5-HT2A Receptor 4 nM (Ki); 5-HT6 Receptor 5-HT7 Receptor mAChR1 9.5 nM (Ki); mAChR4 11 nM (EC50) α2-adrenergic receptor 51 nM (Ki); D2 Receptor 75 nM (Ki)

Clozapine (HF 1854) is a D2 receptor antagonist with a Ki of 75 nM, blocks serotonin 5HT2A receptors with a Ki of 4 nM, inhibits muscarinic M1 receptors with a Ki of 9.5 nM, and blocks α2-adrenergic receptors with a Ki of 51 nM[1]. Clozapine (0-1 μM; 24 h) downregulates 5-HT6 and upregulates 5-HT7 receptors in HeLa cells [2]. Clozapine is a full agonist of the muscarinic M4 receptor (EC50 = 11 nM) expressed in CHO cells [4].

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Clozapine is an atypical antipsychotic with high affinity for several serotonin receptors. This drug causes paradoxical downregulation of 5-hydroxytryptamine(2A) (5-HT)(2A) receptors, but its modulation of other serotonin receptors has not been studied. We examined the effects of clozapine and several other drugs on the regulation of rat 5-HT(6) and 5-HT(7) receptors individually expressed in transfected HeLa cells. Both 5-HT(6) and 5-HT(7) receptor densities (B(max)) were reduced by 5-carboxamidotryptamine, an agonist, and methiothepin, an inverse agonist. Clozapine reduced 5-HT(6) B(max). This suggests that 5-HT(6) receptors are also paradoxically downregulated by the antagonist clozapine. 5-Hydroxytryptamine(7) receptor B(max), on the other hand, was increased by clozapine. Clozapine's modulation of the 5-HT(6) and 5-HT(7) receptor levels may be important in the action of this atypical antipsychotic [2].

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Clozapine was studied in functional assays at human muscarinic M1-M5 receptors expressed in Chinese hamster ovary cells. Clozapine was a full agonist at the muscarinic M4 receptor (EC50 = 11 nM), producing inhibition of forskolin-stimulated cAMP accumulation. In contrast, clozapine potently antagonized agonist-induced responses at the other four muscarinic receptor subtypes. Selective stimulation of M4 receptors may, in part, explain the hypersalivation observed clinically with clozapine. Moreover, the unique overall muscarinic profile of clozapine may contribute to its atypical antipsychotic efficacy [4].

Clozapine (HF 1854) (25 mg/kg/day; intraperitoneal injection; 21 days) showed antipsychotic effects in a mouse model of lysergic acid diethylamide-induced psychosis[3].

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Objective: This study evaluates the effects of chronic treatment with clozapine on the cellular and behavioral responses induced by the hallucinogenic serotonin 5-HT(2A) receptor agonist lysergic acid diethylamide (LSD) as a mouse model of psychosis. Method: Mice were treated chronically (21 days) with 25 mg/kg/day clozapine. Experiments were conducted 1, 7, 14, and 21 days after the last clozapine administration. [(3)H]Ketanserin binding and 5-HT ( 2A ) mRNA expression were determined in mouse somatosensory cortex. Head-twitch behavior, expression of c-fos, which is induced by all 5-HT(2A) agonists, and expression of egr-1 and egr-2, which are LSD-like specific, were assayed. Results: Head-twitch response was decreased and [(3)H]ketanserin binding was downregulated in 1, 7, and 14 days after chronic clozapine. 5-HT ( 2A ) mRNA was reduced 1 day after chronic clozapine. Induction of c-fos, but not egr-1 and egr-2, was rescued 7 days after chronic clozapine. These effects were not observed after short treatment (2 days) with clozapine or chronic haloperidol (1 mg/kg/day). Conclusion: Our findings provide a murine model of chronic atypical antipsychotic drug action and suggest downregulation of the 5-HT(2A) receptor as a potential mechanism involved in these persistent therapeutic-like effects [3].

