Combined in Vitro–in Vivo Approach To Assess the Hepatobiliary Disposition of a Novel Oral Thrombin Inhibitor

Abstract

Two clinical trials and a large set of in vitro transporter experiments were performed to investigate if the hepatobiliary disposition of the direct thrombin inhibitor prodrug AZD0837 is the mechanism for the drug-drug interaction with ketoconazole observed in a previous clinical study. In Study 1, [(3)H]AZD0837 was administered to healthy male volunteers (n = 8) to quantify and identify the metabolites excreted in bile. Bile was sampled directly from the jejunum by duodenal aspiration via an oro-enteric tube. In Study 2, the effect of ketoconazole on the plasma and bile pharmacokinetics of AZD0837, the intermediate metabolite (AR-H069927), and the active form (AR-H067637) was investigated (n = 17). Co-administration with ketoconazole elevated the plasma exposure to AZD0837 and the active form approximately 2-fold compared to placebo, which may be explained by inhibited CYP3A4 metabolism and reduced biliary clearance, respectively. High concentrations of the active form was measured in bile with a bile-to-plasma AUC ratio of approximately 75, indicating involvement of transporter-mediated excretion of the compound. AZD0837 and its metabolites were further investigated as substrates of hepatic uptake and efflux transporters in vitro. Studies in MDCK-MDR1 cell monolayers and P-glycoprotein (P-gp) expressing membrane vesicles identified AZD0837, the intermediate, and the active form as substrates of P-gp. The active form was also identified as a substrate of the multidrug and toxin extrusion 1 (MATE1) transporter and the organic cation transporter 1 (OCT1), in HEK cells transfected with the respective transporter. Ketoconazole was shown to inhibit all of these three transporters; in particular, inhibition of P-gp and MATE1 occurred in a clinically relevant concentration range. In conclusion, the hepatobiliary transport pathways of AZD0837 and its metabolites were identified in vitro and in vivo. Inhibition of the canalicular transporters P-gp and MATE1 may lead to enhanced plasma exposure to the active form, which could, at least in part, explain the clinical interaction with ketoconazole.

Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

“…However, a survey of the UW DIDB showed that of these transporters, only MATE1 has been mentioned as possibly being inhibited to a clinically significant extent by ketoconazole and only hepatic canalicular MATE1 inhibition is implicated in the interaction between ketoconazole and atecegaran metoxil (AZD0837) (Matsson et al, 2013;University of Washington, 2015). Ritonavir and itraconazole had unbound C max /IC 50 values .0.1 only for OCT1, whereas clarithromycin did not have any values .0.1.…”

“…Furthermore, even for those CYP3A4/5 inhibitors evaluated extensively in vitro, there are large interlaboratory differences in reported IC 50 values that are dependent on test system, assay conditions, and probe substrates, among other factors. For instance, the reported range of IC 50 values for ketoconazole varies by 37-fold and 6.4-fold for the inhibition of P-gp and OATP1B1, respectively (Gnoth et al, 2011;Izumi et al, 2013;Matsson et al, 2013;Mikkaichi et al, 2014). Similarly, IC 50 values for ritonavir vary by 85-fold and 117-fold for the inhibition of P-gp and OATP1B1, respectively (Keogh and Kunta, 2006;Herédi-Szabó et al, 2013;Izumi et al, 2013).…”

Exposure–response for biomarkers of anticoagulant effects by the oral direct thrombin inhibitor AZD0837 in patients with atrial fibrillation

“…Verapamil is known to have weaker inhibitory effects on transporters. Similar to the verapamil effect on AZD0837 (33% increase), increases in exposure to the active form of dabigatran have also been observed in AF patients receiving verapamil (23%) and amiodarone (12%), which both are inhibitors of the Pgp transport protein [20]. TG and fibrin D-dimer were used as biomarkers to investigate the anticoagulant effect of AZD0837 and VKA treatments.…”

“…Inhibition of biliary excretion, shown to occur mainly for the active form and mediated by transporter proteins, was suggested to explain a pharmacokinetic interaction with ketoconazole that increased plasma exposure of the active form by approximately two-fold [20]. The active form of AZD0837 was found to be a substrate of both P-glycoprotein (P-gp) and the multidrug and toxin extrusion 1 (MATE1) transporter proteins, which were inhibited by ketoconazole.…”

“…Simulated median steady-state plasma concentration profiles (solid lines) with the 10th and 90th percentiles (shaded area) to illustrate the reference range for a typical AF patient receiving 300 mg once daily and 200 mg twice daily doses Exposure-response of AZD0837 anticoagulant effects in atrial fibrillation AZD0837. Inhibition of biliary excretion, shown to occur mainly for the active form and mediated by transporter proteins, was suggested to explain a pharmacokinetic interaction with ketoconazole that increased plasma exposure of the active form by approximately two-fold [20]. The active form of AZD0837 was found to be a substrate of both P-glycoprotein (P-gp) and the multidrug and toxin extrusion 1 (MATE1) transporter proteins, which were inhibited by ketoconazole.…”