Maria Karlgren

Uppsala University, Uppsala, Uppsala, Sweden

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Publications (26)66.9 Total impact

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    ABSTRACT: 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.
    No preview · Article · Feb 2016 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.
    Full-text · Article · Feb 2016
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    ABSTRACT: Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over six orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of in vivo outcome.
    No preview · Article · Jul 2015 · Journal of Proteome Research
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    ABSTRACT: Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to inter-individual variability in atorvastatin clearance. However, the importance of the bile acid transporter 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 twelve 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 LC-MS/MS. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and inter-individual differences in CLupt of each transporter in vivo. Subsequently, hepatic uptake impairment upon co-administration 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 twelve sample donors. The rank order in expression was OATP1B1 > OATP1B3 ≈ NTCP ≈ 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 between individuals. The inter-individual 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.
    No preview · Article · May 2014 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: Two clinical trials and a large set of in vitro transporter experiments were performed to investigate if the hepatobiliary disposition of the direct thrombin inhibitor prodrug AZD0837 is the mechanism for the drug-drug interaction with ketoconazole observed in a previous clinical study. In Study 1, [(3)H]AZD0837 was administered to healthy male volunteers (n=8) to quantify and identify the metabolites excreted in bile. Bile was sampled directly from the jejunum by duodenal aspiration via an oro-enteric tube. In Study 2, the effect of ketoconazole on the plasma and bile pharmacokinetics of AZD0837, the intermediate metabolite (AR-H069927) and the active form (AR-H067637) was investigated (n=17). Co-administration with ketoconazole elevated the plasma exposure to AZD0837 and the active form approximately two-fold compared to placebo, which may be explained by inhibited CYP3A4 metabolism and reduced biliary clearance, respectively. High concentrations of the active form was measured in bile with a bile-to-plasma AUC ratio of approximately 75, indicating involvement of transporter-mediated excretion of the compound. AZD0837 and its metabolites were further investigated as substrates of hepatic uptake and efflux transporters in vitro. Studies in MDCK-MDR1 cell monolayers and P-glycoprotein (P-gp) expressing membrane vesicles identified AZD0837, the intermediate and the active form as substrates of P-gp. The active form was also identified as a substrate of the multidrug and toxin extrusion 1 (MATE1) transporter and the organic cation transporter 1 (OCT1), in HEK cells transfected with the respective transporter. Ketoconazole was shown to inhibit all of these three transporters; in particular, inhibition of P-gp and MATE1 occurred in a clinically relevant concentration range. In conclusion, the hepatobiliary transport pathways of AZD0837 and its metabolites were identified in vitro and in vivo. Inhibition of the canalicular transporters P-gp and MATE1 may lead to enhanced plasma exposure to the active form, which could, at least in part, explain the clinical interaction with ketoconazole.
    No preview · Article · Sep 2013 · Molecular Pharmaceutics
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    ABSTRACT: The absorption, distribution, metabolism and excretion (ADME) of drugs in vivo are to a large extent dependent on different transport and metabolism routes. Elucidation of this complex transport-metabolism interplay is a major challenge in drug development and at present no in vitro models suitable for this purpose are at hand. The aim of this study was to develop flexible, well-controlled, easy-to-use, integrated cell models, where drug transport and drug metabolism processes could be studied simultaneously. HEK293 cells stably transfected with the organic anion transporting polypeptide 1B1 (OATP1B1) were subjected to either transient transfection or adenoviral infection to introduce the genes expressing cytochrome P450 3A4 (CYP3A4), NADPH cytochrome P450 oxidoreductase (POR), cytochrome b5 (CYB5A), and multidrug resistance protein 1 (MDR1), in different combinations. Thereafter, the time and concentration-dependent transport and metabolism of two well-characterized statins, atorvastatin (acid and lactone forms), and simvastatin (acid form) were determined in the different models. The results show that CYP3A4-dependent metabolism of the more hydrophilic atorvastatin acid was dependent on OATP1B1 uptake and influenced by MDR1 efflux. In contrast, metabolism of the more lipophilic atorvastatin lactone was not affected by active transport, whereas the metabolism of simvastatin acid was less influenced by active transport than atorvastatin acid. Our results, together with the models being applicative for any combination of drug transporters and CYP metabolizing enzymes of choice, provide proof-of-concept for the potential of the new integrated cell models presented as valuable screening tools in drug discovery and development.
