In vitro and in vivo studies on acyl-coenzyme A-dependent bioactivation of zomepirac in rats.
Zomepirac [ZP, 5-(chlorobenzoyl)-1,4-dimethylpyrrole-2-acetic acid] was withdrawn from the market because of unpredictable allergic reactions that may have been caused by ZP-protein adducts formed by reaction of the reactive acyl glucuronide of ZP (ZP-O-G) with endogenous proteins. To test the hypothesis that the reactive ZP acyl coenzyme A thioester (ZP-CoA) was formed and potentially could contribute to formation of ZP-protein adducts, we investigated the acyl CoA-dependent metabolism of ZP in freshly isolated rat hepatocytes (1 mM) and in vivo (100 mg ZP/kg, ip) in rat livers (2 h after dose administration), rat bile (0-4 h), and rat urine (0-24 h). ZP-CoA was detected in freshly isolated hepatocytes and in vivo in rat livers by LC/MS/MS. In addition, the ZP glycine conjugate (ZP-Gly) and ZP taurine conjugates (ZP-Tau) were identified by LC/MS/MS in rat hepatocytes and in vivo in rat livers, rat urine, and rat bile. The identities of ZP-CoA, ZP-Gly, and ZP-Tau were confirmed by comparison of retention times and MS/MS spectra with those of authentic standards. Moreover, the ZP acyl carnitine ester was detected in rat urine and rat bile based upon (i) the chlorine isotope pattern, (ii) MS/MS spectra showing significant ions characteristic for carnitine (m/z 60, 144 and loss of m/z 59) and ZP (m/z 139), and (iii) accurate mass measurements with a mass accuracy of 0.2 ppm. ZP-CoA serves as an obligatory intermediate in the formation of ZP-Gly, ZP-Tau, and ZP carnitine ester, and it is therefore of mechanistic significance that these conjugates were identified. Finally, time-dependent concentration profiles obtained in experiments with rat hepatocytes and in vivo from quantitative analysis of rat livers indicate that ZP-CoA, in addition to ZP-O-G, may contribute to formation of the potentially toxic covalent ZP-protein adducts.
Olsen
Jørgen
J
Department of Analytical Chemistry, The Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark. jorgen.olsen@astrazeneca.com
Li
Chunze
C
Bjørnsdottir
Inga
I
Sidenius
Ulrik
U
Hansen
Steen Honoré
SH
Benet
Leslie Z
LZ
D000105:Acetyl Coenzyme A / Q000502:physiology*; D000818:Animals; D001711:Biotransformation; D002331:Carnitine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013654:Taurine / Q000378:metabolism; D014046:Tolmetin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity
D013654:Taurine; D000105:Acetyl Coenzyme A; C020549:zomepirac; D014046:Tolmetin; D002331:Carnitine; D005998:Glycine