Radioligand binding was analyzed by nonparametric curve fitting using Prism 2.0. The data conformed well to single site models in both 5-HT6 and 5-HT7 cases. KD and Bmax values were calculated for each drug treatment condition within each assay. None of the drug treatments changed the apparent KD; therefore, Bmax values was determined using constrained KD values of 2.4 or 6.3 nM, the experimentally measured averages for control binding with [3H]-5-HT (in 5-HT7 expressing cells) or [3H]-LSD (in 5-HT6 expressing cells), respectively. Two to eight separate binding assays were used to calculate Bmax values for each treatment condition. Statistical analysis of changes in Bmax values was done using Student's two tailed t-test of each drug vs. vehicle determinations. For clarity of data presentation, all Bmax values are expressed as a % of control±SEM [2].

To test the efficiency of clozapine washout from membrane homogenate, clozapine (1 μM) or vehicle was added to membranes prepared from untreated cells in 2× TME and equilibrated at room temperature for 1 h. The membranes were then split into aliquots and incubated at 37°C for 15, 30 or 60 min then pelleted at 20 000×g for 15 min. Membranes were resuspended in fresh 2× TME and specific binding was measured at a single concentration (5 nM [3H]-LSD for 5-HT6 and 1.67 nM [3H]-5-HT for 5-HT7) as described above [2].

Animal/Disease Models: Male 129 S6/Sv mice, lysergic acid diethylamide (LSD)-induced psychosis model[3].
Doses: 25 mg/kg/day.
Route of Administration: Intraperitoneal injection, 21 days.
Experimental Results: Decreased head-twitch response, reduced 5-HT2A mRNA, rescued induction of c-fos, but not egr-1 and egr-2.

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Materials and drug administration [3]
Clozapine, methysergide, haloperidol, Lysergic acid diethylamide (LSD) were used. The injected doses (i.p.), calculated as salts, were clozapine, 1.5, 10.0, or 25.0 mg/kg; haloperidol, 1.0 mg/kg; and LSD, 0.24 mg/kg. These doses were selected based on previous findings with clozapine, haloperidol, and LSD in rodent models (Kuoppamaki et al. 1993; Kontkanen et al. 2002a, b; Gonzalez-Maeso et al. 2008; Gray et al. 2009). These doses represent approximately 12.5, 3.3, and 0.03 % of the LD50 of clozapine (200 mg/kg), haloperidol (30 mg/kg), and LSD (800 mg/kg) after i.p. administration in mice, respectively. Clozapine was injected after suspension in a minimal amount of DMSO supplemented with acetic acid and made up to volume with normal saline. Haloperidol was dissolved in saline. LSD was dissolved in a minimal amount of DMSO and made up to volume with normal saline. Control animals received vehicle injections. Drugs were administered in an injection volume of 100 μl/10 g mouse body weight. All other chemicals were obtained from standard sources.
For effect of chronic treatment with clozapine on head-twitch behavior, chronic (21 days) treatment with clozapine started 3, 4, 5, and 6 weeks before the mice were injected with a single dose of LSD (i.e., 1, 7, 14, or 21 days after the last injection of chronic clozapine). Control mice were chronically (21 days) treated with vehicle and injected on the day of the experiment with a single dose of LSD (i.e., 1 day after the last injection of chronic vehicle). Injection of LSD was performed on the same day in all the groups of mice (i.e., acute LSD 1, 7, 14, or 21 after the last injection of chronic clozapine, and of acute LSD 1 day after the last injection of chronic vehicle).
For the effect of chronic treatment with clozapine on the expression of 5-HT2A receptor (i.e., radioligand binding and mRNA), chronic (21 days) treatment with clozapine started 3, 4, 5, and 6 weeks before the mice were sacrificed (i.e., 1, 7, 14, or 21 days after the last injection of chronic clozapine). Control mice were chronically (21 days) treated with vehicle and sacrificed 1 day after the last injection of chronic vehicle. Mice were sacrificed on the same day in all the groups of mice (i.e., chronic clozapine 1, 7, 14, or 21 days after the last injection and chronic vehicle 1 day after the last injection).
For the effect of chronic treatment with clozapine on the LSD-dependent induction of c-fos, egr-1, and egr-2 in mouse somatosensory cortex, treatment with clozapine or vehicle started 3, 4, 5, and 6 weeks before the mice were injected with a single dose of LSD or vehicle on the day of the experiment (i.e., 1, 7, 14, or 21 days after the last injection of chronic clozapine). Mice were sacrificed 60 min after injection with LSD. Similar conditions were used for chronic treatment with haloperidol. For short-term treatments, mice received one injection of clozapine, or vehicle, per day for 2 days, and experiments were performed 1 day after the last injection.

