Bioactivation of Glafenine by Human Liver Microsomes and Peroxidases: Identification of Electrophilic Iminoquinone Species and GSH Conjugates
ABSTRACT:
Glafenine (Privadol; 2,3-dihydroxypropyl 2-[7-chloro-4-quinoliny]aminolbenzoate) 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.
Introduction:
Glafenine (2,3-dihydroxypropyl 2-[7-chloro-4-quinoliny]aminolbenzoate; Scheme 1) is a non-narcotic analgesic agent widely used in the treatment of pains of various origins. Despite its therapeutic benefits, treatment with glafenine has been overshadowed by rare but severe incidences of hepatic injury as well as a high incidence of anaphylaxis, which eventually led to its withdrawal from the market in most countries. Although the mechanism of glafenine hepatotoxicity is not clearly understood, a probable causal link between glafenine use and the onset of hepatic injury has been established.
In humans, glafenine is rapidly absorbed after oral administration and undergoes extensive hepatic first-pass metabolism mainly by hydrolysis, aromatic hydroxylation, and N-oxidation. One of the primary biotransformation routes of glafenine is the carboxylesterase-catalyzed hydrolysis to the active metabolite glafenic acid (Scheme 1), which is potentially excreted in the urine as an acyl glucuronide conjugate. Glafenic acid was also detected as a major metabolite in plasma, with the ratio of Cmax glafenic acid to Cmax glafenine equal to 18.9 in healthy subjects. A dramatic decrease of first-pass effects was observed in cirrhotic subjects.
Of particular interest in the biotransformation pathways of glafenine in humans is the detection of 5-hydroxy metabolites of glafenine and glafenic acid. As depicted in Scheme 1, glafenine is an anthranilic acid ester derivative and contains a 7-chloroquinoline ring system. 5-Hydroxyglafenine or 5-hydroxy glafenic acid can undergo cytochrome P450 (P450)-mediated two-electron oxidations to form electrophilic quinone imine intermediates that are capable of reacting with cellular proteins and other nucleophiles such as GSH. However, to date, no such reactive intermediates of glafenine and/or their corresponding GSH conjugates has been reported, and the mechanism of bioactivation of glafenine remains unknown.
Materials and Methods:
Materials: The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): glafenine, HRP, MPO, H2O2 (30 wt. % in H2O), L-ascorbic acid, GSH, trichloroacetic acid, and NADPH. Pooled human liver microsomes and Supersomes containing cDNA-baculovirus-insect cell-expressed P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were obtained from BD Gentest (Woburn, MA).
Microsomal Metabolism: All incubations were performed at 37°C in a water bath. Pooled human liver microsomes and the human cDNA-expressed P450 isozymes were carefully thawed on ice before the experiment. Glafenine (10 and 50 μM) was mixed with human liver microsomal proteins (1 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4) supplemented with 1 mM GSH. The total incubation volume was 1 ml. After a 3-min preincubation at 37°C, the incubation reactions were initiated by the addition of 1 mM NADPH.
Results:
Glafenine Metabolites in Human Liver Microsomes: Incubation of glafenine in human liver microsomes with GSH and NADPH generated several products. The major in vitro metabolite was M5 on the basis of the UV chromatogram (Fig. 1). The MS spectrum of M5 showed an [M + H]+ ion at m/z 299. Upon further fragmentation of an ion at m/z 281, the [M + H − H2O]+ ion lost CO (−28) and Cl (−35) to yield ions at m/z 253 and 246, respectively.
The MS spectrum of M3 revealed an [M + H]+ ion at m/z 389 with a chlorine isotope peak at m/z 391, suggesting that M3 is a mono-oxygenated metabolite of glafenine. Fragmentation of the ion at m/z 389 generated product ions at m/z 371, 315, and 297. The MS3 mass spectrum of the product ion of M3 at m/z 297 afforded several fragment ions at m/z 269, 262, and 162. The product ion at m/z 162 suggested that hydroxylation occurred at the benzene ring of glafenine instead of the 7-chloroquinoline ring.
Discussion:
The results from the investigation presented here constitute the first report on the P450- and peroxidase-catalyzed bioactivation of the non-narcotic analgesic glafenine. Two novel GSH conjugates of glafenine, M1 and M2, were formed in the human liver microsomal incubations and characterized by LC/MS/MS and/or NMR experiments. Formation of M1 and M2 was mediated primarily by CYP3A4 relative to other major P450 isoforms. It was found that the 5-hydroxyglafenine metabolite, but not glafenine itself, was oxidized and bioactivated by peroxidases, such as HRP and MPO. These findings are of importance to understand the bioactivation pathways of glafenine and potential links to its mechanism of toxicity.
In conclusion, we found that glafenine undergoes P450-mediated 5-hydroxylation and is subsequently involved in bioactivation and formation of glafenine GSH conjugates in human liver microsomes. GSH addition occurred specifically at the C6 ortho to the carbonyl moiety, presumably because of electronic effects. Upon 5-hydroxylation, M3 was oxidized/bioactivated by P450 enzymes and peroxidases such as HRP and MPO. In summary, findings from this study are of significance in understanding of the bioactivation pathways of glafenine and their potential link to mechanisms of toxicity of glafenine.