Optimization of Methodology for Use of Rat Hepatocytes to Predict Drug Metabolism in vivo
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Rat hepatocytes have been used for several years at Pharmacia & Upjohn to support the screening of promising drug compounds. Previously, little work has been done to investigate the variables inherent to this methodology. In this project, we examined factors such as the method of cell isolation. In particular, the flowrates of the perfusion and digestion steps of the two-step perfusion and the presence of albumin in the incubation medium were investigated to determine if cell viability and yield could be increased. Also, an incubation methodology using a 24-well plate in an incubator designed to increase the number of drugs that could be assayed per hepatocyte preparation was also investigated. The results of this study showed that a faster perfusion rate of 31 mL/min. does a better job of perfusing the liver than the previous flowrate of 26 mL/min. and raises the viability. A slower digestion rate of 21 mL/min. as opposed to the previous rate of 26 mL/min. increased the cell yield. The presence of albumin was found to increase cell viability as well, but whether or not its presence adversely affects a drug's in vitro kinetic behavior was not definitively determined. Finally, it was found that a compound's apparent intrinsic clearance could not be reliably determined using a 24-well plate assay due to difficulties in keeping the temperature of the hepatocytes at 37°C.