Clozapine N-oxide (CNO) (freebase)
Prototypical DREADD activator. Clozapine metabolite.
Clozapine N-oxide CNO) is the prototypical chemical actuator for various DREADDs. It is a metabolite of the atypical antipsychotic clozapine.
‘Excitatory’ (Gq- coupled) DREADDs:
CNO activates the excitatory Gq- coupled DREADDs: hM3Dq, hM1Dq and hM5Dq (pEC50 values are 7.26 and 8.61 at hM3Dq and hM1Dq respectively).
The hM3Dq DREADD is typically used for enhancing neuronal firing and activity (Gq- signaling in neuronal and non-neuronal cells).
‘Inhibitory’ (Gi- coupled) DREADDs:
CNO also activates the inhibitory hM4Di and hM2Di Gi-coupled DREADDs (pEC50 = 6.89 at hM4Di).
The hM4Di DREADD is the most commonly used inhibitory DREADD and is used for neuronal silencing.
Gs and β-arrestin coupled DREADDs:
CNO also activates the Gs- coupled DREADD (GsD) and the β-arrestin preferring DREADD: rM3Darr (Rq(R165L).
Recent findings (Gomez et al 2017) suggest that systemically administered CNO does not readily cross the blood-brain-barrier in vivo, and converts to clozapine which activates DREADDs. Enzymatic and non-enzymatic reduction of CNO to clozapine has been shown in humans, rats, monkeys, guinea pigs and mice.
Care must therefore be taken in experimental design and proper controls should be incorporated, for example, the use of non-DREADD expressing animals may be appropriate (see Mahler and Aston-Jones (2018)).
Jendryka et al (2019) found that in mice, CNO does enter the brain and that unbound CNO is present in the brain at sufficient levels to activate DREADDs directly. Results suggested that CNO is a suitable DREADD agonist but requires between-subject controls for unspecific effects.
CNO has proved to be an effective actuator of muscarinic DREADDs and provided controls are in place, will continue to be an excellent tool. Compound 21 (DREADD agonist 21) represents an alternative to CNO for in vivo studies in which metabolic conversion of CNO to clozapine is an issue (Thompson et al 2019).
In the literature, CNO has been administered intraperitoneally (i.p.), subcutaneously, directly infused intracranially, via drinking water, osmotic mini-pump and recently via eye drops. See our Technical review (table 3) for example administration methods and doses.
Water soluble Clozapine N-oxide (CNO) dihydrochloride is also available:
Clozapine N-oxide (CNO) dihydrochloride is water soluble and easier to solubilise and handle. Our stability studies have found that this product does not precipitate in aqueous solution unlike the freebase form of CNO (which due to its inherent chemical properties requires careful handling, has been shown in the literature to precipitate in solution under certain conditions and batch to batch variation in solubility can occur).
In rhesus macaques, CNO dihydrochloride shows improved bioavailability compared to CNO freebase with less conversion to clozapine
CNO, CNO freebase
Your technical review on CNO stability, solubility and use in the lab has been really helpful to my group. Verified customer
Exactly as described - Clozapine N-oxide (CNO) (freebase) does exactly what it should. Great product and company. Verified customer, Istituto Italiano di Tecnologia
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Storing and Using Your Product
Room temperature (desiccate)
Soluble in DMSO (100mM).
Due to the inherent chemical properties of CNO, this product requires careful handling – our guidelines are detailed below.
Batch to batch variation in solubility can occur. Some batches are water soluble but ongoing solubility in aqueous solutions can be unpredictable.
CNO has been shown in the literature to precipitate in aqueous solution and following further solubility studies that we have undertaken, we have found that precipitation occurred in some concentrated (100mM) samples of CNO dissolved in water.
We therefore recommend using DMSO when working with CNO freebase. If you need to work with aqueous solutions, you should use CNO dihydrochloride which is easier to solubilize and handle than CNO freebase. Our stability studies have found that this product does not precipitate in solution.
Solubilizing in DMSO
CNO (freebase) is soluble in DMSO to 100 mM.
Storage of solutions in DMSO
- Aliquot out the solution into tightly sealed vials for storage.
- Storage of solutions should be at room temperature.
