Hi，

GSK805 and HY-12776 were tested and the following results were obtained:

10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline solution, clear solution, solubility 2.5 mg/ml

The answer to this question is provided by MedChemExpress Technical Support.

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To prepare a GSK805 solution in a mixed solvent system, add the solvents sequentially in the following order:

10% DMSO, 40% PEG300, 5% Tween-80, and finally 45% saline, mixing thoroughly after each addition to achieve a solubility of ≥ 2.5 mg/mL as a clear solution. For example, to make a 1 mL working solution, add 100 μL of 25.0 mg/mL GSK805 in DMSO to 400 μL of PEG300, mix, then add 50 μL of Tween-80 and mix, followed by 450 μL of saline.

Step-by-step protocol

Example for 1 mL solution

• Take 100 μL of GSK805 stock solution (25.0 mg/mL).
• Add 400 μL of PEG300 to it and mix well.
• Add 50 μL of Tween-80 and mix evenly.
• Add 450 μL of saline to make the final volume 1 mL and mix.

Tips for dissolution

• If precipitation or phase separation occurs, use sonication or gentle warming to aid dissolution.
• This protocol can yield a clear solution of GSK805 with a solubility of ≥ 2.5 mg/mL.
• For optimal animal tolerability, keep the proportion of DMSO below 2% in the working solution.

Solubility protocol for a 2.5 mg/mL solution

This is a standard protocol often used for oral administration in animal studies. The percentages represent the final volumetric ratio of the solvents in the working solution.

Individual solvent solubility

• DMSO: GSK805 is highly soluble in DMSO, with a solubility of up to 100 mg/mL.
• PEG300 and Tween-80: While individual solubility isn't typically reported, these are used as co-solvents and surfactants in the combined protocol. PEG300 helps increase the solubility of the mixture, and Tween-80 helps create a stable emulsion.

For GSK805, a formulation of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline is reported to achieve a solubility of at least 2.5 mg/mL, forming a clear solution. This vehicle has been used for in vivo studies and, while commonly considered safe, caution is still warranted for intratracheal (IT) administration.

Solubility protocol

A typical procedure to prepare a 1 mL working solution of 2.5 mg/mL would be:

Safety considerations for intratracheal administration in mice

While the 10% DMSO vehicle is often tolerated in systemic administration, the lungs are particularly sensitive, and researchers have observed signs of toxicity even at these percentages with IT delivery.

• Pulmonary irritation: Both DMSO and Tween-80 can cause irritation in the respiratory system, potentially leading to mild-to-severe toxicity. A study comparing different IT vehicles found that even low concentrations of Tween-80 caused higher eosinophil counts in bronchoalveolar lavage fluid (BALF).
• Alternative formulations: For sensitive animal models, such as immunodeficient or gene knockout mice, it may be advisable to lower the DMSO concentration to 2%, for example, using a formulation of 2% DMSO, 40% PEG300, 5% Tween-80, and 53% saline.
• Reference animal welfare: One study on IT instillation notes that vehicle-treated mice can still show a small increase in neutrophils compared to untreated mice, indicating some level of inflammation is inherent to the procedure or the vehicle itself.

Given the potential for pulmonary irritation from both DMSO and Tween-80 during intratracheal administration, it's a good practice to include a vehicle-only control group in your study. This helps ensure that any observed effects are due to the compound itself, not the vehicle.

Good Luck.

LUCK = Laboring Under Constant Knowledge.

Roger Normand Vendette Hongya Cheng

Thank you for the detailed answers!