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

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 hydrochloride (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) are equally bioavailable relative to a CLOZARIL solution. Following oral administration of clozapine 100 mg twice daily, the average steady-state peak plasma concentration was 319 ng/mL (range: 102 to 771 ng/mL), occurring at the average of 2.5 hours (range: 1 to 6 hours) after dosing. The average minimum concentration at steady state was 122 ng/mL (range: 41 to 343 ng/mL), after 100 mg twice daily dosing.
Approximately 50% of the administered dose is excreted in the urine and 30% in the feces.
The median volume of distribution of clozapine was calculated to be 508 L (272–1290 L).
The median clearance of clozapine is calculated to be 30.3 L/h (14.4–45.2 L/h).
Clozapine is almost completely metabolized prior to excretion and only trace amounts of unchanged drug are detected in the urine and feces. Approximately 50% of the administered dose is excreted in the urine and 30% in the feces. The demethylated, hydroxylated and N-oxide derivatives are components in both urine and feces. Pharmacological testing has shown the desmethyl metabolite to have only limited activity, while the hydroxylated and N-oxide derivatives were inactive.
In man, clozapine tablets (25 mg and 100 mg) are equally bioavailable relative to a clozapine solution. Following a dosage of 100 mg b.i.d., the average steady-state peak plasma concentration was 319 ng/mL (range: 102 to 771 ng/mL), occurring at the average of 2.5 hours (range: 1 to 6 hours) after dosing. The average minimum concentration at steady-state was 122 ng/mL (range: 41 to 343 ng/mL), after 100 mg b.i.d. dosing. Food does not appear to affect the systemic bioavailability of clozapine. Thus, clozapine may be administered with or without food. Clozapine is approximately 97% bound to serum proteins.
Clozapine is rapidly absorbed after both single and repeated oral doses, with steady-state concentrations attained within eight to ten days after beginning therapy.

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Metabolism / Metabolites
Clozapine is almost completely metabolized prior to excretion, and only trace amounts of unchanged drug are detected in the urine and feces. Clozapine is a substrate for many cytochrome P450 isozymes, in particular CYP1A2, CYP2D6, and CYP3A4.The unmethylated, hydroxylated, and N-oxide derivatives are components in both urine and feces. Pharmacological testing has shown the desmethyl metabolite (norclozapine) to have only limited activity, while the hydroxylated and N-oxide derivatives were inactive.
Manic and schizophrenic patients were given neuroleptic clozapine at 300-500 mg daily and metabolites of clozapine were isolated from urine and analyzed by gas chromatography-mass spectrometry. Clozapine was converted into 2 metabolites by replacement of chlorine atom by a hydroxyl or methylsulfide group. Further metabolites were the N-demethyl deriv of 1st two metabolites. A metabolite with an oxidized piperazine ring was also found, and possibility of a metabolite with an oxidized sulfur is suggested.
/Clozapine/ is metabolized to N-oxideclozapine and N-desmethylclozapine, which have less pharmacological activity than the parent compound and are excreted in the urine and, to a lesser extent, in the feces.
Clozapine has known human metabolites that include Clozapine N-glucuronide, Clozapine-N-oxide, and N-Desmethylclozapine.

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Biological Half-Life
The mean elimination half-life of clozapine after a single 75 mg dose was 8 hours (range: 4 to 12 hours), compared to a mean elimination half-life of 12 hours (range: 4 to 66 hours), after achieving a steady state with 100 mg twice daily dosing. A comparison of single-dose and multiple-dose administration of clozapine demonstrated that the elimination half-life increased significantly after multiple dosing relative to that after single-dose administration, suggesting the possibility of concentration-dependent pharmacokinetics.
The mean elimination half-life of clozapine after a single 75 mg dose was 8 hours (range: 4 to 12 hours), compared to a mean elimination half-life, after achieving steady-state with 100 mg b.i.d. dosing, of 12 hours (range: 4 to 66 hours).

