Zotepine targets a broad range of neurotransmitter receptors in the central nervous system, reflecting its complex pharmacodynamic profile. It acts as a potent antagonist at serotonin 5-HT2A receptors with Ki values ranging from 0.69 to 2.6 nM and at dopamine D2 receptors with Ki values of 2.3 to 8 nM. Additionally, Zotepine exhibits high affinity for histamine H1 receptors (IC50 = 8.0 nM) and α1-adrenergic receptors (Kd = 7.3 nM). It also displays antagonistic activity at serotonin 5-HT2C, 5-HT6, and 5-HT7 receptors, as well as dopamine D1 and D3 receptors. The combination of potent 5-HT2A and D2 antagonism is characteristic of atypical antipsychotics and is believed to underlie its therapeutic efficacy in schizophrenia, with the high 5-HT2A/D2 affinity ratio contributing to a lower risk of extrapyramidal side effects compared to typical antipsychotics. Furthermore, the norepinephrine reuptake inhibition mediated by its active metabolite norzotepine adds an antidepressant dimension to its profile, making it particularly useful for patients with schizophrenia who also suffer from depressive symptoms.

Zotepine exhibits multiple antagonistic profiles and strong affinities to α1-adrenergic, α₂-adrenergic, Dopamine D₂, Histamine H1, Muscarinic, 5-HT_1A, 5-HT_1D, 5-HT_2A, and 5-HT_2C receptors, with K_ds of 7.3, 180, 8, 3.3, 330, 280, 80, 2.6, 3.2 nM, respectively[1].
In vitro studies have extensively characterized Zotepine's receptor binding profile and functional activity. Radioligand binding assays have demonstrated its high affinity for serotonin 5-HT2A and dopamine D2 receptors, with Ki values in the low nanomolar range. Functional assays have confirmed that Zotepine acts as an antagonist at these receptors, blocking the downstream signaling pathways activated by serotonin and dopamine. In addition to its receptor antagonism, Zotepine and its metabolite norzotepine have been shown to inhibit norepinephrine reuptake with an IC50 of 51 nM, a property that is not shared by most other atypical antipsychotics. This unique combination of activities has been demonstrated in various in vitro systems, including recombinant cell lines expressing human receptors and synaptosomal preparations for uptake studies. The compound's purity and stability have been validated for research applications, with a purity of ≥99% (HPLC) and a defined molecular weight of 331.86 g/mol (C18H18ClNOS).

Zotepine (1-3 mg/kg; a single i.p.) dose-dependently raises noradrenaline, dopamine, glutamate release, and GABA levels without changing 5-HT levels in the medial prefrontal cortex of rats[2].
In vivo, Zotepine has demonstrated a range of pharmacological effects consistent with its receptor binding profile. It exhibits antipsychotic-like activity in animal models, such as the conditioned avoidance response test and amphetamine-induced hyperactivity models, confirming its D2 receptor antagonism. In addition, Zotepine has been shown to possess anxiolytic and antidepressant-like effects in preclinical models, likely attributable to its 5-HT1A agonism and norepinephrine reuptake inhibition. Clinical studies have confirmed its efficacy in reducing both positive and negative symptoms of schizophrenia. Zotepine has also been investigated as an antimanic agent in acute bipolar mania, further supporting its broad therapeutic potential. Its prolonged half-life of approximately 21 hours allows for once-daily dosing, which is advantageous for patient compliance in chronic psychiatric conditions.

The in vitro enzyme/receptor binding assays for Zotepine involve measuring its affinity for various neurotransmitter receptors using radioligand binding techniques. Membrane preparations from cells or tissues expressing the target receptors are incubated with a radiolabeled ligand (e.g., [3H]ketanserin for 5-HT2A, [3H]spiperone for D2) and varying concentrations of Zotepine. The bound radioactivity is measured, and the Ki values are calculated from competition binding curves. These assays have established Zotepine's high affinity for 5-HT2A (Ki = 0.69 nM), D2 (Ki = 2.3 nM), H1 (IC50 = 8.0 nM), and α1-adrenergic receptors (Kd = 7.3 nM). Functional assays, such as measuring the inhibition of forskolin-stimulated cAMP accumulation, are used to confirm antagonist activity at D2 and 5-HT2A receptors.

