I am working on a drug called Temozolomide in my cell culture experiments. I am having trouble in preparing working concentrations of this drug from 200 to 2000 uM as the drug is soluble in DMSO only and around 0.33 mg/ml in water. The main stock I prepare is around 5 mg/ml (2.5 X 10^4 uM) which when made to a 3000 to 5000 uMI of intermediate stock and working concentrations lower than that; the DMSO percentage always stays higher than 0.22% which is not permissible in cell culture experiments. And if we prepare a stock of 5mg/ml by dissolving the drug in around 150 uL of DMSO and then adjust the volume to 1 ml by 1X PBS; the drug precipitates. Kindly suggest a way prepare the working concentrations.
Have you tried to dilute the temozolomide DMSO solution directly in cell culture medium? For example, you could dilute 500x your DMSO solution of 1000 mM (if it is possible to make it) with a cell culture medium and then you will have 0.2% of DMSO in it.
Anirudha,
It seems that your desired final concentrations are very near (or slightly above) the water solubility limit of this compound. PubChem https://pubchem.ncbi.nlm.nih.gov/compound/Temozolomide
shows the drug MW as 194 g/mol and water solubility as 5 g/L. This latter figure is higher than the maximum solubility that you indicate of 0.33 mg/ml in water or 0.33 g/L. If we assume that your figure of 0.33 g/L is correct for your particular compound, here are the calculations:
Maximum solubility in water:
0.33 g/L divided by 194 g/mol = 1.7 mM
Maximum final DMSO concentration allowed:
0.22% = 0.22 ml/ 100 ml = 2.2 ul/ml
To make 1.7 mM final concentration of your drug (i.e. a saturated soln) in water or buffer, you will need 0.33 g/L = 330 mg/L = 33 mg/100 ml
To achieve this, you must dissolve 33 mg of drug in 0.22 ml of DMSO. This should be possible. This must then be added dropwise, with stirring if possible, to 100 ml of buffer or culture media.
Since this approaches the saturation limit so closely, any minor miscalculation or effects of temperature, salt concentration, or protein in media could easily result in precipitation. Thus, it might be necessary to use a maximum concentration
It seems that 33 mg of Temozolomide in 0.22 ml of DMSO may also be problematic in terms of solubility. Drug solubility in DMSO is 10 mg/ ML according to Sigma and greater than 20 mg/ML according to others on this board. If this is the case, then further downward adjustments in concentration and doses must be made in order to dissolve the drug even in DMSO. Anhydrous ethanol is another possible vehicle, but I don’t see any data regarding solubility in absolute ethanol.
This is a difficult problem because the study requires drug concentrations so close to the solubility limit.
RW
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs