The metabolic activation of abacavir by human liver cytosol and expressed human alcohol dehydrogenase isozymes
Introduction
Adverse drug reactions represent an area of increasing clinical concern for both prescribing physicians and pharmaceutical companies [1], [2]. Approximately 25% of these are designated as idiosyncratic, or hypersensitivity reactions, in that they do not occur in most patients, and do not involve the known pharmacological properties of the drug [3]. There is substantial evidence of immune system mediation, which in turn may be initiated through metabolic activation of the drug to reactive species which covalently bind to proteins [4], [5].
Abacavir (ZIAGEN®, Fig. 1) is a nucleoside reverse transcriptase inhibitor marketed in 1999 for the treatment of HIV-1 infection. Approximately 4% of patients experience a hypersensitivity reaction. The symptoms are varied, but fever and rash are the most common [6], and a small number of fatalities have also been reported. The clinical presentation is consistent with immune mediation, and hence bioactivation is likely to play a role in its origination.
As part of broader investigations to understand the etiology of this hypersensitivity, metabolic investigations were performed to identify potential bioactivation pathways for abacavir, and the enzyme systems involved. While many factors are likely to play a role in idiosyncratic toxicities [7], genetic polymorphisms in drug metabolizing enzymes are factors that can potentially be identified.
The major routes of metabolism of abacavir in humans are conjugation to an ether glucuronide, and oxidation to a carboxylic acid metabolite designated as 2269W93 [8], (Fig. 1). Approximately 15% of the dose is comprised of a number of minor metabolites, but from structural considerations, none of those identified to date indicate the potential for reactive intermediates. In contrast, the metabolism of abacavir to 2269W93 involves a two step oxidation process via an aldehyde intermediate. This aldehyde has not been observed directly as a metabolite of abacavir, and attempts to synthesize it have been unsuccessful due to its apparent instability.
A number of aldehyde metabolites have been previously implicated as reactive and capable of covalent binding to proteins, and this in turn has been suggested to underlay clinical adverse events of the parent compounds. In these cases, the underlying reactivity can be ascribed to one of two mechanisms: Schiff base formation, or a 1,4 addition process. Schiff base formation has been proposed for the aldehyde metabolite of Sorbinil, which shows an incidence of hypersensitivity [9], and for ethanol, in which acetaldyde formation is proposed to underlay the immune hepatitis observed with chronic alcohol consumption [10]. The reversibility of this Schiff base formation has been proposed to be limited either by reduction by ascorbate [11], or for acetaldehyde, cyclization with an amide nitrogen of the protein to form a stable imidazolidinone [12]. Thus mechanisms may exist in vivo for the stabilization of these somewhat unstable adducts. A more widely encountered mechanism is observed for αβ unsaturated aldehydes (Michael type acceptors) and numerous examples exist in the literature of proposals for this type of reactive intermediate being associated with covalent binding and adverse clinical events [13], [14], [15].
As discussed below, both mechanisms might be possible for abacavir. The purposes of the studies described here were to utilize in vitro methods to determine if oxidation of abacavir to the carboxylic acid via an aldehyde intermediate could lead to protein covalent binding, to investigate the mechanism, and to identify the enzyme systems involved.
Section snippets
Chemicals
Human liver microsomes and cytosol were obtained from Xenotech, (Kansas City, KS). NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NAD, 4-MP, methoxylamine, horse liver alcohol dehydrogenase (HLADH), HSA, triethylamine, toluene, isobutylchloroformate, and 7-ethoxycoumarin were obtained from Sigma-Aldrich (St Louis, MO). Laemmli sample buffer was obtained from Bio-Rad (Hercules, CA). Ultima-Flo M was obtained from Packard (Meriden, CT). Gelcode Blue Reagent, SuperBlock Blocking
Microsomal and cytosol incubations
All incubations were performed at physiologically relevant concentrations (∼clinical Cmax). No metabolites were detected in incubations of 14C-abacavir with human liver microsomes at pH 7.4 from 1–20 h. In the human liver cytosolic incubations containing NAD, polar metabolites were detected at 20 h at pH 7.4, including a peak of the same retention time as the major acid metabolite observed in vivo, 2269W93 (Fig. 2). Cytosolic NAD-dependent oxidation of alcohols is indicative of ADH involvement.
Discussion
The metabolism of primary alcohols to carboxylic acids is most commonly mediated by cytosolic ADH or microsomal CYP450 [19], [20]. No metabolites could be detected radiochemically in incubations of abacavir with pooled human liver microsomes, but good conversion to the acid metabolite was observed with human liver cytosol. An aldehyde would be the initial product of such metabolism, and the formation of the acid directly in cytosol might involve a role for aldehyde dehydrogenase. However, ADH
Acknowledgements
Abacavir conjugation to KLH was performed by J. Sailstad. Characterization of human in vivo metabolites was performed by L. St.John. Protein sequence analysis of the αα and γγ isoforms was performed by M. Moyer. We are indebted to Dr T. Hurley, University of Indiana Medical School, for providing the human ADH isozymes.
References (35)
- et al.
Drug-Protein conjugates. XVI. Studies of sorbinil metabolism; formation of 2-hydroxysorbinil and unstable protein conjugates
Biochem. Pharmacol.
(1988) - et al.
The formation and stability of imidazolidinone adducts from acetaldehyde and model peptides. A kinetic study with implications for protein modification in alcohol abuse
Biochem. Pharmacol.
(1996) - et al.
Cytotoxicity of alkylating agents in isolated rat kidney proximal tubular and distal tubular cells
Arch. Biochem. Biophys.
(1991) - et al.
Metabolism of 4-hydroxynonenal a cytotoxic product of lipid peroxidation in rat precision cut liver slices
Toxicol. Lett.
(2000) - et al.
Expression and kinetic characterization of variants of human β1β1 alcohol dehydrogenase containing substitutions at amino acid 47
J. Biol. Chem.
(1990) - et al.
The role of liver alcohol dehydrogenase isozymes in the oxidation of glycol ethers in male and female rats
Toxicol. Appl. Pharmacol.
(1998) - et al.
Aldehyde dismutase activity of human liver alcohol dehydrogenase
FEBS Lett.
(1996) Determination of hemoglobin adducts from aldehydes formed during lipid peroxidation in vitro
Chem-Biol. Interactions
(1992)- et al.
In vitro analysis of metabolic predispositon to drug hypersensitivity reactions
Clin. Exp. Immunol.
(1995) - et al.
The immunological and metabolic basis of drug hypersensitivites
Ann. Rev. Pharmacol.
(1998)