ABSTRACT
Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.
Subject(s)
Doxorubicin/adverse effects , Heart Injuries/chemically induced , Heart Injuries/drug therapy , Molecular Targeted Therapy , Organic Anion Transporters, Sodium-Independent/metabolism , Animals , Child , Gene Expression Regulation , Heart Injuries/physiopathology , Humans , Mice , Myocytes, Cardiac/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Organic Anion Transporters, Sodium-Independent/deficiency , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Sequence Analysis, RNAABSTRACT
Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease susceptibility and drug response. Because only a limited number of transporter substrates are currently known, the function of a large number of SLC transporters is elusive. Here, we describe the proof-of-concept of a novel strategy to identify SLC transporter substrates exemplarily for the proton-coupled peptide transporter (PEPT) 2 (SLC15A2) and multidrug and toxin extrusion (MATE) 1 transporter (SLC47A1), which are important renal transporters of drug reabsorption and excretion, respectively. By combining metabolomic profiling of mice with genetically-disrupted transporters, in silico ligand screening and in vitro transport studies for experimental validation, we identified nucleobases and nucleoside-derived anticancer and antiviral agents (flucytosine, cytarabine, gemcitabine, capecitabine) as novel drug substrates of the MATE1 transporter. Our data confirms the successful applicability of this new approach for the identification of transporter substrates in general, which may prove particularly relevant in drug research.
Subject(s)
Membrane Transport Proteins , Solute Carrier Proteins , Animals , Mice , Ligands , Biological TransportABSTRACT
PURPOSE: Imaging of PARP expression has emerged as valuable strategy for prediction of tumor malignancy. While [18F]PARPi and [18F]FTT are already in clinical translation, both suffer from mainly hepatobiliary clearance hampering their use for detection of abdominal lesions, e.g., liver metastases. Our novel radiotracer [18F]FPyPARP aims to bridge this gap with a higher renal clearance and an easily translatable synthesis route for potential clinical application. METHODS: We developed a less lipophilic variant of [18F]PARPi by exchange of the fluorobenzoyl residue with a fluoronicotinoyl group and automated the radiosyntheses of the three radiotracers. We then conducted a comparative side-by-side study of [18F]PARPi, [18F]FPyPARP, and [18F]FTT in NOD.CB17-Prkdcscid/J mice bearing HCC1937 xenografts to assess xenograft uptake and pharmacokinetics focusing on excretion pathways. RESULTS: Together with decent uptake of all three radiotracers in the xenografts (tumor-to-blood ratios 3.41 ± 0.83, 3.99 ± 0.99, and 2.46 ± 0.35, respectively, for [18F]PARPi, [18F]FPyPARP, and [18F]FTT), a partial shift from hepatobiliary to renal clearance of [18F]FPyPARP was observed, whereas [18F]PARPi and [18F]FTT show almost exclusive hepatobiliary clearance. CONCLUSION: These findings imply that [18F]FPyPARP is an alternative to [18F]PARPi and [18F]FTT for PET imaging of PARP enzymes.
Subject(s)
Liver Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Cell Line, Tumor , Humans , Liver Neoplasms/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Positron-Emission Tomography/methodsABSTRACT
The hepatic Na+-taurocholate cotransporting polypeptide NTCP/SLC10A1 is important for the uptake of bile salts and selected drugs. Its inhibition results in increased systemic bile salt concentrations. NTCP is also the entry receptor for the hepatitis B/D virus. We investigated interindividual hepatic SLC10A1/NTCP expression using various omics technologies. SLC10A1/NTCP mRNA expression/protein abundance was quantified in well-characterized 143 human livers by real-time PCR and LC-MS/MS-based targeted proteomics. Genome-wide SNP arrays and SLC10A1 next-generation sequencing were used for genomic analyses. SLC10A1 DNA methylation was assessed through MALDI-TOF MS. Transcriptomics and untargeted metabolomics (UHPLC-Q-TOF-MS) were correlated to identify NTCP-related metabolic pathways. SLC10A1 mRNA and NTCP protein levels varied 44-fold and 10.4-fold, respectively. Non-genetic factors (e.g., smoking, alcohol consumption) influenced significantly NTCP expression. Genetic variants in SLC10A1 or other genes do not explain expression variability which was validated in livers (n = 50) from The Cancer Genome Atlas. The identified two missense SLC10A1 variants did not impair transport function in transfectants. Specific CpG sites in SLC10A1 as well as single metabolic alterations and pathways (e.g., peroxisomal and bile acid synthesis) were significantly associated with expression. Inter-individual variability of NTCP expression is multifactorial with the contribution of clinical factors, DNA methylation, transcriptional regulation as well as hepatic metabolism, but not genetic variation.
