Effects of Ketoconazole on the In Vivo Biotransformation and Hepatobiliary Transport of the Thrombin Inhibitor AZD0837 in Pigs

Abstract

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.

Metabolic and Pharmaceutical Aspects of Fluorinated Compounds

“…The OCHF 2 motif has been suggested to offer consistent improvements in several metrics, including lower log D and increased passive cellular permeability relative to OCF 3 and slightly lower rates of oxidative metabolism compared with OCH 3 . Examples of clinically evaluated compounds incorporating metabolically stable OCHF 2 substituents are the β-secretase inhibitor 248 ; the antifibrotic agent FT061 ( 250 ), an analogue of the antiallergic drug tranilast ( 249 ) that was effective in a rodent model of diabetic nephropathy; and AZD0837 ( 251 ), the prodrug of a direct inhibitor of thrombin that was evaluated clinically as a treatment for venous thromboembolism. − Additional examples include pantoprazole ( 252 ), an irreversible proton-pump inhibitor marketed for the treatment of gastroesophageal reflux disease (GERD), CHF 6001 ( 253 ), an inhibitor of phosphodiesterase-4 (PDE-4) that was of interest as a potential treatment for pulmonary inflammation, and the long acting PDE-4 inhibitor roflumilast ( 254 ) that is approved to treat chronic obstructive pulmonary disease. − Roflumilast is metabolized to the pyridine N -oxide, an equally active metabolite that is believed to account for 90% of the PDE-4 inhibitory activity of the drug in vivo. The increased electronegativity associated with the OCHF 2 substituent of 248 resulted in a reduction in P-glycoprotein-mediated efflux across the blood–brain barrier, enhancing brain penetration for this centrally acting compound .…”

“…In light of these issues, intratracheal/intranasal administration of CHF 6001 ( 253 ) was explored as a treatment for inflammatory pulmonary disorders, and despite extensive metabolism of the compound in rats prior to disposition, the difluoromethoxy group of [ 14 C]- 253 remained intact in all metabolites identified in the urine and feces . Similarly, metabolite profiling studies of AZD0837 ( 251 ) and pantoprazole ( 252 ) in vivo were completed without any report of biotransformation of the OCHF 2 moieties of these compounds. , Substitution with OCHF 2 was explored further during an effort to produce analogues of the antifibrotic agent tranilast ( 249 ) with improved pharmaceutical properties . While this initiative was able to produce a candidate ( 250 ) with lower clearance in vivo and no associated increase in the volume of distribution, the improvement in pharmacokinetics was ascribed to an increase in the extent of protein binding that helped to sequester the compound from drug-metabolizing enzymes.…”

“…244 Similarly, metabolite profiling studies of AZD0837 (251) and pantoprazole (252) in vivo were completed without any report of biotransformation of the OCHF 2 moieties of these compounds. 242,243 Substitution with OCHF 2 was explored further during an effort to produce analogues of the antifibrotic agent tranilast (249) with improved pharmaceutical properties. 241 While this initiative was able to produce a candidate (250) with lower clearance in vivo and no associated increase in the volume of distribution, the improvement in pharmacokinetics was ascribed to an increase in the extent of protein binding that helped to sequester the compound from drug-metabolizing enzymes.…”

Determination of the thrombin inhibitor AZD0837 and its metabolites in human bile using mixed mode solid phase extraction and LC–MS/MS

“…The method for the determination of AZD0837 and its two metabolites in pig bile was based on a simple dilution of the bile sample followed by a chromatographic method optimized to separate the analytes from the interfering bile components on the LC column [6]. When this method was tested on human bile samples, the chromatogram obtained from a blank bile samples seemed to be free from interfering substances at the retention times of the three analytes.…”

“…Methods for AZD0837 and its two metabolites have earlier been developed for the determination of these analytes in human [3] and animal [6] plasma and pig bile [6].…”