Clozapine N-Oxide (water soluble) - a technical review on stability, solubility and use in the lab
- What should CNO (water soluble) look like?
- What temperature should I store the solid compound at?
- Is the solid powder light sensitive?
- Is the solid powder air sensitive?
- How do I weigh the compound to make my stock solution?
- How do I solubilize CNO (water soluble) to make my stock solution? Which solvent / buffer can I use?
- What should I do if I find precipitate in my stock solution?
- What concentration should my stock solution be?
- What should my stock solution look like?
- Is there a recommended way to solubilize CNO (water soluble)?
- Can I store my stock solution of Clozapine N- oxide (CNO (water soluble))?
- How long does Clozapine N-oxide (CNO water soluble) stay stable in solution?
- Is my stock solution light sensitive?
- Is my stock solution air sensitive?
- Which DREADDs does CNO activate?
- How do I express DREADDs?
- How can I administer CNO?
- Which buffers / solvents can I use to administer CNO?
- What dose of CNO should I give?
- Can CNO be given in drinking water or chow?
- Does CNO taste bitter?
- Can CNO be administered via minipump?
- How long does it take for CNO to become active?
- Does CNO have off-site effects?
- Are there any other DREADD ligands?
- Can DREADDs be used together?
Figure 1 – Chemical structure
of Clozapine N-oxide (CNO). Clozapine N-oxide is an
oxidized piperazine substituted benzodiazepine and a
metabolite of the antipsychotic compound Clozapine
DREADDs are an increasingly popular chemogenetic tool which allows the control of activity of a wide range of cell types (Find out more in our mini-review).
DREADDs are engineered to be activated by synthetic small molecules (designer drugs) such as Clozapine N-Oxide (CNO) which is an oxidized piperazine substituted benzodiazepine and a metabolite of the antipsychotic compound Clozapine. CNO is the most widely used compound to activate DREADDs such as the excitatory hM3Gq DREADD and the inhibitory hM4Gi DREADD.
This technical review aims to answer common questions on how to handle, store and use Clozapine N-Oxide (water soluble) for optimal results, and includes data from stability and solubility studies, using CNO (water soluble) manufactured by Hello Bio.
Hello Bio manufactures high purity (>99%), water soluble CNO and makes it available to researchers at prices around 50% less than other life science suppliers that offer this product.
We have found that the appearance and solubility of CNO from other suppliers may differ due to differing manufacturing procedures. CNO from other suppliers may also contain alcohol impurities such as ethanol/methanol.
A review of the available literature indicates that CNO should be handled, used and stored carefully. Following our manufacture of CNO (water soluble), we have undertaken extensive chemical, stability and solubility studies of this compound to provide a detailed methodology and handling protocol, and to answer questions that researchers may have.
Please note that throughout this review we have used the nomenclature CNO (water soluble) when referring to the CNO manufactured by Hello Bio, to distinguish it from CNO available from other suppliers, which may or may not be water soluble. The handling and storage information outlined in this review is specific to CNO (water-soluble) as manufactured by Hello Bio.
Figure 2 – Picture of solid CNO (water soluble)
CNO (water soluble) is an amorphous, yellow powder (see Fig 2).
We undertake regular quality control analysis of the solid compound and have found no deterioration in product purity when stored at room temperature.
We therefore recommend that you store solid CNO (water soluble) powder from Hello Bio at room temperature (desiccate).
No - we have found no evidence for Hello Bio CNO (water soluble) to be sensitive to light.
No - we have found no evidence for Hello Bio CNO (water soluble) to be sensitive to air.
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.
We have undertaken a detailed solubility study in which Hello Bio CNO (water soluble) was dissolved in either water, DMSO, PBS or saline, at different concentrations, temperatures and for varying durations of time.
The results show that CNO (water souble) was soluble at all the concentrations, buffers and temperatures tested (see Table 1).
CNO (water soluble) is soluble to 100 mM in aqueous media including water, saline, PBS and is also soluble in DMSO (100 mM). However, when 100 mM solutions of CNO (dissolved in water) were stored at 4°C for four days, precipitation was observed in 20% of the samples. This precipitation was readily reversed by warming in a water bath at 40 °C (see Table 1).
Therefore we recommend:
- Make up solutions and use on the same day if possible.
- Ensure that you are working 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.
In line with these findings, different groups also recommend that when possible CNO solutions should be used on the same day and Wirtshaftern and Stratford (2015) mention that “The CNO was prepared immediately before use as it tended to precipitate if stored” .
|Buffer/solvent||Concentration (mM unless|
|Temperature (°C unless|
|Test duration (days)||Result|
|Water||100||4||4||Soluble* (see below)|
|DMSO||50||freezing overnight then|
rethawed, then RT
|DMSO||100||freezing overnight then|
rethawed, then RT
* After 4 days at 4°C, precipitation was observed in 20% of CNO samples dissolved in water (100 mM). Precipitation was readily reversed by warming in a water bath at 40°C.
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.
This depends on your experiment and the working concentration you require.
Please see Table 3 for same examples of different concentrations used in various papers.
When dissolved in water, saline or PBS your solution of CNO (water soluble) should be a clear, yellow to orange color (depending on the concentration) – see Fig 3.
Figure 3 - Hello Bio CNO (water soluble) dissolved in water at 20, 50 and 100mM
Similarly, when dissolved in DMSO, your solution should also be a clear, yellow to orange color (depending on the concentration) – see Fig 4.
Figure 4 - Hello Bio CNO (water soluble) dissolved in DMSO at 50 and 100mM
Whilst Hello Bio CNO (water soluble) is soluble in water, saline, PBS and DMSO, various groups have different approaches to solubilizing CNO. For example, DMSO is commonly used to dissolve CNO before addition to saline for injection.
Smith et al (2016) describe some examples of different group’s approaches on page 13 .
The approach you choose depends on your experimental requirements.
As already indicated, we recommend preparing and using your solutions on the same day. However, if this isn’t possible and you need to prepare stock solutions beforehand, you should aliquot out the solution into tightly sealed vials for storage. Storage of solutions should be at room temperature.
We generally recommend that these will be useable for up to one month. Always check that your product is completely dissolved before use .
Stability studies were performed on 100 mM solutions of Clozapine N-oxide dissolved in water, which were kept at room temperature and ambient lighting for 4 weeks. No special precautions to exclude air were taken.
HPLC analysis of samples at day 1, week 1, week 2 and week 4 showed no deterioration in product purity (>99.60%) (see Fig 5 / Table 2).
Our studies therefore show that CNO (water soluble) is stable in solution at room temperature for at least 4 weeks.
Figure 5 - HPLC analysis of 100 mM CNO solutions dissolved in water after 4 weeks of storage at RT and at ambient lighting. A purity of >99.6% was confirmed after 1 day, 1 week, 2 weeks and 4 weeks of storage, confirming that no deterioration in product purity occurred over 4 weeks.
Purity was determined by HPLC analysis using reversed phase chromatography on a C18 column, using a TFA-acetonitrile/TFA-water gradient and detecting at 240 nm.
Table 2 - Purity of CNO solutions dissolved in water (100 mM) after
No - we have found no evidence that Hello Bio CNO (water soluble) is light sensitive.
No - we have found no evidence that Hello Bio CNO (water soluble) is air sensitive.
Following literature analysis here is some general information for some frequently asked questions relating to the usage of Clozapine N-oxide (water soluble). Do you have any questions that aren’t listed below? If so, contact us on email@example.com and we’ll try our very best to help!
CNO activates various DREADDs including the excitatory Gi-DREADDs (hM3Dq, hM1Dq and hM5Dq), the inhibitory Gq- DREADDs (hM4Di, hM2Di), the Gs -DREADD (GsD) and the β-arrestin preferring DREADD rM3Darr(Rq(R165L)).
DREAADs can be selectively expressed in a targeted cell population by using viral vectors or transgenic mice generated by random or site-specific targeting .
Clozapine N-oxide has high blood brain barrier penetration and is orally bioavailable which allows minimally invasive delivery.
CNO is commonly administered via injection (e.g. intraperitoneal injection [4, 5, 6,7], subcutaneous , or directly infused intracranially [9, 10, 11]). It can also be administered via drinking water , chow  and recently osmotic pump .
Please see Table 3 for same examples of different administration routes used in various papers.
Please consult the literature to choose the most appropriate administration buffer for your experiment.
Sterile saline is most commonly used for i.p. injection. Water, PBS and ACSF have also been used.
CNO has also been added to recording buffer in patch clamp electrophysiology experiments.
Please see Table 3 for same examples of buffers / solvents used in various papers.
The dose of CNO administered in the literature varies and a wide range of doses have been used e.g. 0.2 – 10 mg/kg . We recommend consulting the literature to ascertain the appropriate dose for your experiment.
Maclaren et al (2016) recommend that “using the lowest effectual dose in the assay to be performed, that which in the non-DREADD-expressing animals is experimentally silent, would seem the most straightforward way to minimize any off-target effects of CNO .
Please see Table 3 for same example doses used in various papers.
Although it is difficult to control the amount of CNO ingested and is therefore less precise, CNO can be delivered in a less invasive manner by food or water . This can be useful for long-term studies and if you want to avoid repeated handling and/or injection of animals .
Please see Table 3 for same example papers which given CNO via drinking water [15,16,13].
According to Bryan Roth’s blog a “A small amount of saccharine in the drinking water will mask the slightly bitter taste of CNO”. Milosavljevic et al (2016) mention that their animals were “presented with 0.25 mg/ml CNO in sweetened drinking water (0.2% saccharine and 4% sucrose)” .
There are limited citations using CNO via osmotic pump however, Donato et al (2017) used an osmotic minipump implanted subcutaneously on mice pup's backs to deliver CNO (100 µl of CNO solution (1mg/ml) with a flow of 0.5 µl per hour for up to 7 days .
A thesis by Mason, X et al (2015) also used an osmotic pump to deliver CNO (diluted in 0.9% sterile saline to a concentration of 5mg/mL). ~240 µL of CNO solution was loaded into a subcutaneously implanted Alzet® 2004 pump .
