Phenotype-Genotype Correlation Applying a Cocktail Approach and an Exome Chip Analysis Reveals Further Variants Contributing to Variation of Drug Metabolism.

Although great progress has been made in the fine-tuning of diplotypes, there is still a need to further improve the predictability of individual phenotypes of pharmacogenetically relevant enzymes. The aim of this study was to analyze the additional contribution of sex and variants identified by exome chip analysis to the metabolic ratio of five probe drugs. A cocktail study applying dextromethorphan, losartan, omeprazole, midazolam, and caffeine was conducted on 200 healthy volunteers. CYP2D6, 2C9, 2C19, 3A4/5, and 1A2 genotypes were analyzed and correlated with metabolic ratios. In addition, an exome chip analysis was performed. These SNPs correlating with metabolic ratios were confirmed by individual genotyping. The contribution of various factors to metabolic ratios was assessed by multiple regression analysis. Genotypically predicted phenotypes defined by CPIC discriminated very well the log metabolic ratios with the exception of caffeine. There were minor sex differences in the activity of CYP2C9, 2C19, 1A2, and CYP3A4/5. For dextromethorphan (CYP2D6), IP6K2 (rs61740999) and TCF20 (rs5758651) affected metabolic ratios, but only IP6K2 remained significant after multiple regression analysis. For losartan (CYP2C9), FBXW12 (rs17080138), ZNF703 (rs79707182), and SLC17A4 (rs11754288) together with CYP diplotypes, and sex explained 50% of interindividual variability. For omeprazole (CYP2C19), no significant influence of CYP2C:TG haplotypes was observed, but CYP2C19 rs12777823 improved the predictability. The comprehensive genetic analysis and inclusion of sex in a multiple regression model significantly improved the explanation of variability of metabolic ratios, resulting in further improvement of algorithms for the prediction of individual phenotypes of drug-metabolizing enzymes.

Comparative Intra-Subject Analysis of Gene Expression and Protein Abundance of Major and Minor Drug Metabolizing Enzymes in Healthy Human Jejunum and Liver.

First pass metabolism by phase I and phase II enzymes in the intestines and liver is a major determinant of the oral bioavailability of many drugs. Several studies analyzed expressions of major drug-metabolizing enzymes (DMEs), such as CYP3A4 and UGT1A1 in the human gut and liver. However, there is still a lack of knowledge regarding other DMEs (i.e., "minor" DMEs), although several clinically relevant drugs are affected by those enzymes. Moreover, there is very limited intra-subject data on hepatic and intestinal expression levels of minor DMEs. To fill this gap of knowledge, we analyzed gene expression (quantitative real-time polymerase chain reaction) and protein abundance (targeted proteomics) of 24 clinically relevant DMEs, that is, carboxylesterases (CES), UDP-glucuronosyltransferases (UGT), and cytochrome P450 (CYP)-enzymes. We performed our analysis using jejunum and liver tissue specimens from the same 11 healthy organ donors (8 men and 3 women, aged 19-60 years). Protein amounts of all investigated DMEs, with the exception of CYP4A11, were detected in human liver samples. CES2, CYP2C18, CYP3A4, and UGT2B17 protein abundance was similar or even higher in the jejunum, and all other DMEs were found in higher amounts in the liver. Significant correlations between gene expression and protein levels were observed only for 2 of 15 jejunal, but 13 of 23 hepatic DMEs. Intestinal and hepatic protein amounts only significantly correlated for CYP3A4 and UGT1A3. Our results demonstrated a notable variability between the individuals, which was even higher in the intestines than in the liver. Our intrasubject analysis of DMEs in the jejunum and liver from healthy donors, may be useful for physiologically-based pharmacokinetic-based modeling and prediction in order to improve efficacy and safety of oral drug therapy.

Novel In Vitro Models for Cell Differentiation and Drug Transport Studies of the Human Intestine.