Absorption, Distribution and Excretion
In humans, clozapine tablets (25 mg and 100 mg) have the same bioavailability as clozapine solution. Following a twice-daily oral administration of 100 mg clozapine, the mean steady-state peak plasma concentration is 319 ng/mL (range: 102 to 771 ng/mL), reached on average 2.5 hours after administration (range: 1 to 6 hours). The mean steady-state minimum concentration is 122 ng/mL (range: 41 to 343 ng/mL) at a dose of 100 mg twice daily. Approximately 50% of the administered dose is excreted in the urine and 30% in the feces. The median volume of distribution of clozapine is 508 L (272–1290 L). The median clearance of clozapine is 30.3 L/h (14.4–45.2 L/h). Clozapine is almost completely metabolized before excretion, with only trace amounts of the original drug detected in urine and feces. Approximately 50% of the administered dose is excreted in urine and 30% in feces. Demethylated, hydroxylated, and N-oxide derivatives are found in urine and feces. Pharmacological studies have shown that the demethylated metabolite has limited activity, while the hydroxylated and N-oxide derivatives are inactive. In humans, the bioavailability of clozapine tablets (25 mg and 100 mg) is comparable to that of clozapine solution. After twice-daily administration of 100 mg, the mean steady-state peak plasma concentration is 319 ng/mL (range: 102 to 771 ng/mL), reached on average 2.5 hours after administration (range: 1 to 6 hours). After twice-daily administration of 100 mg, the mean steady-state minimum concentration is 122 ng/mL (range: 41 to 343 ng/mL). Food does not appear to affect the systemic bioavailability of clozapine. Therefore, clozapine can be taken with food or on an empty stomach. Clozapine binds to serum proteins at a rate of approximately 97%. After a single or multiple oral doses, clozapine is rapidly absorbed, reaching steady-state plasma concentrations within 8 to 10 days of starting treatment. Clozapine is almost completely metabolized before excretion, with only trace amounts detected in urine and feces. Clozapine is a substrate for many cytochrome P450 isoenzymes, particularly CYP1A2, CYP2D6, and CYP3A4. Unmethylated, hydroxylated, and N-oxide derivatives are found in urine and feces. Pharmacological studies have shown that the demethylated metabolite (norclozapine) has limited activity, while the hydroxylated and N-oxide derivatives are inactive. Patients with mania and schizophrenia taking 300-500 mg of the antipsychotic clozapine daily have had their clozapine metabolites isolated from urine and analyzed using gas chromatography-mass spectrometry. Clozapine is converted into two metabolites by substituting chlorine atoms with hydroxyl or methyl sulfide groups. Other metabolites are N-demethylated derivatives of the first two metabolites. Studies have also identified a metabolite with an oxidized piperazine ring and suggest the possible existence of a sulfur-oxidized metabolite. Clozapine is metabolized into N-oxyclozapine and N-demethylclozapine, which have lower pharmacological activity than the parent compound and are primarily excreted in the urine, with a small amount excreted in the feces. Known metabolites of clozapine include clozapine N-glucuronide, clozapine N-oxide, and N-demethylclozapine. Biological Half-Life After a single dose of 75 mg clozapine, the mean elimination half-life is 8 hours (range: 4 to 12 hours); while after reaching steady state, the mean elimination half-life of 100 mg twice daily is 12 hours (range: 4 to 66 hours). A comparison of single-dose and multiple-dose administration of clozapine showed that the elimination half-life was significantly prolonged after multiple-dose administration compared to single-dose administration, suggesting a possible concentration-dependent pharmacokinetic pattern. After a single dose of 75 mg clozapine, the mean elimination half-life was 8 hours (range: 4 to 12 hours); while after reaching steady state with twice-daily administration of 100 mg, the mean elimination half-life was 12 hours (range: 4 to 66 hours).

Toxicity Summary
Identification and Uses: Clozapine has demonstrated efficacy in both uncontrolled and controlled studies of patients with schizophrenia and is a broad-spectrum antipsychotic with a relatively rapid onset of action. Clozapine has been used in a small number of patients with advanced idiopathic Parkinson's syndrome to treat dopaminergic psychosis associated with antiparkinsonian therapy; however, adverse reactions such as sedation, confusion, and exacerbation of Parkinson's symptoms may limit the benefit of clozapine treatment in these patients. Clozapine is used to reduce the risk of recurrent suicidal behavior in patients with schizophrenia or schizoaffective disorder who are assessed as having a chronic risk of suicide based on their medical history and recent clinical status. Although the safety and efficacy of clozapine in children and adolescents under the age of 16 have not been established, it has been successfully used to treat a small number of treatment-resistant childhood and adolescent-onset schizophrenia. Clozapine is used for symptomatic treatment of patients with severe schizophrenia whose condition has not responded well to other antipsychotic medications. Human Exposure and Toxicity: The most common adverse reactions of clozapine involve the central and autonomic nervous systems (e.g., somnolence or sedation, excessive salivation) and the cardiovascular system (e.g., tachycardia, hypotension). While some adverse reactions of clozapine (e.g., extrapyramidal reactions, tardive dyskinesia) appear to occur less frequently and are less severe than with other antipsychotics, other potentially serious adverse reactions (e.g., agranulocytosis, seizures) may be more common with clozapine treatment; therefore, the potential risks and benefits should be carefully assessed when considering this medication. Although some studies suggest that the cases in Finland may be related to local genetic or environmental factors, such factors have not been confirmed. In Finland, 18 cases of serious hematologic disorders associated with clozapine were reported within two months (9 of which resulted in death). Agranulocytosis caused 8 deaths, and leukemia may have caused the 9th death. The incidence of agranulocytosis is 0.3‰ in 22 countries outside Finland where clozapine is marketed, while the incidence in Finland is almost 20 times higher, compared to 0.1‰ to 0.8‰ for other tricyclic antipsychotics. Patients treated with flexible-dose clozapine (mean dose: 274.2 mg daily) for approximately 2 years had a 26% lower risk of suicide attempts or hospitalizations for suicide prevention compared to patients treated with flexible-dose olanzapine (mean dose: 16.6 mg daily); treatment resistance was independent of response to clozapine or olanzapine. Animal studies: Repeated oral administration in rats (6 months) and dogs (3 months) reduced weight gain at doses of 20 mg/kg or higher in rats and 10 mg/kg or higher in dogs. Hepatomegaly was not strictly dose-related and was not accompanied by changes in histological or hematological chemistry, and was completely reversible upon discontinuation of the drug. No toxic effects were observed in rats or dogs. Oral administration of clozapine at 20 or 40 mg/kg daily was not teratogenic in rats or rabbits and had no effect on fertility in male and female rats. However, a dose of 40 mg/kg of clozapine inhibited the growth of lactating pups in the test mothers. The fertility of the F1 generation female mice was normal, and no developmental abnormalities were observed in the F2 generation. Clozapine hydrochloride inhibited conditioned avoidance behavior in rats, suppressed benzoquinone-induced writhing syndrome in mice, and lowered body temperature. Clozapine hydrochloride antagonized oxatromoline-induced tremors and lacrimation in mice and reduced the acute toxicity of physostigmine and the level of 5-hydroxyindoleacetic acid ester in the brain.

