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ATP-Binding-Cassette Transporters in Biliary Efflux and Drug-Induced Liver Injury
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. (Drug Delivery)ORCID iD: 0000-0002-9701-4489
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 172
Keyword [en]
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
Pharmaceutics
Identifiers
URN: urn:nbn:se:uu:diva-205355ISBN: 978-91-554-8702-7 (print)OAI: oai:DiVA.org:uu-205355DiVA: diva2:641249
Public defence
2013-09-06, B21, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-08-16 Created: 2013-08-16 Last updated: 2014-04-16
List of papers
1. Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2)
Open this publication in new window or tab >>Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2)
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2008 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 51, no 11, 3275-3287 p.Article in journal (Refereed) Published
Abstract [en]

The chemical space of registered oral drugs was explored for inhibitors of the human multidrug-resistance associated protein 2 (MRP2, ABCC2), using a data set of 191 structurally diverse drugs and drug-like compounds. The data set included a new reference set of 75 compounds, for studies of hepatic drug interactions with transport proteins, CYP enzymes, and compounds associated with liver toxicity. The inhibition of MRP2-mediated transport of estradiol-17 beta-D-glucuronide was studied in inverted membrane vesicles from Sf9 cells overexpressing human MRP2. A total of 27 previously unknown MRP2 inhibitors were identified, and the results indicate an overlapping but narrower inhibitor space for MRP2 compared with the two other major ABC efflux transporters P-gp (ABCB1) and BCRP (ABCG2). In addition, 13 compounds were shown to stimulate the transport of cstradiol-17 beta-D-glucuronide. The experimental results were used to develop a computational model able to discriminate inhibitors from noninhibitors according to their molecular structure, resulting in a predictive power of 86% for the training set and 72% for the test set. The inhibitors were in general larger and more lipophilic and presented a higher aromaticity than the noninhibitors. The developed computational model is applicable in an early stage of the drug discovery process and is proposed as a tool for prediction of MRP2-mediated hepatic drug interactions and toxicity.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-96583 (URN)10.1021/jm7015683 (DOI)000256504800025 ()18457386 (PubMedID)
Available from: 2007-12-20 Created: 2007-12-20 Last updated: 2017-12-14Bibliographically approved
2. Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs
Open this publication in new window or tab >>Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs
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2009 (English)In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 26, no 8, 1816-1831 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: To study the inhibition patterns of the three major human ABC transporters P-gp (ABCB1), BCRP (ABCG2) and MRP2 (ABCC2), using a dataset of 122 structurally diverse drugs. METHODS: Inhibition was investigated in cellular and vesicular systems over-expressing single transporters. Computational models discriminating either single or general inhibitors from non-inhibitors were developed using multivariate statistics. RESULTS: Specific (n = 23) and overlapping (n = 19) inhibitors of the three ABC transporters were identified. GF120918 and Ko143 were verified to specifically inhibit P-gp/BCRP and BCRP in defined concentration intervals, whereas the MRP inhibitor MK571 was revealed to inhibit all three transporters within one log unit of concentration. Virtual docking experiments showed that MK571 binds to the ATP catalytic site, which could contribute to its multi-specific inhibition profile. A computational model predicting general ABC inhibition correctly classified 80% of both ABC transporter inhibitors and non-inhibitors in an external test set. CONCLUSIONS: The inhibitor specificities of P-gp, BCRP and MRP2 were shown to be highly overlapping. General ABC inhibitors were more lipophilic and aromatic than specific inhibitors and non-inhibitors. The identified specific inhibitors can be used to delineate transport processes in complex experimental systems, whereas the multi-specific inhibitors are useful in primary ABC transporter screening in drug discovery settings.

Keyword
ABC transporters, drug transport, inhibition, structure-activity relationships, transport proteins
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-109429 (URN)10.1007/s11095-009-9896-0 (DOI)000267685800003 ()19421845 (PubMedID)
Available from: 2009-10-15 Created: 2009-10-15 Last updated: 2017-12-12Bibliographically approved
3. Biliary Drug Excretion and Drug-drug Interactions in Sandwich Cultured Human Hepatocytes Predicted from Inverted Membrane Vesicles and Targeted Proteomics
Open this publication in new window or tab >>Biliary Drug Excretion and Drug-drug Interactions in Sandwich Cultured Human Hepatocytes Predicted from Inverted Membrane Vesicles and Targeted Proteomics
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(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
Identifiers
urn:nbn:se:uu:diva-204199 (URN)
Available from: 2013-07-23 Created: 2013-07-23 Last updated: 2013-08-19
4. Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP; ABCB11)
Open this publication in new window or tab >>Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP; ABCB11)
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2013 (English)In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 136, no 2, 328-343 p.Article in journal (Other academic) Published
Abstract [en]

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a dataset of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, while positive molecular charge was associated with a lack of inhibition. All approved drugs in the dataset (n=182) were categorized according to DILI warnings in drug labels issued by the FDA and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including nine drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux while BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH  was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI. 

Keyword
Bile Salt Export Pump, ABCB11, BSEP, ABC transporters, drug transport, inhibition, structure-activity relationships, transport proteins, Drug Induced Liver Injury, DILI
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-204168 (URN)10.1093/toxsci/kft197 (DOI)000328695500006 ()
Available from: 2013-07-23 Created: 2013-07-23 Last updated: 2017-12-06Bibliographically approved

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