- Please take care to ensure that your product is completely dissolved in your solution before use.
We do not recommend using CNO freebase with aqueous solutions however, we provide these guidelines if you do:
Solubilizing CNO (freebase) in aqueous solutions (e.g. water / saline)
When working with the compound in aqueous solutions, we recommend:
- Make up solutions and use immediately. Do not store solutions.
- Ensure you work in a dust free environment when preparing and handling solutions.
- Ensure that the product and solvents are at ambient temperature before preparing solutions. This means that you should allow the product to equilibrate to RT for at least one hour before opening and using.
- Please take care to ensure that your product is completely dissolved in your solution before use.
Our stability studies have shown that CNO in solution remains chemically stable (>99% purity by HPLC) for at least 4 weeks at room temperature.
Storage of solutions in aqueous solutions (e.g. water/saline
We recommend that you do not store aqueous solutions of CNO. However, if this is necessary:
- Aliquot out the solution into tightly sealed vials for storage. Storage of solutions should be at room temperature.
- Do not store solutions at temperatures below room temperature (i.e. +4°C/ -20°C) - this is more likely to lead to precipitation of the compound.
- Preferably, solutions should be made in transparent, colorless vials so that any precipitation can be observed and remedied prior to use.
- Always check that your product is completely dissolved before use; if precipitation is found follow the steps below.
If you find precipitate in your solution, gently heat your solution in a water bath to approx 40°C and the compound should readily re-dissolve. Always take care to ensure that the compound is completely dissolved before use.
Water soluble alternatives:
CNO dihydrochloride is now available and is also water soluble. It is easier to solubilise and handle and unlike the freebase form of CNO, our stability studies have found that this product does not precipitate in solution.
This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use.
References for Clozapine N-oxide (CNO) (freebase)
Novel designer receptors to probe GPCR signaling and physiology.Wess et al (2013) Trends Pharmacol Sci. 34(7) : 385-92PubMedID: 23769625
Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.Arbruster et al (2007) Proc Natl Acad Sci U S A. 104(12) : 5163-8PubMedID: 17360345
The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.Weiner et al (2004) Psychopharmacology (Berl). 177(1-2) : 207-16PubMedID: 15258717
A chemical-genetic approach to study G protein regulation of beta cell function in vivo.Guettier et al (2009) Proc Natl Acad Sci U S A 106(45) : 19197-202PubMedID: 19858481
Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.Gomez et al (2017) Science 357(6350) : 503-507PubMedID: 28774929
Chemogenetic inhibition of cells in the paramedian midbrain tegmentum increases locomotor activity in rats.Wirtshafter and Stratford (2016) Brain Res 1632: : 98-106PubMedID: 26707405
CNO Evil? Considerations for the Use of DREADDs in Behavioral Neuroscience.Mahler and Aston-Jones (2018) Neuropsychopharmacology doi: 10.1038 : npp.2017.299PubMedID: 29303143
DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in VivoThompson et al (2018) ACS Pharmacol. Transl. Sci. 10.1021 : /acsptsci.8b00012
A Comparative Study of the Pharmacokinetics of Clozapine N-Oxide and Clozapine N-Oxide Hydrochloride Salt in Rhesus Macaques.Allen et al (2019) J Pharmacol Exp Ther 368(2) : 199-207PubMedID: 30523062
Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice.Jendryka et al (2019) Sci Rep. 9(1) : 4522PubMedID: 30872749
Limiting habenular hyperactivity ameliorates maternal separation-driven depressive-like symptoms.Tchenio et al (2017) Nat Commun 8(1) : 1135PubMedID: 29074844
A synaptic threshold mechanism for computing escape decisionsEvans et al (2018) bioRxiv https://doi.org/ : 10.1101/272492
Circadian clock neurons mediate time-of-day dependent responses to lightJones et al(2018) bioRxiv https://doi.org/ : 10.1101/291955
SCN VIP neurons are essential for normal light-mediated resetting of the circadian system.Jones et al(2018) J Neurosci pii: 1322-18. : doi: 10.1523/JNEUROSCI.1322-18.2PubMedID: 30082421
In vitro and in vivo properties of GLP-1 producing neurons: the brain actions of a gut hormoneMarie Kragelund Bachmann Holt(2017) Thesis : UCL
Cholinergic modulation of spinal motoneurons and locomotor control networks in miceNascimento, F(2018) Thesis : St Andrews University
Chemogenetic Stimulation of Electrically Coupled Midbrain GABA Neurons in Alcohol Reward and DependencePistorius et al (2017) Thesis https://scholarsarchive.byu.edu/etd/6813/ : Brigham Young University
The Neurochemistry and Neural Circuitry of Stress ResilienceBrooke, ND(2018) Thesis 8- : 2018
Nucleus Accumbens Fast-Spiking Interneurons Constrain Impulsive ActionPiskansky et al(2019) bioRxiv : doi: https://doi.org/10.1101/516
Constructing knowledge: Semantic memories in miceSchut, E.H.S.(2019) Thesis, : Radboud University
What should CNO (freebase) look like?
