| 1. |
- Ahlin, Gustav, 1977-, et al.
(författare)
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Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs
- 2009
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 37:12, s. 2275-2283
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Ahlin, Gustav, 1977-, et al.
(författare)
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Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1: : predictions of metformin interactions
- 2011
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Ingår i: The Pharmacogenomics Journal. - : Springer Science and Business Media LLC. - 1470-269X .- 1473-1150. ; 11:6, s. 400-411
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Ahlin, Gustav, 1977-
(författare)
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In vitro and in silico prediction of drug-drug interactions with transport proteins
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Ahlin, Gustav, et al.
(författare)
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Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1
- 2008
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 51:19, s. 5932-5942
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Fridén, Markus, et al.
(författare)
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Measurement of Unbound Drug Exposure in Brain : Modelling of pH Partitioning Explains Diverging Results between the Brain Slice and Brain Homogenate Methods
- 2011
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 39:3, s. 353-362
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Tidskriftsartikel (refereegranskat)abstract
- Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f(u,brain)), determined using the brain homogenate method, with the unbound volume of distribution in brain (V(u,brain)), determined using the brain slice method. Discrepancies were frequent and primarily related to drug pH partitioning, due to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V(u,brain) from measurements of f(u,brain) and drug pKa. This model allowed prediction of V(u,brain) from f(u,brain) within a 2.2-fold error range for 95% of the drugs, as compared to a 4.5-fold error range using the brain homogenate f(u,brain) method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin and prototypic organic cation transporter substrates. It is concluded that intra-brain drug distribution is governed by several diverse mechanisms in addition to non-specific binding and that the slice method is therefore more reliable than the homogenate method. Alternatively, predictions of V(u,brain) can be made from homogenate f(u,brain) using the presented pH partition model, although this model does not take into consideration possible active brain cell uptake.
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| 6. |
- Hilgendorf, Constanze, et al.
(författare)
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Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines
- 2007
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 35:8, s. 1333-1340
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Karlgren, Maria, et al.
(författare)
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In Vitro and In Silico Strategies to Identify OATP1B1 Inhibitors and Predict Clinical Drug-Drug Interactions
- 2012
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Ingår i: Pharmaceutical research. - : Springer Science and Business Media LLC. - 0724-8741 .- 1573-904X. ; 29:2, s. 411-426
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 8. |
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| 9. |
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| 10. |
- Matsson, Pär, et al.
(författare)
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A Global Drug Inhibition Pattern for the Human ATP-Binding Cassette Transporter Breast Cancer Resistance Protein (ABCG2)
- 2007
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Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0022-3565 .- 1521-0103. ; 323:1, s. 19-30
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Tidskriftsartikel (refereegranskat)abstract
- In this article, we explore the entire structural space of registered drugs to obtain a global model for the inhibition of the drug efflux transporter breast cancer resistance protein (BCRP; ABCG2). For this purpose, the inhibitory effect of 123 structurally diverse drugs and drug-like compounds on mitoxantrone efflux was studied in Saos-2 cells transfected with human wild-type (Arg482) BCRP. The search for BCRP inhibitors throughout the drug-like chemical space resulted in the identification of 29 previously unknown inhibitors. The frequency of BCRP inhibition was 3 times higher for compounds reported to interact with other ATP-binding cassette (ABC) transporters than for compounds without reported ABC transporter affinity. An easily interpreted computational model capable of discriminating inhibitors from noninhibitors using only two molecular descriptors, octanol-water partition coefficient at pH 7.4 and molecular polarizability, was constructed. The discriminating power of this two-descriptor model was 93% for the training set and 79% for the test set, respectively. The results were supported by a global pharmacophore model and are in agreement with a two-step mechanism for the inhibition of BCRP, where both the drug's capacity to insert into the cell membrane and to interact with the inhibitory binding site of the transporter are important.
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