ABSTRACT
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
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
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
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
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
Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.
ABSTRACT
Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.
ABSTRACT
Remdesivir has been approved for treatment of COVID-19 and shortens the time to recovery in hospitalized patients. Drug transporters removing remdesivir from the circulation may reduce efficacy of treatment by lowering its plasma levels. Information on the interaction of remdesivir with drug transporters is limited. We therefore assessed remdesivir as substrate and inhibitor of the clinically relevant hepatic drug uptake transporters organic anion transporting poly-peptide (OATP)-1B1 (SLCO1B1), its common genetic variants OATP1B1*1b, OATP1B1*5, OATP1B1*15, as well as OATP1B3 (SLCO1B3), OATP2B1 (SLCO2B1) and organic cation transporter (OCT)-1 (SLC22A1). Previously established transporter-overexpressing cells were used to measure (i) cellular remdesivir uptake and (ii) cellular uptake of transporter probe substrates in the presence of remdesivir. There was a high remdesivir uptake into vector-transfected control cells. Moderate, but statistically significant higher uptake was detected only for OATP1B1-, OATP1B1*1b and OATP1B1*15-expressing cells when compared with control cells at 5 µM. Remdesivir inhibited all investigated transporters at 10 µM and above. In conclusion, the low uptake rates suggest that OATP1B1 and its genetic variants, OATP1B3, OATP2B1 and OCT1 are not relevant for hepatocellular uptake of remdesivir in humans. Due to the rapid clearance of remdesivir, no clinically relevant transporter-mediated drug-drug interactions are expected.
ABSTRACT
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
Organic cation transporter (OCT) 3 (SLC22A3) is a widely expressed drug transporter, handling notably metformin and platinum derivatives, as well as endogenous compounds like monoamine neurotransmitters. OCT3 has been shown to be inhibited by a few marketed tyrosine kinase inhibitors (TKIs). The present study was designed to determine whether additional TKIs may interact with OCT3. For this purpose, the effects of 25 TKIs toward OCT3 activity were analyzed using OCT3-overexpressing HEK293 cells. 13/25 TKIs, each used at 10 µM, were found to behave as moderate or strong inhibitors of OCT3 activity, that is, they decreased OCT3-mediated uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide by at least 50% or 80%, respectively. This OCT3 inhibition was correlated to some molecular descriptors of TKIs, such as the percentage of H atoms and that of cationic forms at pH = 7.4. It was concentration-dependent, notably for brigatinib, ceritinib, and crizotinib, which exhibited low half maximal inhibitory concentration (IC50 ) values in the 28-106 nM range. Clinical concentrations of these three marketed TKIs, as well as those of pacritinib, were next predicted to inhibit in vivo OCT3 activity according to regulatory criteria. Cellular TKI accumulation experiments as well as trans-stimulation assays, however, demonstrated that OCT3 does not transport brigatinib, ceritinib, crizotinib, and pacritinib, thus discarding any implication of OCT3 in the pharmacokinetics of these TKIs. Taken together, these data suggest that some TKIs may act as potent inhibitors of OCT3 activity, which may have consequences in terms of drug-drug interactions and toxicity.