Subject(s)
Organic Anion Transporters, Sodium-Dependent , Symporters , Bile Acids and Salts/metabolism , Chromatography, Liquid , Hepatitis B virus/genetics , Hepatitis Delta Virus/genetics , Humans , Liver/metabolism , Organic Anion Transporters, Sodium-Dependent/biosynthesis , Organic Anion Transporters, Sodium-Dependent/genetics , Organic Anion Transporters, Sodium-Dependent/metabolism , Peptides/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Symporters/biosynthesis , Symporters/genetics , Symporters/metabolism , Tandem Mass Spectrometry , Taurocholic Acid/metabolismABSTRACT
Interactions of the Janus kinase (JAK) inhibitor ruxolitinib with solute carriers (SLCs) remain incompletely characterised. The present study was therefore designed to investigate this issue.The interactions of ruxolitinib with SLCs were analysed using transporter-overexpressing human embryonic kidney HEK293 cells. Substrate accumulation was detected by spectrofluorimetry, liquid chromatography coupled to tandem mass spectrometry or scintillation counting.Ruxolitinib was found to potently inhibit the activities of organic anion transporter 3 (OAT3), organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1) and MATE2-K (half maximal inhibitory concentration (IC50) < 10 µM). It blocked OAT1, OAT4, OATP1B1, OATP1B3, OATP2B1 and OCT3, but in a weaker manner (IC50 > 10 µM), whereas OCT1 was not impacted. No time-dependent inhibition was highlighted. When applying the US Food and Drug Administration (FDA) criteria for transporters-related drug-drug interaction risk, OCT2 and MATE2-K, unlike MATE1 and OAT3, were predicted to be in vivo inhibited by ruxolitinib. Cellular uptake studies additionally indicated that ruxolitinib is a substrate for MATE1 and MATE2-K, but not for OAT3 and OCT2.Ruxolitinib in vitro blocked activities of most of SLC transporters. Only OCT2 and MATE-2K may be however clinically inhibited by the JAK inhibitor, with the caution for OCT2 that in vitro inhibition data were generated with an FDA-non recommended fluorescent substrate. Ruxolitinib MATEs-mediated transport may additionally deserve attention for its possible pharmacological consequences in MATE-positive cells.
Subject(s)
Janus Kinase Inhibitors , Pharmaceutical Preparations , Drug Interactions , HEK293 Cells , Humans , Nitriles , Organic Cation Transport Proteins , Pyrazoles , PyrimidinesABSTRACT
Organic cation transporters (OCTs) of the solute carrier family (SLC) 22 are the subject of intensive research because they mediate the transport of many clinically-relevant drugs such as the antidiabetic agent metformin, the opioid tramadol, and the antimigraine agent sumatriptan. OCT1 (SLC22A1) and OCT2 (SLC22A2) are highly expressed in human liver and kidney, respectively, while OCT3 (SLC22A3) shows a broader tissue distribution. As suggested from studies using knockout mice, particularly OCT2 and OCT3 appear to be of relevance for brain physiological function and drug response. The knowledge of genetic factors and epigenetic modifications affecting function and expression of OCTs is important for a better understanding of disease mechanisms and for personalized treatment of patients. This review briefly summarizes the impact of genetic variants and epigenetic regulation of OCTs in general. A comprehensive overview is given on the consequences of OCT2 and OCT3 knockout in mice and the implications of genetic OCT2 and OCT3 variants on central nervous system function in humans.
Subject(s)
Metformin , Organic Cation Transport Proteins , Animals , Cations , Epigenesis, Genetic , Humans , Hypoglycemic Agents , Mice , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolismABSTRACT
OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.
Subject(s)
Organic Anion Transporters/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Biological Transport , HumansABSTRACT
OBJECTIVE: Drug resistance is a major concern in the treatment of individuals with epilepsy. No genetic markers for resistance to individual antiseizure medication (ASM) have yet been identified. We aimed to identify the role of rare genetic variants in drug resistance for three common ASMs: levetiracetam (LEV), lamotrigine (LTG), and valproic acid (VPA). METHODS: A cohort of 1622 individuals of European descent with epilepsy was deeply phenotyped and underwent whole exome sequencing (WES), comprising 575 taking LEV, 826 LTG, and 782 VPA. We performed gene- and gene set-based collapsing analyses comparing responders and nonresponders to the three drugs to determine the burden of different categories of rare genetic variants. RESULTS: We observed a marginally significant enrichment of rare missense, truncating, and splice region variants in individuals who were resistant to VPA compared to VPA responders for genes involved in VPA pharmacokinetics. We also found a borderline significant enrichment of truncating and splice region variants in the synaptic vesicle glycoprotein (SV2) gene family in nonresponders compared to responders to LEV. We did not see any significant enrichment using a gene-based approach. SIGNIFICANCE: In our pharmacogenetic study, we identified a slightly increased burden of damaging variants in gene groups related to drug kinetics or targeting in individuals presenting with drug resistance to VPA or LEV. Such variants could thus determine a genetic contribution to drug resistance.
Subject(s)
Anticonvulsants/therapeutic use , Drug Resistance/genetics , Epilepsy/drug therapy , Epilepsy/genetics , Pharmacogenomic Variants/genetics , Case-Control Studies , Female , Genotype , Humans , Lamotrigine/therapeutic use , Levetiracetam/therapeutic use , Male , Valproic Acid/therapeutic useABSTRACT
OATP2B1 is an intestinal and hepatic drug uptake transporter. Intestinal OATP2B1 has been elucidated as the mechanism of unexpected clinical drug-drug interactions (DDIs), where drug exposure was unexpectedly decreased with unchanged half-life. Hepatic OATP2B1 may be an understudied clinical DDI mechanism. The aim of the present work was to understand the prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors in marketed drug space. HEK293 cells stably overexpressing human OATP2B1 or vector control were generated and cultured for 72 h in a 96-well format. OATP2B1-mediated uptake of dibromofluorescein (DBF) was found to be optimal at 10 µM concentration and 30 min incubation time. A total of 294 drugs (top 300 marketed drugs, excluding biologics and restricted drugs, supplemented with â¼100 small-molecule drugs) were screened for OATP2B1 inhibition at 10 µM. Drugs demonstrating ≥50% inhibition in this screen were advanced for IC50 determination, which was extrapolated to clinical intestinal and hepatic OATP2B1 inhibition as per 2017 FDA DDI guidance. Of the 294 drugs screened, 67 elicited ≥50% inhibition of OATP2B1-mediated DBF uptake at 10 µM screening concentration. For the 67 drugs flagged in the single-concentration inhibition screen, upon evaluation of a full concentration range, IC50 values could be determined for 58 drugs. OATP2B1 IC50 values established for these 58 drugs were extrapolated as potentially clinically relevant at the intestinal level for 38 orally administered drugs (Igut/IC50 ≥ 10), and 17 were flagged as potential clinical inhibitors of hepatic OATP2B1 uptake (1 + Iin,max,u/IC50 ≥ 1.1). This analysis of 294 drugs demonstrated prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors to be 13 and 6%, respectively. As OATP2B1-inhibitor drugs are not exceedingly rare, these results suggest that clinical OATP2B1 DDIs have been rarely observed because OATP2B1 is uncommonly the predominant determinant of drug disposition.
