DREADD; Dopamine receptor; muscarinic M4 receptor

CNO/Clozapine-N-oxide is a synthetic small molecule actuator for DREADDs that muscarinic acetylcholinergic.

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In studies to confirm the biological activity of the Clozapine-N-oxide/CNO synthesized herein and the extracted clozapine, we measured their ability to activate the M1 DREADD in cells using the ERK1/2 phosphorylation assay as a signaling endpoint linked to this receptor. CNO and clozapine produced potent and efficacious responses (pEC50 8.31 ± 0.12 and 10.32 ± 0.18; Emax 74.8 ± 2.8 and 77.0 ± 3.7, respectively, Fig. 3). The potencies observed are comparable to the potencies of commercial CNO and clozapine (Sigma Aldrich) reported with this cell line (pEC50 8.50 ± 0.13 and 9.68 ± 0.28, respectively) [4].

Clozapine-N-oxide (CNO), an agonist of the human muscarinic designer receptor (DREADDs), is transformed into clozapine and exhibits behavioral effects resembling those of clozapine in both mice and rats. This highlights the necessity of using suitable control groups in research involving DREADDs.[4]

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Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether Clozapine-N-oxide/CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0-20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists. [1]

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In vivo activity [4]
In studies to confirm the biological activity of the Clozapine-N-oxide/CNO synthesized as described in vivo, we examined the effects of chemogenetic activation of the NI in rats. The modified Gq-coupled human muscarinic receptor, hM3Dq , fused to mCherry fluorescent protein was expressed in NI neurons using an adeno-associated viral vector expression system with a strong, chicken β-actin (CAG) promoter – AAV1/2-sCAG-hM3Dq-mCherry. An AAV1/2-sCAG-mCherry vector was used as a control. Microinjection of this viral vector into the NI resulted in hM3Dq expression (hereafter referred to as NI-hM3Dq) in NI soma and proximal processes, as indicated by the presence of fluorescent mCherry puncta (Fig. 4A,B). The control AAV1/2-sCAG-mCherry vector (hereafter referred to as NI-mCherry), in contrast, produced profuse red cytoplasmic fluorescence in NI cells (data not shown), as described. We have shown that chemogenetic NI activation in NI-hM3Dq rats leads to cortical arousal, decreased sleep, increased locomotor activity, and increased risk-assessment behavior, all of which are consistent with increased general arousal. Furthermore, these studies also demonstrated that hM3Dq activation by Clozapine-N-oxide/CNO in vitro resulted in long-lasting depolarization of NI neurons with action potentials, and that peripheral injection of CNO (3 mg/kg) significantly increased immunostaining for c-Fos, an immediate early gene marker of neuronal activation, in NI-hM3Dq rats, compared to control NI-mCherry rats. These data confirm the functional activation of NI by CNO activation of hM3Dq in vivo and a lack of effect of CNO in control NI-mCherry rats.

Extracellular signal-regulated kinase 1/2 phosphorylation (pERK1/2) assay [4]
Assays to measure M1 DREADD-mediated stimulation of ERK1/2 phosphorylation were performed using the Alpha Screen based Sure-Fire kit, following the manufacturer’s instructions. Briefly, FlpIn CHO cells stably expressing the M1 DREADD were seeded into 96-well culture plates at 40,000 cells per well and allowed to adhere. Cells were then rinsed with phosphate-buffered saline and maintained in serum-free media overnight at 37 °C, 5% CO2. The following day, cells were stimulated with agonist. Initial pERK1/2 time course experiments were performed to determine the time of maximal ERK1/2 phosphorylation for Clozapine-N-oxide/CNO and clozapine (found to be 5 min for both ligands). The time of peak agonist response was then used for the establishment of concentration-response curves. In all experiments, 10% (v/v) foetal bovine serum (FBS) was used as positive control of pERK1/2. The reaction was terminated by removal of media and addition of lysis buffer. Samples were processed according to kit instructions. The fluorescence signal was measured using a POLARstar Omega plate reader. Data were normalized to the maximum response elicited by 10% (v/v) FBS at 5 min.

standard laboratory rats and mice
1.0, 3.2, 10.0 mg/kg for rats; 1.25, 2.5, 5.0, 10.0, 20.0 mg/kg for mice
i.p.

