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Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design

BACKGROUND AND PURPOSE OF THE STUDY: Glimepiride (GLM) was chosen as a model substrate in order to determine the kinetic parameters for in vitro metabolism via human liver micrososmes (HLM). We aimed to optimize the turnover of the substrate by the test system in relation to incubation time and HLM...

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Autores principales: Ruikar, Dipti B, Rajput, Sadhana J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555947/
https://www.ncbi.nlm.nih.gov/pubmed/23351782
http://dx.doi.org/10.1186/2008-2231-20-38
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author Ruikar, Dipti B
Rajput, Sadhana J
author_facet Ruikar, Dipti B
Rajput, Sadhana J
author_sort Ruikar, Dipti B
collection PubMed
description BACKGROUND AND PURPOSE OF THE STUDY: Glimepiride (GLM) was chosen as a model substrate in order to determine the kinetic parameters for in vitro metabolism via human liver micrososmes (HLM). We aimed to optimize the turnover of the substrate by the test system in relation to incubation time and HLM concentration in such a way that it was linearly dependent on time and less than 20% of the substrate was consumed which utilized the lowest amount of the HLM. Further we aimed to report K(m) and V(max) values for GLM. METHODS: Linearity of enzyme reactions in microsomal incubations was assessed by monitoring the effect of incubation time (from 5 to 60 min) and HLM concentration (from 0.2 to 0.75 mg/ml) on metabolite formation of GLM. The ideal conditions for turnover of GLM were justified using 3x3 factorial design. F value was calculated to confirm the omission of insignificant terms from the full-model to derive a reduced- model polynomial equation. The regression equation was used to develop a contour plot that showed turnover rate within the limits of this design. The optimized reaction velocity data was extrapolated to carry out the kinetic studies in vitro to generate a saturation curve for the determination of K(m) and V(max) values. RESULTS: The reaction was found to be linear with respect to both incubation time between 24 and 50 min and HLM concentration between 0.3 to 0.65 mg/ml. The K(m) and V(max) values obtained by nonlinear least squares regression method was found to be 28.9 ± 2.97 μMole and 0.559 ± 0.017 μMole respectively. Lineweaver-Burk plot was also used to estimate K(m) and V(max) which yield value of 29.411 ± 1.25 μMole and 0.571 ± 0.020 μMole/min/mg protein respectively. MAJOR CONCLUSION: The statistical approach successfully allows for the optimization of reaction time course experiments. The results obtained with linear as well as the nonlinear transformation were found to be in close agreement with each other which shows the best precision for estimates of K(m) and V(max).
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spelling pubmed-35559472013-01-31 Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design Ruikar, Dipti B Rajput, Sadhana J Daru Research Article BACKGROUND AND PURPOSE OF THE STUDY: Glimepiride (GLM) was chosen as a model substrate in order to determine the kinetic parameters for in vitro metabolism via human liver micrososmes (HLM). We aimed to optimize the turnover of the substrate by the test system in relation to incubation time and HLM concentration in such a way that it was linearly dependent on time and less than 20% of the substrate was consumed which utilized the lowest amount of the HLM. Further we aimed to report K(m) and V(max) values for GLM. METHODS: Linearity of enzyme reactions in microsomal incubations was assessed by monitoring the effect of incubation time (from 5 to 60 min) and HLM concentration (from 0.2 to 0.75 mg/ml) on metabolite formation of GLM. The ideal conditions for turnover of GLM were justified using 3x3 factorial design. F value was calculated to confirm the omission of insignificant terms from the full-model to derive a reduced- model polynomial equation. The regression equation was used to develop a contour plot that showed turnover rate within the limits of this design. The optimized reaction velocity data was extrapolated to carry out the kinetic studies in vitro to generate a saturation curve for the determination of K(m) and V(max) values. RESULTS: The reaction was found to be linear with respect to both incubation time between 24 and 50 min and HLM concentration between 0.3 to 0.65 mg/ml. The K(m) and V(max) values obtained by nonlinear least squares regression method was found to be 28.9 ± 2.97 μMole and 0.559 ± 0.017 μMole respectively. Lineweaver-Burk plot was also used to estimate K(m) and V(max) which yield value of 29.411 ± 1.25 μMole and 0.571 ± 0.020 μMole/min/mg protein respectively. MAJOR CONCLUSION: The statistical approach successfully allows for the optimization of reaction time course experiments. The results obtained with linear as well as the nonlinear transformation were found to be in close agreement with each other which shows the best precision for estimates of K(m) and V(max). BioMed Central 2012-09-10 /pmc/articles/PMC3555947/ /pubmed/23351782 http://dx.doi.org/10.1186/2008-2231-20-38 Text en Copyright ©2012 Ruikar and Rajput; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Ruikar, Dipti B
Rajput, Sadhana J
Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
title Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
title_full Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
title_fullStr Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
title_full_unstemmed Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
title_short Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
title_sort optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555947/
https://www.ncbi.nlm.nih.gov/pubmed/23351782
http://dx.doi.org/10.1186/2008-2231-20-38
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