Subject(s)
Organic Cation Transport Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Biological Transport/drug effects , Crizotinib/pharmacology , HEK293 Cells/drug effects , Humans , Organophosphorus Compounds/pharmacology , Pyrimidines/pharmacology , Sulfones/pharmacologyABSTRACT
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
BACKGROUND AND PURPOSE: The metabolic activity of cytochrome P450 (CYP) 2D6 is highly variable and CYP2D6 genotypes insufficiently explain the extensive and intermediate metabolic phenotypes, limiting the prediction of drug response plus adverse drug reactions. Since CYP2D6 prototypic substrates are positively charged, the aim of this study was to evaluate the organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) as potential contributors to the variability of CYP2D6 hydroxylation of debrisoquine, dextromethorphan, diphenhydramine, perhexiline and sparteine. EXPERIMENTAL APPROACH: OCT1/SLC22A1-, OCT2/SLC22A2-, OCT3/SLC22A3-, MATE1/SLC47A1-, and MATE2K/SLC47A2-overexpressing cell lines were used to investigate the transport of the selected drugs. Individuals from a study cohort, well defined with respect to CYP2D6 genotype and sparteine pharmacokinetics, were genotyped for the common OCT1 variants rs12208357 (OCT1-R61C), rs34130495 (OCT1-G401S), rs202220802 (OCT1-Met420del), rs34059508 (OCT1-G465R), OCT2 variant rs316019 (OCT2-A270S) and MATE1 variant rs2289669. Sparteine pharmacokinetics was stratified according to CYP2D6 and OCT1, OCT2 or MATE1 genotype. KEY RESULTS: OCTs and MATE1 transport sparteine and debrisoquine with high affinity in vitro, but OCT- and MATE1-dependent transport of dextromethorphan, diphenhydramine and perhexiline was not detected. Sparteine and debrisoquine transport depends on OCT1 genotype; however, sparteine pharmacokinetics is independent from OCT1 genotype. CONCLUSIONS AND IMPLICATIONS: Some drugs that are substrates of CYP2D6 are also substrates of OCTs and MATE1, suggesting overlapping specificities. Variability in sparteine hydroxylation in extensive and intermediate metabolizers cannot be explained by OCT1 genetic variants indicating presence of other factors. Dose-dependent toxicities of dextromethorphan, diphenhydramine and perhexiline appear to be independent from OCTs and MATEs.
Subject(s)
Organic Cation Transport Proteins , Pharmaceutical Preparations , Cations , Cytochrome P-450 CYP2D6/genetics , Humans , Organic Cation Transport Proteins/genetics , Organic Cation Transporter 2 , PhenotypeABSTRACT
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
Bone production, maintenance, and modeling are a well-balanced process involving mineralization by osteoblasts and resorption by osteoclasts. Sex steroid hormones, including their conjugated forms, contribute majorly to maintaining this balance. Recently, variants in the SLC22A9 gene have been associated with osteoporosis in Korean females. We had recently shown that SLC22A9, encoding organic anion transporter 7 (OAT7), is an uptake transporter of estrone sulfate and identified several genetic variants in Europeans leading to functional consequences in vitro. We therefore hypothesized that SLC22A9 genetic variants may contribute to the pathophysiology of osteoporosis in Europeans. To test this hypothesis, we examined the associations of SLC22A9 variants with bone quality, fractures, and bone turnover markers. We genotyped SLC22A9 variants in 5,701 (2,930 female) subjects (age range, 20-93 years) extracted from the population-based Study of Health in Pomerania (SHIP and SHIP-TREND) covered by the Illumina Infinium HumanExome BeadChip version v1.0 (Exome Chip). Descriptive data (e.g., history of fractures), ultrasonography of the calcaneus, as well as serum concentrations of carboxy-terminal telopeptide of type I collagen, amino-terminal propeptide of type I procollagen, and vitamin D were determined. Comprehensive statistical analyses revealed no association between low-frequency and rare SLC22A9 variants and bone quality, fractures, and bone turnover markers. Our results indicate that single genetic SLC22A9 variants do not have a major impact on osteoporosis risk prediction in Europeans, yet findings need to be replicated in larger-scale studies.
Subject(s)
Biomarkers/blood , Fractures, Bone/genetics , Genetic Predisposition to Disease , Organic Anion Transporters, Sodium-Independent/genetics , Osteoporosis/epidemiology , Polymorphism, Single Nucleotide , White People/genetics , Adult , Aged , Aged, 80 and over , Bone Density , Bone Remodeling , Case-Control Studies , Cross-Sectional Studies , Europe/epidemiology , Female , Fractures, Bone/blood , Gene Expression Profiling , Humans , Male , Middle Aged , Organic Anion Transporters, Sodium-Independent/blood , Osteoporosis/blood , Osteoporosis/genetics , Osteoporosis/pathology , Prognosis , Young AdultABSTRACT
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
Aim: Pharmacoresistance is a major burden in epilepsy treatment. We aimed to identify genetic biomarkers in response to specific antiepileptic drugs (AEDs) in genetic generalized epilepsies (GGE). Materials & methods: We conducted a genome-wide association study (GWAS) of 3.3 million autosomal SNPs in 893 European subjects with GGE - responsive or nonresponsive to lamotrigine, levetiracetam and valproic acid. Results: Our GWAS of AED response revealed suggestive evidence for association at 29 genomic loci (p <10-5) but no significant association reflecting its limited power. The suggestive associations highlight candidate genes that are implicated in epileptogenesis and neurodevelopment. Conclusion: This first GWAS of AED response in GGE provides a comprehensive reference of SNP associations for hypothesis-driven candidate gene analyses in upcoming pharmacogenetic studies.
