Petter Svanberg

Determination of Human Hepatocyte Intrinsic Clearance for Slowly Metabolized Compounds: Comparison of a Primary Hepatocyte/Stromal Cell Co-culture with Plated Primary Hepatocytes and HepaRG

A key requirement in drug discovery is to accurately define intrinsic clearance (CLint) values of less than 1 µl/min/106 hepatocytes, which requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHHs), and the HµREL human hepatocyte/stromal cell co-cultures for determination of low CLint values. The investigation demonstrated that the systems were capable of providing statistically significant CLint estimations down to 0.2 µl/min/106 cells. The HµREL assay provided a higher level of reproducibility, with repeat significant CLint values being defined in a minimum of triplicate consecutive assays for six of seven of the low CLint compounds compared with four of seven for PHHs and two of seven for HepaRG. The assays were also compared with a suspension assay using drugs with higher CLint values and diverse enzymology. The CLint values from the PHH and HµREL assays were similar to those defined by a hepatocyte suspension assay, indicating that they can be used interchangeably alongside a standard assay. Finally, data from these two assays could also predict in vivo hepatic metabolic CLint to within 3-fold for greater than 70% of the compounds tested, with average fold errors (AFE) of 1.6 and 2.3, respectively, whereas the HepaRG data were predictive to within 3-fold for only 50% of compounds (AFE 2.9). In summary, all systems have utility for low CLint determination, but the HµREL co-culture appears slightly superior regarding overall assay performance.

Development of a Novel Lung Slice Methodology for Profiling of Inhaled Compounds.

The challenge of defining the concentration of unbound drug at the lung target site after inhalation limits the possibility to optimize target exposure by compound design. In this study, a novel rat lung slice methodology has been developed and applied to study drug uptake in lung tissue, and the mechanisms by which this occurs. Freshly prepared lung slices (500 μm) from drug-naive rats were incubated with drugs followed by determination of the unbound drug volume of distribution in lung (Vu,lung), as the total concentration of drug in slices divided by the buffer (unbound) concentration. Vu,lung determined for a set of inhaled drug compounds ranged from 2.21 mL/g for salbutamol to 2970 mL/g for dibasic compound A. Co-incubation with monensin, a modulator of lysosomal pH, resulted in inhibition of tissue uptake of basic propranolol to 13%, indicating extensive lysosomal trapping. Partitioning into cells was particularly high for the cation MPP+ and the dibasic compound A, likely because of the carrier-mediated transport and lysosomal trapping. The results show that different factors are important for tissue uptake and the presented method can be used for profiling of inhaled compounds, leading to a greater understanding of distribution and exposure of drug in the lung.

Predicting Metabolic Clearance for Drugs That Are Actively Transported into Hepatocytes: Incubational Binding as a Consequence of in Vitro Hepatocyte Concentration Is a Key Factor

Incubational binding or the fraction of drug unbound in an in vitro incubation, fuinc, is an important parameter to predict or measure in the pursuit of accurate clearance predictions from in vitro data. Here we describe a method for fuinc determination directly in the hepatocyte intrinsic clearance (CLint) assay with emphasis on compounds that are actively transported into hepatocytes, hypothesizing that for such compounds the typical protocol of 1 million hepatocytes/ml systematically underestimates the maximum attainable unbound intracellular drug concentration. Using the transporter substrate atorvastatin as a test compound, incubations were performed and a mathematical model applied to describe metabolism, distribution, and binding at different hepatocyte concentrations. From these investigations it was evident that, since binding is more extensive intracellularly than in the medium, increased partitioning into the cellular volume, due to active uptake, increases the total amount of atorvastatin bound in the incubation. Consequently, a significant lowering of the hepatocyte concentration impacts the free drug concentration in the incubation and increases the observed rate of metabolism and therefore observed CLint (that is, when viewed from the media drug concentration). The applicability of the findings was tested for a series of 11 actively transported zwitterions for which standard rat hepatocyte metabolic CLint data (1 million cells/ml incubation) poorly predicted in vivo clearance (average fold error of 5.4). Using metabolic CLint determined at a lower hepatocyte concentration (0.125 million cells/ml) considerably improved clearance predictions (average fold error of 2.3).

Optimized Experimental Design for the Estimation of Enzyme Kinetic Parameters: An Experimental Evaluation

A set of compounds (n = 30), including traditional cytochrome P450 substrates and compounds from AstraZenecas compound library, was used in an experimental evaluation of an optimal design approach (ODA) for the estimation of enzyme kinetic parameters (CLint, Vmax, and Km). A depletion method previously shown to provide reliable results, the multiple depletion curves method (MDCM), was used as reference. Experiments were conducted with human liver microsomes, and samples were analyzed using liquid chromatography-tandem mass spectrometry. CLint estimated with the ODA were in >90% of the cases within a 2-fold difference compared with MDCM estimates. In addition, good agreement was generally seen for Vmax and Km estimates between the two methods as >80% of the estimates were within or almost within a 2-fold difference. The variability in Vmax and Km estimates were generally higher than for CLint estimates. In addition, decreased substrate turnover considerably increased the variability in Vmax and Km estimates, whereas only a modest increase was observed for CLint estimates. The experimental design of using multiple starting concentrations for the estimation of enzyme kinetics was shown to be appropriate even when there was a limitation to the number of samples. The method allowed for good estimates of CLint and also for Vmax and Km in many cases. Hence, this approach is a good alternative for the estimation of enzyme kinetic parameters, especially if enzyme saturation and an assessment of a potential risk for nonlinear metabolism are of interest.