Comparison of clearance predictions using primary cultures and suspensions of human hepatocytes

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Abstract
Various incubation conditions of human hepatocytes were compared for their accuracy in predicting the in vivo hepatic clearance (CLH) of model compounds. The test compounds were the highly cleared, low protein bound naloxone (in vivo CLH = 25 ml min−1 kg−1; free fraction = 0.6), the medium clearance, highly protein bound midazolam (CLH = 12 ml min−1 kg−1; free fraction = 0.04) and the low clearance, highly protein bound bosentan (CLH = 3.9 ml min−1 kg−1; free fraction = 0.02). Each compound was tested in three ‘hepatocyte systems’, using resections from three donors, in the presence and absence of human serum. Those hepatocyte systems were: conventional primary cultures, freshly isolated suspensions and cryopreserved suspended hepatocytes. Except for a twofold overestimated CLH for bosentan from conventional primary cultures, and despite variable cryopreservation recoveries, similar predictions of CLH were recorded with all hepatocyte systems. Moreover, the CLH values obtained with cryopreserved suspended hepatocytes were similar to those obtained with freshly isolated suspensions. For midazolam and bosentan, the predicted in vivo CLH was markedly higher in the presence of serum, whereas serum had little influence on the scaled-up CLH of naloxone. In vivo, CLH was properly approached for naloxone and bosentan (particularly from experiments in the presence of serum), but it was strongly underestimated for midazolam (particularly in the absence of serum). Additional compounds need to be investigated to confirm the above findings as well as to assess why the clearances of some highly protein-bound compounds are still considerably underestimated.