RESUMO
Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are broadly applicable to the characterization of in vitro and in vivo models, in vitro to in vivo extrapolation (IVIVE), and interindividual variability prediction. However, the emerging need of DMET quantification in small sample volumes such as organ-on a chip effluent, organoids, and biopsies requires ultrasensitive protein quantification methods. We present an ultrasensitive method that relies on an optimized sample preparation approach involving acetone precipitation coupled with a microflow-based liquid chromatography-tandem mass spectrometry (µLC-MS/MS) for the DMET quantification using limited sample volume or protein concentration, i.e., liver tissues (1-100 mg), hepatocyte counts (~4000 to 1 million cells), and microsomal protein concentration (0.01-1 mg/ml). The method was applied to quantify DMETs in differential tissue S9 fractions (liver, intestine, kidney, lung, and heart) and cryopreserved human intestinal mucosa (i.e., CHIM). The method successfully quantified >75% of the target DMETs in the trypsin digests of 1 mg tissue homogenate, 15,000 hepatocytes, and 0.06 mg/ml microsomal protein concentration. The precision of DMET quantification measured as the coefficient of variation across different tissue weights, cell counts, or microsomal protein concentration was within 30%. The method confirmed significant extrahepatic abundance of non-cytochrome P450 enzymes such as dihydropyridine dehydrogenase (DPYD), epoxide hydrolases (EPXs), arylacetamide deacetylase (AADAC), paraoxonases (PONs), and glutathione S-transferases (GSTs). The ultrasensitive method developed here is applicable to characterize emerging miniaturized in vitro models and small volume biopsies. In addition, the differential tissue abundance data of the understudied DMETs will be important for physiologically-based pharmacokinetic (PBPK) modeling of drugs.
Assuntos
Preparações Farmacêuticas , Espectrometria de Massas em Tandem , Cromatografia Líquida , Hepatócitos , Humanos , Proteínas de Membrana TransportadorasRESUMO
1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.