Subject(s)
Drug Evaluation, Preclinical/methods , Organic Anion Transporters/antagonists & inhibitors , Biological Transport , Cell Survival/drug effects , Drug Interactions , Erlotinib Hydrochloride/pharmacology , Fluoresceins/metabolism , HEK293 Cells , Half-Life , Humans , Inhibitory Concentration 50 , Intestinal Mucosa/metabolism , Liver/metabolism , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , TransfectionABSTRACT
Neurotoxic bilirubin is the end product of heme catabolism in mammals. Bilirubin is solely conjugated by uridine diphospho-glucuronosyltransferase 1A1, which is a membrane-bound enzyme that catalyzes the transfer of glucuronic acid. Due to low function of hepatic and intestinal uridine diphospho-glucuronosyltransferase 1A1 during the neonatal period, human neonates develop mild to severe physiological hyperbilirubinemia. Accumulation of bilirubin in the brain leads to the onset of irreversible brain damage, termed kernicterus. Breastfeeding is one of the most significant factors that increase the risk of developing kernicterus in infants. Why does this most natural way of feeding increase the risk of brain damage or even death? This question leads to the hypothesis that breast milk-induced hyperbilirubinemia might bring certain benefits that outweigh those risks. While bilirubin is neurotoxic and cytotoxic, this compound is also a potent antioxidant. There are studies showing improved clinical conditions in patients with hyperbilirubinemia. Accumulating evidence also shows that genetic polymorphisms linked to hyperbilirubinemia are beneficial against various diseases. In this review article, we first introduce the production, metabolism, and transport of bilirubin. We then discuss the potential benefits of neonatal and adult hyperbilirubinemia. Finally, epigenetic factors as well as metabolomic information associated with hyperbilirubinemia are described. This review article advances the understanding of the physiological importance of the paradoxical compound bilirubin. (Hepatology 2018;67:1609-1619).
Subject(s)
Bilirubin/physiology , Homeostasis/physiology , Hyperbilirubinemia/etiology , Adult , Animals , Humans , Infant, Newborn , MetabolomicsABSTRACT
The efflux transporter breast cancer resistance protein BCRP/ABCG2 is well-known for its contribution to multi-drug resistance in cancer. Its relevance in cancer biology independent from drug efflux remains largely elusive. Our study aimed at elucidating the biological relevance and regulatory mechanisms of BCRP/ABCG2 in clear cell renal cell carcinoma (ccRCC) and disease progression. Two independent ccRCC-cohorts [Cohort 1 (KIRC/TCGA): n = 453, Cohort 2: n = 64] were investigated to elucidate BCRP/ABCG2 mRNA and protein expression and their association with survival. The impact of BCRP/ABCG2 on response to sunitinib treatment was investigated in two independent sunitinib-treated ccRCC-cohorts based on mRNA levels. Moreover, underlying regulatory mechanisms for interindividual variability of BCRP/ABCG2 expression were systematically assessed. Owing to redundant functional properties, mRNA and protein expression of the multidrug resistance protein MDR1/ABCB1 were additionally evaluated in these cohorts. In independent ccRCC-cohorts, low BCRP/ABCG2 and MDR1/ABCB1 mRNA and protein expression were associated with severity (e.g., tumor stage) of ccRCC and poor cancer-specific survival. BCRP/ABCG2 and MDR1/ABCB1 mRNA expression were linked to decreased progression-free survival after sunitinib treatment. Germline and somatic variants influenced interindividual variability of BCRP/ABCG2 expression only moderately. miR-212-3p and miR-132-3p were identified to regulate BCRP/ABCG2 posttranscriptionally by interaction with the ABCG2 3'UTR as confirmed through reporter gene assays in RCC cell lines. In summary, BCRP/ABCG2 expression in ccRCC underlies considerable interindividual variability with impact on patient survival and response to sunitinib treatment. While germline or somatic genetic variants and DNA methylation cannot explain aberrant BCRP/ABCG2 expression, miR-212-3p and miR-132-3p were identified to contribute to posttranscriptional regulation of BCRP/ABCG2.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/metabolism , Neoplasm Proteins/metabolism , 3' Untranslated Regions , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/genetics , Cohort Studies , DNA Methylation , Disease-Free Survival , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Energy Metabolism , Female , Humans , Kidney Neoplasms/drug therapy , Kidney Neoplasms/genetics , Male , MicroRNAs/genetics , Middle Aged , Mutation , Neoplasm Proteins/genetics , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , Severity of Illness Index , Sunitinib/therapeutic useABSTRACT
The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( Vmax = 3299 pmol/million cells/min, Km = 35.9 µM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.
