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

Abstract

Various incubation conditions of human hepatocytes were compared for their accuracy in predicting the in vivo hepatic clearance (CL_H) of model compounds. The test compounds were the highly cleared, low protein bound naloxone (in vivo CL_H = 25 ml min⁻¹ kg⁻¹; free fraction = 0.6), the medium clearance, highly protein bound midazolam (CL_H = 12 ml min⁻¹ kg⁻¹; free fraction = 0.04) and the low clearance, highly protein bound bosentan (CL_H = 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 CL_H for bosentan from conventional primary cultures, and despite variable cryopreservation recoveries, similar predictions of CL_H were recorded with all hepatocyte systems. Moreover, the CL_H values obtained with cryopreserved suspended hepatocytes were similar to those obtained with freshly isolated suspensions. For midazolam and bosentan, the predicted in vivo CL_H was markedly higher in the presence of serum, whereas serum had little influence on the scaled-up CL_H of naloxone. In vivo, CL_H 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.

Key words::

• Human hepatocytes
• in vitro models
• clearance prediction
• cryopreservation
• Viaspan
• serum
• bosentan
• midazolam
• naloxone