uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Biliary Drug Excretion and Drug-drug Interactions in Sandwich Cultured Human Hepatocytes Predicted from Inverted Membrane Vesicles and Targeted Proteomics
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. (Drug Delivery)ORCID iD: 0000-0002-9701-4489
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Bristol-Myers Squibb, New York, USA.
Bristol-Myers Squibb, New York, USA.
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The ABC multidrug resistance proteins in the canalicular membrane are important contributors to the pharmacokinetic properties of drugs and endogenous compounds. In this study, a new approach for determining the individual contribution from three ABC transporters to the biliary clearance was investigated. First, the inhibition of P-glycoprotein (Pgp/ABCB1), Breast Cancer Resistance Protein (BCRP/ABCG2) and Multidrug-Resistance Associated Protein 2 (MRP2/ABCC2) was investigated in inverted membrane vesicles from HEK293 cells overexpressing each of the ABC proteins.  Inhibition profiles of prototypic substrate transport were obtained for 24 compounds and compared with results obtained in alternative expression systems. A common substrate for the three transporters was found in estradiol 17β-glucuronide (E17G). The contribution of each transporter to the E17G biliary efflux was investigated in HEK vesicles and in sandwich cultured human hepatocytes (SCHH), using nine inhibitors with different specificity towards each of the ABC-proteins. After quantification of transport kinetics and protein expression, the maximal transport activity (MTA) of each of the transporters was calculated and their contribution to the biliary clearance of E17G was predicted. The results show a good correlation between HEK-MRP2 and Sf9-MRP2 vesicle data. However, up to 40-fold lower IC50-values were obtained for Pgp and BCRP in the HEK vesicles compared to cellular expression systems. The physiologically more relevant SCHH were found to identify ABC inhibitors observed in the vesicle system that lost their ABC inhibition in this more complex model. SCHH therefore contribute with important additional information impossible to assess in vesicular systems. Finally, the MTA and subsequent of biliary clearance determinations were used to predict the absolute biliary excretion of E17G in SCHH, which was predicted with a prediction accuracy of 90%.

National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-204199OAI: oai:DiVA.org:uu-204199DiVA: diva2:637918
Available from: 2013-07-23 Created: 2013-07-23 Last updated: 2013-08-19
In thesis
1. ATP-Binding-Cassette Transporters in Biliary Efflux and Drug-Induced Liver Injury
Open this publication in new window or tab >>ATP-Binding-Cassette Transporters in Biliary Efflux and Drug-Induced Liver Injury
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes.

The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions.

In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models were developed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 67 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 172
ABC transport protein, Pgp, P-glycoprotein, ABCB1, BCRP, breast cancer resistance protein, ABCG2, MRP2, multidrug resistance-associated protein 2, ABCC2, BSEP, bile salt export pump, ABCB11, sandwich cultured human hepatocytes, SCHH, drug-induced liver injury, DILI, multivariate data analysis, OPLS
National Category
Pharmaceutical Sciences
Research subject
urn:nbn:se:uu:diva-205355 (URN)978-91-554-8702-7 (ISBN)
Public defence
2013-09-06, B21, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Available from: 2013-08-16 Created: 2013-08-16 Last updated: 2014-04-16

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Pedersen, Jenny M.
By organisation
Department of PharmacyDepartment of Surgical Sciences
Pharmaceutical Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 207 hits
ReferencesLink to record
Permanent link

Direct link