Role of ABCB1, ABCG2, ABCC2 and ABCC5 transporters in placental passage of zidovudine

Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP‐binding cassette (ABC) transporters, p‐glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance‐associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co‐administered with AZT, did not affect ABC transporter‐mediated AZT transfer. Copyright © 2016 John Wiley & Sons, Ltd.     

High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transporters

Up-regulation of pump (transporter) expression and selection of resistant cancer cells result in cancer multidrug resistance to diverse substrates of these transporters. While more than 48 members of the ATP binding cassette (ABC) transporter superfamily have been identified, up to now only three human ABC transporters-ABCB1, ABCC1, and ABCG2-have unambiguously been shown to contribute to cancer multidrug resistance. The use of low-toxicity and high-specificity agents as a targeted transporter inhibition strategy is necessary to effectively overcome multiple drug resistance. An objective of the present studies was to develop and validate HyperCyt (IntelliCyt, Albuquerque, NM) flow cytometry high-throughput screeening assays to assess the specificity of test compounds that inhibited transporters as an integral part of the screen. Two separate duplex assays were constructed: one in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate, and the other in which ABCB1 and ABCC1 transporters were evaluated in parallel using fluorescent calcein acetoxymethyl ester as substrate. ABCB1-expressing cells were color-coded to allow their distinction from cells expressing the alternate transporter. The assays were validated in a screen of the Prestwick Chemical Library (Illkirch, France). Three novel selective inhibitors of the ABCC1 transporter were identified in the screen, and the activity of each was confirmed in follow-up chemosensitivity shift and reversal studies. This high-throughput screening assay provides an efficient approach for identifying selective inhibitors of individual ABC transporters, promising as probes of transporter function and therapeutic tools for treating chemotherapy-resistant cancers.     

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI₅₀ was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p < 0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI₅₀ 25.7 and 40.4 nM respectively, p < 0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p < 0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b^?/? as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC_0–6 h 242 vs. 152 μg/L h respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b^?/? and Abcc2^?/? mice, concentrations in the brain were significantly higher at 6 h following drug administration in Abcb1a/1b^?/? mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.     

Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2

ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein) and ABCG2 (breast cancer-resistance protein [BCRP], mitoxantrone-resistance protein [MXR], or ABC transporter in placenta [ABCP]), are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenetics of the ABC transporters ABCB1 and ABCG2, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.     

In vitro methods suitable for the prediction of drug and ABC transporter, especially ABCG2 interactions

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by in vitro systems using transfected insect and/or human cell lines.     

Influence of ABCC2, SLCO1B1, and ABCG2 polymorphisms on the pharmacokinetics of olmesartan

This study was designed to determine whether genetic polymorphisms of multidrug-resistant protein 2 (ABCC2), organic anion transporting polypeptide 1B1 (SLCO1B1), and breast cancer resistance protein (ABCG2) have an effect on olmesartan pharmacokinetics in Korean subjects. Sixty-eight healthy male volunteers who participated in previous pharmacokinetics studies of olmesartan medoxomil (single dose, 20 mg or 40 mg) were enrolled. All subjects were analyzed and grouped according to the genotypes of ABCC2, SLCO1B1, and ABCG2. The dose-normalized peak plasma concentration (C(max)) and area under the plasma concentration-time curve (AUCt) values were analyzed. The dose-normalized mean C(max) and AUC(t) in the ABCC2 -24CT genotype group were higher than those in the -24CC genotype group [C(max,dn): CT 26.1 ± 6.5 (ng/mL)/mg versus CC 22.1 ± 6.7 (ng/mL)/mg, P = 0.010, AUC(t,dn): CT 178.7 ± 45.6 (hr·ng(-1)·mL(-1))/mg versus CC 149.9 ± 39.8 (hr·ng(-1)·mL(-1))/mg, P = 0.010]. The difference in AUC(t,dn) between the ABCC2 -1549GG and -1549GA genotype groups was statistically significant [GG 149.0 ± 41.0 (hr·ng(-1)·mL(-1))/mg versus GA 174.1 ± 43.3 (hr·ng(-1)·mL(-1))/mg, P = 0.019]. No significant differences were observed for any other single nucleotide polymorphism in ABCC2, SLCO1B1, or ABCG2. The ABCC2 -24CC genotype group exhibited lower systemic exposure of olmesartan than the -24CT genotype group, whereas no significant differences were observed in the other transporter genotype groups.     

