Physiologically based pharmacokinetics modeling and transporter proteomics to predict systemic and local liver and muscle disposition of statins.

Statins are used to reduce liver cholesterol levels but also carry a dose-related risk of skeletal muscle toxicity. Concentrations of statins in plasma are often used to assess efficacy and safety, but because statins are substrates of membrane transporters that are present in diverse tissues, local differences in intracellular tissue concentrations cannot be ruled out. Thus, plasma concentration may not be an adequate indicator of efficacy and toxicity. To bridge this gap, we used physiologically based pharmacokinetic (PBPK) modeling to predict intracellular concentrations of statins. Quantitative data on transporter clearance were scaled from in vitro to in vivo conditions by integrating targeted proteomics and transporter kinetics data. The developed PBPK models, informed by proteomics, suggested that organic anion-transporting polypeptide 2B1 (OATP2B1) and multidrug resistance-associated protein 1 (MRP1) play a pivotal role in the distribution of statins in muscle. Using these PBPK models, we were able to predict the impact of alterations in transporter function due to genotype or drug-drug interactions on statin systemic concentrations and exposure in liver and muscle. These results underscore the potential of proteomics-guided PBPK modeling to scale transporter clearance from in vitro data to real-world implications. It is important to evaluate the role of drug transporters when predicting tissue exposure associated with on- and off-target effects.

Optimization of hERG and Pharmacokinetic Properties for Basic Dihydro-8H-purin-8-one Inhibitors of DNA-PK.

The DNA-PK complex is activated by double-strand DNA breaks and regulates the non-homologous end-joining repair pathway; thus, targeting DNA-PK by inhibiting the DNA-PK catalytic subunit (DNA-PKcs) is potentially a useful therapeutic approach for oncology. A previously reported series of neutral DNA-PKcs inhibitors were modified to incorporate a basic group, with the rationale that increasing the volume of distribution while maintaining good metabolic stability should increase the half-life. However, adding a basic group introduced hERG activity, and basic compounds with modest hERG activity (IC50 = 10-15 µM) prolonged QTc (time from the start of the Q wave to the end of the T wave, corrected by heart rate) in an anaesthetized guinea pig cardiovascular model. Further optimization was necessary, including modulation of pK a, to identify compound 18, which combines low hERG activity (IC50 = 75 µM) with excellent kinome selectivity and favorable pharmacokinetic properties.

Resolving the distribution-metabolism interplay of eight OATP substrates in the standard clearance assay with suspended human cryopreserved hepatocytes.

Uptake transporters may act to elevate the intrahepatic exposure of drugs, impacting the route and rate of elimination, as well as the drug-drug interaction potential. We have here extended the assessment of metabolic drug stability in a standard human hepatocyte incubation to allow for elucidation of the distribution-metabolism interplay established for substrates of drug transporters. Cellular concentration-time profiles were obtained from incubations of eight known OATP substrates at 1 µM, each for two different 10-donor batches of suspended cryopreserved human hepatocytes. The kinetic data sets were analyzed using a mechanistic mathematical model that allowed for separate estimation of active uptake, bidirectional diffusion, metabolism and nonspecific extracellular and intracellular binding. The range of intrinsic clearances attributed to active uptake, diffusion and metabolism of the test set spanned more than 2 orders of magnitude each, with median values of 18, 5.3, and 0.5 µL/min/10(6) cells, respectively. This is to be compared with the values for the apparent clearance from the incubations, which only spanned 1 order of magnitude with a median of 2.6 µL/min/10(6) cells. The parameter estimates of the two pooled 10-donor hepatocyte batches investigated displayed only small differences in contrast to the variability associated with use of cells from individual donors reported in the literature. The active contribution to the total cellular uptake ranged from 55% (glyburide) to 96% (rosuvastatin), with an unbound intra-to-extracellular concentration ratio at steady state of 2.1 and 17, respectively. Principal component analysis showed that the parameter estimates of the investigated compounds were largely influenced by lipophilicity. Active cellular uptake in hepatocytes was furthermore correlated to pure OATP1B1-mediated uptake as measured in a transfected cell system. The presented approach enables the assessment of the key pathways regulating hepatic disposition of transporter and enzyme substrates from one single, reproducible and generally accessible human in vitro system.

Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells.

Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system.