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Non-human toxicity values
Rat intravenous LD50: 41.6 mg/kg
Rat subcutaneous LD50: 240 mg/kg
Rat intramuscular LD50: 210 mg/kg
Rat oral LD50: 251 mg/kg

[1]. Clozapine, a fast-off-D2 antipsychotic. ACS Chem Neurosci. 2014 Jan 15;5(1):24-9.

[2]. Clozapine downregulates 5-hydroxytryptamine6 (5-HT6) and upregulates 5-HT7 receptors in HeLa cells. Neurosci Lett. 2000 Jul 21;288(3):236-40.

[3]. Persistent effects of chronic clozapine on the cellular and behavioral responses to LSD in mice. Psychopharmacology (Berl). 2013 Jan;225(1):217-26.

[4]. Clozapine is a potent and selective muscarinic M4 receptor agonist. Eur J Pharmacol. 1994 Nov 15;269(3):R1-2.

Since its first synthesis and testing, clozapine has exhibited a unique property: it has antipsychotic effects without the Parkinsonian motor side effects. The antipsychotic mechanism of clozapine is the transient occupation of dopamine D2 receptors in the human striatum, unlike haloperidol and chlorpromazine, which occupy D2 receptors for a longer period of time. The chemical structure of clozapine allows it to dissociate from D2 receptors relatively quickly. After the transient occupation of D2 receptors, a surge in neural activity increases synaptic dopamine levels, thereby displacing clozapine. Although clozapine may occupy other types of receptors, they may not be very effective in preventing Parkinson's disease. The transient occupation of D2 receptors by clozapine allows patients to move easily and comfortably. [1] Reason: For patients with schizophrenia, the best treatment with antipsychotic drugs requires medication that lasts for weeks to months. However, a single dose of antipsychotic drugs can reverse schizophrenia-like behavioral changes in rodent psychosis models. This raises questions about the physiological relevance of this antipsychotic-like activity. Objective: This study aimed to evaluate the effects of long-term clozapine treatment on cellular and behavioral responses in a mouse model of psychosis induced by the hallucinogenic 5-HT2A receptor agonist lysergic acid diacetamide (LSD). Methods: Mice were treated with clozapine at 25 mg/kg/day for 21 days. Experiments were performed on days 1, 7, 14, and 21 after the last administration. [³H]ketoserin binding and 5-HT₂A mRNA expression levels in the mouse somatosensory cortex were measured. Head twitching behavior, c-fos expression induced by all 5-HT(2A) agonists, and the expression of LSD-like specific receptors egr-1 and egr-2 were also examined. Results: Head twitching response was reduced and [(3)H]ketoserin binding was downregulated on days 1, 7, and 14 after long-term clozapine administration. 5-HT(2A) mRNA levels were reduced one day after long-term clozapine administration. After 7 days of long-term clozapine administration, the induction of c-fos was restored, but the induction of egr-1 and egr-2 was not restored. The above effects were not observed with short-term (2 days) clozapine administration or long-term haloperidol (1 mg/kg/day). Conclusion: Our results provide a mouse model of chronic atypical antipsychotic drug action and suggest that downregulation of 5-HT(2A) receptors may be a potential mechanism for these persistent treatment-like effects. [3]

C18H21CL3N4

399.75

2711603-38-0

Clozapine;5786-21-0;Clozapine (Standard);5786-21-0;Clozapine hydrochloride;54241-01-9

Typically exists as solids at room temperature

HF 1854 dihydrochloride; 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine,dihydrochloride; 2711603-38-0; Clozapine (dihydrochloride); HY-14539B; 3-chloro-6-(4-methylpiperazin-1-yl)-11H-benzo[b][1,4]benzodiazepine;dihydrochloride

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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