Certain classes of drug molecules are potentially labile to the plasma enzymes. For instance, structures containing ester or amide-linked groups are prone to hydrolysis by esterase, amidase or protease enzymes. On the other hand, some prodrugs become biologically active as a result of the same enzymatic reactions. In either case, plasma enzymes can significantly alter the bioavailability of the active compounds, and this should be evaluated at early stages of drug discovery process. Determining plasma stability of the tested compounds is typically performed using HPLC-MS.
The examples of plasma stability data for Verapamil (good plasma stability) and Propantheline (poor plasma stability) are shown on graphs below.
Incubations are carried out in 96-well polypropylene plates in 5 aliquots of 70 μL each (one for each time point). Test compounds (10 μM, final solvent concentration 1 %) are incubated at 37°C. Five time points over 120 minutes are analyzed (0,20,40,60 and 120 min). All incubations are performed in duplicates. The samples are analyzed by HPLC-MS (API3000, AB Sciex). The percentage of parent compound remaining after incubation in plasma is plotted versus incubation time, and plasma half-life (T½) is calculated from the obtained curve.
The percentage of parent compound remaining after incubation in plasma is plotted versus incubation time, and plasma half-life (T½) is calculated from the obtained curve. Full study report is provided.
2 µmol of a dry compound or 200 µL of 10 mM DMSO stock solutions can be used.