Cholesterol and non-cholesterol sterol transporters: ABCG5, ABCG8 and NPC1L1: a review

1.?Whole-body sterol (cholesterol and xenosterol) balance is delicately regulated by the gastrointestinal tract and liver, which control sterol absorption and excretion, respectively, in addition to the contribution to the cholesterol pool by whole-body cholesterol synthesis. In the past ten years enormous strides have been made not only in establishing that specific transporters mediate the entry and exit of sterols and how these may regulate selective sterol access to the body pools, but also in how these pathways operate to integrate these physiological pathways.

2.?The entry of sterols from the gastrointestinal and biliary canalicular lumen into the body is mediated by NPC1L1, which was discovered by a novel method, via a genomics–bioinformatics approach.

3.?Identification of the genetic basis responsible for causing sitosterolaemia, characterized by plant sterol accumulation, led to the identification of two half-transporters (ABCG5 and ABCG8) that normally efflux plant sterols (and cholesterol) into the intestinal and biliary lumen for faecal excretion.

4.?The objective of this review is to provide up-to-date knowledge on genomics, proteomics and function of these two transporter systems.     

Cholesterol and non-cholesterol sterol transporters: ABCG5, ABCG8 and NPC1L1: a review

1. Whole-body sterol (cholesterol and xenosterol) balance is delicately regulated by the gastrointestinal tract and liver, which control sterol absorption and excretion, respectively, in addition to the contribution to the cholesterol pool by whole-body cholesterol synthesis. In the past ten years enormous strides have been made not only in establishing that specific transporters mediate the entry and exit of sterols and how these may regulate selective sterol access to the body pools, but also in how these pathways operate to integrate these physiological pathways. 2. The entry of sterols from the gastrointestinal and biliary canalicular lumen into the body is mediated by NPC1L1, which was discovered by a novel method, via a genomics-bioinformatics approach. 3. Identification of the genetic basis responsible for causing sitosterolaemia, characterized by plant sterol accumulation, led to the identification of two half-transporters (ABCG5 and ABCG8) that normally efflux plant sterols (and cholesterol) into the intestinal and biliary lumen for faecal excretion. 4. The objective of this review is to provide up-to-date knowledge on genomics, proteomics and function of these two transporter systems.     

Pharmacogenomics of MRP transporters (ABCC1-5) and BCRP (ABCG2)

Elucidation of the key mechanisms that confer interindividual differences in drug response remains an important focus of drug disposition and clinical pharmacology research. We now know both environmental and host genetic factors contribute to the apparent variability in drug efficacy or in some cases, toxicity. In addition to the widely studied and recognized genes involved in the metabolism of drugs in clinical use today, we now recognize that membrane-bound proteins, broadly referred to as transporters, may be equally as important to the disposition of a substrate drug, and that genetic variation in drug transporter genes may be a major contributor of the apparent intersubject variation in drug response, both in terms of attained plasma and tissue drug level at target sites of action. Of particular relevance to drug disposition are members of the ATP Binding Cassette (ABC) superfamily of efflux transporters. In this review a comprehensive assessment and annotation of recent findings in relation to genetic variation in the Multidrug Resistance Proteins 1-5 (ABCC1-5) and Breast Cancer Resistance Protein (ABCG2) are described, with particular emphasis on the impact of such transporter genetic variation to drug disposition or efficacy.     

Pharmacogenomics of MRP Transporters (ABCC1-5) and BCRP (ABCG2)

Elucidation of the key mechanisms that confer interindividual differences in drug response remains an important focus of drug disposition and clinical pharmacology research. We now know both environmental and host genetic factors contribute to the apparent variability in drug efficacy or in some cases, toxicity. In addition to the widely studied and recognized genes involved in the metabolism of drugs in clinical use today, we now recognize that membrane-bound proteins, broadly referred to as transporters, may be equally as important to the disposition of a substrate drug, and that genetic variation in drug transporter genes may be a major contributor of the apparent intersubject variation in drug response, both in terms of attained plasma and tissue drug level at target sites of action. Of particular relevance to drug disposition are members of the ATP Binding Cassette (ABC) superfamily of efflux transporters. In this review a comprehensive assessment and annotation of recent findings in relation to genetic variation in the Multidrug Resistance Proteins 1–5 (ABCC1-5) and Breast Cancer Resistance Protein (ABCG2) are described, with particular emphasis on the impact of such transporter genetic variation to drug disposition or efficacy.     

