Determination of the epimeric composition of ibuprofenyl-CoA

Abstract

Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of ( R)-, ( S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C 18 reverse-phase column was developed to quantitate “total” ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP α 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing ( R)- and ( S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for ( R)- and ( S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70–80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.