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The multiple depletion curves method provides accurate estimates of intrinsic clearance (CLint), maximum velocity of the metabolic reaction (Vmax), and Michaelis constant (Km): accuracy and robustness evaluated through experimental data and Monte Carlo simulations
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
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2009 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 1, 47-58 p.Article in journal (Refereed) Published
Abstract [en]

The use of multiple depletion curves for the estimation of maximum velocity of the metabolic reaction (V(max)), the Michaelis constant (K(m)), and intrinsic clearance (CL(int)) was thoroughly evaluated by means of experimental data and through a series of Monte Carlo simulations. The enzyme kinetics of seven compounds were determined using the multiple depletion curves method (MDCM), the traditional initial formation rate of metabolite method (IFRMM), and the "in vitro t(1/2)" method, and the parameter estimates that were derived from the three methods were compared. The impact of a change in enzyme activity during the incubation period on the parameter estimates and the possibility to correct for this were also investigated. The MDCM was in good overall agreement with the IFRMM. Correction for a change in enzyme activity was possible and resulted in a better concordance in CL(int) estimates. The robustness of the method in coping with different rates of substrate turnover and variable starting concentrations were also demonstrated through Monte Carlo simulations. Furthermore, the limitations imposed by assumptions inherent in the in vitro t(1/2) method were demonstrated both experimentally and by simulations. This study demonstrates that the MDCM is a robust and efficient method for estimating enzyme kinetic variables with high accuracy and precision. The method may potentially be used in a wide range of applications, from pure enzyme kinetics to in vitro-based predictions of the pharmacokinetics of compounds with multiple and/or unknown metabolic pathways.

Place, publisher, year, edition, pages
2009. Vol. 37, no 1, 47-58 p.
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-87652DOI: 10.1124/dmd.108.021477ISI: 000261928100009PubMedID: 18824525OAI: oai:DiVA.org:uu-87652DiVA: diva2:132982
Available from: 2009-01-07 Created: 2009-01-07 Last updated: 2011-01-13Bibliographically approved
In thesis
1. Hepatic Disposition of Drugs and the Utility of Mechanistic Modelling and Simulation
Open this publication in new window or tab >>Hepatic Disposition of Drugs and the Utility of Mechanistic Modelling and Simulation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The elimination of drugs from the body is in many cases performed by the liver. Much could be gained if an accurate prediction of this process could be made early in the development of new drugs. However, for the elimination to occur, the drug molecule needs first to get inside the liver cell.

Disposition is the expression used to encapsulate both elimination and distribution. This thesis presents novel approaches and models based on simple in vitro systems for the investigation of processes involved in the hepatic drug disposition.

An approach to the estimation of enzyme kinetics based on substrate depletion data from cell fractions was thoroughly evaluated through experiments and simulations. The results that it provided were confirmed to be accurate and robust. In addition, a new experimental setup suitable for a screening environment, i.e., for a reduced number of samples, was generated through optimal experimental design. The optimization suggested that sampling at late time points over a wide range of concentration was the most advantageous.

A model, based on data from primary hepatocytes in suspension, for the investigation of cellular disposition of metabolized drugs was developed. Information on the relative importance of metabolism and membrane protein related distribution was obtained by analysis of changes in the kinetics by specific inhibition of the various processes. The model was evaluated by comparing the results to those obtained from an in vivo study analyzed with an especially constructed mechanistic PBPK model. These investigations showed that the suggested model produced good predictions of the relative importance of metabolism and carrier mediated membrane transport for hepatic disposition.

In conclusion, new approaches for the investigation of processes involved in hepatic disposition were developed. These methods were shown to be robust and increased the output of information from already commonly implemented in vitro systems.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 132
Keyword
Hepatic disposition, pharmacokinetics, mechanistic modelling, drug-drug interactions, enzyme kinetics, Vmax, Km, CLint, carrier-mediated transport, active transport, modelling, in vitro-in vivo extrapolation, physiologically based pharmacokinetic model, optimal experimental design, experimental optimization, data analysis optimization
National Category
Pharmaceutical Sciences Pharmaceutical Sciences
Research subject
Biopharmaceutics
Identifiers
urn:nbn:se:uu:diva-132571 (URN)978-91-554-7934-3 (ISBN)
Public defence
2010-12-10, B:21, Uppsala Biomedicinska Centrum - BMC, Husargatan 3, Uppsala, 09:15 (English)
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Available from: 2010-11-18 Created: 2010-10-21 Last updated: 2011-01-13Bibliographically approved

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