Cytochrome P450 2C9 based microreactor combined with capillary electrophoresis for on-line kinetic and inhibition studies
| Authors | |
|---|---|
| Year of publication | 2015 |
| Type | Conference abstract |
| Citation | |
| Description | In pre-clinical stage of new drug development pharmacokinetic and pharmacodynamic studies play a crucial role. Essential part of these studies is determination of kinetic parameters and inhibition potential of the drug candidate towards cytochromes P450 (CYP), as these liver enzymes are responsible for majority of biotransformation processes leading to deactivation and excretion almost all of xenobiotics, including drugs. For those studies high throughput and cost-effectiveness are the ultimate objectives driving the search for novel methods and techniques. In this work we present immobilized enzymatic microreactor (IMER) coupled with capillary electrophoresis (CE) in on-line configuration, which enables rapid analysis with miniscule sample consumption and reuse of targeted enzyme. In our study the CYP 2C9 isoform was immobilized on magnetic microparticles coated with carboxyl groups using standard coupling agents 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysulfosuccinimide and introduced into capillary, where the particles formed an IMER in magnetic field created by two Nd magnets. The capillary thus served both as the reaction chamber and separation medium resulting in short incubation and analysis time about 20 minutes in total, while consuming only tens of nl of sample and repeated use of enzyme for 6 effective measurements. For the kinetic and inhibition study the Food and Drug Administration recommended substrate diclofenac and inhibitor sulfaphenazole were used, the biotransformation product 4’-hydroxydiclofenac was quantified using UV-VIS detection. The optimized CE system provided sufficient sensitivity and repeatability to obtain kinetic and inhibition parameters which were in good correlation to literature. Unfortunately being this a novel approach many hurdles were encountered: i) first the enzyme deactivation was observed and characterised leading to employment of reference measurement to ensure repeatability and ii) second the determination of effective concentration of reactants inside the reactor proved exacting, therefore an adjusted model based on Hagen-Poiseuille law was proposed. |
| Related projects: |