Quantitative Contribution of Six Major Transporters to the Hepatic Uptake of Drugs: “SLC-Phenotyping” Using Primary Human Hepatocytes

Abstract

Hepatic uptake transporters [solute carriers (SLCs)], including organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, sodium-dependent taurocholate cotransporting polypeptide (NTCP), and organic anion (OAT2) and organic cation (OCT1) transporters, play a key role in determining the systemic and liver exposure of chemically diverse drugs. Here, we established a phenotyping approach to quantify the contribution of the six SLCs, and passive diffusion, to the overall uptake using plated human hepatocytes (PHHs). First, selective inhibitor conditions were identified by screening about 20 inhibitors across the six SLCs using single-transfected human embryonic kidney 293 cells. Data implied rifamycin SV (20 mM) inhibits three OATPs, while rifampicin (5 mM) inhibits OATP1B1/1B3 only. Further, hepatitis B virus myristoylated-preS1 peptide (0.1 mM), quinidine (100 mM), and ketoprofen (100-300 mM) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported (f t) by the individual SLCs was characterized for 20 substrates with PHH. Generally, extended clearance classification system class 1A/3A (e.g., warfarin) and 1B/3B compounds (e.g., statins) showed predominant OAT2 and OATP1B1/1B3 contribution, respectively. OCT1mediated uptake was prominent for class 2/4 compounds (e.g., metformin). Third, in vitro f t values were corrected using quantitative proteomics data to obtain "scaled f t ." Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/1B3 f t was assessed, leveraging statin clinical drug-drug interaction data with rifampicin as the perpetrator. Finally, we outlined a novel stepwise strategy to implement phenotypic characterization of SLC-mediated hepatic uptake for new molecular entities and drugs in a drug discovery and development setting.

Dose‐Dependent Inhibition of OATP1B by Rifampicin in Healthy Volunteers: Comprehensive Evaluation of Candidate Biomarkers and OATP1B Probe Drugs

“…20%) to the systemic elimination, 20 and suggested involvement of other transporters in hepatic uptake. 27 Hence, the dynamic range of AUCR at high levels of hepatic OATP1B inhibition depends on the contribution of non-OATP1B-mediated clearance mechanisms even if they only make a small contribution (e.g., 10-20%) to overall clearance. This is the first demonstration that valsartan AUC 0-24h discriminated the dose-dependent effects of rifampicin as atorvastatin and pitavastatin did (Figure 2), although valsartan is not listed as clinical substrate for OATP1B (https ://www.fda.…”

Identification of Glycochenodeoxycholate 3‐O‐Glucuronide and Glycodeoxycholate 3‐O‐Glucuronide as Highly Sensitive and Specific OATP1B1 Biomarkers

“…In vitro studies Both GCDCA-3G and GDCA-3G were screened in vitro as substrates of various human liver solute carriers individually transfected into Human Embryonic Kidney (HEK) 293 cells as previously described. 37 In this instance, however, additional uptake studies were conducted with OATP1B1*5 and OATP1B1*15 similarly transfected into HEK293 cells (provided by Yuichi Sugiyama, Tokyo University, Japan), and OATP1B1*14 transfected into HEK293 cells (acquired from Absorption Systems, Exton, PA). OATP1B1*1A expressed in HEK293 cells was prepared in-house as described in Supplemental Methods.…”

“…Sample processing and bioanalysis of rosuvastatin, taurocholic acid, metformin, fluvastatin, and cyclic guanosine monophosphate has already been described. 37 Both GCDCA-3G and GDCA-3G (~ 0.01 to 100 µM) were also studied as in vitro inhibitors of OATP1B1*1A, OATP1B3, and OATP2B1 (expressed in HEK293 cells) using rosuvastatin (0.5 µM) as substrate. Where possible, the concentration of glucuronide rendering 50% inhibition of rosuvastatin uptake (IC 50 ) was determined.…”

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs

“…However, the remaining uptake clearances of PTV and RSV in the presence of rifamycin SV (30 μM) showed no declining trend (Table I ). Of note, the recent studies reported that the complete inhibition of active influx transporters (both OATP1Bs and non-OATP1B transporters) in hepatocytes requires the rifamycin SV concentrations of 1 mM or higher ( 15 , 19 ). Thus, the measured uptake clearances of PTV and RSV in the presence of rifamycin SV (30 μM) likely include the uptake clearance via active influx by non-OATP1B transporters as well as passive diffusion.…”