27 research outputs found
Quantification of Hepatic Organic Anion Transport Proteins OAT2 and OAT7 in Human Liver Tissue and Primary Hepatocytes
Organic anion transporter (OAT) 2
and OAT7 were recently shown
to be involved in the hepatic uptake of drugs; however, there is limited
understanding of the population variability in the expression of these
transporters in liver. There is also a need to derive relative expression-based
scaling factors (REFs) that can be used to bridge in vitro functional
data to the in vivo drug disposition. To this end, we quantified OAT2
and OAT7 surrogate peptide abundance in a large number of human liver
tissue samples (<i>n</i> = 52), as well as several single-donor
cryopreserved human hepatocyte lots (<i>n</i> = 30) by a
novel, validated liquid chromatography tandem mass spectrometry (LC–MS/MS)
method. The average surrogate peptide expression of OAT2 and OAT7
in the liver samples was 1.52 ± 0.57 and 4.63 ± 1.58 fmol/μg
membrane protein, respectively. While we noted statistically significant
differences (<i>p</i> < 0.05) in hepatocyte and liver
tissue abundances
for both OAT2 and OAT7, the differences were relatively small (1.8-
and 1.5-fold difference in median values, respectively). Large interindividual
variability was noted in the hepatic expression of OAT2 (16-fold in
liver tissue and 23-fold in hepatocytes). OAT7, on the other hand,
showed less interindividual variability (4-fold) in the
livers, but high variability for the hepatocyte lots (27-fold). A
significant positive correlation in OAT2 and OAT7 expression was observed,
but expression levels were neither associated with age nor sex. In
conclusion, our data suggest marked interindividual variability in
the hepatic expression of OAT2/7, which may contribute to the pharmacokinetic
variability of their substrates. Because both transporters were less
abundant in hepatocytes than livers, a REF-based approach is recommended
when scaling in vitro hepatocyte transport data to predict hepatic
drug clearance and liver exposure of OAT2/7 substrates
Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1‑<i>O</i>‑β-Glucuronide
Gemfibrozil (GEM), which decreases
serum triglycerides and low
density lipoprotein, perpetrates drug–drug interactions (DDIs)
with several drugs. These DDIs are primarily attributed to the inhibition
of drug transporters and metabolic enzymes, particularly cytochrome
P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-<i>O</i>-β-glucuronide (GG). Here, we characterized the transporter-mediated
hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes
(SCHH) and transporter-transfect systems. Significant active uptake
was noted in SCHH for the metabolite. GG, but not GEM, showed substrate
affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3,
and 2B1. In SCHH, glucuronidation was characterized affinity constants
(<i>K</i><sub>m</sub>) of 7.9 and 61.4 μM, and biliary
excretion of GG was observed. Furthermore, GG showed active basolateral
efflux from preloaded SCHH and ATP-dependent uptake into membrane
vesicles overexpressing multidrug resistance-associated protein (MRP)
2, MRP3, and MRP4. A mathematical model was developed to estimate
hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies.
Collectively, the hepatic transporters play a key role in the disposition
and thus determine the local concentrations of GEM and more so for
GG, which is the predominant inhibitory species against CYP2C8 and
OATP1B1
Role of Hepatic Organic Anion Transporter 2 in the Pharmacokinetics of <i>R</i>- and <i>S</i>‑Warfarin: In Vitro Studies and Mechanistic Evaluation
Interindividual
variability in warfarin dose requirement demands personalized medicine
approaches to balance its therapeutic benefits (anticoagulation) and
bleeding risk. Cytochrome P450 2C9 (<i>CYP2C9</i>) genotype-guided
warfarin dosing is recommended in the clinic, given the more potent <i>S</i>-warfarin is primarily metabolized by CYP2C9. However,
only about 20–30% of interpatient variability in <i>S</i>-warfarin clearance is associated with <i>CYP2C9</i> genotype.
We evaluated the role of hepatic uptake in the clearance of <i>R</i>- and <i>S</i>-warfarin. Using stably transfected
HEK293 cells, both enantiomers were found to be substrates of organic
anion transporter (OAT)2 with a Michaelis–Menten constant (<i>K</i><sub>m</sub>) of ∼7–12 μM but did not
show substrate affinity for other major hepatic uptake transporters.
Uptake of both enantiomers by primary human hepatocytes was saturable
(<i>K</i><sub>m</sub> ≈ 7–10 μM) and
inhibitable by OAT2 inhibitors (e.g., ketoprofen) but not by OATP1B1/1B3
inhibitors (e.g., cyclosporine). To further evaluate the potential
role of hepatic uptake in <i>R</i>- and <i>S</i>-warfarin pharmacokinetics, mechanistic modeling and simulations
were conducted. A “bottom-up” PBPK model, developed
assuming that OAT2–CYPs interplay, well recovered clinical
pharmacokinetics, drug–drug interactions, and <i>CYP2C9</i> pharmacogenomics of <i>R</i>- and <i>S</i>-warfarin.
Clinical data were not available to directly verify the impact of
OAT2 modulation on warfarin pharmacokinetics; however, the bottom-up
PBPK model simulations suggested a proportional change in clearance
of both warfarin enantiomers with inhibition of OAT2 activity. These
results suggest that variable hepatic OAT2 function, in conjunction
with CYP2C, may contribute to the high population variability in warfarin
pharmacokinetics and possibly anticoagulation end points and thus
warrant further clinical investigation