The Research Of Drug Metabolism Via Cytochrome P450 Enzyme Based On Graphene Composites

Cytochrome P450 enzymes (CYP450s) with an active center of iron protoheme are involved in most clinical drugs metabolism process. Drugs metabolism via CYP450s can cause drug-drug, drug-food and drug-biomolecule interactions which would result in toxicities and undesired consequences. Thus, to develop and establish a system for the study of drug metabolism via CYP450 enzymes in vitro is very urgent, especially for the assessment of drug interactions, the development of new drugs and the selection of many more drugs.In this paper, a system of CYP450s catalytic drug metabolism was established successfully. Graphene composites were used as the electrode matrix for the immobilization of CYP450s, and the electrode was used to replace expensive coenzyme for providing two electrons to initiate CYP450s catalysis. The direct electron transfer mechanism between CYP450s and the electrode, and the electrochemical behavior of CYP450s catalyzing drug metabolism were studied by electrochemical analysis methods. And the electrochemical driven various drugs metabolism via CYP450s was achieved. Details are presented as follows:(1) Polyethylenimine functionalized graphene composites (PEI-RGO) were prepared by the method of chemical reduction together with non-covalent modification. Ultraviolet spectroscopy, Raman spectroscopy, transmission electron microscope and energy dispersive spectroscopy were used to characterize the structure, surface morphology and elements of PEI-RGO. The graphene composites possessing good properties of dispersion, conduction, biocompatibility and film-forming, could be applied to construction of electrochemical biosensors.(2) A system of electrochemical driven tramadol metabolism via CYP2D6 was constructed by immobilizing CYP2D6 on the PEI-RGO modified glassy carbon electrode through covalent bonding and electrostatic interaction. Direct and reversible electron transfer of the immobilized CYP2D6 with the direct electron transfer constant of 0.47 s-1 and midpoint potential of -0.483 V was obtained. In the presence of substrate tramadol, the electrochemical driven CYP2D6 mediated catalytic behavior toward the conversion of tramadol to o-demethyl-tramadol was confirmed. The Michaelis-Menten constant and heterogeneous reaction rate constant during the metabolism of tramadol were calculated to be 23.85 μM and 1.96 cm s-1, respectively. High performance liquid chromatography-mass spectrometry measurements (HPLC-MS) confirmed that the products by electrochemical driven tramadol metabolism strategy were in accordance with previous reports on the metabolic reaction of tramadol catalyzed by CYP2D6 in vivo. Furthermore, the inhibition effect of quinidine on CYP2D6 catalyze-cycle was also investigated with the IC50 value of 0.693 μM. It implied that this strategy could maintain the biological activity and catalytic property of CYP2D6 very well.(3) The above system of electrochemical driven CYP2D6 mediated tramadol metabolism was applied to various clinical drugs metabolism. The electrochemical responses of the immobilized CYP2D6 to various drugs were monitored by cyclic voltammetry. And the Michaelis-Menten constant values of these drugs were compared with the corresponding values in previous reports. HPLC-MS strategy confirmed the products of every drug that generated by electrochemical driven drugs metabolism via CYP2D6. All product yields estimated from different perspectives of electrochemistry and chromatography were compared and analyzed. This electrochemical platform of CYP450s not only has potentials for the assessment of drug interactions, but also offers value in building other biomolecules system in vitro.