Pär Nordell

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.

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).

Transition State of Rare Event Base Pair Opening Probed by Threading into Looped DNA

King Abdullah University of Science and Technology is gratefully acknowledged for an award to B.N. including the Ph.D. student position of M.K.

Montelukast Disposition: No Indication of Transporter-Mediated Uptake in OATP2B1 and OATP1B1 Expressing HEK293 Cells.

Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast.

Determination of Incubational Binding in In Vitro Microsomal and Hepatocyte Metabolic Stability Incubations: A Comparison of Methods.

The fraction of unbound drug (fuinc) in in vitro intrinsic clearance (CLint) incubation is an important parameter in the pursuit of accurate clearance predictions and is often predicted using algorithms based on drug lipophilicity measures. However, analysis of an AstraZeneca database suggests that simple lipophilicity alone is a relatively poor predictor of fuinc measured using equilibrium dialysis. He fuinc value can also be measured directly in CLint assays using multiple concentrations of hepatocytes or microsomal protein. Since this approach informs of the unbound drug concentration in the assay used to predict in vivo clearance, it should be considered the gold standard method. As a starting point for building better predictive algorithms we aimed to determine if equilibrium dialysis really is an appropriate assay for assessing fuinc. Employing a large number of compounds with a wide range of lipophilicities, experiments were performed to measure fuinc using rat hepatocytes (RH) and human liver microsomes (HLM) in both assay formats. A high percentage (94% and 93% for HLM and RH, respectively) of the fuinc values were within 2-fold when the compound distribution coefficient describing the ratio of compound concentration in octanol and pH 7.4 buffer when the test system is at equilibrium (lipophilicity measure) (logD7.4) values were less than 3.5. However, with logD7.4 values greater than these, the agreement was considerably worse. Additional experimental data generated indicated that this discrepancy was likely due to failings in the direct method when drug binding is high. Thus, we conclude that unbound CLint can be indeed calculated indirectly by incorporating equilibrium dialysis data with measured CLint but that simple lipophilicity descriptors alone may be inadequate for predicting fuinc.The fraction of unbound drug (fu inc ) in in vitro intrinsic clearance (CL int ) incubation is an important parameter in the pursuit of accurate clearance predictions and is often predicted using algorithms based on drug lipophilicity measures. However, analysis of an AstraZeneca database suggests that simple lipophilicity alone is a relatively poor predictor of fu inc measured using equilibrium dialysis. He fu inc value can also be measured directly in CL int assays using multiple concentrations of hepatocytes or microsomal protein. Since this approach informs of the unbound drug concentration in the assay used to predict in vivo clearance, it should be considered the gold standard method. As a starting point for building better predictive algorithms we aimed to determine if equilibrium dialysis really is an appropriate assay for assessing fu inc . Employing a large number of compounds with a wide range of lipophilicities, experiments were performed to measure fu inc using rat hepatocytes (RH) and human liver microsomes (HLM) in both assay formats. A high percentage (94% and 93% for HLM and RH, respectively) of the fu inc values were within 2-fold when the compound distribution coefficient describing the ratio of compound concentration in octanol and pH 7.4 buffer when the test system is at equilibrium (lipophilicity measure) (logD 7.4 ) values were less than 3.5. However, with logD 7.4 values greater than these, the agreement was considerably worse. Additional experimental data generated indicated that this discrepancy was likely due to failings in the direct method when drug binding is high. Thus, we conclude that unbound CL int can be indeed calculated indirectly by incorporating equilibrium dialysis data with measured CL int but that simple lipophilicity descriptors alone may be inadequate for predicting fu inc .

Finding at-DNA – Kinetic Recognition of Long Adenine-Thymine Stretches by Metal-Ligand Complexes

High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.