    Full-text · Article · Jul 2013 · Molecular Pharmaceutics
  • Pär Matsson · Per Artursson · Maria Karlgren

    No preview · Chapter · Jul 2013
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    ABSTRACT: 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.
    Full-text · Article · Apr 2012 · Journal of Medicinal Chemistry
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    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 μM. 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.
    Full-text · Article · Aug 2011 · Pharmaceutical Research
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    ABSTRACT: The cytochromes P450 (CYPs) are very efficient catalysts of foreign compound metabolism and are responsible for the major part of metabolism of clinically important drugs. The enzymes are important in cancer since they (a) activate dietary and environmental components to ultimate carcinogens, (b) activate or inactivate drugs used for cancer treatment, and (c) are potential targets for anticancer therapy. The genes encoding the CYP enzymes active in drug metabolism are highly polymorphic, whereas those encoding metabolism of precarcinogens are relatively conserved. A vast amount of literature is present where investigators have tried to link genetic polymorphism in CYPs to cancer susceptibility, although not much conclusive data have hitherto been obtained, with exception of CYP2A6 polymorphism and tobacco induced cancer, to a great extent because of lack of important functional polymorphisms in the genes studied. With respect to anticancer treatment, the genetic CYP polymorphism is of greater importance, where treatment with tamoxifen, but also with cyclophosphamide and maybe thalidomide is influenced by CYP genetic variants. In the present review we present updates on CYP genetics, cancer risk and treatment and also epigenetic aspects of interindividual variability in CYP expression and the use of these enzymes as targets for cancer therapy. We conclude that the CYP polymorphism does not predict cancer susceptibility to any large extent but that this polymorphism might be an important factor for optimal cancer therapy using selected anticancer agents.
    No preview · Article · Oct 2009 · Human Genetics
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    ABSTRACT: Cytochrome P450 (CYP) enzymes are important for drug metabolism. A novel cytochrome P450 enzyme, CYP2W1, has recently been identified. This enzyme is mainly found in foetal colon tissue and in tumour tissue. In this pilot study, we have investigated the expression of CYP2W1 in 162 tumours from patients with stages II and III colorectal cancer. The expression of CYP2W1 enzyme was immunohistochemically detected using a polyclonal antibody. Staining intensity was defined using a visual grading scale from 0 to 3. Grades 0-2 were classified as low, and grade 3 was classified as high expression of CYP2W1. About 64% of the tumours expressed a low level of CYP2W1-expression, and 36% expressed a high level. CYP2W1-expression was an independent prognostic factor for overall survival (p=0.007), where a high expression was associated with a worse clinical outcome. Immunohistochemically assessed expression of CYP2W1 is an independent prognostic factor in patients with stages II and III colorectal cancer.
    No preview · Article · Mar 2009 · European journal of cancer (Oxford, England: 1990)
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    ABSTRACT: CYP2W1 is a novel enzyme shown to be selectively expressed in rat fetal colon and in human colon cancer and has previously been suggested as a potential drug target for cancer therapy. Here, the expression and gene methylation of CYP2W1 were analyzed in human colon carcinoma cell lines, colon tumors and in corresponding normal colon tissue. CYP2W1 mRNA and protein expression in HepG2 and Caco-2TC7 cells and normal colon and colon tumor tissue samples were analyzed using real-time PCR and Western blotting. CYP2W1 gene methylation status in the same samples was analyzed using the sodium bisulfite sequencing method. CYP2W1 mRNA was detected in all (n = 39) tumor samples analyzed. Moreover, in 60% (12/20) of the colon tumors, CYP2W1 mRNA levels were substantially higher than in corresponding normal tissues. CYP2W1 protein was detected in most of the colon tumor samples analyzed (n = 16), which appeared to be of two apparent phenotypes: those with five- to ten-fold induced CYP2W1 (approximately 50% of the tumors), and those with low expression, harboring similar or only slightly higher amounts of CYP2W1 as compared with surrounding control tissue. Methylation analysis of the CpG island in the exon 1-intron 1 junction of the CYP2W1 gene from both cell lines, tumors and normal tissues revealed that demethylated CpG dinucleotides appeared as a requirement for high CYP2W1 gene expression. The expression of CYP2W1 is colon tumor-specific and is associated with methylation status of the CYP2W1 gene, suggesting a potential causal link between the gene hypomethylation and its enhanced expression.
