Inactivation of CYP2C8 by gemfibrozil 1-O-B-glucuronide accumulation in human hepatocytes: Implications for clinical DDI predictions

CYP2C8 inactivation by gemfibrozil glucuronide has been associated with clinical drug-drug interactions (DDIs) observed between gemfibrozil and cerivastatin. Since hepatocytes can be used to evaluate oxidative and conjugative metabolism, we investigated the possibility that hepatocytes, but not microsomes, could detect inactivation of CYP2C8 when incubated with gemfibrozil using amodiaquine as a specific CYP2C8 substrate. Using hepatocytes, the K_I and the k_inact values determined for inactivation of CYP3A4 by gemfibrozil were 4.12 µM and 0.04 min^-1, respectively. Unexpectedly, the K_I value was 5-fold lower than what has previously been reported for the inactivation of CYP2C8 by gemfibrozil glucuronide in human liver microsomes. We hypothesized that in hepatocytes, the rate of gemfibrozil formation would exceed its rate of efflux leading to the intracellular accumulation of gemfibrozil glucuronide and that the lower K_I value would reflect the higher concentration of gemfibrozil glucuronide available to the enzyme. To investigate this hypothesis, the intracellular concentration of gemfibrozil glucuronide in suspended human hepatocytes was estimated using LC/MS/MS and microscopically determining the average hepatocyte volume.  Surprisingly, the intracellular concentration of gemfibrozil glucuronide was 191-fold higher than the extracellular concentration. However, the fraction of CYP2C8 metabolizing cerivastatin (0.61) was not high enough to allow the resulting inactivation parameters and accumulation to improve the DDI prediction observed between gemfibrozil and cerivastatin Consequently,these results suggest the involvement of another mechanism contributing to the observed DDIs for gemfibrozil and cerivastatin.