The SmartAmp Method: Rapid Detection of SNPs in Thiopurine S-Methyltransferase and ABC Transporters ABCC4 and ABCG2

Genetic polymorphisms of drug transporters as well as drug metabolizing enzymes have been documented to play a significant role in patients' responses to medication. A key requirement for advancing personalized medicine is the ability to rapidly and conveniently test for patients' genetic polymorphisms. We have recently developed a rapid and cost-effective method for single nucleotide polymorphism (SNP) detection, named Smart Amplification Process (SmartAmp), which enables us to detect genetic polymorphisms or mutations in 30 to 45 min under isothermal conditions without the need for DNA isolation and PCR amplification. This article presents the SmartAmp-based detection of SNPs in the thiopurine S-methyltransferase gene as well as in the ATP-binding cassette (ABC) transporter ABCC4 and ABCG2 genes that are critically involved in drug-induced adverse reactions. The SmartAmp method is expected to provide a practical and cost-effective tool for pharmacogenomics-based personalized medicine.     

Genetic variations and haplotype structures of the ABC transporter gene ABCC1 in a Japanese population

Multidrug resistance-related protein 1 (MRP1), an ATP-binding cassette transporter encoded by the ABCC1 gene, is expressed in many tissues, and functions as an efflux transporter for glutathione-, glucuronate- and sulfate-conjugates as well as unconjugated substrates. In this study, the 31 exons and their flanking introns of ABCC1 were comprehensively screened for genetic variations in 153 Japanese subjects to elucidate the linkage disequilibrium (LD) profiles and haplotype structures of ABCC1 that is necessary for pharmacogenetic studies of the substrate drugs. Eighty-six genetic variations including 31 novel ones were found: 1 in the 5'-flanking region, 1 in the 5'-untranslated region (UTR), 20 in the coding exons (9 synonymous and 11 nonsynonymous variations), 4 in the 3'-UTR, and 60 in the introns. Of these, eight novel nonsynonymous variations, 726G>T (Trp242Cys), 1199T>C (Ile400Thr), 1967G>C (Ser656Thr), 2530G>A (Gly844Ser), 3490G>A (Val1164Ile), 3550G>A (Glu1184Lys), 3901C>T (Arg1301Cys), and 4502A>G (Asp1501Gly), were detected with an allele frequency of 0.003. Based on the LD profiles, the analyzed regions of the gene were divided into five LD blocks (Blocks -1 and 1 to 4). The multiallelic repeat polymorphism in the 5'-UTR was defined as Block -1. For Blocks 1, 2, 3 and 4, 32, 23, 23 and 13 haplotypes were inferred, and 9, 7, 7 and 6 haplotypes commonly found on > or = 10 chromosomes accounted for > or = 91% of the inferred haplotypes in each block. Haplotype-tagging single nucleotide polymorphisms for each block were identified to capture the common haplotypes. This study would provide fundamental and useful information for the pharmacogenetic studies of MRP1-dependently effluxed drugs in Japanese.     

Expression and functional characterization of human ABC transporter ABCG2 variants in insect cells

Hitherto three variant forms of ABCG2 have been documented on the basis of their amino acid moieties (i.e., Arg, Gly, and Thr) at the position 482. In the present study, we have generated those variants of ABCG2 by site-directed mutagenesis and expressed them in Sf9 insect cells. The apparent molecular weight of the expressed ABCG2 variants was 130,000 under non-reductive conditions, whereas it was reduced to 65, 000 by treatment with mercaptoethanol. It is suggested that ABCG2 exists in the plasma membrane of Sf9 cells as a homodimer bound through cysteinyl disulfide bond(s). Both ATPase activity and drug transport of ABCG2 variants were examined by using plasma membrane fractions prepared from ABCG2-overexpressing Sf9 cells. The ATPase activity of the plasma membrane expressing ABCG2 (Gly-482) was significantly enhanced by prazosin. In contrast, ABCG2 (Arg-482) transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly-482 and Thr-482). It is strongly suggested that the amino acid moiety at the position of 482 is critical for the substrate specificity of ABCG2.     

Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications

Pharmacogenomics, the study of the influence of genetic factors on drug action, is increasingly important for predicting pharmacokinetics profiles and/or adverse reactions to drugs. Drug transporters, as well as drug metabolism play pivotal roles in determining the pharmacokinetic profiles of drugs and their overall pharmacological effects. There is an increasing number of reports addressing genetic polymorphisms of drug transporters. However, information regarding the functional impact of genetic polymorphisms in drug transporter genes is still limited. Detailed functional analysis in vitro may provide clear insight into the biochemical and therapeutic significance of genetic polymorphisms. This review addresses functional aspects of the genetic polymorphisms of human ATP-binding cassette transporters, ABCB1 and ABCG2, which are critically involved in the pharmacokinetics of drugs.