    Full-text · Article · Oct 2007 · Pharmacogenomics
  • Maria Karlgren · Magnus Ingelman-Sundberg
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    ABSTRACT: Recently, a new cytochrome P450, designated CYP2W1, was identified. This enzyme is expressed in transformed tissues and during fetal life, whereas in human adult tissues only low levels of expression have been detected. CYP2W1 has been shown to metabolise arachidonic acid and benzfetamine, as well as being able to metabolically activate several procarcinogens, including polycyclic aromatic hydrocarbon dihydrodiols, aflatoxin B1 and sterigmatocystin. The gene expression is governed by gene methylation. The selective expression in some forms of cancers and the low expression in normal tissues render CYP2W1 as a possible drug target during cancer therapy. Here, the authors review the data currently available for this enzyme and discuss its potential as a drug target.
    No preview · Article · Feb 2007 · Expert Opinion on Therapeutic Targets
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    ABSTRACT: The 16th International Symposium on Microsomes and Drug Oxidations (MDO2006) in Budapest, Hungary, had almost 400 attendees and was nicely organized by Laszlo Vereczkey and colleagues. The meeting had a very high standard in the field of drug metabolism, drug transport and related areas and in addition, the social events were much appreciated. At the meeting 70 invited lectures were presented in plenary sessions and in three parallel symposia sessions, and about 178 posters were shown, among them 26 posters in the young investigators workshop. The review herein is given of a majority (57) of the lectures presented at the Symposium.
    No preview · Article · Jan 2007 · Drug News & Perspectives
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    ABSTRACT: A novel human cytochrome P450, CYP2W1, was cloned and expressed heterologously. No or very low CYP2W1 mRNA levels were detected in fetal and adult human tissues, expression was however seen in 54% of human tumor samples investigated (n=37), in particular colon and adrenal tumors. Western blotting also revealed high expression of CYP2W1 in some human colon tumors. In rat tissues, CYP2W1 mRNA was expressed preferentially in fetal but also in adult colon. The CYP2W1 gene was shown to encompass one functional CpG island in the exon 1-intron 1 region which was methylated in cell lines lacking CYP2W1 expression, but unmethylated in cells expressing CYP2W1. Re-expression of CYP2W1 was seen following demethylation by 5-Aza-2'-deoxycytidine. Transfection of HEK293 cells with CYP2W1 caused the formation of a properly folded enzyme, which was catalytically active with arachidonic acid as a substrate. It is concluded that CYP2W1 represents a tumor-specific P450 isoform with potential importance as a drug target in cancer therapy.
