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Customized Micro Total Analysis Systems to Study Human Phase I Metabolism

Final Report Summary - CUMTAS (Customized Micro Total Analysis Systems to Study Human Phase I Metabolism)

The CUMTAS project focused on development of low-cost microfluidic technology for rapid screening of molecular-level metabolic interactions associated with cytochrome P450 (CYP) enzyme mediated drug elimination. CYP system is the main detoxification mechanism for both therapeutic drugs and many other chemical additives for which we are continuously exposed to. Since the majority of these compounds are eliminated via only a handful of different CYP isoenzymes, the body's drug elimination capacity is easily interfered by other intentionally (e.g. nutrients) or unintentionally (e.g. preservatives) uptaken chemicals, which may lead to suboptimal drug levels in vivo. The chemicals may either inhibit each other's elimination leading to accumulation of therapeutic drugs in the body so that they exceed toxic levels, or they may induce the expression of more enzymes leading to too fast drug elimination and thus loss of efficacy. In addition to other chemicals, also genetic factors as well as age, sex or disease state (e.g. inflammation) may alter the elimination speed of therapeutic drugs. In this project, two different concepts, including paper microfluidic lateral flow assays and droplet-based assays, feasible for direct determination of the CYP enzyme activity of liver enzymes in ultimately small volumes were developed with a view to enabling, at best, determination of personalized drug clearance capacity in the human body. In addition, through-flow immobilized enzyme assays were developed and thoroughly validated for more in-depth study of the mechanistic basis of drug-drug interactions, for example, discrimination of time-dependent (irreversible) and reversible enzyme inhibiting compounds from each other.
As the current bottleneck in screening of such interactions is associated with the speed of the analytical process flow, the microsystems technology developed in the CUMTAS project may also improve the throughput of drug-drug interaction screening by enabling parallelization of multiple analytical units side by side and integration of the drug metabolising unit with other analytical unit operations, such as separation of the metabolic reaction products from each other. To overcome the often limited detection sensitivity of the microfluidic assays in comparison to current standard technologies (i.e. well-plate based fluorescence screening assays and liquid chromatography mass spectrometry), several miniaturized sensor elements including micromirrors, microlenses and electrochemical detecting elements, were developed and integrated with the microfluidic separation systems. In parallel to scientific aims, a range of new polymer-based manufacturing materials and low-cost, non-cleanroom-based manufacturing methods, as well as new enzyme immobilization strategies for both soluble and membrane-bound enzymes, were introduced to support the technological development of the microfluidic bioanalytical platforms in general terms.