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

Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5′-Adenosine Monophosphate-Activated Protein Kinase (AMPK)

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (<b>1</b>) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds <b>10</b> (PF-06679142) and <b>14</b> (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3

Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5′-Adenosine Monophosphate-Activated Protein Kinase (AMPK)

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (<b>1</b>) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds <b>10</b> (PF-06679142) and <b>14</b> (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3