Subject(s)
Antipsychotic Agents/pharmacology , Clozapine/pharmacology , Schizophrenia/drug therapy , Solute Carrier Proteins/metabolism , Antipsychotic Agents/therapeutic use , Brain/cytology , Brain/metabolism , Cell Line, Tumor , Clozapine/therapeutic use , Endothelial Cells/metabolism , HEK293 Cells , Hepatocytes/metabolism , Humans , Primary Cell Culture , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solute Carrier Proteins/isolation & purificationABSTRACT
Multidrug and toxin extrusion (MATE; SLC47A) proteins are membrane transporters mediating the excretion of organic cations and zwitterions into bile and urine and thereby contributing to the hepatic and renal elimination of many xenobiotics. Transported substrates include creatinine as endogenous substrate, the vitamin thiamine and a number of drug agents with in part chemically different structures such as the antidiabetic metformin, the antiviral agents acyclovir and ganciclovir as well as the antibiotics cephalexin and cephradine. This review summarizes current knowledge on the structural and molecular features of human MATE transporters including data on expression and localization in different tissues, important aspects on regulation and their functional role in drug transport. The role of genetic variation of MATE proteins for drug pharmacokinetics and drug response will be discussed with consequences for personalized medicine.
Subject(s)
Drug Therapy , Organic Cation Transport Proteins/metabolism , Precision Medicine , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Computational Biology , Humans , Models, Biological , Models, Molecular , Organ Specificity , Organic Cation Transport Proteins/antagonists & inhibitors , Organic Cation Transport Proteins/chemistry , Organic Cation Transport Proteins/genetics , Pharmaceutical Preparations/chemistry , Phosphorylation , Sequence Alignment , Species Specificity , Substrate SpecificityABSTRACT
Organic anion transporters play an essential role in the distribution and excretion of numerous endogenous metabolic products and exogenous organic anions, including a host of widely prescribed drugs. The expression and activity of these transporters is influenced by several conditions, including transcriptional regulation, gender-dependent regulation, and genetic variation. In addition, the interaction of these transporters with several drugs and endogenous substrates has been well documented and may play a significant role in drug disposition and development of various disease states, such as nephrotoxicity and familial idiopathic hypouricemia. Members of this family of transporters have been localized mainly to the renal epithelia of various species. Much of the early research in this field has focused on their role in renal drug transport, yet increasing research on this family of transporters has localized them to various other epithelial tissues, including liver, brain, and placenta. Thus, an understanding of the role of these transporters in drug interaction and disposition in the kidney and other tissues may help in the determination of individual drug response, susceptibility to drug toxicity, and chemical carcinogenesis. This review seeks to summarize current knowledge of the molecular function and substrate profile of cloned organic anion transporters and to discuss recent progress in the understanding of the impact of interindividual variability, transcriptional regulation, and tissue distribution on individual drug response.