CNO (freebase) is an amorphous, yellow powder
What temperature should I store the solid compound at?
We undertake regular quality control analysis of the solid compound and have found no deterioration in product purity when stored at room temperature.
We recommend that you store solid CNO (freebase) powder at room temperature (desiccate).
Is the solid powder light sensitive?
No - we have found no evidence for Hello Bio CNO (free base) to be sensitive to light.
Is the solid powder air sensitive?
No - we have found no evidence for Hello Bio CNO (freebase) to be sensitive to air.
How do I weigh the compound to make my stock solution?
You should weigh out the quantity of product that you require for your experiment as the amount of product in our vials isn’t weighed out accurately enough for direct addition of solution.
How do I solubilize CNO to make my stock solution? Which solvent / buffer can I use?
Batch to batch variation in the solubility of CNO (freebase) can occur. Although some batches are soluble in aqueos soution, the ongoing solubility of these solutions can be unpredictable. This has been shown in the literature and in our stability studies.
Therefore, when working with CNO (freebase) we recommending using DMSO (100mM).
Various groups have different approaches to solubilizing CNO. DMSO is commonly used to dissolve CNO before addition to saline for injection.
What should I do if I find precipitate in my stock solution?
Gently heat your solution in a water bath to ~40°C and the compound should readily re-dissolve. Always take care to ensure that the compound is completely dissolved before use.
What concentration should my stock solution be?
This depends on your experiment and the working concentration you require.
What should my stock solution look like?
When dissolved your solution of CNO (freebase) should be a clear, yellow to orange color (depending on the concentration)
Can I store my stock solution of Clozapine N- oxide (CNO (freebase)?
We recommend making up solutions and using them immediately. Do not store solutions.
How long does Clozapine N-oxide (CNO freebase) stay stable in solution?
Our stability studies have shown that CNO in solution remains chemically stable (>99% purity by HPLC) for at least 4 weeks at room temperature. Please see stabiltiy data below.
Is my stock solution light sensitive?
No - we have found no evidence that Hello Bio CNO (freebase) is light sensitive.
Is my stock solution air sensitive?
No - we have found no evidence that Hello Bio CNO (freebase) is air sensitive.
We used Compound 21 (DREADD agonist 21) dihydrochloride (water soluble) (1mg/Kg) and CNO in rats (1mg/Kg) and we found a great effect on cells infected with dreadds-Gs. Both products are really easy to dissolve in saline!
Hello Bio's great pricing for CNO has enabled our small lab to greatly expand our DREADD experiments beyond what we initially thought we could afford. We are using CNO (doses up to 3 mg/kg IP) in drug self-administration paradigms in rats. Following the very helpful guides Hello Bio have available on their website, we easily dissolve CNO in saline for injection purposes, 20-30 minutes prior to testing. We do see some non-specific effects of CNO (in DREADD-free animals, likely due to clozapine metabolisation), although nothing that can't be controlled for with careful experimental design. In light of this, Hello Bio generously offered a free sample of the novel DREADD agonist Compound 21, which shouldn't metabolise to clozapine, and which we are now testing in our behavioural paradigms in contrast to CNO. Great products, great staff and support!
Exactly as described - Clozapine N-oxide (CNO) (freebase) does exactly what it should. Great product and company.