Cellular assays for Zotepine are conducted to evaluate its effects on receptor-mediated signaling pathways in living cells. Cells expressing human serotonin or dopamine receptors are treated with Zotepine, and the downstream signaling is measured. For example, the antagonist activity at dopamine D2 receptors is assessed by measuring the reversal of dopamine-induced inhibition of cAMP production in CHO cells expressing human D2 receptors. Similarly, the antagonist activity at 5-HT2A receptors is evaluated by measuring the inhibition of phospholipase C activation and calcium mobilization. The norepinephrine reuptake inhibition by norzotepine can be assessed in synaptosomal preparations or in cells expressing norepinephrine transporters, where the uptake of radiolabeled norepinephrine is measured in the presence of the compound. These cell-based assays provide functional confirmation of the binding data and help elucidate the compound's mechanism of action.

Male Sprague-Dawley rats (250-300 g)
1, 3 mg/kg
A single i.p.
In vivo animal experiments for Zotepine are typically conducted in rodent models of schizophrenia and depression. The antipsychotic efficacy is evaluated using the conditioned avoidance response test, where rats are trained to avoid a foot shock by moving to a safe compartment, and the ability of Zotepine to suppress avoidance behavior is measured. The amphetamine-induced hyperactivity model is another standard assay, where Zotepine's ability to reverse the hyperlocomotion induced by amphetamine is assessed. For antidepressant-like effects, the forced swim test or tail suspension test in mice can be used. In these models, Zotepine is administered orally or intraperitoneally, and the duration of immobility is measured. The compound's effects on extracellular levels of monoamines, GABA, and glutamate in the prefrontal cortex have also been studied using microdialysis techniques in freely moving rats. These in vivo experiments provide a comprehensive picture of Zotepine's pharmacological profile and its potential clinical applications.

Absorption, Distribution and Excretion
Preclinical pharmacokinetic studies showed that when zotepine was administered at doses of 25-100 mg, plasma concentrations increased in a dose-dependent manner, with a time to peak concentration (tmax) of 2-4 hours and a peak plasma concentration (Cmax) of 6.9-19.6 ng/ml. After reaching peak concentration, plasma concentrations declined slowly. Preclinical studies indicated that after oral administration of zotepine, the drug was rapidly and almost completely absorbed from the gastrointestinal tract. The parent drug and its metabolites were rapidly distributed to tissues. Only a small amount of parent drug zotepine was excreted in the urine; the parent drug and its metabolites were primarily excreted in the feces via bile. The apparent volume of distribution of zotepine was 109 L/kg. The apparent oral clearance of zotepine was 4.6 mg/h·kg. Metabolism/Metabolites Zotepine is well metabolized in vivo, undergoing extensive first-pass metabolism to produce the metabolite norzotepine and several inactive metabolites. The main enzymes involved in the metabolism of zotepine are CYP1A2 and CYP3A4. Some major metabolic pathways include N-demethylation and oxidation of the N or S atom, hydroxylation of the aromatic ring, and subsequent conjugation reactions. Known metabolites of zotepine include 3-hydroxyzotepine, zotepine N-oxide, zotepine-oxide, norzotepine, and 2-hydroxyzotepine. The biological half-life of zotepine has been reported to be 21 hours.
Zotepine is well-absorbed after oral administration and has a prolonged half-life of approximately 21 hours, supporting once-daily dosing. It has a molecular weight of 331.86 g/mol and a molecular formula of C18H18ClNOS. The compound is metabolized extensively in the liver, with norzotepine being a major active metabolite that contributes to its overall pharmacological profile. Norzotepine is a potent inhibitor of norepinephrine reuptake (IC50 = 51 nM), which distinguishes Zotepine from many other atypical antipsychotics and may contribute to its antidepressant-like effects. Zotepine is poorly soluble in water but soluble in organic solvents, which affects its formulation for research and clinical use. The compound should be stored at room temperature under inert gas to maintain its stability.

Protein Binding
Zothipin and its main active metabolite, norzothipin, have a plasma protein binding rate of 97% of the administered dose.
Zotepine has a well-characterized toxicity profile from both preclinical and clinical studies. Common side effects include sedation, weight gain, and extrapyramidal symptoms, although the latter are less frequent than with typical antipsychotics due to its 5-HT2A antagonism. Zotepine may also be associated with QT prolongation, requiring monitoring of cardiac function in clinical use. Preclinical toxicology studies have evaluated its safety in various animal models, and the compound has been shown to be generally well-tolerated at therapeutic doses. However, as with all antipsychotics, the risk-benefit ratio must be carefully considered for each patient.

[1]. Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds. Life Sci. 2000 Nov 24;68(1):29-39.

[2]. Effects of zotepine on extracellular levels of monoamine, GABA and glutamate in rat prefrontal cortex. Br J Pharmacol. 2009 Jun;157(4):656-65.

