NameClozapine N-oxide (CNO) (freebase)
Short descriptionPrototypical DREADD activator. Clozapine metabolite.
Biological descriptionClozapine 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.
The hM3Dq DREADD is typically used for enhancing neuronal firing and activating (Gq- signaling in neuronal and non-neuronal cells.
‘Inhibitory’ (Gi- coupled) DREADDs:
CNO also activates the inhibitory hM4Di and hM2Di Gi-coupled DREADDs.
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).
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.
Recent findings suggest that systemically administered CNO does not readily cross the blood-brain-barrier in vivo, and converts to clozapine which activates DREADDs. Care must be taken in experimental design and proper controls should be incorporated.
Clozapine N-oxide (CNO) dihydrochloride is also available. Clozapine N-oxide (CNO) dihydrochloride is also water soluble, but is easier to solubilise and handle and is more stable in solution than the freebase form of CNO (which due to its inherent chemical properties requires careful handling and has been shown in the literature to precipitate in solution under certain conditions).
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
Citations1 citation Submit Your Citation Now
Chemical name8-Chloro-11-(4-methyl-4-oxido-1-pip erazinyl)-5H-dibenzo[b,e][1,4]diazepine
Storing and Using Your Product
Storage instructionsRoom temperature (desiccate)
Solubility overviewSoluble in water (100mM)* see handling instructions | DMSO (100mM).
HandlingDue to the inherent chemical properties of CNO, this product requires careful handling – our guidelines are detailed below.
CNO will dissolve in aqueous solutions (water, saline) however its ongoing solubility is unpredictable. CNO has been shown in the literature to precipitate in 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.
CNO dihydrochloride is now available. Our stability studies have not found this product to precipitate in solution.
When working with the compound in aqueous solutions, we recommend:
Solubilizing CNO (freebase) in aqueous solutions (e.g. water / saline)
- 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.
StabilityOur 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 waterWe recommend that you do not store aqueous solutions (e.g. water/saline) 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.
PrecipitationIf 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.
CNO dihydrochloride is now available and is also water soluble. It is easier to solubilise and handle and is more stable in solution than the freebase form of CNO. Unlike the freebase form of CNO, our stability studies have found that this product does not precipitate in solution.
Water soluble alternatives:
CNO (freebase) is soluble in DMSO to 100 mM.
Solubilizing in DMSO
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.
ImportantThis 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: https://www.ncbi.nlm.nih.gov/pub
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
Limiting habenular hyperactivity ameliorates maternal separation-driven depressive-like symptoms.Tchenio et al(2017) Nat Commun 8(1) : 1135PubMedID: 29074844
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.
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)
Is there a recommended way to solubilize CNO (freebase)?
Although, CNO will dissolve in aqueous solutions (e.g. water, saline) its ongoing solubility is unpredictable. Various groups have different approaches to solubilizing CNO. For example, DMSO is commonly used to dissolve CNO before addition to saline for injection.
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.
- 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.
- Working better! Since my last review, we have continued using CNO for conditioned place preference, and results are looking better as we continue to figure out parameters.