A common question is how long does CNO stay stable in solution? As mentioned above, our stability studies showed that CNO was stable in solution at room temperature for at least 4 weeks. HPLC analysis during this time showed no deterioration in product purity.
DREADD activation with CNO occurs after 30 min and lasts for about 2 hours . Although plasma levels of CNO rapidly decline, behavioural effects may still be evident for up to 6h .
Clozapine N-oxide lacks appreciable affinity (Ki = 1 µM) at relevant native CNS targets and is not subject to significant metabolic transformation in mice . However, CNO is known to back-metabolize to clozapine in humans and guinea pigs .
Recent studies have demonstrated that CNO may exert behavioural effects on rats that do not express DREADDs  and Salomon et al (2016) have also demonstrated biological activity of DREADDs in the absence of CNO .
Care must therefore be taken in experimental design and proper controls should be incorporated.
Maclaren et al (2016) recommend that all experiments which utilize CNO should verify that the administration of CNO does not have effects in the species and strain being used. Importantly, they emphasize the need for a CNO-only DREADD-free control group when designing DREADD-based experiments .
Yes – the clozapine analog Compound 21 (DREADD agonist 21) acts as a highly potent hM3Dq agonist and does not convert to Clozapine as it is not metabolized by the same pathways as CNO . It has been indicated that Compound 21 (DREADD agonist 21) has equivalent potency in vivo when compared with clozapine N-oxide. Compound 21 therefore represents an alternative to CNO.
Perlapine is a novel, potent and selective hM3Dq DREADD receptor agonist and is better suited to translational studies .
Additionally, the inert ligand Salvinorin B (SALB) acts at the inhibitory KORD DREADD .
Figure 6 – Different designer compounds (CNO, perlapine, Compound 21 and SALB) are used to activate different DREADDs.
Yes - due to the existence of multiple DREADDs which can be activated by different ligands, DREADDs can be used in a ‘multiplexed’ approach in which DREADDs which respond to CNO (e.g. hM3Dq / hM4Di) and the SALB responsive KORD DREADD can be expressed in the same animal to enable bidirectional control of neuronal activity , 22, 3, 23].
|Reference||In vitro or in vivo usage?||Administration method / usage||Dosage (mg/kg)||Administration vehicle||Final concentration||Solvent used for stock solution||Final DMSO concentration||Stock concentration (mg/ml)|
|21||In vitro||Electrophysiology||Recording buffer||10uM||DMSO||100mM|
|19||In vitro||Electrophysiology||Saline||500nM - 1uM|
|6||In vitro||Electrophysiology||30uM||Extracellular solution|
|15||In vivo||Drinking water||5-6.25||Water (0.2% saccharine and 4% surcose added)|
|16||In vivo||Drinking water||0.25mg/ml||Water|
|13||In vivo||Drinking water||0.25mg/ml||Water|
|24||In vivo||Drinking water||5||Water||40mg/l||Water|
|9||In vivo||Intracranial injection||0.3||Saline||0.3mg/ml||DMSO||0.50%|
|1||In vivo||Intracranial injection||2.5||Water||2.5 & 10mg/ml||DMSO||20%|
|10||In vivo||Intracranial injection||ACSF||100uM & 1mM|
|25||In vivo||Intraperitoneal (i.p.)||10||Saline||1mg/ml||Saline|
|5||In vivo||Intraperitoneal (i.p.)||1||Saline||1, 2 and 5 mg/ml||DMSO||0.50%|
|15||In vivo||Intraperitoneal (i.p.)||5|
|16||In vivo||Intraperitoneal (i.p.)|
|13||In vivo||Intraperitoneal (i.p.)||2||PBS||0.2mg/ml||PBS|
|9||In vivo||Intraperitoneal (i.p.)||3||Saline||DMSO||0.50%|
|21||In vivo||Intraperitoneal (i.p.)||3||Saline||DMSO||100 mM|
|18||In vivo||Intraperitoneal (i.p.)||1|
|26||In vivo||Intraperitoneal (i.p.)||1||Saline||Saline|
|23||In vivo||Intraperitoneal (i.p.)||3||Saline||Saline|
|19||In vivo||Intraperitoneal (i.p.)||0.1, 0.3, 0.5, 1 & 5||Saline|
|24||In vivo||Intraperitoneal (i.p.)||2 & 5|
|6||In vivo||Intraperitoneal (i.p)||1|
|10||In vivo||Intraperitoneal (i.p.)||0.1, 1, 10 & 20||Saline||DMSO||5%||1ml/kg|
|27||In vivo||Intraperitoneal (i.p.)||1|
|28||In vivo||Intraperitoneal (i.p.)||0.1, 1 and 10||Saline||5uM||2.50%||10mg/ml|
|17||In vivo||Osmotic minipump|
|14||In vivo||Osmotic minipump||Saline||5mg/ml|
|8||In vivo||Subcutaneous (s.c.)||3 & 10||PBS||10mg/ml||DMSO||15%||66mg/ml|
|29||In vivo||Subcutaneous (s.c.)||1||Saline|
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