Toxicity Summary
IDENTIFICATION AND USE: Clozapine has been shown to be an effective, relatively rapid-acting, broad-spectrum antipsychotic agent in both uncontrolled and controlled studies of patients with schizophrenia.Clozapine has been used in a limited number of patients with advanced, idiopathic parkinsonian syndrome for the management of dopaminomimetic psychosis associated with antiparkinsonian drug therapy, but adverse effects such as sedation, confusion, and increased parkinsonian manifestations may limit the benefit of clozapine therapy in these patients. Clozapine is used to reduce the risk of recurrent suicidal behavior in patients with schizophrenia or schizoaffective disorder who are judged to be at chronic risk for such behavior, based on history and recent clinical state. Although the safety and efficacy of clozapine in children and adolescents younger than 16 years of age have not been established, the drug has been successfully used for the management of childhood-onset schizophrenia in a limited number of treatment-resistant children and adolescents. Clozapine is used for the symptomatic management of schizophrenia in severely ill patients whose disease fails to respond adequately to other antipsychotic therapy. HUMAN EXPOSURE AND TOXICITY: The most frequent adverse effects of clozapine involve the central and autonomic nervous systems (e.g., drowsiness or sedation, hypersalivation) and the cardiovascular system (e.g., tachycardia, hypotension). While the frequency and severity of some adverse effects (e.g., extrapyramidal reactions, tardive dyskinesia) appear to be less with clozapine than with other antipsychotic agents, other potentially serious adverse effects (e.g., agranulocytosis, seizures) may occur more frequently with clozapine therapy, and the potential risks and benefits should be evaluated carefully whenever therapy with the drug is considered. Although it has been suggested that a local genetic or environmental factor or factors may have been involved in the Finnish cases, the existence of such a factor has not been documented. During a 2 month period in Finland there were 18 reports of severe blood disorders (9 fatal) associated with clozapine. Agranulocytosis accounted for 8 of the deaths and leukemia probably for the ninth. Experience in 22 other countries outside Finland where clozapine had been marketed indicated an incidence of agranulocytosis of 0.3 per 1000 compared with an incidence almost 20 times as high in Finland and with 0.1 to 0.8 per 1000 for other tricyclic neuroleptics. patients who received flexible dosages of clozapine (mean dosage: 274.2 mg daily) for approximately 2 years had a 26% reduction in their risk for suicide attempts or hospitalization to prevent suicide compared with those who received flexible dosages of olanzapine (mean dosage: 16.6 mg daily); the treatment-resistant status of patients was not predictive of response to clozapine or olanzapine. ANIMAL STUDIES: Repeated oral administration to rats (6 months) and to dogs (3 months) decreased wt gain with doses of 20 mg/kg or more in rats and of 10 mg/kg or more in dogs. Hepatic hypertrophy, which was not strictly dose-dependent, was not associated with either histological changes or changes in blood chemistry and was completely reversible after discontinuation of treatment. No toxic signs were observed in rats or in dogs. Clozapine in daily oral doses of 20 or 40 mg/kg to rats and rabbits had no teratogenic effects and no influence on the fertility of male and female rats. At 40 mg/kg, however, clozapine inhibited growth of suckling young of treated mothers. Fertility of F1 treated mothers was normal and development of F2 showed no abnormalities. Clozapine hydrochloride inhibited conditioned avoidance behavior in rats, also inhibited writhing syndrome induced by phenylbenzoquinone in mice, and decreased body temp. Clozapine hydrochloride antagonized tremor and lacrimation induced by oxotremorine in mice, decreased the acute toxicity of physostigmine and 5-hydroxyindol acetate level in brain.

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Non-Human Toxicity Values
LD50 Rat iv 41.6 mg/kg
LD50 Rat sc 240 mg/kg
LD50 Rat im 210 mg/kg
LD50 Rat oral 251 mg/kg

[1]. Seeman P, et al. 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.

Ever since clozapine was first synthesized and tested, it showed the unique property of having antipsychotic action but no Parkinson-like motor side effects. The antipsychotic basis of clozapine is to transiently occupy dopamine D2 receptors in the human striatum, in contrast to haloperidol and chlorpromazine, which have a prolonged occupation of D2 receptors. The chemical structure of clozapine facilitates a relatively rapid dissociation from D2 receptors. After short-term occupation of D2 receptors, peak neural activity raises synaptic dopamine, which then displaces clozapine. While clozapine also occupies other types of receptors, they may not have a significant role in preventing parkinsonism. Clozapine's transient occupation of D2 receptors permits patients to move easily and comfortably. [1]

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Rationale: In schizophrenia patients, optimal treatment with antipsychotics requires weeks to months of sustained drug therapy. However, single administration of antipsychotic drugs can reverse schizophrenia-like behavioral alterations in rodent models of psychosis. This raises questions about the physiological relevance of such antipsychotic-like activity. 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]

C18H20CL2N4

363.28

C, 59.51; H, 5.55; Cl, 19.52; N, 15.42

54241-01-9

54241-01-9 (HCl); 34233-69-7 (N-oxide); 5786-21-0

Typically exists as solids at room temperature

CN1CCN(CC1)C2=NC3=C(C=CC(=C3)Cl)NC4=CC=CC=C42.Cl

HF 1854 hydrochloride; Clozapine hydrochloride; 54241-01-9; Clozapine (hydrochloride); CLZ-ChemoNM; 80862U56A3; 3-chloro-6-(4-methylpiperazin-1-yl)-11H-benzo[b][1,4]benzodiazepine;hydrochloride; CHEMBL538973; Clozapine HCl;

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.)