Stability of Water-Soluble DREADD ligands in Solution: A Technical Review

Background

DREADDs are engineered to be activated by synthetic small molecules (designer drugs) such as Clozapine N-Oxide (CNO) which is a metabolite of the antipsychotic compound Clozapine. Until recently, CNO (freebase) was the most widely used compound to activate the excitatory hM3Dq DREADD and the inhibitory hM4Di DREADD. However, new tools are emerging that offer alternatives to CNO – and that offer advantages in terms of ease of use, solubility, stability and metabolism.

Hello Bio offers a wide and unique range of water-soluble DREADDs ligands. These include:

• Clozapine N-oxide dihydrochloride
• Clozapine dihydrochloride
• Perlapine dihydrochloride
• Compound 21 (DREADD agonist 21) dihydrochloride (water soluble)

Many of these tools are novel, and our chemists have carried out detailed stability and solubility testing to assess these products. The data can help researchers make an informed decision regarding which ligand to choose for their particular assay or experiment.

This technical review aims to answer common questions on how to handle, store and use the water-soluble DREADD ligands clozapine N-oxide dihydrochloride, clozapine dihydrochloride, perlapine dihydrochloride, and compound 21 (DREADD agonist 21) dihydrochloride (water soluble) for optimal results, and includes data from preliminary stability and solubility studies.

Please note, that the products used in this study were manufactured by Hello Bio, and our recommendations are for Hello Bio products only. Our chemists have not carried out any testing of products manufactured by other suppliers, whose properties may differ due to differences in purity, or manufacturing processes.

Storage & stability of the solid powder

The stability of the solid forms of clozapine N-oxide dihydrochloride, clozapine dihydrochloride, perlapine dihydrochloride, and compound 21 (DREADD agonist 21) dihydrochloride (water soluble) were tested using HPLC, over a period of 1 month, at room temperature and at  -20^oC.

Key findings:

• No deterioration in quality was observed for any of the products during this 1 month period.
  
  
• There were no indications that the solid powders were sensitive to light or air.
  
  
• It is important to note that all of the dihydrochloride salts are hygroscopic (that is, they have a tendency to absorb water). Contact with air may therefore may cause these products to become sticky. Whilst product performance / biological activity should not be affected, we do recommend storing these products in tightly sealed jars.
  
  

Solubility

All of the products tested (clozapine N-oxide dihydrochloride, clozapine dihydrochloride, perlapine dihydrochloride, and compound 21 (DREADD agonist 21) dihydrochloride (water soluble)) were readily soluble in water, up to a concentration of 100 mM. This means that they went into solution easily – gentle whirilimix / vortexing was used to dissolve them, and no warming was required.

No precipitation of any of these solutions were observed during the stability and solubility testing - this contrasts with CNO (freebase), where precipitation is seen under certain conditions – this may make it a more difficult product to work with in the laboratory (see Clozapine N-Oxide (freebase) - a technical review on stability, solubility and use in the lab)

Stability of solutions at room temperature

Clozapine N-oxide dihydrochloride, clozapine dihydrochloride, perlapine dihydrochloride, and compound 21 (DREADD agonist 21) dihydrochloride (water soluble) were each dissolved in water, at a concentration of 100 mM.

The solutions were kept at room temperature (25^oC) with ambient lighting, and purity was tested by HPLC (at 210 nm) at 0, 5, 24, 48, 72 and 96 hours. No special precautions were taken to exclude air. The preliminary results are shown in Figure 1.

Key findings:

• Solutions of perlapine dihydrochloride demonstrated stability over the 96 hour test period
  
  
• Solutions of the three other ligands tested all exhibited a reduction in purity over the 96 hour test period.
  
  

Stability of solutions at -20^oC

There was no change in purity of any of the compounds in solution, when stored at -20^oC after 24 hours or after 1 month.

Using solutions at room temperature – our recommendations

In light of the findings, our recommendations for use of these DREADDs ligands are as follows:

Perlapine dihydrochloride

Whilst solutions of this product are stable at room temperature, we recommend storing solutions at 4^oC if they are to be used within 96 hours. If more long-term storage of solutions is required, you should aliquot the solution into tightly sealed vials and store at -20°C for up to one month. Allow the product to equilibrate to RT for at least one hour before opening and using. 

Compound 21 (DREADD agonist 21) dihydrochloride, clozapine dihydrochloride and clozapine N-oxide dihydrochloride

• Make up solutions and use immediately if possible, and certainly within 48h. Our studies shows that after 96 hours, the purity of the compounds in solution drops to around 95-97% and will continue to drop over time.
  
  
• If storage of solutions is required, you should aliquot the solution into tightly sealed vials and store at -20°C for up to one month. Allow the product to equilibrate to RT for at least one hour before opening and using.
  
  

Summary

Whilst the novel water-soluble DREADD ligands are easier to solubilize and handle than the traditionally used clozapine N-oxide (freebase), they do vary in their stability in solution, at room temperature. However, these stability issues can be easily mitigated by taking some basic precautions when using and storing these solutions.

For further information, please contact the technical support team at Hello Bio.