Glafenine (Privadol; 2,3-dihydroxypropyl 2-[(7-chloro-4-quinolinyl) amino]
benzoate) is a non-
narcotic analgesic agent widely used for the treatment of pains of various origins. Severe liver toxicity and a high incidence of
anaphylaxis were reported in patients treated with
glafenine, eventually leading to its withdrawal from the market in most countries. It is proposed that bioactivation of
glafenine and subsequent binding of reactive metabolite(s) to critical cellular
proteins play a causative role. The study described herein aimed at characterizing pathways of
glafenine bioactivation and the metabolic
enzymes involved. Two GSH conjugates of
glafenine were detected in human liver microsomal incubations using liquid chromatography tandem mass spectrometry. The structures of detected conjugates were determined as GSH adducts of 5-hydroxyglafenine (M3) and 5-hydroxy
glafenic acid (M4), respectively. GSH conjugation took place with a strong preference at C6 of the
benzene ring of
glafenine, ortho to the carbonyl moiety. These findings are consistent with a bioactivation sequence involving initial
cytochrome P450-catalyzed 5-hydroxylation of the
benzene ring of
glafenine, followed by two electron oxidations of M3 and M4 to form corresponding para-
quinone imine intermediates that react with GSH to form GSH adducts M1 and M2, respectively. Formation of M1 and M2 was primarily catalyzed by heterologously expressed recombinant
CYP3A4 and to a lesser extent,
CYP2C19 and
CYP2D6. We demonstrated that M3 can also be bioactivated by
peroxidases, such as
horseradish peroxidase and
myeloperoxidase. In summary, these findings have significance in understanding the bioactivation pathways of
glafenine and their potential link to mechanisms of toxicity of
glafenine.