To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an experimental model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n ‫؍‬ 5) or together with single-dose ketoconazole (600 mg) (n ‫؍‬ 6). The prodrug (n ‫؍‬ 2) and its active metabolite (n ‫؍‬ 2) were also administered intravenously to provide reference doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concentrations in the bile with low variability (Ae bile ‫؍‬ 53 ؎ 6% of the enteral dose), showing that the compound had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concentration-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metabolism. Reduced metabolism also seemed to explain the increased plasma exposure of the active compound because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion experiments for AR-H067637 in pig liver microsomes with and without the addition of ketoconazole.

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.

The effect of a single intrajejunal dose of gemfibrozil (600 mg) on the plasma pharmacokinetics and biliary excretion of a single intrajejunal dose of rosuvastatin (20 mg) was investigated by using a multichannel catheter positioned in the distal duodenum-proximal jejunum in 8 healthy volunteers. Bile and plasma samples were collected every 20 minutes for 200 minutes, with additional plasma samples being drawn for up to 48 hours. Gemfibrozil did not affect the bioavailability of rosuvastatin, although it increased the apparent absorption phase during the initial 200 minutes (AUC(plasma,200min)) by 1.56-fold (95% confidence interval, 1.14-2.15). The interaction was less pronounced in this single-dose study than in a previous report when gemfibrozil was administered repeatedly; nevertheless, the interaction coincided with the highest exposure to gemfibrozil. The plausible reason why the interaction in this investigation was only minor is the low exposure to gemfibrozil (and its metabolites), suggesting that the total plasma concentration of gemfibrozil needs to be above 20 µM to affect the disposition of rosuvastatin. This study demonstrates the value of monitoring the plasma pharmacokinetics of the inhibitor, and not only the drug under investigation, to improve the mechanistic interpretation.

The biliary excretion of the oral thrombin inhibitor ximelagatran and its metabolites was investigated by using duodenal aspiration in healthy volunteers following intraintestinal dosing. In the first investigation, radiolabeled [(14)C]ximelagatran was administered, enabling quantification of the biliary excretion and identification of metabolites in the bile. In the second study, the effect of erythromycin on the biliary clearance of ximelagatran and its metabolites was investigated to clarify the reported ximelagatran-erythromycin interaction. Approximately 4% of the intraintestinal dose was excreted into bile with ximelagatran and its active form, melagatran, being the most abundant compounds. Four novel ximelagatran metabolites were identified in bile (<0.1% of dose). Erythromycin changed the pharmacokinetics of ximelagatran and its metabolites, with an elevated ximelagatran (78% increase), OH-melagatran (89% increase), and melagatran (86% increase) plasma exposure and higher peak plasma concentrations of the compounds being measured. In parallel, the biliary clearance was moderately reduced. The results suggest that inhibition of hepatobiliary transport is a likely mechanism for the interaction between erythromycin and ximelagatran. Furthermore, the study demonstrated the value of direct bile sampling in humans for the identification of primary biliary metabolites.