Shaking may have very minor contribution in diffusing the drug through the membrane. Rather it depend on ionizability and lipophilicity of drug candidate.

Dear Dr. Lohidakshan,

Thank you for taking time out to open a discussion on this topic. I thought the same that shaking might not affect the ability of microsome to metabolize drug substrates. Here as I am performing in vitro metabolism using human liver micrsome where the CYP enzymes are acting on the drugs to metabolize them, I am not sure if membrane permeability would be a limiting factor as there are no cells present.

Thank you so much for your kind advises.

P450 are slow enzymes and diffusion is unlikely to be limiting. Moderate shaking is preferred to assure everything is mixed and reduce variability. Try pipetting a DMSO stock into buffer and you will see it sink to the bottom. Other solvents may reduce but not eliminate this problem. For short incubations I would expect similar values if you mix the sample well by pipetting up and down a few times versus modest orbital shaking. Intense agitation is likely to denature protein and have a negative impact on the incubation.

Dear. Dr. Cameron,

Thank you so much for taking time out to look at my question and provide your valuable advise. I have been performing overnight incubation with microsome in 384 well plates without any shaking. Drugs are dissolved in 20% Acetonitrile+ 80% water, and before incubation i am spinning the plates for short time and the drug concentration are 0.2 mM. We have been checking the metabolic efficiency by LC-MS with 6 control CYP substrates by checking the percent of parent drug metabolized and considering how fast or slow metabolizers the specific CYP enzymes are for the standard drugs, we are getting about 50-100% parent disappearance.

Thank you.