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In vitro and in silico prediction of drug-drug interactions with transport proteins
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. (Läkemedelsformulering)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities.

The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined.

The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins.

The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed.

In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2009. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 104
Keywords [en]
Solute carrier, SLC transporter, OCT1, Organic cation transporter 1, SLC22A1, OATP1B1, SLCO1B1, Organic anion transporting peptide 1B1, Drug transport, Active transport, Genetic polymorphism, Cell lines, Gene expression, Multivariate data analysis, OPLS
National Category
Pharmaceutical Sciences Pharmaceutical Sciences
Research subject
Pharmaceutics; Biopharmaceutics
Identifiers
URN: urn:nbn:se:uu:diva-107492ISBN: 978-91-554-7589-5 (print)OAI: oai:DiVA.org:uu-107492DiVA, id: diva2:231916
Public defence
2009-10-02, B21, Biomedical Center, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-09-10 Created: 2009-08-13 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines
Open this publication in new window or tab >>Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines
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2007 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 35, no 8, p. 1333-1340Article in journal (Refereed) Published
Abstract [en]

This study was designed to quantitatively assess the mRNA expression of 36 important drug transporters in human jejunum, colon, liver, and kidney. Expression of these transporters in human organs was compared with expression in commonly used cell lines (Caco-2, HepG2, and Caki-1) originating from these organs to assess their value as in vitro transporter system models, and was also compared with data obtained from the literature on expression in rat tissues to assess species differences. Transporters that were highly expressed in the intestine included HPT1, PEPT1, BCRP, MRP2, and MDR1, whereas, in the liver, OCT1, MRP2, OATP-C, NTCP and BSEP were the main transporters. In the kidney, OAT1 was expressed at the highest levels, followed by OAT3, OAT4, MCT5, MDR1, MRP2, OCT2, and OCTN2. The best agreement between human tissue and the representative cell line was observed for human jejunum and Caco-2 cells. Expression in liver and kidney ortholog cell lines was not correlated with that in the associated tissue. Comparisons with rat transporter gene expression revealed significant species differences. Our results allowed a comprehensive quantitative comparison of drug transporter expression in human intestine, liver, and kidney. We suggest that it would be beneficial for predictive pharmacokinetic research to focus on the most highly expressed transporters. We hope that our comparison of rat and human tissue will help to explain the observed species differences in in vivo models, increase understanding of the impact of active transport processes on pharmacokinetics and distribution, and improve the quality of predictions from animal studies to humans.

Keywords
Urinary system, Digestive system, Cell line, Established cell line, In vitro, Kidney, Liver, Gut, Human, Genetics, Gene, Carrier protein, Drug
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-11385 (URN)10.1124/dmd.107.014902 (DOI)000248200000013 ()17496207 (PubMedID)
Available from: 2007-09-11 Created: 2007-09-11 Last updated: 2018-01-12Bibliographically approved
2. Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs
Open this publication in new window or tab >>Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs
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2009 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 12, p. 2275-2283Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to investigate the gene and protein expression profiles of important drug transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters (HeLa, HEK293) and leukaemia cell lines used to study drug resistance by ABC-transporters (HL-60, K562) were investigated, and compared with organotypic cell lines (HepG2, Saos-2, Caco-2 and Caco-2 TC7). For gene expression studies, real-time PCR was used, while monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression and nine for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1; SLC16A1 was investigated using 14C-lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression and the expression patterns were barely affected by transfection. The leukaemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, while the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. Importantly, the monocarboxylic acid transporting protein MCT1 was significantly expressed in all, and functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.

Keywords
Cell lines, Caco-2, HEK293, HeLa, Saos-2, HL-60, K562, HepG2, Gene expression, Protein expression, MCT1
National Category
Pharmaceutical Sciences
Research subject
Biopharmaceutics; Pharmaceutics
Identifiers
urn:nbn:se:uu:diva-107571 (URN)10.1124/dmd.109.028654 (DOI)000271935200002 ()19741037 (PubMedID)
Available from: 2009-08-17 Created: 2009-08-17 Last updated: 2018-01-13Bibliographically approved
3. Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1
Open this publication in new window or tab >>Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1
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2008 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 51, no 19, p. 5932-5942Article in journal (Refereed) Published
Abstract [en]

