Clozapine N-oxide (CNO) dihydrochloride (water soluble)(HB6149)
Clozapine N-oxide (CNO) dihydrochloride (water soluble)
Dihydrochloride salt of CNO - the prototypical DREADD activator
Clozapine N-oxide dihydrochloride (CNO) is the dihydrochloride salt of CNO which is the prototypical chemical DREADDs activator. It is a metabolite of clozapine. In rhesus macaques, CNO dihydrochloride shows improved bioavailability compared to CNO freebase with less conversion to 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.
Handling & stabliity
Our stability studies have shown that CNO dihydrochloride is easier to solubilize and handle and have found that this product does not precipitate in solution unlike 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 aqueous solution under certain conditions). Please see our handling guidelines below.
CNO dihydrochloride, CNO.2HCl
We have been using Clozapine N-oxide (CNO) dihydrochloride (CNO.2HCl) from Hello Bio. We are fully satisfied with this product: it is readily water soluble and stable. We have administered CNO-2HCl to animals via IP injection and intracranial microinjection; and we have observed CNO-induced cellular and behavioral effects fully consistent with its expected activation of DREADD receptors." Verified customer, UTHSC
Great product. Great company and product. Better pricing & similar quality compared to the most of the other companies. We used CNO dihydrochloride for both i.p. and drinking water delivery, and it worked fine. Nice service. Verified customer, University of Minnesota
Works as expected, and significantly cheaper than alternatives. Will use this for future CNO purchases Verified customer, UCSF
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Other Non-CNO DREADD activators available:
- Water soluble Compound 21 (DREADD agonist 21) hydrochloride has minimal off-target activity and is indicated not to metabolize to clozapine. The freebase Compound 21 (DREADD agonist 21) is also available.
- Perlapine hydrochloride (water soluble) is a potent and selective hM3Dq DREADD agonist. Perlapine freebase is also available.
- Salvinorin B (SALB) Salvinorin B (SALB) potently activates the inhibitory KORD DREADD to induce neuronal inhibition.
Storing and Using Your Product
Soluble in water (100 mM). Always store solutions at -20°C.
Storage of solid
- Store at -20°C.
- Please note that the compound is a hydroscopic solid and contact with air may cause material to become sticky. Product performance should not be affected but we recommend storing the material in a sealed jar.
Storage of solutions
- Make up solutions and use immediately.
- If storage of solutions is required, you should aliquot out the solution into tightly sealed vials and store at -20°C and store these for up to one month.
- Allow the product to equilibrate to RT for at least one hour before opening and using.
Storage of solutions at room temperature
- We recommend only keeping solutions at room temperature (25°C) for a few days as our studies have shown that after 96 hours the purity of the compound in solution drops to ~95% and will continue to drop over time.
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) dihydrochloride (water soluble)
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
Differential regulation of rat 5-HT2A and 5-HT2C receptors after chronic treatment with clozapine, chlorpromazine and three putative atypical antipsychotic drugs.Kuoppamã?ki M et al (1995) Neuropsychopharmacology 13(2) : 139-50.PubMedID: 8597525
Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.Gomez et al (2017) Science 357(6350) : 503-507PubMedID: 28774929
CNO Evil? Considerations for the Use of DREADDs in Behavioral Neuroscience.Mahler and Aston-Jones (2018) Neuropsychopharmacology doi: 10.1038 : /npp.2016.251PubMedID: 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
cAMP-producing chemogenetic activation of indirect pathway striatal projection neurons and the downstream effects on the globus pallidus and subthalamic nucleus in freely moving mice.Bouabid and Zhou (2018) J Neurochem doi: 10.1111/jnc.14331. : [Epub ahead of print]PubMedID: 29500819
cAMP-producing chemogenetic and adenosine A2a receptor activation inhibits the inwardly rectifying potassium current in striatal projection neurons.Wang and Zhou(2019) Neuropharmacology : pii: S0028-3908(18)30555-0.PubMedID: 30659840
Midbrain Contributions to Innate Defense and Anxiety BehaviorsAguilar et al (2019) Georgetown University : Thesis
Chemogenetic activation of orexin/hypocretin neurons ameliorates aging induced changes in behavior and energy expenditure.Stanojlovic et al (2019) Am J Physiol Regul Integr Comp Physiol. : . doi: 10.1152/ajpregu.00383.201PubMedID: 30726119
Chemogenetic activation of oxytocin neurons: temporal dynamics, hormonal release, and behavioral consequencesGrund et al (2019) Psychoneuroendocrinology : https://doi.org/10.1016/j.psyneuPubMedID: 30954921
Chemogenetic stimulation of the infralimbic cortex reverses alcohol-induced fear memory overgeneralization.Scarlata et al (2019) Sci Rep. 9(1) : 6730PubMedID: 31040357
Chemogenetic stimulation of the infralimbic cortex reverses alcohol-induced fear memory overgeneralizationScarlata MJ et al(2019) Sci Rep 9(1) : 6730PubMedID: 31040357
Regulation of perineuronal nets in the adult cortex by the electrical activity of parvalbumin interneuronsDevienne et al (2019) bioRxiv : 10.1101/671719
Central Amygdala Prepronociceptin-Expressing Neurons Mediate Palatable Food Consumption and RewardHardaway JA et al(2019) Neuron 102(5) : 1037-1052.e7PubMedID: 31029403
Activating newborn neurons suppresses depression and anxiety-like behaviorsTunc-Ozcan E et al(2019) Nat Commun 10(1) : 3768PubMedID: 31434877