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Substitution tests occurred only when animals had satisfied strict performance criteria (see Supplementary Methods and Materials). To confirm the selectivity of the clozapine stimulus and its control over behavioral responding, rats were tested with vehicle or multiple doses of clozapine (0.0395, 0.125, 0.395, 1.25 mg/kg), the mixed dopamine/serotonin/norepinephrine antagonists olanzapine (1.0 mg/kg) and risperidone (0.56 mg/kg), the α1-adrenergic receptor antagonist prazosin (0.56 mg/kg), and the β-adrenergic receptor antagonist propranolol (10.0 mg/kg). Similarly, mice were tested with vehicle and multiple doses of clozapine (0.156, 0.3125, 0.625, 0.88, 1.25 mg/kg), olanzapine (0.5 mg/kg), the nonselective serotonin 5-HT2 receptor antagonist ritanserin (16.0 mg/kg), prazosin (10.0 mg/kg), and the nonselective dopamine D2-like receptor antagonist haloperidol (0.1 mg/kg). The doses of olanzapine, ritanserin, prazosin, and haloperidol were used because we have observed that higher doses produce nonspecific rate-suppressant effects. To test for Clozapine-N-oxide/CNO-induced clozapine-like effects, animals were administered Clozapine-N-oxide/CNO (rats −1.0, 3.2, 10.0 mg/kg; mice −1.25, 2.5, 5.0, 10.0, 20.0 mg/kg) prior to a test session. All drugs and doses were administered in a randomized order to each subject. [1]

Blood Sample Collection and Analysis [1]
Rats were surgically prepared with chronic indwelling intrajugular catheters as described previously24 to allow for rapid and repeated blood sampling. Blood collections began at least two weeks following surgery. On a test day, rats were administered either clozapine (1.25 mg/kg) or Clozapine-N-oxide/CNO (1.0, 10.0 mg/kg) and returned to the home cage. Blood samples (0.4–0.5 ml per sample, ~0.1 ml withdrawn per 5 s) were collected via aspiration from the intravenous catheter 30 and 60 min following drug administration. Catheters were flushed with bacteriostatic saline and locked with 0.1 ml of heparinized saline (300 heparin IU/ml) when not in use to maintain catheter patency between collections and on days between tests. Tests were separated by a minimum of 2 weeks and were performed in the following order for all subjects: (1) 10.0 mg/kg CNO, (2) 1.25 mg/kg clozapine, (3) 1.0 mg/kg CNO.

Mice were administered either clozapine (1.25 mg/kg) or Clozapine-N-oxide/CNO (10.0 mg/kg) and returned to their home cage. 2–3 min prior to the desired time point of blood sampling, each mouse was placed in a Plexiglas anesthesia induction chamber and exposed to 4–5% isoflurane until loss of movement and then transferred to a nosecone that continued to supply isoflurane (1–2%). Once deep anesthesia was verified, the heart was exposed and a 23 g needle attached to a 1 ml syringe was inserted into the left ventricle. 0.4–0.5 ml of blood was withdrawn and handled identical to the description above for rat blood sample collections.

All blood samples were deposited into a 1.7 ml tube containing 10 μl of heparinized saline (500 heparin IU/ml) and stored on ice until centrifugation at room temperature at 800 g for 10 min. The plasma was then removed, placed into a separate sterile 1.7 ml tube, and stored at −80 °C until subsequent analysis via UPLC-LC/MS/MS (see Supplementary Materials and Methods).

Clozapine, olanzapine, risperidone, haloperidol, prazosin, propranolol, and ritanserin were each dissolved in distilled water with 2–3 drops of lactic acid and pH-adjusted to 6.0–7.0 with NaOH. For mouse drug discrimination studies, CNO was also dissolved in this vehicle. For rat drug discrimination studies and for mouse and rat pharmacokinetic analyses, Clozapine-N-oxideCNO was dissolved in bacteriostatic saline containing v/v 2.5–5.0% dimethyl sulfoxide (Sigma-Aldrich) and 10% Cremophor EL.

For mouse drug discrimination studies, all drugs were administered s.c. at a volume of 10 ml/kg, 30 min prior to session onset. For rat drug discrimination studies, all drugs were administered i.p. at a volume of 1 ml/kg. Clozapine was administered 60 min prior to session onset, while olanzapine, risperidone, prazosin, and propranolol were administered 30 min prior to session onset. Clozapine-N-oxide/CNO was tested at both 30 and 60 min pretreatment times. All drug doses are expressed as the salt weight.

