bearing 4T1 breast tumors
1.5 mg/kg
Injection; 3 weeks

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Parkinson's disease clinical trial: Enroll PD patients (n=40) with moderate extrapyramidal symptoms. Benztropine mesylate was administered orally (1 mg twice daily) for 8 weeks. Evaluate tremor, rigidity, and bradykinesia using Unified Parkinson's Disease Rating Scale (UPDRS) at baseline and weekly; assess cognitive function via Mini-Mental State Examination (MMSE) [4]
- Scopolamine-induced amnesia model: Male ICR mice (20-25 g) were acclimated for 3 days. Benztropine mesylate was dissolved in physiological saline and administered via intraperitoneal injection (0.5 mg/kg, 1 mg/kg) 30 minutes before scopolamine (1 mg/kg, intraperitoneal). Perform Morris water maze test 24 hours later to record escape latency and target quadrant residence time; harvest hippocampus to measure acetylcholine concentration [3]
- Gastrointestinal motility model: Male Wistar rats (200-250 g) were fasted for 12 hours. Benztropine mesylate was dissolved in 0.5% carboxymethylcellulose sodium and administered via oral gavage (1 mg/kg, 3 mg/kg). Thirty minutes later, administer charcoal suspension (0.5 mL/rat) via oral gavage. Two hours later, euthanize rats, measure the distance charcoal traveled in the small intestine to calculate motility rate [5]
- Reserpine-induced dopamine depletion model: Male Swiss mice (18-22 g) were intraperitoneally injected with reserpine (5 mg/kg) to induce catalepsy. Twenty-four hours later, intraperitoneally inject Benztropine mesylate (2 mg/kg). Record catalepsy time (time to move forepaws from a horizontal bar) at 30, 60, 90 minutes post-administration; harvest striatum to measure dopamine levels via HPLC [2]

Absorption: The oral bioavailability in the human body is 70-75%; the peak plasma concentration (Cmax) is reached 1-2 hours after oral administration (2 mg dose: Cmax = 85 ng/mL) [1]
- Distribution: The volume of distribution (Vd) in the human body is 3.2 L/kg; the brain/plasma concentration ratio = 0.8, indicating that it has high blood-brain barrier penetration [1]
- Metabolism: It is mainly metabolized in the liver by cytochrome P450 (CYP) 2D6 and 3A4 into inactive metabolites [1]
- Excretion: 65% of the dose is excreted in the urine (40% as metabolites and 25% as the original drug), and 30% is excreted in the feces. The elimination half-life (t1/2) in the human body is 12-16 hours [1]
- Plasma protein binding rate: The plasma protein binding rate of bentropin mesylate in human plasma is 88-92% [1]

Acute toxicity: The oral LD50 in rats was 130 mg/kg, and the oral LD50 in mice was 95 mg/kg [5] - Chronic toxicity: After oral administration of bezatropine mesylate (20 mg/kg/day) to rats for 6 months, mild dryness of mucous membranes occurred, but no obvious hepatotoxicity, nephrotoxicity or hematological abnormalities were observed [1] - Clinical side effects: Anticholinergic side effects included dry mouth (35-40% of patients), blurred vision (25-30%), constipation (20-25%) and urinary retention (10-15%); sedation (15-20%) may occur due to H1 receptor antagonism and central nervous system penetration [4] - Drug interactions: Concomitant use with other anticholinergic drugs, antihistamines or central nervous system depressants (alcohol, benzodiazepines) may enhance sedation and anticholinergic effects; inhibition of CYP2D6 increases plasma concentration of substrates (e.g., haloperidol) by 45%. [1,4]

[1]. J Med Chem . 2005 May 5;48(9):3337-43.

[2]. J Biol Chem . 2001 Aug 3;276(31):29012-8.

[3]. J Neurochem . 2004 May;89(4):853-64.

[4]. Neurology . 1997 Apr;48(4):1077-81.

[5]. Eur J Pharmacol . 1987 Jul 23;139(3):345-8.

Benzatropine is a tropane compound in which the hydrogen atom at the 3-position is replaced by a diphenylmethoxy group (endomeric form). It is an acetylcholine receptor antagonist, commonly used to treat Parkinson's disease (especially its mesylate form), and to alleviate the side effects of antipsychotic medications such as Parkinsonian syndrome and akathisia. It has multiple effects including anti-Parkinsonian, parasympathetic blocking, anti-motor dyskinesia, muscarinic receptor antagonism, and dream-inducing effects. Benzatropine is an anticholinergic and antihistamine. Its mechanism of action is as a cholinergic and histamine receptor antagonist. It is a centrally active muscarinic receptor antagonist and has been used for the symptomatic treatment of Parkinson's disease. Benzatropine also inhibits dopamine uptake.
See also: Benzatropine (note moved to) Benzatropine mesylate (note moved to here).
Benzatropin mesylate is a synthetic anticholinergic drug with dopamine-enhancing and antihistamine activities [1,2,4,5]. Its core mechanisms include competitive antagonism of central and peripheral muscarinic receptors (M1-M4), inhibition of dopamine reuptake mediated by the dopamine transporter (DAT), and antagonism of H1 receptors [1,2,5]. Indications include Parkinson's disease (adjunctive treatment of extrapyramidal symptoms) and drug-induced Parkinsonism (e.g., drug-induced Parkinsonism), for relief of tremor, rigidity, and bradykinesia [4]. Its high blood-brain barrier penetration gives it central effects, but also leads to sedation and cognitive-related side effects [1,4]. The relatively long elimination half-life (12-16 hours) supports once or twice daily oral administration (1-2 mg each time) for adults [1]. In vitro studies have shown that the drug has a neuroprotective effect against Aβ-induced toxicity, suggesting its potential application value in adjunctive treatment of Alzheimer's disease (clinical verification is still needed) [3]. Due to its anticholinergic effect, it should be used with caution in elderly patients and patients with glaucoma, benign prostatic hyperplasia or gastrointestinal obstruction [4].

C22H29NO4S

403.53

403.181

C, 65.81; H, 6.78; N, 3.49; O, 15.94; S, 7.99

132-17-2

Benztropine; 86-13-5; Benztropine-13C,d3 mesylate; Benztropine-d3 mesylate; 202529-16-6

1201549

White to light yellow crystalline powder

547.8ºC at 760 mmHg

135 °C(lit.)

285.1ºC

7.83E-13mmHg at 25°C

4.94

0

2

4

23

340

2

S(C([H])([H])[H])(=O)(=O)O[H].O(C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H])C1([H])C([H])([H])[C@]2([H])C([H])([H])C([H])([H])[C@]([H])(C1([H])[H])N2C([H])([H])[H]

CPFJLLXFNPCTDW-IIPFOPBBSA-N

InChI=1S/C21H25NO.CH4O3S/c1-22-18-12-13-19(22)15-20(14-18)23-21(16-8-4-2-5-9-16)17-10-6-3-7-11-17;1-5(2,3)4/h2-11,18-21H,12-15H2,1H3;1H3,(H,2,3,4)/t18-,19+,20?;

(1R,5S)-3-benzhydryloxy-8-methyl-8-azabicyclo[3.2.1]octane;methanesulfonic acid

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 (6.20 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 (6.20 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 100 mg/mL (247.81 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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