The liver-specific organic cation transport protein (OCT1; SLC22A1) transports several cationic drugs including the antidiabetic drug metformin and the anticancer agents oxaliplatin and imatinib. In this study, we explored the chemical space of registered oral drugs with the aim of studying the inhibition pattern of OCT1 and of developing predictive computational models of OCT1 inhibition. In total, 191 structurally diverse compounds were examined in HEK293-OCT1 cells. The assay identified 47 novel inhibitors and confirmed 15 previously known inhibitors. The enrichment of OCT1 inhibitors was seen in several drug classes including antidepressants. High lipophilicity and a positive net charge were found to be the key physicochemical properties for OCT1 inhibition, whereas a high molecular dipole moment and many hydrogen bonds were negatively correlated to OCT1 inhibition. The data were used to generate OPLS-DA models for OCT1 inhibitors; the final model correctly predicted 82% of the inhibitors and 88% of the noninhibitors of the test set.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-86815 (URN)10.1021/jm8003152 (DOI)000259760500010 ()18788725 (PubMedID)
Available from: 2008-12-08 Created: 2008-12-08 Last updated: 2017-12-14Bibliographically approved
4. Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1:: predictions of metformin interactions
Open this publication in new window or tab >>Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1:: predictions of metformin interactions
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2011 (English)In: The Pharmacogenomics Journal, ISSN 1470-269X, E-ISSN 1473-1150, Vol. 11, no 6, p. 400-411Article in journal (Refereed) Published
Abstract [en]

Common genetic variants of the liver-specific human organic cation transporter 1 (OCT1; SLC22A1) have reduced transport capacity for substrates such as the antidiabetic drug metformin. The effect of the reduced OCT1 function on drug interactions associated with OCT1 has not been investigated and was, therefore, the focus of the study presented here. HEK293 cells expressing human OCT1-reference or the variants R61C, V408M, M420del and G465R were first used to study the kinetics and inhibition pattern of the OCT1 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). In the second part OCT1-mediated (14)C-metformin uptake was studied in the presence of drugs administered concomitantly with metformin. Transport studies using ASP(+) showed that the function of the variants decreased in the following order: OCT1-reference = V408M = M420del >R61C > >G465R. Variants M420del and R61C were more sensitive to drug inhibition, with IC(50) values up to 23 times lower than those of the OCT1-reference. Uptake studies using (14)C-metformin were in qualitative agreement with those using ASP(+), with the exception that a larger reduction in transport capacity was observed for M420del. Concomitantly administered drugs, such as verapamil and amitriptyline, revealed potential drug-drug interactions at clinical plasma concentrations of metformin for OCT1-M420del.

Keywords
OCT1, polymorphism, metformin, drug-drug intaeractions, transport protein
National Category
Pharmaceutical Sciences
Research subject
Biopharmaceutics; Pharmaceutics
Identifiers
urn:nbn:se:uu:diva-107572 (URN)10.1038/tpj.2010.54 (DOI)000297506500003 ()
Available from: 2009-08-17 Created: 2009-08-17 Last updated: 2018-01-13Bibliographically approved
5. In Vitro and In Silico Strategies to Identify OATP1B1 Inhibitors and Predict Clinical Drug-Drug Interactions
Open this publication in new window or tab >>In Vitro and In Silico Strategies to Identify OATP1B1 Inhibitors and Predict Clinical Drug-Drug Interactions
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2012 (English)In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 29, no 2, p. 411-426Article in journal (Other academic) Published
Abstract [en]

To establish in vitro and in silico models that predict clinical drug-drug interactions (DDIs) with the OATP1B1 (SLCO1B1) transporter. The inhibitory effect of 146 drugs and drug-like compounds on OATP1B1-mediated transport was studied in HEK293 cells. A computational model was developed to predict OATP1B1 inhibition. Concentration-dependent effects were investigated for six compounds; clinical DDIs were predicted by calculating change in exposure (i.e. R-values) in eight different ways. Sixty-five compounds were identified as OATP1B1 inhibitors at 20 mu M. The computational model predicted the test set with 80% accuracy for inhibitors and 91% for non-inhibitors. In vitro-in vivo comparisons underscored the importance of using drugs with known clinical effects as references. Thus, reference drugs, cyclosporin A, gemfibrozil, and fenofibrate, provided an inhibition interval to which three antiviral drugs, atazanavir, lopinavir, and amprenavir, could be compared and their clinical DDIs with OATP1B1 classified. Twenty-two new OATP1B1 inhibitors were identified, a predictive OATP1B1 inhibition in silico model was developed, and successful predictions of clinical DDIs were obtained with OATP1B1.

Keywords
in silico, in vitro-in vivo extrapolation, inhibition, MRP2, OATP1B1
National Category
Pharmaceutical Sciences
Research subject
Biopharmaceutics; Pharmaceutics
Identifiers
urn:nbn:se:uu:diva-107573 (URN)10.1007/s11095-011-0564-9 (DOI)000299506700007 ()
Available from: 2009-08-17 Created: 2009-08-17 Last updated: 2018-01-13Bibliographically approved

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