RESUMO
"Personalized medicine" is a new concept in health care, one aspect of which defines the specificity and dosage of drugs according to effectiveness and safety for each patient. Dosage strongly depends from the rate of metabolism which is primarily regulated by the activity of cytochrome P450. In addition to the need for a genetic characterization of the patients, there is also the necessity to determine the drug-clearance properties of the polymorphic P450 enzyme. To address this issue, human P450 2D6 and 2C9 were engineered and covalently linked to an electrode surface allowing fast, accurate, and reliable measurements of the kinetic parameters of these phase-1 drug metabolizing polymorphic enzymes. In particular, the catalytic activity of P450 2C9 on the electrode surface was found to be improved when expressed from a gene-fusion with flavodoxin from Desulfovibrio vulgaris (2C9/FLD). The results are validated using marker drugs for these enzymes, bufuralol for 2D6, and warfarin for 2C9/FLD. The platform is able to measure the same small differences in K(M), and it allows a fast and reproducible mean to generated the product identified by HPLC from which the k(cat) is calculated.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Preparações Farmacêuticas/metabolismo , Polimorfismo Genético , Alelos , Biocatálise , Sistema Enzimático do Citocromo P-450/química , Eletroquímica , Genótipo , Humanos , Modelos Moleculares , Oxirredução , Medicina de Precisão , Conformação Proteica , Engenharia de ProteínasRESUMO
This paper is the first report of a P450-electrode in a microfluidic format. A 30 µL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(M) values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron > nifedipine > quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Técnicas Eletroquímicas/métodos , Microfluídica/métodos , Carbolinas/análise , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Microssomos Hepáticos/metabolismo , Nifedipino/análise , Ondansetron/análise , Quinidina/análise , Especificidade por SubstratoRESUMO
This communication reports on the first electrochemical study of the human flavin-containing monooxygenase 3 (hFMO3) either absorbed or covalently linked to different electrode surfaces. Glassy carbon and gold electrodes gave reversible electrochemical signals of an active hFMO3. The midpoint potential measured for the immobilized enzyme on a glassy carbon electrode was -445 +/- 8 mV (versus Ag/AgCl). A monolayer coverage was obtained on gold functionalized with dithio-bismaleimidoethane that covalently linked surface accessible cysteines of hFMO3. A structural model of the enzyme was generated to rationalize electrochemistry results. The turnover of the active enzyme was measured with two specific drugs: tamoxifen and benzydamine. For tamoxifen, 1.7 and 8.0 microM of its N-oxide product were formed by the enzyme immobilized on glassy carbon and gold electrodes, respectively. In the case of benzydamine, a K(M) of 44 +/- 5 microM was measured upon application of a -600 mV bias to the enzyme immobilized on the glassy carbon electrode that is in good agreement with the values published for microsomal hFMO3 where NADPH is the electron donor.