Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity

Abstract

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in human. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the inter-individual variation in one of the four model reaction activities, i.e., ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 g microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.

Cytochrome P450 Enzymes and Drug Metabolism in Humans

“…CYPs play important roles in the maintenance of general human health, particularly as they relate to the metabolism of pharmaceuticals ( Supplementary Table S1, see Supplementary Materials ). Of salient interest for CYPs in drug metabolism is the varying response of individual patients to administered pharmaceuticals [ 16 ]. It is known that some individuals metabolize drugs relatively rapidly, while other individuals metabolize the same drugs relatively slowly [ 17 ].…”

Effects of ginkgo leaf tablet on the pharmacokinetics of rosiglitazone in rats and its potential mechanism

“…One of the most critical biological processes impacting the blood concentration of drugs in vivo is the intrinsic metabolic clearance mediated by the hepatic CYP450 enzymes (Sathyanarayanan et al. 2020 ). Drugs are mainly used as substrates, inhibitors, or inducers of CYP450.…”

Microfluidic systems in clinical diagnosis

“…Enzyme activity is a common analysis in clinical diagnosis. A recent example was reported by Sathyanarayanan et al [69] with the assessment of liver function and drug clearance by analyzing liver biopsy samples. The target of this system was the enzyme hepatic cytochrome P450 (CYP), which is a major player in the elimination of clinical drugs in the body.…”

“…The metabolic activity of CYP has also been studied in devices aimed to reproduce the metabolic dynamics of the human body [70]. The goal of this system [69] was to provide a personalized quantification of CYP activity per patient, allowing then for personalized and accurate dosing of necessary drugs. Patients can be slow metabolizers or rapid metabolizers, and drug dosage has to be adjusted accordingly to maintain an adequate concentration of the drug in the blood.…”