Clozapine N-Oxide (water soluble) - a technical review on stability, solubility and use in the lab

Helpful tips for using Clozapine N-oxide (water soluble)Introduction

Background

Working with CNO (water soluble) as a solid

Solubilizing / dissolving CNO (water soluble)

Storing CNO (water soluble)

Using CNO (water soluble) in the laboratory

Clozapine N-Oxide structure

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

Introduction

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 hM3Dq DREADD and the inhibitory hM4Di 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.

Background

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.

Working with CNO (water soluble) as a solid

Picture of solid CNO (water soluble)

Figure 2 – Picture of solid CNO (water soluble)

What should CNO (water soluble) look like?

CNO (water soluble) is an amorphous, yellow powder (see Fig 2).

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 therefore recommend that you store solid CNO (water soluble) powder from Hello Bio at room temperature (desiccate).

Is the solid powder light sensitive?

No - we have found no evidence that Hello Bio CNO (water soluble) is sensitive to light.

Is the solid powder air sensitive?

No - we have found no evidence that Hello Bio CNO (water soluble) is 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.

Solubilizing / dissolving CNO (water soluble)

How do I solubilize CNO (water soluble) to make my stock solution? Which solvent / buffer can I use?

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 soluble) 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 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; Wirtshaftern and Stratford (2015) state that “The CNO was prepared immediately before use as it tended to precipitate if stored” [1].

Table 1 - Summary of solubility studies undertaken on stock solutions of CNO (water soluble) in different buffers under different storage temperatures
Buffer/solventConcentration (mM unless
otherwise stated)
Temperature (°C unless
otherwise stated)
Test duration (days)Result
Saline100RT-Soluble
PBS100RT-Soluble
Water100RT7Soluble*
Water1mg/mlRT7Soluble
Water20RT7Soluble
Water50RT7Soluble
Water100RT7Soluble
Water20377Soluble
Water50377Soluble
Water100377Soluble
Water1mg/mL47Soluble
Water2047Soluble
Water5047Soluble
Water10047Soluble* 
DMSO50freezing overnight then
rethawed, then RT
7Soluble
DMSO100freezing overnight then
rethawed, then RT
7Soluble
DMSO50RT7Soluble
DMSO100RT7Soluble

* After 4 days, 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.

What should I do if I find precipitate in my stock 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.

What concentration should my stock solution be?

This depends on your experiment and the working concentration you require.

Please see Table 3 for same examples of different concentrations reported in various papers.

What should my stock solution look like?

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.

solution of CNO in water at various concentrations

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.

50mM solution of CNO in DMSO

Figure 4 - Hello Bio CNO (water soluble) dissolved in DMSO at 50 and 100mM

Is there a recommended way to solubilize CNO (water soluble)?

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 [2].

The approach you choose depends on your experimental requirements.

Storing CNO (water soluble)

Can I store my stock solution of Clozapine N- oxide (CNO (water soluble))?

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.

How long does Clozapine N-oxide (CNO water soluble) stay stable in solution?

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.

HPLC traces from stability study

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.

Time (weeks)Purity
0>99.6%
1>99.6%
2>99.6%
4>99.6%

Table 2 - Purity of CNO solutions dissolved in water (100 mM) after
1 day, 1 week, 2 weeks and 4 weeks of storage at RT

Is my stock solution light sensitive?

No - we have found no evidence that Hello Bio CNO (water soluble) is light sensitive.

Is my stock solution air sensitive?

No - we have found no evidence that Hello Bio CNO (water soluble) is air sensitive.

Using CNO (water soluble) in the laboratory

Based on a review of the literature, the following is a summary of useful information 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 technicalhelp@hellobio.com and we’ll try our very best to help!

Which DREADDs does CNO activate?

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)).

How do I express DREADDs?

DREAADs can be selectively expressed in a targeted cell population by using viral vectors or transgenic mice generated by random or site-specific targeting [3].

How can I administer CNO?

There is some debate in the literature regarding the brain penetrant properties of CNO – some groups report high BBB penetration [4], whilst a recent study suggests that CNO systemically administered does not readily cross the blood-brain-barrier in vivo, and converts to clozapine which activates DREADDs [5]. Care must be taken in experimental design and proper controls should be incorporated

CNO has been commonly administered via injection (e.g. intraperitoneal injection [6,7,8,9], subcutaneous [10], or directly infused intracranially [12,13]). It has been administered via drinking water [14], chow [15] and recently osmotic pump [16].

Please see Table 3 for some examples of different administration routes used in various papers.

Which buffers / solvents are used to administer CNO?

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.

What doses of CNO are used?

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 [7]. We recommend consulting the literature to ascertain the appropriate dose for your experiment.

Please see Table 3 for same example doses used in various papers.