Subject(s)
Anticonvulsants/therapeutic use , Epilepsy, Generalized/drug therapy , Epilepsy, Generalized/genetics , Genome-Wide Association Study/methods , Adolescent , Case-Control Studies , Child , Cohort Studies , Epilepsy, Generalized/epidemiology , Europe/epidemiology , Female , Humans , Lamotrigine/therapeutic use , Levetiracetam/therapeutic use , Male , Retrospective Studies , Treatment Outcome , Valproic Acid/therapeutic useABSTRACT
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
Systemic therapy of advanced hepatocellular carcinoma (HCC) with the small-molecule multikinase inhibitor sorafenib is associated with large interindividual pharmacokinetic variability and unpredictable side effects potentially requiring dose reduction or treatment termination. Organic cation transporter (OCT1; gene SLC22A1) has been proposed as a clinical biomarker of HCC response. Because proof is lacking that OCT1 transports sorafenib, we used a combinatorial approach to define how OCT1 contributes to sorafenib transport. Overexpression of functional OCT1 protein in Xenopus laevis oocytes and mammalian cell lines did not facilitate sorafenib transport. Otherwise, sorafenib considerably accumulated in liver cancer cell lines despite negligible OCT1 mRNA and protein levels. Sorafenib pharmacokinetics was independent of OCT1 genotype in mice. Finally, SLC22A1 mRNA expression was significantly reduced by DNA methylation in The Cancer Genome Atlas HCC cohort. These results clearly demonstrate OCT1-independent cellular sorafenib uptake indicating that OCT1 is apparently not a valid biomarker of sorafenib response in HCC.
Subject(s)
Antineoplastic Agents/administration & dosage , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Octamer Transcription Factor-1/genetics , Sorafenib/administration & dosage , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Biological Transport , Cell Line, Tumor , Female , Genotype , HEK293 Cells , Hep G2 Cells , Humans , Mice , Mice, Knockout , Oocytes/metabolism , Sorafenib/pharmacokinetics , Sorafenib/pharmacology , Xenopus laevisABSTRACT
Carcinogenic heterocyclic aromatic amines (HAAs) interact with some drug transporters, like the efflux pump BCRP and the organic anion transporters OAT1 and OAT3. The present study was designed to determine whether they can also target activities of the organic cation transporters (OCTs), using mainly OCT1-, OCT2- and OCT3-overexpressing HEK293 cells. Fifteen HAAs were demonstrated to differently alter OCT activities; with a cut-off of at least 50% reduction of transporter activity by 100⯵M HAAs, 5/15 HAAs, including Trp-P-1 and Trp-P-2, inhibited activities of OCT1, OCT2 and OCT3, whereas 7/15 HAAs, including PhIP and MeIQx, blocked those of OCT2 and OCT3, 1/15 HAAs reduced those of OCT1 and OCT2 and 2/15 HAAs, including AαC, only that of OCT2. IC50 values of Trp-P-1 and Trp-P-2 towards OCT activities were found to be in the 2-6⯵M range, likely not relevant for human exposure to HAAs through smoking or the diet. Trp-P-1 and Trp-P-2 additionally failed to trans-stimulate OCT1 and OCT2 activities and exhibited similar accumulation in OCT1/2-transduced HEK293 cells and control HEK293-MOCK cells. These data demonstrate that HAAs, notably Trp-P-1 and Trp-P-2, interact with OCT1/2, without however being transported, thus likely discarding a major role for OCT1/2 in HAA systemic toxicokinetics.