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In vitro and in silico Predictions of Hepatic Transporter-Mediated Drug Clearance and Drug-Drug Interactions in vivo
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The liver is the major detoxifying organ, clearing the blood from drugs and other xenobiotics. The extent of hepatic clearance (CL) determines drug exposure and hence, the efficacy and toxicity associated with exposure. Drug-drug interactions (DDIs) that alter the hepatic CL may cause more or less severe outcomes, such as adverse drug reactions. Accurate predictions of drug CL and DDI risk from in vitro data are therefore crucial in drug development.

Liver CL depends on several factors including the activities of transporters involved in the hepatic uptake and efflux. The work in this thesis aimed at developing new in vitro and in silico methods to predict hepatic transporter-mediated CL and DDIs in vivo. Particular emphasis was placed on interactions involving the hepatic uptake transporters OATP1B1, OATP1B3, and OATP2B1. These transporters regulate the plasma concentration-time profiles of many drugs including statins.

Inhibition of OATP-mediated transport by 225 structurally diverse drugs was investigated in vitro. Several novel inhibitors were identified. The data was used to develop in silico models that could predict OATP inhibitors from molecular structure. Models were developed for static and dynamic predictions of in vivo transporter-mediated drug CL and DDIs. These models rely on a combination of in vitro studies of transport function and mass spectrometry-based quantification of protein expression in the in vitro models and liver tissue. By providing estimations of transporter contributions to the overall hepatic uptake/efflux, the method is expected to improve predictions of transporter-mediated DDIs. Furthermore, proteins of importance for hepatic CL were quantified in liver tissue and isolated hepatocytes. The isolation of hepatocytes from liver tissue was found to be associated with oxidative stress and degradation of transporters and other proteins expressed in the plasma membrane. This has implications for the use of primary hepatocytes as an in vitro model of the liver. Nevertheless, by taking the altered transporter abundance into account using the method developed herein, transport function in hepatocyte experiments can be scaled to the in vivo situation. The concept of protein expression-dependent in vitro-in vivo extrapolations was illustrated using atorvastatin and pitavastatin as model drugs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 81 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 193
Keyword [en]
OATP1B1, OATP1B3, OATP2B1, NTCP, drug transporters, human hepatocytes, atorvastatin, pitavastatin, proteomics, sandwich-cultured human hepatocytes, SCHH, mechanistic modeling, in vitro-in vivo extrapolation, transport inhibition, hepatic uptake, hepatocyte isolation, transporter contribution
National Category
Pharmaceutical Sciences
Research subject
Biopharmaceutics
Identifiers
URN: urn:nbn:se:uu:diva-241376ISBN: 978-91-554-9141-3 (print)OAI: oai:DiVA.org:uu-241376DiVA: diva2:778815
Public defence
2015-02-27, Sal B41, Biomedicinskt centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-02-06 Created: 2015-01-12 Last updated: 2015-03-09
List of papers
1. Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug - Drug Interactions
Open this publication in new window or tab >>Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug - Drug Interactions
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2012 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 10, 4740-4763 p.Article in journal (Refereed) Published
Abstract [en]

The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug-drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1, The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-176227 (URN)10.1021/jm300212s (DOI)000304338800017 ()
Available from: 2012-06-19 Created: 2012-06-18 Last updated: 2017-12-07Bibliographically approved
2. Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions
Open this publication in new window or tab >>Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions
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2014 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 7, 1210-1218 p.Article in journal (Refereed) Published
Abstract [en]

Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to interindividual variability in atorvastatin clearance. However, the importance of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) in atorvastatin uptake clearance (CLupt) is not yet clarified. To elucidate this issue, we investigated the relative contribution of NTCP, OATP1B1, OATP1B3, and OATP2B1 to atorvastatin CLupt in 12 human liver samples. The impact of inhibition on atorvastatin CLupt was also studied, using inhibitors of different isoform specificities. Expression levels of the four transport proteins were quantified by liquid chromatography tandem mass spectrometry. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and interindividual differences in CLupt of each transporter in vivo. Subsequently, hepatic uptake impairment on coadministration of five clinically interacting drugs was predicted using in vitro inhibitory potencies. NTCP and OATP protein expression varied 3.7- to 32-fold among the 12 sample donors. The rank order in expression was OATP1B1 > OATP1B3 approximate to NTCP approximate to OATP2B1. NTCP was found to be of minor importance in atorvastatin disposition. Instead, OATP1B1 and OATP1B3 were confirmed as the major atorvastatin uptake transporters. The average contribution to atorvastatin uptake was OATP1B1 > OATP1B3 >> OATP2B1 > NTCP, although this rank order varied among individuals. The interindividual differences in transporter expression and CLupt resulted in marked differences in drug-drug interactions due to isoform-specific inhibition. We conclude that this variation should be considered in in vitro to in vivo extrapolations.

National Category
Pharmacology and Toxicology Basic Medicine
Identifiers
urn:nbn:se:uu:diva-229430 (URN)10.1124/dmd.113.056309 (DOI)000338341300016 ()
Available from: 2014-08-08 Created: 2014-08-07 Last updated: 2017-12-05Bibliographically approved
3. Comparative proteomics of human liver tissue and hepatocytes reveal systematic differences in proteins determining hepatic drug exposure
Open this publication in new window or tab >>Comparative proteomics of human liver tissue and hepatocytes reveal systematic differences in proteins determining hepatic drug exposure
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(English)Manuscript (preprint) (Other academic)
Keyword
Quantitative proteomics, Oxidative stress, plasma membrane protein, drug transport and metabolism, pitavastatin, Total Protein Approach, ADME, OATP, CYP, UGT
National Category
Pharmaceutical Sciences
Research subject
Biopharmaceutics
Identifiers
urn:nbn:se:uu:diva-241374 (URN)
Available from: 2015-01-12 Created: 2015-01-12 Last updated: 2015-03-09
4. Mechanistic Modeling of Pitavastatin Disposition in Sandwich-Cultured Human Hepatocytes: A Proteomics-Informed Bottom-Up Approach
Open this publication in new window or tab >>Mechanistic Modeling of Pitavastatin Disposition in Sandwich-Cultured Human Hepatocytes: A Proteomics-Informed Bottom-Up Approach
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2016 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 44, no 4, 505-516 p.Article in journal (Refereed) Published
Abstract [en]

Isolated human hepatocytes are commonly used to predict transporter-mediated clearance in vivo. Such predictions, however, do not provide estimations of transporter contributions and consequently do not allow predictions of the outcome resulting from a change in the activity of a certain transporter, e.g., by inhibition or a genetic variant with reduced function. Pitavastatin is a drug that is heavily dependent on hepatic transporters for its elimination and it is mainly excreted as unchanged drug in the bile. For this reason, pitavastatin was used as a model drug to demonstrate the applicability of a bottom-up approach to predict transporter-mediated disposition in sandwich-cultured human hepatocytes (SCHH), allowing for the estimation of transporter contributions. Transport experiments in transfected HEK293 cells and inverted membrane vesicles overexpressing each of the relevant transport proteins were used to generate parameter estimates for the mechanistic model. By adjusting for differences in transporter abundance between the in vitro systems and individual SCHH batches, the model successfully predicted time profiles of medium and intracellular accumulation. Our predictions of pitavastatin bile accumulation could, however, not be confirmed due to a very low biliary excretion of pitavastatin in relation to the hepatic uptake in our SCHH. This study is, to our knowledge, the first to successfully simulate transporter-mediated processes in a complex system such as SCHH at the level of individual transport proteins using a bottom-up approach.

Keyword
pitavastatin, SCHH, hepatocytes, drug transport, OATP, NTCP, hepatic uptake, Pgp, BCRP, MRP2, MRP3
National Category
Pharmaceutical Sciences
Research subject
Biopharmaceutics
Identifiers
urn:nbn:se:uu:diva-241375 (URN)10.1124/dmd.115.066746 (DOI)000372880600005 ()26842596 (PubMedID)
Funder
Swedish Research Council, 2822Lars Hierta Memorial Foundation
Available from: 2015-01-12 Created: 2015-01-12 Last updated: 2017-12-05Bibliographically approved

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