The metabolic activation of abacavir by human liver cytosol and expressed human alcohol dehydrogenase isozymes

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Abstract

Abacavir (ZIAGEN®) is a reverse transcriptase inhibitor marketed for the treatment of HIV-1 infection. A small percentage of patients experience a hypersensitivity reaction indicating immune system involvement and bioactivation. A major route of metabolism for abacavir is oxidation of a primary βγ unsaturated alcohol to a carboxylic acid via an aldehyde intermediate. This process was shown to be mediated in vitro by human cytosol and NAD, and subsequently the αα and γ2γ2 human isoforms of alcohol dehydrogenase (ADH). The αα isoform effected two sequential oxidation steps to form the acid metabolite and two isomers, qualitatively reflective of in vitro cytosolic profiles. The γ2γ2 isozyme generated primarily an isomer of abacavir, which was minor in the αα profiles. The aldehyde intermediate could be trapped in incubations with both isozymes as an oxime derivative. These metabolites can be rationalized as arising via the aldehyde which undergoes isomerization and further oxidation by the αα enzyme or reduction by the γ2γ2 isozyme. Non-extractable abacavir protein residues were generated in cytosol, and with αα and γ2γ2 incubations in the presence of human serum albumin (HSA). Metabolism and residue formation were blocked by the ADH inhibitor 4-methyl pyrazole (4-MP). The residues generated by the αα and γ2γ2 incubations were analyzed by SDS-PAGE with immunochemical detection. The binding of rabbit anti-abacavir antibody to abacavir-HSA was shown to be dependent on metabolism (i.e. NAD-dependent and 4-MP sensitive). The mechanism of covalent binding remains to be established, but significantly less abacavir-protein residue was detected with an analog of abacavir in which the double bond was removed, suggestive of a double bond migration and 1,4 addition process.

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.

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