Zotepine is a dibenzothiophene compound and a tertiary amine compound. It is both an alpha-adrenergic and a serotonergic drug, belonging to the second generation of antipsychotics. Zotepine, with the chemical formula 2-chloro-11-(2-dimethylaminoethoxy)-dibenzothiophene, is a neuroleptic. It was designed and synthesized by Fujisawa Pharmaceutical Co., Ltd. Since the 1980s, it has been used as an antipsychotic in Japan, India, and parts of Europe, including the UK and Germany. Zotepine has never been approved by the U.S. Food and Drug Administration (FDA). In 1993, it was classified as an inactive pharmaceutical ingredient (Class I, Type II). In 1995, the FDA investigated the manufacturing process of zotepine tablets in Germany, but its status remained inactive. In 2016, when the FDA re-evaluated antipsychotics, zotepine did not meet the threshold effect for further research. By 2015, the European Medicines Agency (EMA) had conducted pharmacovigilance studies on it. Potential treatment for acute renal failure.

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Drug Indications
Zotepine, like other atypical antipsychotics, is considered a first-line treatment for newly diagnosed schizophrenia. It is generally considered the preferred treatment for acute schizophrenic episodes when communication with the patient is impossible. Zotepine, as an atypical antipsychotic, is used to treat patients who cannot tolerate the side effects of conventional antipsychotics, or to treat patients with relapsed and poorly controlled schizophrenia. It is important to note that the above indications are all related to atypical antipsychotics. Zotepine has not yet been approved by the U.S. Food and Drug Administration (FDA), the Canadian Medicines Agency (EMA), or the European Medicines Agency (EMA), and studies have not shown any additional benefit compared to other approved atypical antipsychotics. Schizophrenia is a chronic and severe mental disorder that affects a patient's thinking, feelings, and behavior. It is typically characterized by blurred perception of reality, accompanied by hallucinations, delusions, and thought and motor disturbances.

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Mechanism of Action
Zotepine is a dopamine antagonist with high affinity for D1 and D2-like receptors. It exhibits potent antagonism against multiple serotonin receptors, such as 5-HT2a, 5-HT2c, 5-HT6, and 5-HT7. Zotepine's activity is also associated with the inhibition of norepinephrine reuptake and serotonergic activity. All these effects enable zotepine to improve negative and cognitive symptoms of schizophrenia.

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Pharmacodynamics
In preclinical studies, zotepine demonstrated stronger anti-serotonergic activity compared to other antipsychotics. It has also been reported to increase the seizure threshold of the amygdala. When the effects of zotepine were analyzed via electroencephalography, typical responses to less potent antipsychotics were observed. Zotepine is a sedative. After taking zotepine, patients showed improvements in numerical and complex reaction abilities. However, these effects are often accompanied by increased heart rate, elevated prolactin levels, and some typical antipsychotic side effects.
Zotepine is an atypical antipsychotic that has been used clinically in several countries for the treatment of schizophrenia. It has also been investigated for acute bipolar mania. Its unique pharmacological profile, including potent 5-HT2A and D2 antagonism, along with norepinephrine reuptake inhibition via its active metabolite norzotepine, distinguishes it from other antipsychotics. Zotepine has demonstrated clinical efficacy comparable to haloperidol and chlorpromazine for positive symptoms while showing superior efficacy against negative symptoms. Despite its promising profile, Zotepine is not approved in all countries, and its use has been limited by the availability of newer atypical antipsychotics. Nevertheless, it remains a valuable pharmacological tool for studying schizophrenia and bipolar disorder, and its multi-target mechanism continues to be of interest in psychopharmacology research.

C18H18CLNOS

331.86

331.079

C, 65.15; H, 5.47; Cl, 10.68; N, 4.22; O, 4.82; S, 9.66

26615-21-4

5736

White to off-white solid powder

1.3±0.1 g/cm3

478.4±45.0 °C at 760 mmHg

90-91 °C

243.2±28.7 °C

0.0±1.2 mmHg at 25°C

1.656

6.57

0

3

4

22

401

0

CN(C)CCOC1=CC2=CC=CC=C2SC3=C1C=C(C=C3)Cl

HDOZVRUNCMBHFH-UHFFFAOYSA-N

InChI=1S/C18H18ClNOS/c1-20(2)9-10-21-16-11-13-5-3-4-6-17(13)22-18-8-7-14(19)12-15(16)18/h3-8,11-12H,9-10H2,1-2H3

2-(3-chlorobenzo[b][1]benzothiepin-5-yl)oxy-N,N-dimethylethanamine

Zotepine

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)

DMSO: ~25 mg/mL (~75.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)