Individual differences in pharmacokinetics may cause extensive variability in drug efficacy, toxicity and adverse drug reactions, and represent a major concern in drug development. The overall aim of this thesis was to evaluate the hepatic transport and metabolism in in vitro models used in assessments of drug pharmacokinetics and toxicity, and to investigate the contribution of membrane transporters and metabolizing enzymes to in vivo pharmacokinetic variability.
In plated primary human hepatocytes, uptake kinetics studies of OATP1B1/1B3-mediated transport showed an extensive and variable decrease in OATP1B1/1B3 activity and increased passive diffusion over time in two-dimensional (2D) culture. In three-dimensional (3D) bioreactor cultures of primary human hepatocytes, OATP1B1 activity was observed for at least 7 days, while CYP3A4 activity was observed at day 3 and 4 in culture. The activity data were in agreement with immunohistochemical stainings which showed OATP1B1 and CYP3A4 protein expression for at least 9 days in culture. In bioreactor cultures of differentiated HepaRG cells, the observed CYP3A4 activity was comparable to primary human hepatocytes, while OATP1B1 activity could not be detected later than day 2. In 2D cultures of hepatocytes derived from human embryonic and induced pluripotent stem cells, OATP1B1 and CYP activities were very low compared to plated cryopreserved human hepatocytes, but moderate activity of the hepatic transporters NTCP and BSEP was observed. Finally, an in vivo study investigating the relationship between expression of OATP1B1, MDR1 and CYP3A4 and the pharmacokinetics of atorvastatin in 21 obese patients with paired biopsies from liver and intestinal segments showed a significant positive correlation between OATP1B1 expression and oral clearance (CL/F) of atorvastatin, while no association was observed with CYP3A4 or MDR1.
In conclusion, plated primary human hepatocytes are a useful in vitro model for OATP1B1/1B3-mediated uptake studies, but only for a restricted period of time in culture. The preserved OATP1B1 and CYP3A4 activity in bioreactor cultures of primary human hepatocytes allows long-term in vitro studies of hepatic drug clearance and toxicity in this system. Differentiated HepaRG cells cultured in the same 3D system represents a useful in vitro tool for long-term studies of slowly metabolized drugs, but the low OATP1B1 activity is a major limitation of this model compared to human hepatocytes. Furthermore, stem-cell derived human hepatocytes represent a potential alternative to human hepatocytes, but additional refinements of the derivation process are required in order to obtain fully functional human hepatocytes applicable in drug disposition and metabolism studies in vitro. Finally, the in vivo study shows that uptake transporters could be more important than metabolizing enzymes for the pharmacokinetic variability of certain drugs.
List of papers. The papers are removed from the thesis due to publisher restrictions.
I. Ulvestad M, Björquist P, Molden E, Asberg A, Andersson TB. OATP1B1/1B3 activity in plated primary human hepatocytes over time in culture. Biochem Pharmacol. 2011; 82(9): 1219-26. doi:10.1016/j.bcp.2011.07.076
II. Ulvestad M, Darnell M, Molden E, Ellis E, Åsberg A, Andersson TB. Evaluation of organic anion-transporting polypeptide 1B1 and CYP3A4 activities in primary human hepatocytes and HepaRG cells cultured in a dynamic three-dimensional bioreactor system. J Pharmacol Exp Ther 2012; 343(1): 145-56. doi:10.1124/jpet.112.195750
III. Ulvestad M, Nordell P, Asplund A, Rehnström M, Karlsson SJ, Holmgren G, Davidson L, Brolén G, Edsbagge J, Björquist P, Küppers-Munther B, Andersson TB. Drug metabolizing enzymes and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells. Biochem Pharmacol. 2013; 86(5):691-702. doi:10.1016/j.bcp.2013.06.029
IV. Ulvestad M, Skottheim IB, Jakobsen GS, Bremer S, Molden E, Asberg A, Hjelmesæth J, Andersson TB, Sandbu R, Christensen H. Impact of OATP1B1, MDR1, and CYP3A4 Expression in Liver and Intestine on Interpatient Pharmacokinetic Variability of Atorvastatin in Obese Subjects. Clin Pharmacol Ther 2013; 93(3): 275-82. doi:10.1038/clpt.2012.261