Can CNO be given in drinking water or chow?

CNO has been delivered by food or water [3]. This can be useful for long-term studies and if you want to avoid repeated handling and/or injection of animals [14].

Please see Table 3 for same example papers which given CNO via drinking water [17,18,15].

Does CNO taste bitter?

According to Bryan Roth’s blog a “A small amount of saccharine in the drinking water will mask the slightly bitter taste of CNO” [33].  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)” [17].

Administration via minipump

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 [16].

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). Approx 240 µL of CNO solution was loaded into a subcutaneously implanted Alzet® 2004 pump [19].

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.

Does CNO have off-site effects?

Clozapine N-oxide lacks appreciable affinity (Ki = 1 µM) at relevant native CNS targets [2]. However, CNO is known to back-metabolize to clozapine in humans, guinea pigs [2], primates and mice [5].

Recent studies have demonstrated that CNO may exert behavioural effects on rats that do not express DREADDs [7] and Salomon et al (2016) have also demonstrated biological activity of DREADDs in the absence of CNO [23].

Gomez et al (2017) have reported that CNO has a low affinity for DREADDs in comparison with clozapine, and that systemically administered CNO does not readily cross the blood-brain-barrier in vivo, and converts to clozapine which activates DREADDs [5].

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 [7].

Care must therefore be taken in experimental design and proper controls should be incorporated.

Are there any other DREADD ligands?

Yes – the clozapine analog Compound 21 (which is also available in a water soluble form) acts as a highly potent hM3Dq agonist. There are indications that Compound 21 may not be metabolized to clozapine [22] and may have equivalent potency in vivo when compared with CNO.

Perlapine is a novel, potent and selective hM3Dq DREADD receptor agonist and is better suited to translational studies [22].

Additionally, the inert ligand Salvinorin B (SALB) acts at the inhibitory KORD DREADD [3].

DREADD ligands
Figure 6 – Different designer compounds (CNO, perlapine, Compound 21 and SALB) are used to activate different DREADDs.

Can DREADDs be used together?

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 [27, 24, 3, 25].

Table 3 – An overview table providing example usages, administration methods & vehicles, dosages, concentrations, stock solvents and concentrations of CNO used in different papers and experiments
ReferenceIn vitro or in vivo usage?Administration method / usageDosage (mg/kg)Administration vehicleFinal concentrationSolvent used for stock solutionFinal DMSO concentrationStock concentration (mg/ml)
21In vitroElectrophysiologyRecording buffer10uMDMSO100mM
19In vitroElectrophysiologySaline500nM - 1uM
24In vitroElectrophysiology10uM
6In vitroElectrophysiology30uMExtracellular solution
25In vitroElectrophysiology10N/A1uMSaline
16In vitro1uM
15In vivoDrinking water5-6.25Water (0.2% saccharine and 4% surcose added)
16In vivoDrinking water0.25mg/mlWater
13In vivoDrinking water0.25mg/mlWater
24In vivoDrinking water5Water40mg/lWater
11In vivoIntracranial injectionSaline3 μMSaline
1In vivoIntracranial injection2.5Water2.5 & 10mg/mlDMSO20%
10In vivoIntracranial injectionACSF100uM & 1mM
25In vivoIntraperitoneal (i.p.)10Saline1mg/mlSaline
5In vivoIntraperitoneal (i.p.)1Saline1, 2 and 5 mg/mlDMSO0.50%
15In vivoIntraperitoneal (i.p.)5
16In vivoIntraperitoneal (i.p.)
13In vivoIntraperitoneal (i.p.)2PBS0.2mg/mlPBS
9In vivoIntraperitoneal (i.p.)3SalineDMSO0.50%
21In vivoIntraperitoneal (i.p.)3SalineDMSO100 mM
18In vivoIntraperitoneal (i.p.)1
26In vivoIntraperitoneal (i.p.)1SalineSaline
23In vivoIntraperitoneal (i.p.)3SalineSaline
19In vivoIntraperitoneal (i.p.)0.1, 0.3, 0.5, 1 & 5Saline
24In vivoIntraperitoneal (i.p.)2 & 5
6In vivoIntraperitoneal (i.p)1
10In vivoIntraperitoneal (i.p.)0.1, 1, 10 & 20SalineDMSO5%1ml/kg
27In vivoIntraperitoneal (i.p.)1
28In vivoIntraperitoneal (i.p.)0.1, 1 and 10Saline5uM2.50%10mg/ml
17In vivoOsmotic minipump
14In vivoOsmotic minipumpSaline5mg/ml
8In vivoSubcutaneous (s.c.)3 & 10PBS10mg/mlDMSO15%66mg/ml
29In vivoSubcutaneous (s.c.)1Saline

References

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