Influence of structural variations in peptidomimetic 4-amidinophenylalanine-derived thrombin inhibitors on plasma clearance and biliary excretion in rats

Purpose. Systemic and hepato-biliary clearance of peptidomimetic thrombin inhibitors of the 4-amidinophenylalanine amide-type, derived from NAPAP (Nalpha-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) by substituting Gly in P2 for natural and unnatural amino acids or by varying the C- and N-terminal moieties, resp., were investigated. Methods. Concentrations of the compounds administered as intravenous bolus injection at a dose of 1 mg/kg to bile duct-cannulated rats were determined in plasma and bile samples collected over 4 hours using reversed-phase HPLC. Results. NAPAP and the derivatives with additional charged groups are comparatively hydrophilic compounds. For NAPAP and most of the derivatives the biliary clearance accounted for a high percentage of the rapid systemic plasma clearance. Derivatives 2a-c with a second basic group in P2 position showed lower systemic and biliary clearance compared to NAPAP, whereas their cumulative biliary excretion after a period of 120 min was less affected. Bis-benzamidine derivatives 4a and 5 with the second amidino group in the N-terminal moiety had the lowest biliary clearance. Additional carboxylic groups reduced the systemic and biliary clearance only as free amidinophenylalanine carboxyl in 3a and 5. No influence compared to NAPAP was observed for 2d with a free carboxyl group in P2 position. Conclusions. The weak correlation of the log P values of the compounds with the clearance parameters indicates the influence of structural variations, especially of charged groups, in this series of compounds rather than overall lipophilicity on hepato-biliary elimination mediated by hepatocellular transporters.