Meeting the Challenge of Predicting Hepatic Clearance of Compounds Slowly Metabolized by Cytochrome P450 Using a Novel Hepatocyte Model, HepatoPac

Abstract

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4-6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min per kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 10 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 76% (13 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/ min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 60% within 2-fold and 90% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

“…The twofold lower underprediction average for predicted clearances in HepatoPac® was in line with the twofold higher metabolic activity in HepatoPac® as compared to the suspension cultures from the same donor. Our results agree well with the finding of Chan et al (10), who also reported a twofold higher metabolic capacity in HepatoPac® compared to suspended hepatocytes from a different donor and using different model compounds. For low-clearance compounds, warfarin and tolbutamide, the long-term system was clearly superior for the prediction of hepatic clearances as no clearance values could be determined in the suspension cultures.…”

“…The in vitro clearances were then scaled by applying the well-stirred model using the conventional approach as described in BMATERIALS AND METHODS^(Eqs. [8][9][10][11][12]. Fraction unbound values in the incubation media and human plasma were experimentally determined and are listed in Table III together with the blood-to-plasma ratios.…”

“…[8][9][10][11][12][13][14][15][16]. For that, the fractions unbound in the incubation media and human plasma were experimentally determined and are listed in Table III together with the blood-to-plasma ratios.…”

“…Longterm in vitro liver models, such as the hepatic stem cell line HepaRG™ (6), micropatterned HepatoPac® co-cultures of human hepatocytes with mouse embryonic 3T3 fibroblasts cells (7), and HμREL™ hepatocyte/fibroblast co-culture plates (8), as well as alternative approaches for low clearance determination, such as the relay method with suspension hepatocyte cultures (9), are being intensively explored. Chan et al (10) studied the Christophe Meille was a Roche employee during the conduct of this research. He is now employed at Novartis Pharma, Basel, Switzerland.…”

Addressing the Challenges of Low Clearance in Drug Research

“…When cultured in this way, it was shown that drug-metabolizing enzyme activities were sustained for several days without a change in medium, thereby yielding the potential to carry out long drug metabolism incubations potentially ideal for measuring reliable consumption rates of low-CL int compounds. This was demonstrated by Chan et al (22), for several drugs, including low-CL int drugs theophylline, diazepam, tolbutamide, meloxicam, among others using hepatocytes from three individual donors. As with the Liverchip method, it was low-CL int drugs that were best predicted, with high-CL int drugs underpredicted.…”