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

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.