The most common in vitro model for absorption, distribution, metabolism, and excretion (ADME) purposes is currently the Caco-2 cell line. However, clear differences in gene and protein expression towards the small intestine and an, at best, fair prediction accuracy of intestinal drug absorption restrict the usefulness of a model for intestinal epithelial cells. To overcome these limitations, we evaluated a panel of low-passaged patient-derived colorectal cancer cell lines of the HROC collection concerning similarities to small intestinal epithelial cells and their potential to predict intestinal drug absorption. After initial screening of a larger panel, ten cell lines with confluent outgrowth and long-lasting barrier-forming potential were further characterized in close detail. Tight junctional complexes and microvilli structures were detected in all lines, anda higher degree of differentiation was observed in 5/10 cell lines. All lines expressed multiple transporter molecules, with the expression levels in three lines being close to those of small intestinal epithelial cells. Compared with the Caco-2 model, three HROC lines demonstrated both higher similarity to jejunal epithelial tissue cells and higher regulatory potential of relevant drug transporters. In summary, these lines would be better-suited human small intestinal epithelium models for basic and translational research, especially for ADME studies.

Protein Abundance of Drug Metabolizing Enzymes in Human Hepatitis C Livers.

Hepatic drug metabolizing enzymes (DMEs), whose activity may be affected by liver diseases, are major determinants of drug pharmacokinetics. Hepatitis C liver samples in different functional states, i.e., the Child-Pugh class A (n = 30), B (n = 21) and C (n = 7) were analyzed for protein abundances (LC-MS/MS) and mRNA levels (qRT-PCR) of 9 CYPs and 4 UGTs enzymes. The protein levels of CYP1A1, CYP2B6, CYP2C8, CYP2C9, and CYP2D6 were not affected by the disease. In the Child-Pugh class A livers, a significant up-regulation of UGT1A1 (to 163% of the controls) was observed. The Child-Pugh class B was associated with down-regulation of the protein abundance of CYP2C19 (to 38% of the controls), CYP2E1 (to 54%), CYP3A4 (to 33%), UGT1A3 (to 69%), and UGT2B7 (to 56%). In the Child-Pugh class C livers, CYP1A2 was found to be reduced (to 52%). A significant trend in down-regulation of the protein abundance was documented for CYP1A2, CYP2C9, CYP3A4, CYP2E1, UGT2B7, and UGT2B15. The results of the study demonstrate that DMEs protein abundances in the liver are affected by hepatitis C virus infection and depend on the severity of the disease.

Importance of ACE2 for SARS-CoV-2 Infection of Kidney Cells.

In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SARS-CoV-2 into human kidney cells. First, the expression of the cellular receptor ACE2 was demonstrated to be decisive for viral SARS-CoV-2 cell entry in human AB8 podocytes, whereas the presence of the transmembrane protease serine 2 (TMPRSS2) was dispensable. Moreover, the ACE2 protein amount was well detectable by mass spectrometry analysis in human kidneys, while TMPRSS2 could be detected only in a few samples. Additionally, a negative correlation of the ACE2 protein abundance to male sex and elderly aged females in human kidney tissues was demonstrated in this work. Last, the possibility of a direct infection of kidney tubular renal structures by SARS-CoV-2 was demonstrated.

Kidney Drug Transporters in Pharmacotherapy.

The kidney functions not only as a metabolite elimination organ but also plays an important role in pharmacotherapy. The kidney tubule epithelia cells express membrane carriers and transporters, which play an important role in drug elimination, and can determine drug nephrotoxicity and drug-drug interactions, as well as constituting direct drug targets. The above aspects of kidney transport proteins are discussed in the review.

Establishment and Characterization of Novel Human Intestinal In Vitro Models for Absorption and First-Pass Metabolism Studies.

Commonly used intestinal in vitro models are limited in their potential to predict oral drug absorption. They either lack the capability to form a tight cellular monolayer mimicking the intestinal epithelial barrier or the expression of cytochrome P450 3A4 (CYP3A4). The aim of this study was to establish a platform of colorectal cancer patient-derived cell lines for evaluation of human intestinal drug absorption and metabolism. We characterized ten 2D cell lines out of our collection with confluent outgrowth and long-lasting barrier forming potential as well as suitability for high throughput applications with special emphasis on expression and inducibility of CYP3A4. By assessment of the transepithelial electrical resistance (TEER) the cells barrier function capacity can be quantified. Very high TEER levels were detected for HROC60. A high basal CYP3A4 expression and function was found for HROC32. Eight cell lines showed higher CYP3A4 induction by stimulation via the vitamin D receptor compared to Caco-2 cells (5.1- to 16.8-fold change). Stimulation of the pregnane X receptor led to higher CYP3A4 induction in two cell lines. In sum, we identified the two cell lines HROC183 T0 M2 and HROC217 T1 M2 as useful tools for in vitro drug absorption studies. Due to their high TEER values and inducibility by drug receptor ligands, they may be superior to Caco-2 cells to analyze oral drug absorption and intestinal drug-drug interactions. Significance statement: Selecting appropriate candidates is important in preclinical drug development. Therefore, cell models to predict absorption from the human intestine are of the utmost importance. This study revealed that the human cell lines HROC183 T0 M2 and HROC217 T1 M2 may be better suited models and possess higher predictive power of pregnane X receptor- and vitamin D-mediated drug metabolism than Caco-2 cells. Consequently, they represent useful tools for predicting intestinal absorption and simultaneously enable assessment of membrane permeability and first-pass metabolism.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) is localized in lysosomes and mediates resistance against kinase inhibitors.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3-mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance. Significance Statement We describe the characterization of a splice variant of the liver-type uptake transporter OATP1B3 expressed in several tumor entities. This variant is localized in lysosomes mediating resistance against kinase inhibitors which are substrates of this transport protein by transporting them into lysosomes and thereby reducing the cytoplasmic concentration of these antitumor agents. Therefore, the expression of the Ct-OATP1B3 protein is associated with a better survival of cells revealing a new mechanism of drug resistance.

Protein Abundance of Drug Transporters in Human Hepatitis C Livers.

Transmembrane drug transport in hepatocytes is one of the major determinants of drug pharmacokinetics. In the present study, ABC transporters (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, and BSEP) and SLC transporters (MCT1, NTCP, OAT2, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3) were quantified for protein abundance (LC-MS/MS) and mRNA levels (qRT-PCR) in hepatitis C virus (HCV)-infected liver samples from the Child-Pugh class A (n = 30), B (n = 21), and C (n = 7) patients. Protein levels of BSEP, MRP3, MCT1, OAT2, OATP1B3, and OCT3 were not significantly affected by HCV infection. P-gp, MRP1, BCRP, and OATP1B3 protein abundances were upregulated, whereas those of MRP2, MRP4, NTCP, OATP2B1, and OCT1 were downregulated in all HCV samples. The observed changes started to be seen in the Child-Pugh class A livers, i.e., upregulation of P-gp and MRP1 and downregulation of MRP2, MRP4, BCRP, and OATP1B3. In the case of NTCP, OATP2B1, and OCT1, a decrease in the protein levels was observed in the class B livers. In the class C livers, no other changes were noted than those in the class A and B patients. The results of the study demonstrate that drug transporter protein abundances are affected by the functional state of the liver in hepatitis C patients.

Gene Expression and Protein Abundance of Nuclear Receptors in Human Intestine and Liver: A New Application for Mass Spectrometry-Based Targeted Proteomics.

BACKGROUND: Unwanted drug-drug interactions (DDIs), as caused by the upregulation of clinically relevant drug metabolizing enzymes and transporter proteins in intestine and liver, have the potential to threaten the therapeutic efficacy and safety of drugs. The molecular mechanism of this undesired but frequently occurring scenario of polypharmacy is based on the activation of nuclear receptors such as the pregnane X receptor (PXR) or the constitutive androstane receptor (CAR) by perpetrator agents such as rifampin, phenytoin or St. John's wort. However, the expression pattern of nuclear receptors in human intestine and liver remains uncertain, which makes it difficult to predict the extent of potential DDIs. Thus, it was the aim of this study to characterize the gene expression and protein abundance of clinically relevant nuclear receptors, i.e., the aryl hydrocarbon receptor (AhR), CAR, farnesoid X receptor (FXR), glucocorticoid receptor (GR), hepatocyte nuclear factor 4 alpha (HNF4α), PXR and small heterodimer partner (SHP), in the aforementioned organs. METHODS: Gene expression analysis was performed by quantitative real-time PCR of jejunal, ileal, colonic and liver samples from eight human subjects. In parallel, a targeted proteomic method was developed and validated in order to determine the respective protein amounts of nuclear receptors in human intestinal and liver samples. The LC-MS/MS method was validated according to the current bioanalytical guidelines and met the criteria regarding linearity (0.1-50 nmol/L), within-day and between-day accuracy and precision, as well as the stability criteria. RESULTS: The developed method was successfully validated and applied to determine the abundance of nuclear receptors in human intestinal and liver samples. Gene expression and protein abundance data demonstrated marked differences in human intestine and liver. On the protein level, only AhR and HNF4α could be detected in gut and liver, which corresponds to their highest gene expression. In transfected cell lines, PXR and CAR could be quantified. CONCLUSIONS: The substantially different expression pattern of nuclear receptors in human intestinal and liver tissue may explain the different extent of unwanted DDIs in the dependence on the administration route of drugs.

Regulation of Drug Transport Proteins-From Mechanisms to Clinical Impact: A White Paper on Behalf of the International Transporter Consortium.

Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, alternative splicing, post-translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation-mediated changes in transporter function on drug disposition and response.

The reference liver-CYP450 and UGT enzymes in healthy donor and metastatic livers: the impact of genotype.

BACKGROUND: Hepatic enzymes involved in drug metabolism vary markedly in expression, abundance and activity, which affects individual susceptibility to drugs and toxicants. The present study aimed to compare mRNA expression and protein abundance of the most pharmacologically relevant drug-metabolizing enzymes in two main sources of the control liver samples that are used as the reference, i.e. organ donor livers and non-tumorous tissue from metastatic livers. An association analysis of the most common genetic variants with mRNA and protein levels was also performed. METHODS: The CYP450 and UGT enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, UGT1A1, UGT1A3, UGT2B7 and UGT2B15) were analyzed for mRNA (qPCR) and protein abundance (LC-MS/MS) in healthy donors (n = 11) and metastatic (n = 13) livers. Genotyping was performed by means of TaqMan assays and pyrosequencing. RESULTS: Significantly higher protein abundance in the metastatic livers was observed in case of CYP2C9, CYP2D6, and UGT2B7, and for UGT1A3 the difference was only significant at mRNA level. For all the enzymes except CYP2E1 some significant correlation between mRNA and protein content was observed, and for UGT1A1 an inverse correlation with age was noted. CYP2C19, CYP3A5 and CYP2D6 were significantly affected by genotype. CONCLUSION: The selection of a control group for the study on drug-metabolizing enzymes (e.g. in pathological states) may possibly affect its conclusions on differences in mRNA and protein content. Genotyping for common functional variants of CYP450 enzymes should be performed in all studies on drug-metabolizing enzymes.

Gene Expression and Protein Abundance of Hepatic Drug Metabolizing Enzymes in Liver Pathology.

Hepatic drug metabolizing enzymes (DMEs) markedly affect drug pharmacokinetics. Because liver diseases may alter enzymatic function and in turn drug handling and clinical efficacy, we investigated DMEs expression in dependence on liver pathology and liver failure state. In 5 liver pathologies (hepatitis C, alcoholic liver disease, autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis) and for the first time stratified according to the Child-Pugh score, 10 CYPs (CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and 4 UGTs (UGT1A1, UGT1A3, UGT2B7 and UGT2B) enzymes were quantified for protein abundance (LC-MS/MS) and gene expression (qRT-PCR). CYP2E1 was the most vulnerable enzyme, and its protein levels were significantly reduced just in Child-Pugh class A livers. The protein abundance of CYP1A1, CYP2B6, CYP2C19, CYP2D6 as well as UGT1A1, UGT1A3 and UGT2B15 was relatively stable in the course of progression of liver function deterioration. Alcoholic liver disease and primary biliary cholangitis were involved in the most prominent changes in the protein abundances, with downregulation of 6 (CYP1A2, CYP2C8, CYP2D6, CYP2E1, CYP3A4, UGT2B7) and 5 (CYP1A1, CYP2B6, CYP2C8, CYP2E1, CYP3A4) significantly downregulated enzymes, respectively. The results of the study demonstrate that DMEs protein abundance is affected both by the type of liver pathology as well as functional state of the organ.

Impact of kidney dysfunction on hepatic and intestinal drug transporters.

Emerging information suggests that pathology of the kidney may not only affect expression and function of membrane transporters in the organ, but also in the gastrointestinal tract and the liver. Transporter dysfunction may cause effects on handling of drug as well as endogenous compounds with subsequent clinical consequences. A literature search was conducted on Ovid and PubMed databases to select relevant in vitro, animal and human studies that have reported expression, protein abundance and function of the gastrointestinal and liver localized ABC transporters and SLC carriers in kidney dysfunction or uremia states. The altered function of drug transporters in the liver and intestines in kidney failure subjects may provide compensatory activity in handling endogenous compounds (e.g. uremic toxins), which is expected to affect drug pharmacokinetics and local drug actions.

Mass spectrometry-based targeted proteomics method for the quantification of clinically relevant drug metabolizing enzymes in human specimens.

Biotransformation by phase I and II metabolizing enzymes represents the major determinant for the oral bioavailability of many drugs. To estimate the pharmacokinetics, data on protein abundance of hepatic and extrahepatic tissues, such as the small intestine, are required. Targeted proteomics assays are nowadays state-of-the-art for absolute protein quantification and several methods for quantification of drug metabolizing enzymes have been published. However, some enzymes remain still uncovered by the analytical spectra of those methods. Therefore, we developed and validated a quantification assay for two carboxylesterases (CES-1, CES-2), 17 cytochrome P450 enzymes (CYP) (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F12, CYP4A11) and five UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT2B7, UGT2B15, UGT2B17). Protein quantification was performed by analyzing proteospecific surrogate peptides after tryptic digestion with stable isotope-labelled standards. Chromatographic separation was performed on a Kinetex® 2.6 µm C18 100 Å core-shell column (100 × 2.1 mm) with a gradient elution using 0.1% formic acid and acetonitrile containing 0.1% formic acid with a flow rate of 200 µl/min. Three mass transitions were simultaneously monitored with a scheduled multiple reaction monitoring (sMRM) method for each analyte and standard. The method was partly validated according to current bioanalytical guidelines and met the criteria regarding linearity (0.1-25 nmol/L), within-day and between-day accuracy and precision as well as multiple stability criteria. Finally, the developed method was successfully applied to determine the abundance of the aforementioned enzymes in human intestinal und liver microsomes. Our work offers a new fit for purpose method for the absolute quantification of CES, CYPs and UGTs in various human tissues and can be used for the acquisition of data for physiologically based pharmacokinetic modelling.

Comparative Hepatic and Intestinal Efflux Transport of Statins.

Previous studies have shown that lipid-lowering statins are transported by various ATP-binding cassette (ABC) transporters. However, because of varying methods, it is difficult to compare the transport profiles of statins. Therefore, we investigated the transport of 10 statins or statin metabolites by six ABC transporters using human embryonic kidney cell-derived membrane vesicles. The transporter protein expression levels in the vesicles were quantified with liquid chromatography-tandem mass spectrometry and used to scale the measured clearances to tissue levels. In our study, apically expressed breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) transported atorvastatin, fluvastatin, pitavastatin, and rosuvastatin. Multidrug resistance-associated protein 3 (MRP3) transported atorvastatin, fluvastatin, pitavastatin, and, to a smaller extent, pravastatin. MRP4 transported fluvastatin and rosuvastatin. The scaled clearances suggest that BCRP contributes to 87%-91% and 84% of the total active efflux of rosuvastatin in the small intestine and the liver, respectively. For atorvastatin, the corresponding values for P-gp-mediated efflux were 43%-79% and 66%, respectively. MRP3, on the other hand, may contribute to 23%-26% and 25%-37% of total active efflux of atorvastatin, fluvastatin, and pitavastatin in jejunal enterocytes and liver hepatocytes, respectively. These data indicate that BCRP may play an important role in limiting the intestinal absorption and facilitating the biliary excretion of rosuvastatin and that P-gp may restrict the intestinal absorption and mediate the biliary excretion of atorvastatin. Moreover, the basolateral MRP3 may enhance the intestinal absorption and sinusoidal hepatic efflux of several statins. Taken together, the data show that statins differ considerably in their efflux transport profiles. SIGNIFICANCE STATEMENT: This study characterized and compared the transport of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin acid and four atorvastatin metabolites by six ABC transporters (BCRP, MRP2, MRP3, MRP4, MRP8, P-gp). Based on in vitro findings and protein abundance data, the study concludes that BCRP, MRP3, and P-gp have a major impact in the efflux of various statins. Together with in vitro metabolism, uptake transport, and clinical data, our findings are applicable for use in comparative systems pharmacology modeling of statins.

