Expression and Induction Potential of Cytochromes P450 in Human Cryopreserved Hepatocytes

Abstract

ABSTRACT:Fresh human hepatocytes are still considered as the "gold standard" to screen in vitro for cytochrome P450 (P450) induction. However, sparse availability of good quality human liver tissue for research purposes and the demand for standardized cell populations, together with the need for proper storage of the cells not immediately required, have resulted in the development of cryopreservation techniques that provide adequate viability and plateability of hepatocytes after thawing. This study aimed at validating cryopreserved human hepatocytes as a model to investigate P450 induction. Cryopreserved cells from four different donors were plated and cultured for 48 h, followed by incubation in the presence of typical P450 inducers. During the experiments, quality of the cultured cells was monitored both physiologically and morphologically. Concomitantly, the activity of CYP1A2, 2B6, 2C9, 2E1, and 3A4 was measured together with their mRNA and protein expression. Determination of CYP1A2, 2B6, 2C9, 2E1, and 3A4 activity in control versus prototypical inducer-treated hepatocytes revealed a maximal significant mean 11.6-, 2.8-, 1.9-, 1.5-, and 9.0-fold induction over their basal expression, respectively. Protein expression analysis of these P450s confirmed these results. Moreover, a mean 44.9-, 3.5-, 3.2-, and 13.8-fold induction of CYP1A2, 2B6, 2C9, and 3A4 mRNA was observed. Our data demonstrate that cryopreserved human hepatocytes are a valuable tool to study the induction of CYP1A2, 2B6, 2C9, 2E1, and 3A4.

Current Industrial Practices in Assessing CYP450 Enzyme Induction: Preclinical and Clinical

“…The drug metabolizing enzymes remain inducible after cryopreservation, and due to the significant variation in activities of drug metabolizing enzymes between individual human livers, certain lots of cryopreserved cells can generate results essentially indistinguishable from freshly isolated cells (35,46). mRNA, protein expression and activities of CYP1A2, 2B6, 2C9, 2E1 and 3A4 in cryopreserved hepatocytes are inducible by prototypical inducers (47), as is the activity of various UDP-glucuronosyltransferases, carboxylesterases, and sulfotransferases (48,49). The advantage cryopreserved cells offer over fresh isolates is that experiments can be planned ahead and are not dependent on the sporadic availability of fresh primary hepatocytes.…”

Evaluation of 23 Lots of Commercially Available Cryopreserved Hepatocytes for Induction Assays of Human Cytochromes P450

“…Moreover, CYP2Cs are responsible for metabolizing approximately 20% of all prescribed drugs, including tolbutamide, phenytoin, warfarin, and ibuprofen (Goldstein, 2001). Despite the importance of CYP2Cs for drug metabolism, only a limited number of papers has described induction of human CYP2C8, CYP2C9, and CYP2C19 using fresh hepatocytes (Gerbal-Chaloin et al, 2001;Raucy et al, 2002), or induction of CYP2C9 using cryopreserved hepatocytes (Roymans et al, 2005). Moreover, the reports on induction of CYP2Cs are contradictory.…”

“…Induction of various P450 mRNAs, including CYP1A2, CYP2B6, CYP2C, and CYP3A4 mRNA, has been largely demonstrated mostly using fresh hepatocytes (Raucy et al, 2002;Madan et al, 2003). In contrast, only a limited number of papers has reported such induction results using cryopreserved hepatocytes (Roymans et al, 2005;Hewitt et al, 2007). For P450 induction assays at pharmaceutical companies, the cryopreserved hepatocytes are often used after selection by evaluating a large number of the hepatocyte lots for P450 inducibility prior to the experiments.…”

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

“…Induction is generally evaluated by measuring enzyme activity as activity represents the most relevant end point for drug-drug interaction. Both freshly isolated and plateable cryopreserved human hepatocytes can be used for the induction study (Li, 2007;Roymans et al, 2004Roymans et al, , 2005.…”