Biological Activity

Bioactivity / Mechanism

Clozapine N-oxide (CNO) can activate the inhibitory DREADD hM4Di in cultured neurons. In a calcium-oscillation assay, CNO suppressed network activity with an EC50 of 8.1 nM; this functional value is assay-specific and is not a binding-affinity measurement. [1]

Literature-reported observations and conditions; not Adooq-tested protocols or general treatment recommendations.

In Vitro Studies

In primary rat cortical cultures, CNO reduced spontaneous calcium-oscillation frequency when hM4Di was expressed. Untransduced cultures showed no corresponding response under the tested conditions. The assay used buffer controls and retigabine as a positive control. [1]

CNO suppresses calcium oscillations in hM4Di-expressing primary neurons

Cell-based study · Primary cortical neuronal cultures from embryonic day 18 Sprague-Dawley rats; hM4Di-expressing cultures compared with untransduced controls. [1]

CNO reduced the frequency of spontaneous calcium oscillations in hM4Di-expressing cultures. The assay measures network calcium activity indirectly, not receptor-binding affinity. Untransduced cultures did not show this response; retigabine was an assay positive control.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Reduction of normalized spontaneous calcium-oscillation frequency in hM4Di-expressing neurons	EC50 8.1 nM	[1]
Effect in untransduced cultures and assay controls	Control responses No effect of the DREADD agonists alone on calcium oscillations in untransduced cultures; retigabine (30 μM) blocked oscillations.	[1]

Literature-Reported Treatment Conditions

Treatment concentration: CNO concentration-response testing; Methods specify 0.3 nM–1 μM. EC50 is an assay endpoint, not a treatment concentration.
Exposure duration: 5-min baseline followed by 5-min recording after compound addition.
Method: Automated compound addition during FLIPR calcium-fluorescence recording.
Vehicle: Assay-buffer control wells (HBSS with 10 mM HEPES, pH 7.4).
Co-treatment: None; CNO, clozapine and compound 21 were tested separately.
Reported limits: Independent culture-preparation count and compound stock solvent for this cellular assay are not specified in the selected methods; plate/duplicate counts do not establish biological replicate count.

Methods and source locations

Compound reported in the paper: Clozapine N-oxide; Compound label in the paper: CNO

Evidence concerns the reported CNO material and assay/model conditions; it does not validate an Adooq lot.

Figure 1a–b and caption; Results: In vitro potency of CNO, CLZ and Cmpd-21 at the hM4Di receptor; Methods: FLIPR assay and Data analysis

Reduction of normalized spontaneous calcium-oscillation frequency in hM4Di-expressing neurons: Fluo-4 FLIPR; Hill-function fit. Each compound dose repeated on three individual plates in duplicate; figure error bars are SEM. No uncertainty interval reported for this EC50. — Figure 1a–b and caption; Results: In vitro potency of CNO, CLZ and Cmpd-21 at the hM4Di receptor; Methods: FLIPR assay and Data analysis

Effect in untransduced cultures and assay controls: Assay-buffer wells used for normalization; untransduced cultures test receptor dependence. — Figure 1a–b and caption; Results: In vitro potency of CNO, CLZ and Cmpd-21 at the hM4Di receptor; Methods: FLIPR assay and Data analysis

In Vivo Studies

After a single 3.5 mg/kg intraperitoneal CNO dose in C57BL/6J mice, both parent CNO and converted clozapine were detected in cortical tissue. CNO was also measurable in CSF at 15 and 30 min, whereas clozapine remained below the CSF assay detection limit. Total tissue exposure and CSF exposure differed markedly, and these measurements do not determine which compound dominates in-vivo DREADD activation. [1]

Parent CNO and converted clozapine show different CNS exposure profiles in mice

Animal study · 14–16-week-old C57BL/6J mice; plasma, CSF and anterior cortical brain tissue sampled after CNO administration. Sex is not explicitly given for this pharmacokinetic cohort in Subjects. [1]

After 3.5 mg/kg intraperitoneal CNO, parent CNO was measured in CSF and cortex, while converted clozapine accumulated in brain tissue. Clozapine remained below the CSF detection limit. These exposure data do not quantify the relative contributions of parent and metabolite to in-vivo DREADD activation.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
CNO concentration in CSF after CNO injection	Mean concentration 11.2 nM at 15 min; 10.8 nM at 30 min	[1]
CNO concentration in cortical tissue after CNO injection	Mean concentration 23.8 nM at 15 min; 18.5 nM at 30 min; not detected at 60 min	[1]
Converted clozapine in CSF after CNO injection	Detection threshold Below 1–2.5 nM detection limit at all sampling points (15, 30 and 60 min)	[1]
Converted clozapine in cortical tissue after CNO injection	Mean concentration Approximately 427 nM at 15 min; mean 406 nM at 60 min	[1]

