Evaluation of Cryopreserved Human Hepatocytes as an Alternative in Vitro System to Microsomes for the Prediction of Metabolic Clearance

Abstract

ABSTRACT:Human liver microsomes have typically resulted in marked underprediction of in vivo human intrinsic clearance (CL int ); therefore, the utility of cryopreserved hepatocytes as an alternative in vitro system has become an important issue. In this study, 10 compounds (tolbutamide, diclofenac, S-warfarin, S-mephenytoin, dextromethorphan, bufuralol, quinidine, nifedipine, testosterone, and terfenadine) were selected as substrate probes for CYP2C9, 2C19, 2D6, and 3A4, and the kinetics of metabolite formation (n ‫؍‬ 14 pathways) were investigated in three individual lots of cryopreserved hepatocytes and in a pool of human liver microsomes. For the majority of the compounds, lower unbound K M or S 50 values were observed in hepatocytes compared with microsomes, on average by 50% over a 200-fold range (0.5-140 M). Expressed on an equivalent liver weight basis, a good correlation between microsomal and hepatocyte V max values was observed for most pathways greater than 5 orders of magnitude (0.16-216 nmol/min/g liver). Unbound hepatocyte CL int (CL int,u ) values, when scaled to the whole liver (range 0.38-4000 ml/min/kg), were on average 2.5-fold higher than microsomal CL int,u values, with the exception of tolbutamide and diclofenac, for which lower hepatocellular CL int,u values were observed. Hepatocyte predicted CL int values were compared with human in vivo CL int values, and to supplement our data, in vitro data from cryopreserved hepatocytes were collated from four other published sources. These data show that for 37 drugs, there is, on average, a 4.5-fold under-prediction of the in vivo CL int using cryopreserved hepatocytes, representing a significant reduction in prediction bias compared with human microsomes.Human liver microsomes have traditionally been the most commonly used in vitro system for the prediction of metabolic clearance, in particular, for new chemical entities within drug discovery programs in the pharmaceutical industry. However, the use of this system has typically resulted in an underestimation of clearance, as illustrated by a data set of 55 compounds in which a 9-fold under-prediction of the in vivo intrinsic clearance (CL int ) was observed . As a result of this, attention in recent years has been placed on the use of alternative in vitro systems for clearance prediction. Fresh human hepatocytes are envisaged as a potentially more accurate system because of the full complement of both phase I and phase II metabolizing enzymes, along with the presence of transporter proteins, which should result in drug concentrations within the hepatocyte that are equivalent to in vivo concentrations within the liver. However, the limited availability of fresh human tissue and the cost implications involved in the preparation of freshly isolated human hepatocytes have resulted in cryopreserved hepatocytes emerging as the favored alternative, which also have the added advantage of being readily available commercially and more convenient to use (Li et al., 1999).Two major issues concerning the use ...

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

“…Although no attempt was made to measure nonspecific binding in the present study, previous reports indicate that the concentration of unbound drug in the incubation medium for alprazolam, midazolam , and tolbutamide (Brown et al, 2007) would have been no more than marginally affected by such binding at the concentrations used; although no equivalent data for phenacetin were available, the log P of this compound indicates marginal binding also.…”

“…The CL int and CL max values were compared with the equivalent values obtained using the commonly used suspended human cryopreserved hepatocyte system for phenacetin (Stringer et al, 2008), tolbutamide (Brown et al, 2007), alprazolam (Brown et al, 2007), and midazolam (Brown et al, 2007) after multiplication by the appropriate RAF (above). The CL int and CL max values scaled to in vivo were compared with the equivalent human in vivo CL int values for phenacetin (Stringer et al, 2008), tolbutamide (Brown et al, 2007), alprazolam (Brown et al, 2007), and midazolam (Brown et al, 2007).…”

“…Technical advances in freezing protocols have made cryopreserved human hepatocytes avail-able from commercial suppliers, which has notably enabled their application for hepatic clearance studies (Bachmann et al, 2003;Naritomi et al, 2003;McGinnity et al, 2004). Compared with microsomes, hepatocytes seem to provide significant improvement in the accuracy of clearance predictions because of the reduced bias (Brown et al, 2007). However, cryopreserved hepatocytes are not fully quantitative for predictions as a systematic underprediction of the in vivo value is obtained Brown et al, 2007).…”

“…Compared with microsomes, hepatocytes seem to provide significant improvement in the accuracy of clearance predictions because of the reduced bias (Brown et al, 2007). However, cryopreserved hepatocytes are not fully quantitative for predictions as a systematic underprediction of the in vivo value is obtained Brown et al, 2007).…”

Using Simcyp to project human oral pharmacokinetic variability in early drug research to mitigate mechanism‐based adverse events

“…Following the processing of the mass spectral data to afford a potentiator concentration for each incubation time point sample using peak area ratios (analyte/IS), the compound elimination rate constant (k el ) was determined by linear regression of the natural log concentration versus time data, and a half-life (t 1/2 ) was calculated as (ln2)/k el . Using the respective t 1/2 and particular incubation parameters, a potentiator-specific apparent intrinsic clearance (CL int,app ) was determined for each studied in vitro system, with further scaling of liver CL int,app to hepatic blood clearance (CL H ) using the well-stirred model without (HLM and human hepatocytes) and with (HLM) all human binding factors [11,12]. All calculations were performed using the BioBook module in the E-Workbook Suite (IDBS Ltd, Guildford, Surrey, UK) or by manual calculation in Microsoft Excel (Microsoft Corp., Redmond, WA).…”