Abstract
Acyl glucuronides are intrinsically reactive metabolites of carboxylate drugs, capable of undergoing hydrolysis, intramolecular rearrangement (isomerization via acyl migration), and intermolecular transacylation reactions. Transacylation with nucleophilic groups located on protein molecules leads to covalent drug-protein adducts. Protein adducts can also form from the rearrangement isomers via a glycation mechanism. In this study, the isolated perfused rat liver preparation was used to separately trace the dispositions of the nonsteroidal anti-inflammatory drug diflunisal (DF), its reactive acyl glucuronide metabolite (DAG), and a mixture of DAG rearrangement isomers (iso-DAG), each administered at 30-μg DF equivalents/ml perfusate (four recirculating perfusions each group). After administration of DF, the drug was eliminated in a log linear manner over 3 hr, with apparent elimination half-life (t½) of 2.6 ± 0.4 hr. The sulfate conjugate (DS), excreted almost exclusively into perfusate, accounted for 14.2% of the dose, with the phenolic glucuronide (DPG) and DAG (11.1 and 7.9% of dose, respectively) excreted primarily in bile. Only a small portion (2.3%) of the dose was recovered as novel “diglucuronides” (D-2G, arising from phenolic glucuronidation of iso-DAG), excreted exclusively in bile. Covalent DF-protein adducts were found in both perfusate (0.98%) and liver (0.14%). After administration of DAG, rapid hydrolysis occurred (initial DAGt½ 17.3 ± 4.2 min). At 3 hr, recoveries (in comparison to DF-dosed perfusions) were similar for DF (51.7%) and DAG (8.3%), significantly decreased for DS (10.6%) and DPG (6.4%), and significantly increased for iso-DAG (0.8%), D-2G (9.1%), and covalent adducts in perfusate (1.49%) and liver (0.30%). After administration of iso-DAG, elimination from perfusate was slower (t½ 55 ± 15 min), and hydrolysis to DF was modest by comparison with DAG-dosed perfusions. Recoveries as iso-DAG and D-2G in bile were greatly enhanced (8.2 and 36.4%, respectively). Adduct formation was higher in liver (0.76% of dose) but not in perfusate (1.03%). Immunoblots of liver homogenates revealed drug-modified proteins at ca. 110 and 120 kDa. The results show that (a) DAG undergoes avid systemic deconjugation-conjugation cycling and isomerization to iso-DAG; (b) iso-DAG is more resistant to hydrolysis, is readily taken up by hepatocytes and undergoes novel metabolism (phenolic glucuronidation); and (c) the glycation pathway (i.e. using iso-DAG as substrate) plays a major role in formation of covalent DF-protein adducts in liver.
Footnotes
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Send reprint requests to: Dr. Ron Dickinson, Department of Medicine, Clinical Sciences Building, Royal Brisbane Hospital, Qld 4029 Australia.
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This work was supported by a project grant from the National Health and Medical Research Council of Australia.
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↵2 M. Wang and R.G. Dickinson, unpublished data.
- Abbreviations used are::
- DF
- diflunisal
- DAG
- diflunisal acyl glucuronide, iso-DAG, mixture of isomers of diflunisal acyl glucuronide formed by acyl migration
- DPG
- diflunisal phenolic glucuronide
- DS
- diflunisal sulfate
- D-2G
- mixture of phenolic glucuronides of isomers of diflunisal acyl glucuronide
- BSA
- bovine serum albumin
- ECL
- enhanced chemiluminescence
- TBS
- Tris-buffered saline
- SDS-PAGE
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Received May 27, 1997.
- Accepted October 7, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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