Studies on the reactivity of acyl glucuronides--V. Glucuronide-derived covalent binding of diflunisal to bladder tissue of rats and its modulation by urinary pH and beta-glucuronidase

Abstract

Acyl glucuronide conjugates of acidic drugs have been shown to be reactive metabolites capable of undergoing non-enzymic hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions with plasma protein. In an earlier study (King and Dickinson, Biochem Pharmacol 45: 1043-1047, 1993), we documented formation of covalent adducts of diflunisal (DF), a salicylate derivative which is metabolized in part to a reactive acyl glucuronide (DAG), with liver, kidney, skeletal muscle and small and large intestine (in addition to plasma protein) of rats given the drug i.v. twice daily at 50 mg DF/kg for 7 days. The present study shows that covalent adducts of DF were also formed with urinary bladder tissue of these rats, achieving concentrations (ca. 5 micrograms DF equivalents/g tissue) higher than those found in the other tissues noted above. After cessation of dosing, the adduct concentrations declined with an apparent T 1/2 value of ca. 20 hr. Adducts were also formed ex vivo in excised rat bladders in which DAG or a prepared mixture of its acyl migration isomers (iso-DAG) were incubated at pH 5.0, 6.5 and 8.0. After 8 hr incubation, the highest concentrations (ca. 11 micrograms DF equivalents/g) were produced with iso-DAG at pH 5.0, and the lowest (ca. 2.3 micrograms DF equivalents/g) with DAG at pH 5.0. However, a major competing reaction for DAG (at least at pH 5.0) was hydrolysis by beta-glucuronidases originating from bladder tissue. By contrast, iso-DAG was quite resistant to such hydrolysis. The phenolic glucuronide conjugate, another important metabolite of DF, was hydrolysed only slowly. Similar results were obtained in fresh rat urine adjusted to pH 5.0. The results support covalent DF adduct formation in rat bladder originating from both DAG and iso-DAG as ultimate reactants, though the extent of binding is modulated by both urinary pH and beta-glucuronidases.