    No preview · Article · Apr 2006 · Biochemical and Biophysical Research Communications
  • Maria Karlgren · Alvin Gomez · Magnus Ingelman-Sundberg
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    ABSTRACT: A novel human cytochrome P450 cDNA, designated CYP2W1, was cloned (Karlgren et al., 2006). CYP2W1 showed high mRNA expression in HepG2 cells, whereas no or very low expression levels was seen in any adult or fetal tissue or in other cell lines examined. Analysis of its expression in specimens from many human tissues revealed a rather selective expression in human tumor samples where 54 % of the samples investigated had significant expression. Highest amounts of CYP2W1 mRNA were seen in colon and adrenal tumors. Western-blotting also revealed high expression of CYP2W1 in HepG2 cells and also in some human colon tumors, whereas no expression was seen in human liver microsomes. In rat tissues CYP2W1 mRNA was expressed in fetal colon and also in colon from younger animals but was not seen at any significant amounts in other fetal or adult tissues. The CYP2W1 gene was shown to encompass one functional CpG island in the exon 1- intron 1 region (+55 to +305 bp) which was methylated in cell lines lacking CYP2W1 expression, e.g. B16A2 hepatoma cells, but unmethylated in HepG2 cells expressing CYP2W1. Reexpression of CYP2W1 in the silent B16A2 cells was obtained following demethylation by 5-Aza-2'-deoxycytidine. This indicates that the CYP2W1 gene expression to some extent is governed by gene methylation. Transfection of HEK293 cells with a CYP2W1 cDNA caused formation of a properly folded enzyme, as revealed from the CO-reduced difference spectra, which was catalytically active with arachidonic acid as a substrate. Using expressed modified CYP2W1 in E. coli Wu et al. showed that CYP2W1 has a broad substrate specificity, and apart from arachidonic acid also metabolises benzphetamine and several procarcinogens (Wu et al. 2006). CYP2W1 was also expressed in E. coli by Yoshioka et al. (2006). Taken together, CYP2W1 represents a new catalytically active extrahepatic P450, selectively expressed in tumor tissues, with a potential importance as a drug target in cancer therapy. References: Karlgren M, Gomez A, Stark K, Svard J, Rodriguez-Antona C, Oliw E, Bernal ML, Ramon y Cajal S, Johansson I, Ingelman-Sundberg M. Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1. Biochemical and Biophysical Research Communications. 341: 451-458. 2006. Wu ZL, Sohl CD, Shimada T, Guengerich FP. Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1. Molecular Pharmacology. 69: 2007-2014. 2006. Yoshioka H, Kasai N, Ikushiro S, Shinkyo R, Kamakura M, Ohta M, Inouye K, Sakaki T. Enzymatic properties of human CYP2W1 expressed in Escherichia coli. Biochemical and Biophysical Research Communications. 345: 169-174. 2006.
    No preview · Conference Paper · Jan 2006
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    ABSTRACT: The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis.
    No preview · Article · Oct 2005 · Toxicology and Applied Pharmacology
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    ABSTRACT: The 15th International Symposium on Microsomes and Drug Oxidations: Chemical Biology in the Postgenomic Era--New Approaches and Applications in Mainz, Germany, July 4-9, 2004, had 570 attendees and was nicely organized by Franz and Barbara Oesch and colleagues. At the meeting, 73 different lectures were presented and about 240 posters were shown. This review discusses most of the lectures presented at the Symposium.
    No preview · Article · Dec 2004 · Drug News & Perspectives
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    ABSTRACT: A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.
    No preview · Article · Apr 2004 · Biochemical and Biophysical Research Communications
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    ABSTRACT: The hepatic organic anion transporting polypeptides (OATP) 1B1, 1B3 and 2B1 influence the pharmacokinetics of drug classes like statins and angiotensin II receptor antagonists and are involved in many clinical drug-drug interactions (DDIs). Predicting potential interactions with OATPs during drug discovery is, therefore, of value. Here, we developed in vitro and in silico models for the identification and prediction of specific and general inhibitors of the OATPs. The maximal transport activity (MTA) of each transporter in the human liver was predicted from transport kinetics and protein quantification in vitro and in vivo. We used this in vitro to in vivo extrapolation to quantify the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a dataset of 225 drug-like compounds, 91 OATP inhibitors were identified, of which 38 were novel inhibitors. The in silico model identified lipophilicity and polar surface area as key molecular features of OATP inhibition. Predictions of MTA of each OATP identified OATP1B1 and OATP1B3 as the major contributors to atorvastatin uptake in vivo. The relative contributions of OATPs to overall hepatic uptake were, however, changed when inhibitors with different isoform specificities were used in the DDI predictions.
    No preview · Conference Paper ·