Application of sweeping - MEKC combination for monitoring of
diclofenac reaction with cytochrome P450 2C9 isoform

D 2009

Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform

ŘEMÍNEK, Roman, Jiří KONEČNÝ and Zdeněk GLATZ

Basic information

Original name

Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform

Name in Czech

Využití kombinace sweeping - MEKC pro studium reakce diclofenacu scytochromem P450 2C9 isoform

Authors

ŘEMÍNEK, Roman, Jiří KONEČNÝ and Zdeněk GLATZ

Edition

první. Seville, Spain, Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology, p. 125-125, 1 pp. 2009

Publisher

Casa de la Ciencia CSIC

Other information

Language

English

Type of outcome

Stať ve sborníku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Spain

Confidentiality degree

není předmětem státního či obchodního tajemství

Organization unit

Faculty of Science

Keywords (in Czech)

sweeping; MEKC; diclofenac; P450

Keywords in English

sweeping; MEKC; diclofenac; P450

Abstract

ORIG CZ

V originále

During the last decades a large number of new drugs have been introduced into clinical practice. All these new entities had to undergo the complicated time and money consuming developing process. Besides adsorption, distribution, excretion and toxicological studies, this process also includes the study of their metabolism, the affinity to certain drug-metabolising enzymes and the investigation of drug-drug interactions. A number of analytical approaches have been applied for these purposes mostly based on radiometric, colorimetric, fluorescent and high-performance liquid chromatography/mass spectrometry. In addition to these well established methods capillary electrophoresis is gaining the position in this field. The major aim of this work was to demonstrate the possibility of using MEKC for kinetic study of cytochrome P450 2C9 (CYP2C9), one of the most important isoforms in human liver, with diclofenac as a probe substrate. In view of the fact that our recent study 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 8.3 was used as a background electrolyte. 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 results 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 as it was established in the case of CYP3A4 isoform.

In Czech

Byla vypracována metoda kombinující MEKC a sweeping pro studium reakce diclofenacu s cytocromem P450 2C9.

Links

LC06023, research and development project

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

Investor: Ministry of Education, Youth and Sports of the CR

MSM0021622413, plan (intention)

Name: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministry of Education, Youth and Sports of the CR, Proteins in metabolism and interaction of organisms with the environment

Citovat

ŘEMÍNEK, Roman, Jiří KONEČNÝ and Zdeněk GLATZ. Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform. In Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology. první. Seville, Spain: Casa de la Ciencia CSIC, 2009, p. 125-125.

@inproceedings{852830,
   author = {Řemínek, Roman and Konečný, Jiří and Glatz, Zdeněk},
   address = {Seville, Spain},
   booktitle = {Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology},
   edition = {první},
   keywords = {sweeping; MEKC; diclofenac; P450},
   language = {eng},
   location = {Seville, Spain},
   pages = {125-125},
   publisher = {Casa de la Ciencia CSIC},
   title = {Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform},
   year = {2009}
}

TY  - JOUR
ID  - 852830
AU  - Řemínek, Roman - Konečný, Jiří - Glatz, Zdeněk
PY  - 2009
TI  - Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform
PB  - Casa de la Ciencia CSIC
CY  - Seville, Spain
KW  - sweeping
KW  - MEKC
KW  - diclofenac
KW  - P450
N2  - During the last decades a large number of new drugs have been introduced into clinical practice. All these new entities had to undergo the complicated time and money consuming developing process. Besides adsorption, distribution, excretion and toxicological studies, this process also includes the study of their metabolism, the affinity to certain drug-metabolising enzymes and the investigation of drug-drug interactions. A number of analytical approaches have been applied for these purposes mostly based on radiometric, colorimetric, fluorescent and high-performance liquid chromatography/mass spectrometry. In addition to these well established methods capillary electrophoresis is gaining the position in this field. The major aim of this work was to demonstrate the possibility of using MEKC for kinetic study of cytochrome P450 2C9 (CYP2C9), one of the most important isoforms in human liver, with diclofenac as a probe substrate. In view of the fact that our recent study 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 8.3 was used as a background electrolyte. 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 results 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 as it was established in the case of CYP3A4 isoform.
ER  -

ŘEMÍNEK, Roman, Jiří KONEČNÝ and Zdeněk GLATZ. Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform. In \textit{Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology}. první. Seville, Spain: Casa de la Ciencia CSIC, 2009, p.~125-125.

Detailed Information on Publication Record