FULLY AUTOMATED ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM FOR CYP1A1 ACTIVITY MONITORING

Introduction
Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The identification of the enzymes involved in drug metabolism is thus of critical importance for the design of further clinical studies. The availability of specifically expressed human CYPs, namely supersomes, allows the investigation of the contribution of a single metabolic enzyme to the biotransformation pathway of the compound under investigation. CYP1A1, a member of the cytochrome P450 superfamily, was studied in this project. Interestingly, it has been described to be over expressed in various types of cancer. Consequently, CYP1A1 has emerged as a particularly interesting target for cancer therapy.

Methods
All the experiments were carried out on a HP3DCE system equipped with an on-column DAD. The EMMA procedure was performed by injecting a plug containing CYP1A1 supersomes, followed by a plug that contained the co-factor and the substrate, then another plug of CYP1A1 supersomes. The reaction was triggered by the application of a voltage switch. The voltage was then turned off to allow the metabolic reaction to occur. The separation of the components was then performed at -25 kV.

Results
The present study describes the development of a fully automatized in-capillary method to follow metabolization of 7-hydroxycoumarin and screen CYP1A1 inhibitors.
After preliminary studies, satisfying results were obtained using CYP1A1 at a concentration of 200 pmol/mL, while the incubation time was settled to 15 min. Equal reactant plugs were injected at -50 mbar for 6 sec. The short-end injection performed gave rise to a baseline separation of the molecules (substrate, product, CYP1A1 and NADPH) in less than 2 minutes. Adequate plugs overlap was obtained using electrophoretic mixing. The DoE performed highlighted that the voltage switch has a great impact on the metabolite formation.
The amount of product obtained in the optimal conditions was found to be comparable to the one detected after conventional off-line metabolization.
Besides the interest of developing an automatized CE approach for metabolisation studies, we also wanted to investigate the potentiality of this approach to screen CYP1A1 inhibitors. The ability of our system to monitor CYP1A1 inhibition was undertaken with apigenin, a well-known inhibitor.
It is noteworthy that the compatibility of our system with MEKC ensures its applicability to a large variety of molecules.

Novel aspect
Monitoring CYP1A1 activity using a rapid and fully automated EMMA method that
could be used for new anticancer agents screening.