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

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide–Peptoid Hybrid CXCR7 Modulators

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide–peptoid hybrids with high CXCR7 binding affinities (<i>K</i>_i < 100 nM) and measurable passive permeability (<i>P</i>_app > 5 × 10^–6 cm/s). Moreover, bioactive peptide <b>25</b> (<i>K</i>_i = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration