The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics.

Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.

Advances in drug resistance of osteosarcoma caused by pregnane X receptor.

Osteosarcoma (OS) is a prevalent malignancy among adolescents, commonly manifesting during childhood and adolescence. It exhibits a high degree of malignancy, propensity for metastasis, rapid progression, and poses challenges in clinical management. Chemotherapy represents an efficacious therapeutic modality for OS treatment. However, chemotherapy resistance of OS is a major problem in clinical treatment. In order to treat OS effectively, it is particularly important to explore the mechanism of chemotherapy resistance in OS.The Pregnane X receptor (PXR) is a nuclear receptor primarily involved in the metabolism, transport, and elimination of xenobiotics, including chemotherapeutic agents. PXR involves three stages of drug metabolism: stage I: drug metabolism enzymes; stage II: drug binding enzyme; stage III: drug transporter.PXR has been confirmed to be involved in the process of chemotherapy resistance in malignant tumors. The expression of PXR is increased in OS, which may be related to drug resistance of OS. Therefore, wereviewed in detail the role of PXR in chemotherapy drug resistance in OS.

The fluorescence-based competitive counterflow assay developed for organic anion transporting polypeptides 1A2, 1B1, 1B3 and 2B1 identifies pentamidine as a selective OATP1A2 substrate.

Organic anion transporting polypeptides OATP1A2, OATP1B1, OATP1B3 and OATP2B1 are Na+ - and ATP-independent exchangers of large, organic compounds, encompassing structurally diverse xenobiotics, including various drugs. These OATPs influence intestinal absorption (OATP2B1), hepatic clearance (OATP1B1/3) and blood to brain penetration (OATP1A2, OATP2B1) of their drug substrates. Consequently, OATP-mediated drug or food interactions may lead to altered pharmacokinetics and toxicity. During drug development, investigation of hepatic OATP1B1 and OATP1B3 is recommended by international regulatory agencies. Most frequently, OATP-drug interactions are investigated in an indirect assay, i.e., by examining uptake inhibition of a radioactive or fluorescent probe. However, indirect assays do not distinguish between transported substrates and non-transported OATP inhibitors. To fill this hiatus, a novel assay, termed competitive counterflow (CCF) has been developed and has since been applied for several OATPs to differentiate between substrates and non-transported inhibitors. However, previous OATP CCF assays, with the exception of that for OATP1B1, used radioactive probes. In the current study, we demonstrate that sulforhodamine 101 or pyranine can be used as fluorescent probes in a CCF assay to identify transported substrates of OATP1A2, or OATPs 1B1, 1B3 and 2B1, respectively. With the help of the newly developed fluorescence-based CCF method, we identify the FDA-approved anti-protozoal drug, pentamidine as a unique substrate of OATP1A2. Furthermore, we confirm the selective, OATP1A2-mediated uptake of pentamidine in a cytotoxicity assay. Based on our results, OATP1A2 may be an important determinant of pentamidine transport through the blood-brain barrier.

Characterization of Chemoresistance in Pancreatic Cancer: A Look at MDR-1 Polymorphisms and Expression in Cancer Cells and Patients.

Pancreatic malignancy is the fourth cause of cancer-related death in Western countries and is predicted to become the second leading cause of cancer-related mortality by 2030. The standard therapies (FOLFIRINOX and gemcitabine with nab-paclitaxel) are not resolutive because this type of cancer is also characterized by a high chemoresistance, due in part to the activity of the ATP Binding Cassette (ABC) pumps accounting for the reduction in the intracellular concentration of the drugs. In this work, we analyze the occurrence of single-nucleotide polymorphisms (SNPs) in the MDR-1 gene, in different pancreatic cancer cell lines, and in tissues from pancreatic cancer patients by DNA sequencing, as well as the expression levels of MDR-1 mRNA and protein, by qRT-PCR and Western Blot analysis. We found that gemcitabine-resistant cells, in conjunction with homozygosis of analyzed SNPs, showed high MDR-1 basal levels with further increases after gemcitabine treatment. Nevertheless, we did not observe in the human PDAC samples a correlation between the level of MDR-1 mRNA and protein expression and SNPs. Preliminary, we conclude that in our small cohort, these SNPs cannot be used as molecular markers for predicting the levels of MDR-1 mRNA/protein levels and drug responses in patients with PDAC.

Effect of genetic variations in drug transporters, metabolizing enzyme and regulatory genes on the development of HIV-associated lipodystrophy.

Adipocytes play a crucial role in the metabolism of lipids and sugars. Their response varies depending on the circumstances or other factors influenced by physiological and metabolic stresses. People living with HIV (PLWH) experience different effects of HIV and highly active antiretroviral therapy (HAART) on their body fat. Some patients respond well to antiretroviral therapy (ART), while others taking similar regimens do not. The genetic makeup of patients has been strongly linked to the variable responses to HAART among PLWH. The cause of HIV-associated lipodystrophy syndrome (HALS) is not well understood, but it may be influenced by genetic variations in the host. The metabolism of lipid effectively modulates plasma triglyceride and high-density lipoprotein cholesterol levels in PLWH. Genes related to drug metabolism and transport play an important role in the transportation and metabolism of ART drugs. Genetic variation in metabolizing enzyme genes of antiretroviral drugs, lipid transport and transcription factor-related genes could interfere with fat storage and metabolism, contributing to the development of HALS. Hence we examined the impact of genes associated with transport, metabolism and various transcription factors in metabolic complications, and their impact on HALS. A study using databases such as PubMed, EMBASE and Google Scholar was conducted to understand the impact of these genes on metabolic complications and HALS. The present article discuss the changes in the expression and regulation of genes and their involvement in the lipid metabolism, lipolysis and lipogenesis pathways. Moreover, alteration of the drug transporter, metabolizing enzyme and various transcription factors can lead to HALS. Single-nucleotide polymorphisms in genes that play an essential role in drug metabolism and drug and lipid transportation may also contribute to individual differences in the emergence of metabolic and morphological alterations during HAART treatment.

Semi-automated, high-content imaging of drug transporter knockout sea urchin (Lytechinus pictus) embryos.

A defining feature of sea urchins is their extreme fecundity. Urchins produce millions of transparent, synchronously developing embryos, ideal for spatial and temporal analysis of development. This biological feature has been effectively utilized for ensemble measurement of biochemical changes. However, it has been underutilized in imaging studies, where single embryo measurements are used. Here we present an example of how stable genetics and high content imaging, along with machine learning-based image analysis, can be used to exploit the fecundity and synchrony of sea urchins in imaging-based drug screens. Building upon our recently created sea urchin ABCB1 knockout line, we developed a high-throughput assay to probe the role of this drug transporter in embryos. We used high content imaging to compare accumulation and toxicity of canonical substrates and inhibitors of the transporter, including fluorescent molecules and antimitotic cancer drugs, in homozygous knockout and wildtype embryos. To measure responses from the resulting image data, we used a nested convolutional neural network, which rapidly classified embryos according to fluorescence or cell division. This approach identified sea urchin embryos with 99.8% accuracy and determined two-cell and aberrant embryos with 96.3% and 89.1% accuracy, respectively. The results revealed that ABCB1 knockout embryos accumulated the transporter substrate calcein 3.09 times faster than wildtypes. Similarly, knockouts were 4.71 and 3.07 times more sensitive to the mitotic poisons vinblastine and taxol. This study paves the way for large scale pharmacological screens in the sea urchin embryo.

Functional Characterization of Six SLCO1B1 (OATP1B1) Variants Observed in Finnish Individuals with a Psychotic Disorder.

Variants in the SLCO1B1 (solute carrier organic anion transporter family member 1B1) gene encoding the OATP1B1 (organic anion transporting polypeptide 1B1) protein are associated with altered transporter function that can predispose patients to adverse drug effects with statin treatment. We explored the effect of six rare SLCO1B1 single nucleotide variants (SNVs) occurring in Finnish individuals with a psychotic disorder on expression and functionality of the OATP1B1 protein. The SUPER-Finland study has performed exome sequencing on 9381 individuals with at least one psychotic episode during their lifetime. SLCO1B1 SNVs were annotated with PHRED-scaled combined annotation-dependent (CADD) scores and the Ensembl variant effect predictor. In vitro functionality studies were conducted for the SNVs with a PHRED-scaled CADD score of >10 and predicted to be missense. To estimate possible changes in transport activity caused by the variants, transport of 2',7'-dichlorofluorescein (DCF) in OATP1B1-expressing HEK293 cells was measured. According to the findings, additional tests with rosuvastatin and estrone sulfate were conducted. The amount of OATP1B1 in crude membrane fractions was quantified using a liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomics analysis. Six rare missense variants of SLCO1B1 were identified in the study population, located in transmembrane helix 3: c.317T>C (p.106I>T), intracellular loop 2: c.629G>T (p.210G>V), c.633A>G (p.211I>M), c.639T>A (p.213N>L), transmembrane helix 6: 820A>G (p.274I>V), and the C-terminal end: 2005A>C (p.669N>H). Of these variants, SLCO1B1 c.629G>T (p.210G>V) resulted in the loss of in vitro function, abolishing the uptake of DCF, estrone sulfate, and rosuvastatin and reducing the membrane protein expression to 31% of reference OATP1B1. Of the six rare missense variants, SLCO1B1 c.629G>T (p.210G>V) causes a loss of function of OATP1B1 transport in vitro and severely decreases membrane protein abundance. Carriers of SLCO1B1 c.629G>T might be susceptible to altered pharmacokinetics of OATP1B1 substrate drugs and might have increased likelihood of adverse drug effects such as statin-associated musculoskeletal symptoms.

Androgen-Dependent Expression of CUX2 mRNA in the Pig Liver Is Associated with That of Drug Metabolizing Enzymes and Drug Transporters.

We previously identified androgen-dependent sex differences in the mRNA expression of drug metabolizing enzymes (DMEs), including CYPs, sulfotransferases and uridine 5'-diphospho-glucuronosyltransferases, and drug transporters in the pig liver and kidney. To elucidate the mechanism for such sex differences in pigs, we herein focused on the key regulators cut-like homeobox 2 (Cux2), B-cell lymphoma 6 (Bcl6), and signal transducer and activator of transcription 5b (Stat5b), which are reported to be responsible for the sex-biased gene expression of Cyps in the mouse liver. We used real-time RT-PCR to examine androgen-dependent sex differences in the mRNA levels of these regulators in the liver and kidney basically using Meishan and Landrace pigs. Significant sex differences (male > female) in the level of CUX2 mRNA were detected in the liver of both breeds, and levels were significantly decreased in males by castration and increased in castrated males and intact females by administering testosterone propionate. No such clear androgen-dependent sex differences in hepatic BCL6 or STAT5B mRNA expression were observed in either breed. In the kidney, androgen-dependent gene expression of these regulators was not observed. In the liver, CUX2 mRNA expression closely correlated with that of DMEs and drug transporters, which were previously shown to have androgen-dependent expression. Together, these findings demonstrate that hepatic CUX2 mRNA is expressed in an androgen-dependent manner, and strongly suggest that CUX2 plays a key role in the androgen-dependent gene expression of hepatic DMEs and drug transporters.

Effect of Growth Hormone Receptor Deficiency on Androgen-Associated Gene Expression of Hepatic Drug Metabolizing Enzymes and Drug Transporters in Pigs.

Growth hormone receptor (GHR)-deficient pigs were generated using the CRISPR/Cas9 system to investigate the involvement of GHR-mediated growth hormone (GH) signaling in androgen-associated gene expression of hepatic drug metabolizing enzymes (DMEs) and drug transporters. We initially confirmed that no wild-type GHR mRNA was present in GHR-/- (GHR-KO) pigs; in addition, as previously reported, those pigs exhibited decreases in body weight and serum insulin-like growth factor-1 concentration and an increase in serum GH concentration compared with the levels in GHR-/+ and GHR+/+ pigs with a wild-type GHR mRNA. The real-time RT-PCR results on the mRNA levels of hepatic DMEs and drug transporters in the GHR-KO pigs and the pigs with a wild-type GHR mRNA revealed that, among the examined hepatic DMEs, the mRNA levels of CYP1A2, CYP2A19, sulfotransferase (SULT) 1A1, and SULT2A1 were higher in GHR-KO pigs than in the pigs with a wild-type GHR mRNA, whereas the opposite trend was observed for the mRNA level of uridine 5'-diphospho-glucuronosyltransferase 1A6. No such significant differences in the mRNA levels of three hepatic drug transporters including multidrug resistance protein 1 were observed. In addition, the mRNA level of hepatic cut-like homeobox 2 (CUX2), which is expressed in an androgen-dependent manner and associated with the hepatic mRNA expression of several DMEs, was significantly decreased in GHR-KO pigs. The present findings strongly suggest that not only serum androgen but also GHR-mediated GH signaling contributes to the mRNA expression of several DMEs and CUX2, but not transporters, in the pig liver.

Interactions of organophosphate flame retardants with human drug transporters.

Organophosphate flame retardants (OPFRs) are environmental pollutants of increasing interest, widely distributed in the environment and exerting possible deleterious effects towards the human health. The present study investigates in vitro their possible interactions with human drug transporters, which are targets for environmental chemicals and actors of their toxicokinetics. Some OPFRs, i.e., tris(2-butoxyethyl) phosphate (TBOEP), tris(1,3-dichloroisopropyl) phosphate (TDCPP), tri-o-cresyl phosphate (TOCP) and triphenyl phosphate (TPHP), were found to inhibit activities of some transporters, such as organic anion transporter 3 (OAT3), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2 (OCT2) or breast cancer resistance protein (BCRP). These effects were concentration-dependent, with IC50 values ranging from 6.1 µM (for TDCPP-mediated inhibition of OCT2) to 51.4 µM (for TOCP-mediated inhibition of BCRP). OPFRs also blocked the transporter-dependent membrane passage of endogenous substrates, notably that of hormones. OAT3 however failed to transport TBOEP and TPHP. OPFRs additionally repressed mRNA expressions of some transporters in cultured human hepatic HepaRG cells, especially those of OAT2 and OCT1 in response to TOCP, with IC50 values of 2.3 µM and 2.5 µM, respectively. These data therefore add OPFRs to the expanding list of pollutants interacting with drug transporters, even if OPFR concentrations required to impact transporters, in the 2-50 µM range, are rather higher than those observed in humans environmentally or dietarily exposed to these chemicals.

Renal Organic Anion Transporters 1 and 3 In Vitro: Gone but Not Forgotten.

Organic anion transporters 1 and 3 (OAT1 and OAT3) play a crucial role in kidney function by regulating the secretion of multiple renally cleared small molecules and toxic metabolic by-products. Assessing the activity of these transporters is essential for drug development purposes as they can significantly impact drug disposition and safety. OAT1 and OAT3 are amongst the most abundant drug transporters expressed in human renal proximal tubules. However, their expression is lost when cells are isolated and cultured in vitro, which is a persistent issue across all human and animal renal proximal tubule cell models, including primary cells and cell lines. Although it is well known that the overall expression of drug transporters is affected in vitro, the underlying reasons for the loss of OAT1 and OAT3 are still not fully understood. Nonetheless, research into the regulatory mechanisms of these transporters has provided insights into the molecular pathways underlying their expression and activity. In this review, we explore the regulatory mechanisms that govern the expression and activity of OAT1 and OAT3 and investigate the physiological changes that proximal tubule cells undergo and that potentially result in the loss of these transporters. A better understanding of the regulation of these transporters could aid in the development of strategies, such as introducing microfluidic conditions or epigenetic modification inhibitors, to improve their expression and activity in vitro and to create more physiologically relevant models. Consequently, this will enable more accurate assessment for drug development and safety applications.

Cell State Transitions and Phenotypic Heterogeneity in Luminal Breast Cancer Implicating MicroRNAs as Potential Regulators.

Luminal breast cancer subtypes respond poorly to endocrine and trastuzumab treatments due to cellular heterogeneity arising from the phenotype transitions, accounted for mainly by the loss of receptor expression. The origins of basal-like and human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer subtypes have been attributed to genetic and protein modifications in stem-like cells and luminal progenitor cell populations, respectively. The post-transcriptional regulation of protein expression is known to be influenced by microRNAs (miRNAs) that are deemed to be master regulators of several biological processes in breast tumorigenesis and progression. Our objective was to identify the fractions of luminal breast cancer cells that share stemness potentials and marker profiles and to elucidate the molecular regulatory mechanism that drives transitions between fractions, leading to receptor discordances. Established breast cancer cell lines of all prominent subtypes were screened for the expression of putative cancer stem cell (CSC) markers and drug transporter proteins using a side population (SP) assay. Flow-cytometry-sorted fractions of luminal cancer cells implanted in immunocompromised mice generated a pre-clinical estrogen receptor alpha (ERα+) animal model with multiple tumorigenic fractions displaying differential expression of drug transporters and hormone receptors. Despite an abundance of estrogen receptor 1 (ESR1) gene transcripts, few fractions transitioned to the triple-negative breast cancer (TNBC) phenotype with a visible loss of ER protein expression and a distinct microRNA expression profile that is reportedly enriched in breast CSCs. The translation of this study has the potential to provide novel therapeutic miRNA-based targets to counter the dreaded subtype transitions and the failure of antihormonal therapies in the luminal breast cancer subtype.

Zebrafish (Danio rerio) larva as an in vivo vertebrate model to study renal function.

There is an increasing interest in using zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition. As the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in this study whether 3- to 4-day-old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cutoff of 4.4-7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of organic anion transporter oat (slc22) multidrug resistance-associated protein mrp1 (abcc1), mrp2 (abcc2), mrp4 (abcc4), and zebrafish larva p-glycoprotein analog abcb4 was shown. In addition, nonrenal clearance of fluorescent substrates and plasma protein binding characteristics were assessed in vivo. The results of transporter experiments were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 h postfertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.NEW & NOTEWORTHY The study of renal function remains a challenge. In vitro cell-based assays are approved to study, e.g., ABC/SLC-mediated drug transport but do not cover other renal functions such as glomerular filtration. Here, in vivo studies combined with in vitro assays are needed, which are time consuming and expensive. In view of these limitations, our proof-of-concept study demonstrates that the zebrafish larva is a translational in vivo test model that allows for mechanistic investigations to study renal function.

Amiloride is a suitable fluorescent substrate for the study of the drug transporter human multidrug and toxin extrusion 1 (MATE1).

Human multidrug and toxin extrusion 1 (MATE1; SLC47A1) is highly expressed in the kidneys and the liver. It plays a significant role in drug and endogenous compound disposition, and therefore, a rapid evaluation of its inhibition is important for drug development and for the understanding of renal and hepatic physiology. Amiloride is a potassium-sparing diuretic used for treating hypertension; it also demonstrates strong fluorescence in organic solvent or detergent solutions. In this study, we investigated the transport characteristics of amiloride by human MATE1. Cellular accumulation of amiloride was evaluated in control vector- or MATE1-transfected HEK293 cells. Cells were lysed with 1% sodium dodecyl sulfate, and fluorescence was measured using a microplate reader at wavelengths of 364ex and 409em. With ammonium prepulse-induced intracellular acidification, MATE1 transported amiloride at an extracellular pH of 7.4. The uptake demonstrated an overshoot phenomenon and saturated, with the Km and Vmax being 23.5 µM and 1.01 nmol/mg/min, respectively. MATE1-mediated amiloride transport also presented with a bell-shaped pH profile that reached a maximum pH value of 7.4. The inhibitor sensitivity of MATE1-facilitated amiloride transport was similar to those of known substrates, such as tetraethylammonium and metformin. Among the tested inhibitors, pyrimethamine demonstrated the most potent inhibition with an IC50 value of 0.266 µM. Furthermore, MATE1 was found to be inhibited by fampridine, which was previously considered to be a non-inhibitor of MATE1. This study demonstrates that amiloride is a suitable fluorescent substrate for the in vitro study of the transport activity of MATE1.

Sex, Organ, and Breed Differences in the mRNA Expression of Drug Transporters in the Liver and Kidney of Pigs.

Domestic pigs are attractive as an animal model for humans because of their anatomical and physiological similarities to humans. In this study, sex, organ, and breed differences in the mRNA expression of drug transporters such as breast cancer resistance protein (BCRP), multidrug resistance protein 1 (MDR1), multidrug resistance associated protein 2 (MRP2), organic anion porting polypeptide 1B3 (OATP1B3), organic anion transporters (OAT1, OAT2, and OAT3), and organic cation transporters (OCT1 and OCT2) were examined by RT-PCR in the liver and kidney of 5-month-old Meishan and Landrace pigs. No sex differences in the amount of BCRP mRNA were observed in both breeds. In Meishan pigs, sex differences (male < female) in the mRNA amounts of MDR1, OATP1B3, and OCT1 were observed in the liver. Similarly, sex differences in the mRNA amounts of MRP2, OAT1, OAT2, OAT3, and OCT2 were observed in the kidney of Meishan pigs: male > female for MRP2, OAT3, and OCT2, and male < female for OAT1 and OAT2. However, no such sex differences were observed in Landrace pigs. In addition, regardless of breed, hepatic OAT1, OAT3, and OCT2 mRNAs and renal OATP1B3 mRNA were not detected. Thus, organ and breed differences in the expression of drug transporters suggest the existence of genetically controlled organ-selective factors. Furthermore, additional experiments in castrated and/or testosterone propionate-treated pigs strongly suggested that sex and breed differences in the gene expression of drug transporters, especially hepatic OCT1 and renal OAT1, were primarily due to the difference in serum testosterone concentration.

Highly sensitive simultaneous quantification of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid in human plasma using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry.

Indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid are uremic toxins that accumulate in renal failure and have been reported to decrease the activities of the drug-metabolizing enzyme cytochrome P450 3A and the drug transporter organic anion transporting polypeptides 1B, respectively. In this study, we established and validated an assay for simultaneous quantification of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid in human plasma. The samples were pretreated by solid-phase extraction, and measured by ultra-high-performance liquid chromatography-tandem mass spectrometry. The validation results for this assay were within the acceptable limits recommended by the US Food and Drug Administration, with a lower limit of quantitation of 0.05 µg/mL for both indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. Recovery rates of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid corrected by internal standard were 100.7-101.9 and 100.2-101.3%, respectively. Matrix effects of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid corrected by internal standard were 101.1-105.5 and 97.0-103.8%, respectively. The validated assay was used to analyze indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid concentrations in the plasma samples of healthy volunteers and patients with chronic kidney disease. All the measured plasma indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid concentrations were within the calibration ranges. This novel method may contribute to predicting the activities of drug-metabolizing enzymes and drug transporters in individual patients.

Interactions of organophosphorus pesticides with ATP-binding cassette (ABC) drug transporters.

Although pharmaceutical companies have to study drug-transporter interaction, environmental contaminant interactions with these transporters are not well characterised. In this study, we demonstrated using in vitro transfected cell line that some organophosphorus pesticides are able to interact with drug efflux transporters like P-glycoprotein, BCRP and MRPs.According to our results, dibrom was found to inhibit only Hoechst binding site of P-gp with an IC50 closed to 77 µM, phosmet inhibited BCRP efflux with an IC50 of 42 µM and only profenofos was able to inhibit BCRP, MRPs and P-gp at two binding sites. As profenofos appeared to be a potent ABC transporter inhibitor, we studied its potential substrate property towards P-gp.Using a docking approach, we developed an in silico tool to study pesticide properties to be a probe or inhibitor of P-gp transporter. From both in silico and in vitro results, profenofos was not considered as a P-gp substrate.Combining both in vitro and docking methods appears to be an attractive approach to select pesticides that would not pass into the blood systemic circulation.

In Vitro Characterization of Renal Drug Transporter Activity in Kidney Cancer.

The activity of drug transporters is central to the secretory function of the kidneys and a defining feature of renal proximal tubule epithelial cells (RPTECs). The expression, regulation, and function of these membrane-bound proteins is well understood under normal renal physiological conditions. However, the impact of drug transporters on the pathophysiology of kidney cancer is still elusive. In the present study, we employed different renal cell carcinoma (RCC) cell lines and a prototypical non-malignant RPTEC cell line to characterize the activity, expression, and potential regulatory mechanisms of relevant renal drug transporters in RCC in vitro. An analysis of the uptake and efflux activity, the expression of drug transporters, and the evaluation of cisplatin cytotoxicity under the effects of methylation or epidermal growth factor receptor (EGFR) inhibition showed that the RCC cells retained substantial drug transport activity. In RCC cells, P-glycoprotein was localized in the nucleus and its pharmacological inhibition enhanced cisplatin toxicity in non-malignant RPTECs. On the other hand, methylation inhibition enhanced cisplatin toxicity by upregulating the organic cation uptake activity in RCC cells. Differential effects of methylation and EGFR were observed in transporter expression, showing regulatory heterogeneity in these cells. Interestingly, the non-malignant RPTEC cell line that was used lacked the machinery responsible for organic cation transport, which reiterates the functional losses that renal cells undergo in vitro.

Differences in Transporters Rather than Drug Targets Are the Principal Determinants of the Different Innate Sensitivities of Trypanosoma congolense and Trypanozoon Subgenus Trypanosomes to Diamidines and Melaminophenyl Arsenicals.

The animal trypanosomiases are infections in a wide range of (domesticated) animals with any species of African trypanosome, such as Trypanosoma brucei, T. evansi, T. congolense, T. equiperdum and T. vivax. Symptoms differ between host and infective species and stage of infection and are treated with a small set of decades-old trypanocides. A complication is that not all trypanosome species are equally sensitive to all drugs and the reasons are at best partially understood. Here, we investigate whether drug transporters, mostly identified in T. b. brucei, determine the different drug sensitivities. We report that homologues of the aminopurine transporter TbAT1 and the aquaporin TbAQP2 are absent in T. congolense, while their introduction greatly sensitises this species to diamidine (pentamidine, diminazene) and melaminophenyl (melarsomine) drugs. Accumulation of these drugs in the transgenic lines was much more rapid. T. congolense is also inherently less sensitive to suramin than T. brucei, despite accumulating it faster. Expression of a proposed suramin transporter, located in T. brucei lysosomes, in T. congolense, did not alter its suramin sensitivity. We conclude that for several of the most important classes of trypanocides the presence of specific transporters, rather than drug targets, is the determining factor of drug efficacy.

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.