Identification of alcohol dehydrogenase (ADH) isoforms catalyzing hydroxyzine oxidation using recombinant human ADH: Serendipitous finding of potent ADH inhibition by organic solvents.

Hydroxyzine, an aliphatic alcohol, is metabolized to the carboxylic acid cetirizine via 2 sequential oxidation steps involving the formation of an aldehyde intermediate. The initial aldehyde formation is considered the rate-limiting step in cetirizine production and is suggested to be catalyzed by NAD+-dependent alcohol dehydrogenase (ADH), which oxidizes aliphatic alcohols. However, the specific ADH isoform(s) involved remain unidentified. Identifying these enzyme(s) is essential for understanding interindividual variability in drug pharmacokinetics, efficacy, and toxicity. The aim of this study was to identify the ADH isoforms responsible for catalyzing hydroxyzine aldehyde formation using recombinant human ADHs. Hydroxyzine aldehyde formation in the human liver S9 fraction was significantly higher when NAD+ was used as a cofactor than with NADP+ or NADPH, supporting the involvement of ADH. Among the recombinant hepatic human ADH isoforms (ADH1A, ADH1B, ADH1C, ADH4, and ADH5), all except ADH1A catalyzed hydroxyzine aldehyde formation. Furthermore, hydroxyzine aldehyde formation in human liver cytosol was significantly inhibited by 4-methylpyrazole, a specific inhibitor of ADH1, consistent with a predominant role for ADH1B and ADH1C. DMSO, used as the solvent for 4-methylpyrazole, strongly inhibited the hydroxyzine aldehyde formation, with IC50 values of 0.29% ± 0.056% for ADH1B and 0.06% ± 0.004% for ADH1C. Additional solvent screening revealed that even at a concentration of 0.5%, methanol and isopropanol also strongly inhibited ADH isoforms-excluding ADH5-when hydroxyzine or abacavir was used as the substrate. These findings highlight the potent inhibitory effects of DMSO and several other organic solvents on ADH activity. SIGNIFICANCE STATEMENT: Through the identification of alcohol dehydrogenase (ADH) isoforms catalyzing hydroxyzine aldehyde formation by recombinant human ADH, the study revealed that several common organic solvents strongly inhibit ADH1. This highlights the need to consider the unintended inhibitory effects of such solvents on ADH activity when solubilizing lipophilic compounds.