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

Evaluating the Role of Multidrug Resistance Protein 3 (MDR3) Inhibition in Predicting Drug-Induced Liver Injury Using 125 Pharmaceuticals

The role of bile salt export protein (BSEP) inhibition in drug-induced liver injury (DILI) has been investigated widely, while inhibition of the canalicular multidrug resistant protein 3 (MDR3) has received less attention. This transporter plays a pivotal role in secretion of phospholipids into bile and functions coordinately with BSEP to mediate the formation of bile acid-containing biliary micelles. Therefore, inhibition of MDR3 in human hepatocytes was examined across 125 drugs (70 of Most-DILI-concern and 55 of No-DILI-concern). Of these tested, 41% of Most-DILI-concern and 47% of No-DILI-concern drugs had MDR3 IC₅₀ values of <50 μM. A better distinction across DILI classifications occurred when systemic exposure was considered where safety margins of 50-fold had low sensitivity (0.29), but high specificity (0.96). Analysis of physical chemical property space showed that basic compounds were twice as likely to be MDR3 inhibitors as acids, neutrals, and zwitterions and that inhibitors were more likely to have polar surface area (PSA) values of <100 Ų and cPFLogD values between 1.5 and 5. These descriptors, with different cutoffs, also highlighted a group of compounds that shared dual potency as MDR3 and BSEP inhibitors. Nine drugs classified as Most-DILI-concern compounds (four withdrawn, four boxed warning, and one liver injury warning in their approved label) had intrinsic potency features of <20 μM in both assays, thereby reinforcing the notion that multiple inhibitory mechanisms governing bile formation (bile acid and phospholipid efflux) may confer additional risk factors that play into more severe forms of DILI as shown by others for BSEP inhibitors combined with multidrug resistance-associated protein (MRP2, MRP3, MRP4) inhibitory properties. Avoiding physical property descriptors that highlight dual BSEP and MDR3 inhibition or testing drug candidates for inhibition of multiple efflux transporters (e.g., BSEP, MDR3, and MRPs) may be an effective strategy for prioritizing drug candidates with less likelihood of causing clinical DILI

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human

<p>1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug–drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment.</p> <p>2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC₅₀ values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH.</p> <p>3. In the absence of preincubation, the IC₅₀ values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC₅₀ were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem).</p> <p>4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.</p

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>_m) 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>_m ≈ 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

<i>In vitro</i> studies with two human organic anion transporters: OAT2 and OAT7

<p>1.Penciclovir, ganciclovir, creatinine, <i>para</i>-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7.</p> <p>2.For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS.</p> <p>3.Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC₅₀ of 17, 22, 23, 24, 35 and 586 μM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC₅₀ = 27 µM; >90% inhibition) by plated human hepatocytes (PHH).</p> <p>4.It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.</p