Subject(s)
Organic Anion Transporters , Pharmaceutical Preparations/metabolism , Xenobiotics/pharmacokinetics , Animals , Biological Transport , Drug Interactions , Drug Therapy , Drug-Related Side Effects and Adverse Reactions , Female , Gene Expression Regulation , Humans , Male , Organ Specificity , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Organic Anion Transporters/physiology , Sex Characteristics , Substrate Specificity , Tissue Distribution , Xenobiotics/toxicityABSTRACT
Targeted chemotherapy for hepatocellular carcinoma (HCC) is impaired by intrinsic and/or acquired drug resistance. Because drugs used in HCC therapy (e.g., anthracyclines or the tyrosine kinase inhibitor sorafenib) are substrates of uptake and/or efflux transporters, variable expression of these transporters at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. In this study, the variability of expression of uptake transporters [organic cation transporter (OCT) 1 and OCT3] and efflux transporters [multidrug resistance 1 (MDR1)/P-glycoprotein, multidrug resistance protein (MRP) 1, MRP2, and breast cancer resistance protein (BCRP)], selected for their implication in transporting drugs used in HCC therapy, was investigated. HCC and corresponding nontumor tissue samples were collected from 24 Japanese patients at the time of surgery. Protein expression was determined by immunohistochemistry. Expression data were correlated with clinicopathological characteristics and patients' outcome (median follow-up, 53 months). Generally, expression was highly variable among individual tumor samples. Yet median expression of OCT1, OCT3, and MDR1 in HCC was significantly lower (1.4-, 2.7-, and 2-fold, respectively) than in nontumor tissue, while expression of MRP2 persisted and BCRP showed a trend of increased levels in HCC. Patients with low BCRP expression had significantly shorter overall and recurrence-free survival times. Results suggest different expression patterns of drug transporters in HCC, which are associated only in part with clinicopathological characteristics. Detailed information on expression of drug transporters in HCC may be promising for individualization and optimization of drug therapy for liver cancer.
Subject(s)
Asian People/genetics , Biological Transport/genetics , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Membrane Transport Proteins/genetics , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Middle Aged , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Neoplasm Proteins/genetics , Octamer Transcription Factor-3/genetics , Organic Cation Transporter 1/geneticsABSTRACT
BACKGROUND AND PURPOSE: The ATP-dependent biliary efflux transporter ABCC2, also known as multidrug resistance protein 2 (MRP2), is essential for the cellular disposition and detoxification of various xenobiotics including drugs as well as endogenous metabolites. Common functionally relevant ABCC2 genetic variants significantly alter drug responses and contribute to side effects. The aim of this study was to determine functional consequences of rare variants identified in subjects with European ancestry using in silico tools and in vitro analyses. EXPERIMENTAL APPROACH: Targeted next-generation sequencing of the ABCC2 gene was used to identify novel variants in European subjects (n = 143). Twenty-six in silico tools were used to predict functional consequences. For biological validation, transport assays were carried out with membrane vesicles prepared from cell lines overexpressing the newly identified ABCC2 variants and estradiol ß-glucuronide and carboxydichlorofluorescein as the substrates. KEY RESULTS: Three novel rare ABCC2 missense variants were identified (W227R, K402T, V489F). Twenty-five in silico tools predicted W227R as damaging and one as potentially damaging. Prediction of functional consequences was not possible for K402T and V489F and for the common linked variants V1188E/C1515Y. Characterisation in vitro showed increased function of W227R, V489F and V1188E/C1515Y for both substrates, whereas K402T function was only increased for carboxydichlorofluorescein. CONCLUSION AND IMPLICATIONS: In silico tools were unable to accurately predict the substrate-dependent increase in function of ABCC2 missense variants. In vitro biological studies are required to accurately determine functional activity to avoid misleading consequences for drug therapy.
ABSTRACT
OBJECTIVE: To study histamine transport and metabolism of salivary gland (SG) epithelial cells in healthy controls and SS patients. METHODS: Enzymes and transporters involved in histamine metabolism were analysed in cultured human submandibular salivary gland (HSG) epithelial cells and tissue sections using quantitative real-time PCR and immunostaining. HSG cells were used to study [(3)H]histamine uptake [(±1-methyl-4-phenylpyridinium (MPP)] and efflux by liquid scintillation counting. RESULTS: mRNA levels of l-histidine decarboxylase (HDC) and histamine-N-methyltransferase (HNMT) were similar in the control and SS glands, but diamine oxidase was not expressed at all. Organic cation transporter 3 (OCT3) in healthy SG was localized in the acinar and ductal cells, whereas OCT2 was restricted to the myoepithelial cells. Both transporters were significantly decreased in SS at mRNA and protein levels. OCT3-mRNA levels in HSG cells were significantly higher than those of the other studied transporters. Uptake of [(3)H]histamine was inhibited by MPP in a time-dependent manner, whereas [(3)H]histamine-preloaded HSG cells released it. CONCLUSION: Ductal epithelial cells are non-professional histamine-producing cells able to release histamine via OCTs at the resting state up to â¼100 nM, enough to excite H3R/H4R(+) epithelial cells, but not H1R, which requires burst release from mast cells. At the stimulated phase, 50-60 µM histamine passes from the interstitial fluid through the acinar cells to saliva, whereas uptake by ductal cells leads to intracellular degradation by HNMT. OCT3/histamine/H4R-mediated cell maintenance and down-regulation of high histamine levels fail in SS SGs.