Hepatic drug-metabolizing enzymes and drug transporters in Wilson's disease patients with liver failure.

BACKGROUND: Wilson's disease is a genetic disorder inherited in a recessive manner, caused by mutations in the copper-transporter ATP7B. Although it is a well-known disease, currently available treatments are far from satisfactory and their efficacy varies in individual patients. Due to the lack of information about drug-metabolizing enzymes and drug transporters profile in Wilson's disease livers, we aimed to evaluate the mRNA expression and protein abundance of selected enzymes and drug transporters in this liver disorder. METHODS: We analyzed gene expression (qPCR) and protein abundance (LC-MS/MS) of 14 drug-metabolizing enzymes and 16 drug transporters in hepatic tissue from Wilson's disease patients with liver failure (n = 7, Child-Pugh class B and C) and metastatic control livers (n = 20). RESULTS: In presented work, we demonstrated a downregulation of majority of CYP450 and UGT enzymes. Gene expression of analyzed enzymes ranged between 18 and 65% compared to control group and significantly lower protein content of CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP3A4 and CYP3A5 enzymes was observed in Wilson's disease. Moreover, a general decrease in hepatocellular uptake carriers from SLC superfamily (significant at protein level for NTCP and OATP2B1) was observed. As for ABC transporters, the protein abundance of BSEP and MRP2 was significantly lower, while levels of P-gp and MRP4 transporters were significantly higher in Wilson's disease. CONCLUSIONS: Altered hepatic expression of drug-metabolizing enzymes and drug transporters in Wilson's disease patients with liver failure may result in changes of drug pharmacokinetics in that group of patients.

Organic Cation Transporter 1 an Intestinal Uptake Transporter: Fact or Fiction?

Intestinal transporter proteins are known to affect the pharmacokinetics and in turn the efficacy and safety of many orally administered drugs in a clinically relevant manner. This knowledge is especially well-established for intestinal ATP-binding cassette transporters such as P-gp and BCRP. In contrast to this, information about intestinal uptake carriers is much more limited although many hydrophilic or ionic drugs are not expected to undergo passive diffusion but probably require specific uptake transporters. A transporter which is controversially discussed with respect to its expression, localization and function in the human intestine is the organic cation transporter 1 (OCT1). This review article provides an up-to-date summary on the available data from expression analysis as well as functional studies in vitro, animal findings and clinical observations. The current evidence suggests that OCT1 is expressed in the human intestine in small amounts (on gene and protein levels), while its cellular localization in the apical or basolateral membrane of the enterocytes remains to be finally defined, but functional data point to a secretory function of the transporter at the basolateral membrane. Thus, OCT1 should not be considered as a classical uptake transporter in the intestine but rather as an intestinal elimination pathway for cationic compounds from the systemic circulation.

Membrane Carriers and Transporters in Kidney Physiology and Disease.

The growing information suggests that chronic kidney disease may affect expression and function of membrane carriers and transporters in the kidney. The dysfunction of carriers and transporters entails deficient elimination of uremic solutes as well as xenobiotics (drugs and toxins) with subsequent clinical consequences. The renal carriers and transporters are also targets of drugs used in clinical practice, and intentional drug-drug interactions in the kidney are produced to increase therapeutic efficacy. The understanding of membrane carriers and transporters function in chronic kidney disease is important not only to better characterize drug pharmacokinetics, drug actions in the kidney, or drug-drug interactions but also to define the organ pathophysiology.