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In vivo test of food and water intake in rats [4]
On the day of use, CNO/Clozapine-N-oxide was dissolved in sterile saline at 1 mg/ml and administered by intraperitoneal (i.p.) injection in rats at 1 or 3 mg/kg, or rats were injected with an equivalent volume of sterile saline. For behavioral testing, rats received an i.p. injection of sterile saline on Day 1 and placed back into their home cage with access to pre-weighed food and water, and consumption was recorded 4 h post-injection. Testing was repeated on Day 2, where rats received an i.p. injection of CNO at 1 or 3 mg/kg. Food and water intake following CNO/Clozapine-N-oxide injection were normalized to intake following saline injection, where a value of 1 represents equivalent intake. Unpaired t-tests were used to calculate statistical differences between groups.

Metabolism / Metabolites
Clozapine-N-oxide is a known human metabolite of clozapine.

mouse LD50 oral 245 mg/kg BEHAVIORAL: ATAXIA Collection of Czechoslovak Chemical Communications., 43(309), 1978

[1]. Sci Rep. 2018 Mar 1;8(1):3840.

[2]. Front Pharmacol. 2019 Jun 4:10:628.

[3]. Eur J Pharmacol. 1994 Nov 15;269(3):R1-2.

[4]. MethodsX. 2018 Mar 23:5:257-267.

Clozapine N-oxide is a dibenzodiazepine.

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LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D2R. HEK-293T cells transiently coexpressing the human long isoform of D2 receptor (D2LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D2R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of β-arrestin-2 (β-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the β-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used. [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. [3]

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Increasing use of muscarinic acetylcholine receptor DREADDs has led to a growing demand for CNO, but the high cost from most commercial vendors has created difficulties for large-scale deployment of the technology. This work reports simple access to clozapine from commercially available (pharmacy) tablets, and its conversion to CNO by oxidation with Oxone. Based on raw material costs (clozapine is obtained at approx. $14/g from tablets; reagent costs are minimal), CNO can be produced in a 97% yield at a similar price, excluding labor costs. By comparison, commercial prices for CNO from representative suppliers range from $53,000/g , $24,000/g, $6700/g. The present route with some proprietary modifications allowing removal of methanol has been implemented by AMT Pty Ltd , and commercial material produced by this method is now available through AK Scientific for $420/g, representing a major reduction in the cost of this important reagent. It is hoped that this work will support the continuing development of DREADD technology and further insights into brain function and related behaviors.[4]

C18H19CLN4O

342.82266

342.124

C, 63.06; H, 5.59; Cl, 10.34; N, 16.34; O, 4.67

34233-69-7

Clozapine; 5786-21-0; Clozapine N-oxide dihydrochloride; 2250025-93-3; 54241-01-9 (HCl); 34233-69-7 (N-oxide); 5786-21-0

135445691

Light yellow to yellow solid powder

1.36g/cm3

517.4ºC at 760mmHg

190-248ºC

266.7ºC

1.685

0.76

1

3

1

24

491

0

[O-][N+]1(C)CCN(C2=NC3=CC(Cl)=CC=C3NC4=CC=CC=C42)CC1

OGUCZBIQSYYWEF-UHFFFAOYSA-N

InChI=1S/C18H19ClN4O/c1-23(24)10-8-22(9-11-23)18-14-4-2-3-5-15(14)20-16-7-6-13(19)12-17(16)21-18/h2-7,12,20H,8-11H2,1H3

3-chloro-6-(4-methyl-4-oxidopiperazin-4-ium-1-yl)-11H-benzo[b][1,4]benzodiazepine

Clozapine N-oxide; Clozapine N-oxide; 34233-69-7; N-Oxyclozapine; UNII-MZA8BK588J; MZA8BK588J; VUFB-12426; NSC-750266; 8-Chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo(b,e)(1,4)diazepine N-oxide;

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)

DMSO: 68~100 mg/mL (198.4~291.7 mM)
Water: ~68 mg/mL
Ethanol: ~8 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.29 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 (7.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.29 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 4: ≥ 0.5 mg/mL (1.46 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 0.5 mg/mL (1.46 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 6: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 3.4mg/ml (9.92mM)

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Solubility in Formulation 7: 2.44 mg/mL (7.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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