The utility of the bile-exteriorized rat as a source of reactive acyl glucuronides: studies with zomepirac.

Acyl glucuronide conjugates of acidic drugs are chemically unstable metabolites, able to undergo a number of reactions including covalent binding interactions with proteins. The question of whether any toxicological or immunological responses result from such covalent modification of native proteins in vivo is topical. Study of acyl glucuronide reactivity thus requires a convenient source of these metabolites. The utility of the bile-exteriorized rat for this purpose is highlighted herein using the formerly marketed nonsteroidal antiinflammatory agent zomepirac. Zomepirac was injected i.v. at 60 mg/kg four times into bile-exteriorized rats at 6-h intervals. The 24-h bile samples contained ca. 24% of zomepirac doses as zomepirac acyl glucuronide (ZAG). Purification was achieved by washing of the acidified bile with etherhexane, extraction into ethyl acetate, semipreparative HPLC, and crystallization. Overall recovery through the purification procedure was ca. 50%. Identity as ZAG was confirmed by mass spectrometry. The approach takes advantage of the robust glucuronidation capacity of the rat, especially at higher drug doses, and of its ability to preferentially excrete hepatically formed drug glucuronides into bile rather than into urine via blood. Prior to this work, ZAG was presumed to be only a minor metabolite of zomepirac in rats, based on early urinary recovery studies. Thus, measurement of urinary acyl glucuronide conjugates in the rat may severely underestimate their true formation in this species.