title: Reliable Rate Measurements for Active and Passive Hepatic Uptake Using Plated Human Hepatocytes.
abstract: Transporter-mediated hepatic uptake is proven to be the rate-determining step in the systemic clearance of several drugs. Therefore, accurate measurement of active and passive uptake clearances in vitro is critical to facilitate pharmacokinetics and drug-drug interaction predictions. Here, we evaluated the plated human hepatocytes (PHH) and studied the effect of incubation temperature and inhibitor concentration on uptake measurements, in order to reliably estimate hepatic uptake components. Uptake rates measured using PHH, at 37°C without and with rifamycin SV, were comparable with those obtained from suspension hepatocytes and sandwich-cultured hepatocytes for a set of 10-13 compounds. Apparent permeability across monolayers of low-efflux Madin-Darby canine kidney cells was measured at 4, 10, and 37°C. Of the 23 compounds evaluated, 13 compounds showed >2-fold reduction in passive permeability at 4°C compared to 37°C, inferring that low-temperature incubations may underestimate passive uptake. Inhibition studies using transporter-transfected cells suggested that ∼20 μM rifamycin SV completely inhibited organic anion-transporting polypeptides (OATPs), while no significant inhibition was noted for other hepatic uptake transporters. On the basis of inhibition profiles, the contribution of active versus passive and OATP versus non-OATP transport to the PHH uptake was discerned for various endogenous substrates and statins. With the exception of fluvastatin, the statins studied were predominantly transported by OATPs in PHH and the non-OATP transporters, such as Na+-taurocholate co-transporting polypeptide, played a minimal role. In conclusion, PHH is useful for uptake measurements, and rifamycin SV employed at different concentrations can reliably estimate active and passive uptake and characterize OATP-dependent active uptake.
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Yi-An
authors.initials: YA
authors.lastname: Bi
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.|Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, Princeton, New Jersey, USA.
authors.forename: Renato J
authors.initials: RJ
authors.lastname: Scialis
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Lazzaro
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Sumathy
authors.initials: S
authors.lastname: Mathialagan
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Emi
authors.initials: E
authors.lastname: Kimoto
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Keefer
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Vildhede
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Chester
authors.initials: C
authors.lastname: Costales
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: A David
authors.initials: AD
authors.lastname: Rodrigues
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Larry M
authors.initials: LM
authors.lastname: Tremaine
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut, USA. manthena.v.varma@pfizer.com.
authors.forename: Manthena V S
authors.initials: MVS
authors.lastname: Varma
mesh_terms: D000818:Animals; D004285:Dogs; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics*; D061985:Madin Darby Canine Kidney Cells; D027361:Organic Anion Transporters / Q000378:metabolism; D012294:Rifamycins
chemical_list: D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D027361:Organic Anion Transporters; D012294:Rifamycins; C023808:rifamycin SV
keywords: drug transporters; hepatic uptake; organic anion-transporting polypeptide; pharmacokinetics; plated human hepatocytes