Subject(s)
Biological Transport/physiology , Epithelial Cells/metabolism , Histamine/metabolism , Sjogren's Syndrome/metabolism , Submandibular Gland/metabolism , Cells, Cultured , Down-Regulation , Histamine N-Methyltransferase/genetics , Histamine N-Methyltransferase/metabolism , Histidine Decarboxylase/genetics , Histidine Decarboxylase/metabolism , Humans , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 2ABSTRACT
Organic cation transporters (OCT), organic anion transporting polypeptides (OATP) and organic anion transporters (OAT) from the solute carrier (SLC) family play an essential role in the uptake of endogenous compounds and drugs into the hepatocytes and other cell types. The well-documented interindividual variability of expression and activity of these transporters translates into interindividual variability in drug pharmacokinetics and drug response. It is therefore important to elucidate mechanisms affecting membrane transporter expression and function. These mechanisms include transcriptional regulation, disease-dependent regulation and genetic variation. In this review, we will summarize the current knowledge of the molecular functions and substrate profiles of cloned hepatic OCTs, OATPs and OATs and discuss recent advances in understanding variable expression and function. Finally, the role of genetic variation in these transporters on drug exposure will be presented with implications for individual drug response.
Subject(s)
Organic Anion Transporters , Cations/metabolism , Genetic Variation , Humans , Liver/metabolism , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Peptides/metabolismABSTRACT
Multidrug and toxin extrusion proteins (MATEs; SLC47A) are mammalian transporters being predominately expressed in the brush-border membrane of proximal tubule epithelial cells in the kidney and the canalicular membrane of hepatocytes. Functionally, MATEs act as efflux transporters for organic compounds, thereby mediating the elimination process. Two isoforms, MATE1 and 2, have been identified, and, so far, only a limited number of substrates, including clinically used drugs such as metformin and cimetidine, are known. A knockout mouse model has been established, as well, and is a valuable tool for further systematic pharmacokinetic analyses. In this review, we summarize the progress in MATE research on structural, molecular, functional, and pathophysiological aspects. Consequences of genetic variants for pharmacokinetic alterations and drug therapy are discussed.
Subject(s)
Epithelial Cells/metabolism , Hepatocytes/metabolism , Kidney Tubules, Proximal/metabolism , Organic Cation Transport Proteins/metabolism , Xenobiotics/metabolism , Animals , Bile Canaliculi/metabolism , Biological Transport , Genetic Variation , Humans , Mice , Mice, Knockout , Microvilli/metabolism , Organic Cation Transport Proteins/genetics , Xenobiotics/pharmacokineticsABSTRACT
Organic cation transporters (OCTs) of the solute carrier family (SLC) 22 and multidrug and toxin extrusion (MATE) transporters of the SLC47 family have been identified as uptake and efflux transporters, respectively, for xenobiotics including several clinically used drugs such as the antidiabetic agent metformin, the antiviral agent lamivudine, and the anticancer drug oxaliplatin. Expression of human OCT1 (SLC22A1) and OCT2 (SLC22A2) is highly restricted to the liver and kidney, respectively. By contrast, OCT3 (SLC22A3) is more widely distributed. MATEs (SLC47A1, SLC47A2) are predominantly expressed in human kidney. Data on in vitro studies reporting a large number of substrates and inhibitors of OCTs and MATEs are systematically summarized. Several genetic variants of human OCTs and in part of MATE1 have been reported, and some of them result in reduced in vitro transport activity corroborating data from studies with knockout mice. A comprehensive overview is given on currently known genotype-phenotype correlations for variants in OCTs and MATE1 related to protein expression, pharmacokinetics/-dynamics of transporter substrates, treatment outcome, and disease susceptibility.