Abstract

Many carboxylic acid-containing drugs undergo conjugation with D-glucuronic acid in humans, leading to the formation of acyl glucuronides, which are excreted into urine. However, these metabolites can be hydrolyzed back to the parent aglycon; this reaction can be accelerated by human serum albumin (HSA). Although this phenomenon of interaction between the acyl glucuronide and HSA has been described for various drugs, the kinetics of the protein have not been characterized. The aim of this study was to investigate the HSA-mediated mechanism involved in the in vitro hydrolysis by albumin of the acyl glucuronides of (R)- and (S)-ketoprofen (a nonsteroidal anti-inflammatory drug), as model compounds. The conjugates of both ketoprofen enantiomers were incubated, separately or together, with increasing concentrations of albumin (14.5-145 microM) at pH 7.4 and 37 degrees. The reaction followed Michaelis-Menten kinetics and was stereoselective; the (R)-ketoprofen glucuronide was a better substrate than the S-conjugate. To identify the HSA domain involved in the hydrolysis reaction, specific probes of HSA binding sites were used as potential inhibitors. These probes, added at an equimolar probe/glucuronide ratio (145 microM), slightly decreased the hydrolysis (by up to 30%). They affected the reversible binding of (R)-ketoprofen glucuronide to HSA, as shown by CD studies. Because iodoacetic acid did not modify the single free cysteine residue on HSA, this amino acid residue cannot be the reactive one. In addition, the chemical modification of a single tyrosine residue (probably Tyr-411) on HSA by diisopropyl fluorophosphate significantly but weakly affected the hydrolysis of (R)-ketoprofen glucuronide, suggesting that this residue also is not involved in the catalysis. In contrast, the R-conjugate was not bound to modified albumin, as revealed in CD experiments. These results support the existence of distinct sites on HSA for reversible binding and hydrolysis of (R)-ketoprofen glucuronide.