Kinetic study of cytochrome P450 2C9 reaction with diclofenac
by MEKC combined with a sweeping technique

D 2009

Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique

ŘEMÍNEK, Roman, Jiří KONEČNÝ a Zdeněk GLATZ

Základní údaje

Originální název

Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique

Název česky

Kinetická studie reakce cytochromu P450 2C9 s diclofenacem pomocí sweeping-MEKC kombinace

Autoři

ŘEMÍNEK, Roman, Jiří KONEČNÝ a Zdeněk GLATZ

Vydání

Innsbruck, Rakousko, Proceedeing of XVI. Euroanalysis symposium, od s. 310-310, 1 s. 2009

Nakladatel

Congress and Messe Innsbruck GMbH

Další údaje

Jazyk

angličtina

Typ výsledku

Stať ve sborníku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Rakousko

Utajení

není předmětem státního či obchodního tajemství

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova česky

cytochrom P450 2C9; diclofenac; sweeping; MEKC;

Klíčová slova anglicky

cytochrome P450 2C9; diclofenac; sweeping; MEKC;

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 9. 4. 2010 09:10, prof. RNDr. Zdeněk Glatz, CSc.

Anotace

ORIG CZ

V originále

The early knowledge about the metabolism of a new drug and its affinity to certain drug-metabolising enzymes are especially important in the drug development process. The drug discovery thus continually creates a demand for a development of a new generation of high throughput assays. The majority of these studies are oriented on cytochromes P450 (CYP) because these enzymes play a key role in drug metabolism at human. The aim of this work was to demonstrate the possibility of using MEKC for kinetic study of CYP2C9, one of the most important isoforms in human liver, with diclofenac as a probe substrate. In view of the fact that several studies showed that diclofenac hydroxylation deviated slightly from typical Michaelis-Menten kinetics at low substrate concentration, the combination of sweeping with MEKC was applied in this study. The enzymatic reaction thus could be simply monitored by the analysis of the reaction mixture without any pre-treatment; moreover high sensitivity and repeatability of the separations were preserved. A 50 um fused silica capillary (56 cm effective length) was used to carry out all separations. 60 mM SDS in 20 mM phosphate 20 mM tetraborate buffer pH 8.6 was used as BGE. Injection was accomplished by an application of 50 mbar pressure to the sample vial for 48 s. Separation was performed at 24 kV (positive polarity), with the temperature of capillary 25 oC and detection at 200 nm. As the result Michaelis constant 4.62 uM, maximum reaction velocity 18.35 nmol.min-1.nmol-1 and Hill coefficient 1.22 were determined, which were in agreement with the literature data. Value of Hill coefficient confirms a presence of weak positive cooperativity in low substrate concentration region. The explanation could be found in binding of two substrates in or near the active site of CYP2C9.

Česky

Byla provedena kinetická studie reakce cytochromu P450 2C9 s diclofenacem pomocí sweeping-MEKC kombinace.

Návaznosti

LC06023, projekt VaV

Název: Integrované bioanalytické technologie pro mikroanalýzy a diagnostiku s využitím LIF a hmotnostní spektrometrie

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Integrované bioanalytické technologie pro mikroanalýzy a diagnostiku s využitím LIF a hmotnostní spektrometrie

MSM0021622413, záměr

Název: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Proteiny v metabolismu a při interakci organismů s prostředím

Citovat

ŘEMÍNEK, Roman, Jiří KONEČNÝ a Zdeněk GLATZ. Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique. In Proceedeing of XVI. Euroanalysis symposium. Innsbruck, Rakousko: Congress and Messe Innsbruck GMbH, 2009, s. 310-310.

@inproceedings{845499,
   author = {Řemínek, Roman and Konečný, Jiří and Glatz, Zdeněk},
   address = {Innsbruck, Rakousko},
   booktitle = {Proceedeing of XVI. Euroanalysis symposium},
   keywords = {cytochrome P450 2C9; diclofenac; sweeping; MEKC;},
   language = {eng},
   location = {Innsbruck, Rakousko},
   pages = {310-310},
   publisher = {Congress and Messe Innsbruck GMbH},
   title = {Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique},
   year = {2009}
}

TY  - JOUR
ID  - 845499
AU  - Řemínek, Roman - Konečný, Jiří - Glatz, Zdeněk
PY  - 2009
TI  - Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique
PB  - Congress and Messe Innsbruck GMbH
CY  - Innsbruck, Rakousko
KW  - cytochrome P450 2C9
KW  - diclofenac
KW  - sweeping
KW  - MEKC;
N2  - The early knowledge about the metabolism of a new drug and its affinity to certain drug-metabolising enzymes are especially important in the drug development process. The drug discovery thus continually creates a demand for a development of a new generation of high throughput assays. The majority of these studies are oriented on cytochromes P450 (CYP) because these enzymes play a key role in drug metabolism at human. The aim of this work was to demonstrate the possibility of using MEKC for kinetic study of CYP2C9, one of the most important isoforms in human liver, with diclofenac as a probe substrate. In view of the fact that several studies showed that diclofenac hydroxylation deviated slightly from typical Michaelis-Menten kinetics at low substrate concentration, the combination of sweeping with MEKC was applied in this study. The enzymatic reaction thus could be simply monitored by the analysis of the reaction mixture without any pre-treatment; moreover high sensitivity and repeatability of the separations were preserved. A 50 um fused silica capillary (56 cm effective length) was used to carry out all separations. 60 mM SDS in 20 mM phosphate 20 mM tetraborate buffer pH 8.6 was used as BGE. Injection was accomplished by an application of 50 mbar pressure to the sample vial for 48 s. Separation was performed at 24 kV (positive polarity), with the temperature of capillary 25 oC and detection at 200 nm. As the result Michaelis constant 4.62 uM, maximum reaction velocity 18.35 nmol.min-1.nmol-1 and Hill coefficient 1.22 were determined, which were in agreement with the literature data. Value of Hill coefficient confirms a presence of weak positive cooperativity in low substrate concentration region. The explanation could be found in binding of two substrates in or near the active site of CYP2C9.
ER  -

ŘEMÍNEK, Roman, Jiří KONEČNÝ a Zdeněk GLATZ. Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique. In \textit{Proceedeing of XVI. Euroanalysis symposium}. Innsbruck, Rakousko: Congress and Messe Innsbruck GMbH, 2009, s.~310-310.

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