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

Introduction

Background

Working with CNO (freebase) as a solid

• What should CNO (freebase) 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?

Solubilizing / dissolving CNO (freebase)

• How do I solubilize CNO (freebase) 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 (freebase)?

Storing CNO (freebase)

• Can I store my stock solution of Clozapine N- oxide (CNO (freebase))?
• How long does Clozapine N-oxide (CNO freebase) stay stable in solution?
• Is my stock solution light sensitive?
• Is my stock solution air sensitive?

Using CNO (freebase) in the laboratory

• Which DREADDs does CNO activate?
• How do I express DREADDs?
• How can I administer CNO?
• Which buffers / solvents are used to administer CNO?
• What doses of CNO are used?
• Can CNO be given in drinking water or chow?
• Does CNO taste bitter?
• Administration via minipump
• Does CNO have off-site effects?
• Are there any other DREADD ligands?
• Can DREADDs be used together?

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.

Background

Hello Bio manufactures high purity (>99%), CNO freebase 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 (freebase), 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 (freebase). The handling and storage information outlined in this review is specific to CNO (freebase) as manufactured by Hello Bio.

Working with CNO (freebase) as a solid

Solubilizing / dissolving CNO (freebase)

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

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 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 (precipitation was observed in 20% of 100 mM solutions of CNO (dissolved in water) which were stored for four days. This precipitation was readily reversed by warming in a water bath at 40° (see Table 1)).

We therefore recommend using DMSO when working with CNO freebase. If you need to work with aqueous solutions, you should use CNO dihydrochloride.

Solubilizing in DMSO

CNO (freebase) is soluble in DMSO to 100 mM.

Storage of solutions in DMSO

1. Aliquot out the solution into tightly sealed vials for storage.
2. Storage of solutions should be at room temperature.
3. Please take care to ensure that your product is completely dissolved in your solution before use.
   
   

Solubilizing CNO (freebase) in aqueous solutions (e.g. water / saline)

We do not recommend using CNO freebase with aqueous solutions however, we provide these guidelines if you do:

1. Make up solutions and use immediately. Do not store solutions.
2. Ensure you work in a dust free environment when preparing and handling solutions.
3. 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.
4. 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].

Stability 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.

Storage of aqueous solutions

We recommend that you do not store aqueous solutions (e.g. water/saline) of CNO. However, if this is necessary:

1. 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.
2. Always check that your product is completely dissolved before use; if precipitation is found follow the steps below.

Precipitation

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.

 

Table 1 - Summary of solubility studies undertaken on stock solutions of CNO (freebase) in different buffers under different storage temperatures

Buffer/solvent	Concentration (mM unless otherwise stated)	Temperature (°C unless otherwise stated)	Test duration (days)	Result
Saline	100	RT	-	Soluble
PBS	100	RT	-	Soluble
Water	100	RT	7	Soluble*
Water	1mg/ml	RT	7	Soluble
Water	20	RT	7	Soluble
Water	50	RT	7	Soluble
Water	100	RT	7	Soluble
Water	20	37	7	Soluble
Water	50	37	7	Soluble
Water	100	37	7	Soluble
Water	1mg/mL	4	7	Soluble
Water	20	4	7	Soluble
Water	50	4	7	Soluble
Water	100	4	7	Soluble*
DMSO	50	freezing overnight then rethawed, then RT	7	Soluble
DMSO	100	freezing overnight then rethawed, then RT	7	Soluble
DMSO	50	RT	7	Soluble
DMSO	100	RT	7	Soluble

* 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 i scompletely 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 (freebase) should be a clear, yellow to orange color (depending on the concentration) – see Fig 3.

Figure 3 - Hello Bio CNO (freebase) 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 (freebase) dissolved in DMSO at 50 and 100mM

Is there a recommended way to solubilize CNO (freebase)?

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 (freebase)

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

Storage of solutions in DMSO

1. Aliquot out the solution into tightly sealed vials for storage.
2. Storage of solutions should be at room temperature.
3. Please take care to ensure that your product is completely dissolved in your solution before use.

We recommend that you do not store aqueous solutions (e.g. water/saline) of CNO. However, if this is necessary:

1. 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.
2. Always check that your product is completely dissolved before use; if precipitation is found follow the steps below.

Precipitation

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.

How long does Clozapine N-oxide (CNO freebase) 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 (freebase) is chemically 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.

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 (freebase) is light sensitive.

Is my stock solution air sensitive?

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

Using CNO (freebase) 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 (CNO) (freebase). 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 chemically 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 (K_i = 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.

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

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
24	In vitro	Electrophysiology			10uM
6	In vitro	Electrophysiology			30uM	Extracellular solution
25	In vitro	Electrophysiology	10	N/A	1uM	Saline
16	In vitro				1uM
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
11	In vivo	Intracranial injection		Saline	3 μM	Saline
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