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Hepatic Disposition of Drugs and the Utility of Mechanistic Modelling and Simulation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. (Biofarmaci)
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. , p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 132
Keywords [en]
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: urn:nbn:se:uu:diva-132571ISBN: 978-91-554-7934-3 (print)OAI: oai:DiVA.org:uu-132571DiVA, id: diva2:359336
Public defence
2010-12-10, B:21, Uppsala Biomedicinska Centrum - BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-11-18 Created: 2010-10-21 Last updated: 2018-01-12Bibliographically approved
List of papers
1. 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
Open this publication in new window or tab >>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
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2009 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 1, p. 47-58Article 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.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-87652 (URN)10.1124/dmd.108.021477 (DOI)000261928100009 ()18824525 (PubMedID)
Available from: 2009-01-07 Created: 2009-01-07 Last updated: 2018-01-13Bibliographically approved
2. Optimal experimental design for assessment of enzyme kinetics in a drug discovery screening environment
Open this publication in new window or tab >>Optimal experimental design for assessment of enzyme kinetics in a drug discovery screening environment
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2011 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 5, p. 858-863Article in journal (Refereed) Published
Abstract [en]

A penalized ED-optimal design with a discrete parameter distribution was used to find an optimal experimental design for assessment of enzyme kinetics in a screening environment. A data set for enzyme kinetic data (Vmax and Km) was collected from previously reported studies and every Vmax/Km pair (n=76) was taken to represent a unique drug compound. The design was restricted to 15 samples, an incubation time of up to 40 min and starting concentrations (C0) for the incubation between 0.01 and 100 µM. The optimization was performed by finding the sample times and C0 returning the lowest uncertainty (SE) of the model parameter estimates. Individual optimal designs (I-OD), one general optimal design (G-OD) and one for laboratory practice pragmatically modified design (OD) were obtained. In addition, a standard design (STD-D), representing a commonly applied approach for metabolic stability investigations, was constructed. Simulations were performed for OD and STD-D using the Michaelis-Menten (MM) equation and enzyme kinetic parameters were estimated both with MM and a mono exponential (EXP) decay. OD generated a better result (RSE) for 99% of the compounds and an equal or better result (RMSE) for 78% of the compounds. Furthermore, high-quality estimates (RMSE <30%) of both Vmax and Km could be obtained for a considerable number (26%) of the investigated compounds. The results presented in this study demonstrate that the output could generally be improved when compared to that obtained from the standard approaches used today.

Keywords
enzyme kinetics, drug discovery screen, optimal experimental design, CLint, Vmax, Km
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-132347 (URN)10.1124/dmd.110.037309 (DOI)000289619600017 ()21289074 (PubMedID)
Available from: 2010-10-21 Created: 2010-10-18 Last updated: 2018-01-12Bibliographically approved
3. Hepatic disposition of ximelagatran and its metabolites in pig; prediction of the impact of membrane transporters through a simple disposition model
Open this publication in new window or tab >>Hepatic disposition of ximelagatran and its metabolites in pig; prediction of the impact of membrane transporters through a simple disposition model
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2010 (English)In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 27, no 4, p. 597-607Article in journal (Refereed) Published
Abstract [en]

Purpose: The double prodrug ximelagatran is bioconverted, via the intermediates ethylmelagatran and N-hydroxymelagatran, to the direct thrombin inhibitor melagatran. The aims of this study were 1) to investigate the hepatic metabolism and disposition of ximelagatran and the intermediates in pig; and 2) to test a simple in vitro methodology for quantitative investigations of membrane transporters impact on the disposition of metabolized drugs. Methods: Porcine S1 (supernatant fraction obtained by centrifuging at 1000g for 10 min) liver fractions and hepatocytes were incubated in absence and presence of known membrane transporter inhibitors. The in vitro kinetics and disposition were determined by simultaneously fitting of the disappearance of ximelagatran and appearance of ethylmelagatran, N-hydroxymelagatran and melagatran. Results: In S1 liver fractions, the metabolism was significant inhibited by co-incubation of verapamil and ketoconazole but not by erythromycin, quinine and quinidine. The disposition of ximelagatran and the intermediate metabolites in hepatocytes were influenced, to various degrees, by carrier-mediated distribution processes. Conclusion: This work demonstrates that it is possible to obtain profound information of the general mechanisms important in the drug liver disposition with the combination of common in vitro systems and the simple disposition model proposed in this study.

Keywords
melagatran, prodrug, hepatic disposition, kinetic modeling, hepatocytes
National Category
Pharmacology and Toxicology
Research subject
Drug Metabolism
Identifiers
urn:nbn:se:uu:diva-110319 (URN)10.1007/s11095-009-0016-y (DOI)000275556000007 ()20140637 (PubMedID)
Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2018-01-12Bibliographically approved
4. The pharmacokinetics and hepatic disposition of repaglinide in pigs: mechanistic modeling of metabolism and transport
Open this publication in new window or tab >>The pharmacokinetics and hepatic disposition of repaglinide in pigs: mechanistic modeling of metabolism and transport
2012 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 9, no 4, p. 823-841Article in journal (Refereed) Published
Abstract [en]

The predictive power of using in vitro systems in combination with physiologically based pharmacolcinetic (PBPK) modeling to elucidate the relative importance of metabolism and carrier-mediated transport for the pharmacokinetics was evaluated using repaglinide as a model compound and pig as the test system. Repaglinide was chosen as model drug as previous studies in humans have shown that repaglinide is subject to both carrier-mediated influx to the liver cells and extensive hepatic metabolism. A multiple sampling site model in pig was chosen since it provides detailed in vivo information about the liver disposition. The underlying assumption was that both metabolism and carrier-mediated transport are also important for the hepatic disposition of repaglinide in pigs. Microsomes and primary hepatocytes were used for in vitro evaluation of enzyme kinetics and cellular disposition, respectively. In vitro data were generated both with and without metabolism inhibitors (ketoconazole, bezafibrate and trimethoprim) and transport inhibitors (diclofenac and quinine) providing input into a semi-PBPK model. In vivo data were also generated with and without the same enzyme and transporter inhibitors, alone and in combination. The pigs were given repaglinide as intravenous infusions with and without inhibitors in a sequential manner, i.e., a control phase and a test phase. Parameters describing the passive and carrier-mediated flux as well as metabolism were estimated in the control phase. The result from test phase was used to gain further knowledge of the findings from the control phase. The in vivo pig model enabled simultaneous sampling from plasma (pre- and postliver and peripheral) as well as from bile and urine. A semi-PBPK model consisting of 11 compartments (6 tissues + 5 sampling sites) was constructed for the mechanistic elucidation of the liver disposition, in vitro based in vivo predictions, sensitivity analyses and estimations of individual pharmacolcinetic parameters. Both in vitro and in vivo results showed that carrier-mediated influx was important for the liver disposition. The in vivo findings were supported by the result from the test phase where hepatic clearance (4.3 mL min(-1) kg(-1)) was decreased by 29% (metabolism inhibition), 43% (transport inhibition) and 57% (metabolism + transport inhibition). These effects were in good agreement with predicted levels. This study suggests that both metabolism and carrier-mediated uptake are of significant importance for the liver disposition of repaglinide in pigs.

Keywords
PBPK, mechanistic modelling, pharmacokinetics, hepatic disposition, repaglinide
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-132346 (URN)10.1021/mp200218p (DOI)000302186700014 ()
Available from: 2010-10-21 Created: 2010-10-18 Last updated: 2018-01-12Bibliographically approved

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