Conditions shared by the study groups

Route: Intraperitoneal
Frequency: Single administration
Treatment course: 15–60 min sampling window

Study groups and reported outcomes
Group / dose	Comparator	Observed result	Reference
Single-dose CNO pharmacokinetic cohort 3.5 mg/kg	Analytical internal standard and concentration calibration; no vehicle-treated PK comparator is described for Figure 2a–c.	Parent CNO detected in CSF and cortex early after dosing; converted clozapine detected in cortex but below CSF assay detection limit.	[1]

Single-dose CNO pharmacokinetic cohort: Model: C57BL/6J mice, 14–16 weeks; caption reports n=5 at 15 min and n=6 at 30 and 60 min. Matrix-specific sample accounting is not fully consistent (see missing information).; Reported limits: Figure 2b marks two failed CSF samplings and four clozapine non-detections at 15 min, which does not reconcile directly with the caption n=5. No exact available CSF denominator is inferred. One CSF sampling failed at 30 min. PK cohort sex and explicitly PK-specific vehicle are not supplied.

Methods and source locations

Compound reported in the paper: Clozapine N-oxide; Compound label in the paper: CNO

Evidence concerns the reported CNO material and assay/model conditions; it does not validate an Adooq lot.

Figure 2a–c and caption; Results: Pharmacokinetic profile of CNO and CLZ after CNO application in mice; Methods: Subjects and Assessment of pharmacokinetic properties of CNO, CLZ, and Cmpd-21

CNO concentration in CSF after CNO injection: LC–MS analysis. Means explicitly stated in Results. Figure 2b indicates two failed CSF samplings at 15 min and one at 30 min; do not treat injected group counts as available CSF sample counts. — Figure 2a–c and caption; Results: Pharmacokinetic profile of CNO and CLZ after CNO application in mice; Methods: Subjects and Assessment of pharmacokinetic properties of CNO, CLZ, and Cmpd-21

CNO concentration in cortical tissue after CNO injection: LC–MS analysis; Results supplies means and describes final-time-point non-detection. Figure 2c has one CNO non-detection at 15 min and one at 30 min. — Figure 2a–c and caption; Results: Pharmacokinetic profile of CNO and CLZ after CNO application in mice; Methods: Subjects and Assessment of pharmacokinetic properties of CNO, CLZ, and Cmpd-21

Converted clozapine in CSF after CNO injection: Detection limit depends on sample quality. Non-detection is not zero exposure and does not exclude a pharmacologically relevant free clozapine concentration. — Figure 2a–c and caption; Results: Pharmacokinetic profile of CNO and CLZ after CNO application in mice; Methods: Subjects and Assessment of pharmacokinetic properties of CNO, CLZ, and Cmpd-21

Converted clozapine in cortical tissue after CNO injection: Values stated in Results. Total tissue concentration is distinct from freely available concentration. — Figure 2a–c and caption; Results: Pharmacokinetic profile of CNO and CLZ after CNO application in mice; Methods: Subjects and Assessment of pharmacokinetic properties of CNO, CLZ, and Cmpd-21

Single-dose CNO pharmacokinetic cohort · Figure 2a–c and caption; Results: Pharmacokinetic profile of CNO and CLZ after CNO application in mice; Methods: Subjects and Assessment of pharmacokinetic properties of CNO, CLZ, and Cmpd-21

Specifications

Manufacturer	Adooq Bioscience
Actions	Antagonist
CAS Number	34233-69-7
Applications	5-HT1C receptor antagonist
Formula	C₁₈H₁₉ClN₄O
Molecular Weight	342.82
SMILES	C[N+]1(CCN(CC1)C2=C3C=CC=CC3=NC4=C(N2)C=C(C=C4)Cl)[O-]

Storage & Solubility

In Vitro Solubility

Reported Solubility

Solvent	Reported Solubility
DMSO	63 mg/mL (183.77 mM)
Water	63 mg/mL (183.77 mM)
Ethanol	7 mg/mL (20.41 mM)

Preparing Stock Solutions

Stock preparation is unavailable for this product molecular weight.

Calculated stock concentrations do not establish solvent solubility. Refer to the reported solubility data for experimental guidance.

Storage

Store powder at -20°C, keep desiccated.
In powder form, the chemical is stable for 36 months.
In solution, store at -20°C and use within 1 month to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

Research References

[1] Martin Jendryka, Monika Palchaudhuri, Daniel Ursu, et al. Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice Scientific Reports (2019). PMID: 30872749 DOI: 10.1038/s41598-019-41088-2 Source material

Publications

1 publication citing this product

Kazuhei Niitani, .et al. , J Pharmacol Sci, 2025, Sep;159(1):1-7 PMID: 40713343

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Clozapine N-oxide

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