Transporter-mediated uptake into cellular compartments

Active transport across biological membranes represents a critical step in the disposition of many drugs. It is now well-established that different efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) or organic anion transporting polypeptides (OATPs) are involved in the overall disposition and efficacy of numerous compounds. These proteins are mainly expressed at physiological sites of drug absorption and elimination, thus leading to diminished absorption and/or increased transporter-facilitated excretion. Moreover, drug transporters are known to be of protective significance in blood–organ barriers. On the contrary, only little is known about the relevance of transporter function on drug levels within tissues and cellular compartments, i.e. the site of action for many substances. Moreover, the pharmacokinetic processing inside the cell is characterized by uptake, metabolism and elimination. It is gradually being recognized that active uptake and/or efflux transporters may modify target concentrations at the subcellular receptor sites which in turn may have an influence on drug effects. This review will summarize current knowledge about the impact of transporter proteins on drug availability within pharmacologically relevant cellular compartments and tissues as hepatocytes, enterocytes, different blood cell types, brain, and the heart with emphasis on the potential clinical significance of these transporters.     

Drug transporters in the human blood-placental barrier

Studies on the increasing number of transporters found in the placental barrier are gaining momentum, because of their tissue-specific expression, significance in physiology and disease, and the possible utilization of the emerging knowledge in pharmacology. In the placenta, both syncytiotrophoblast and fetal capillary endothelium express transporters. Fetal exposure is determined by the net effect of combination of transporters, their nature and localization in relation to placental cells and their substrate specificity. Although the significance of placental transporters on human fetal drug exposure is almost an unstudied field so far, their potential use to design drugs that do not cross the placenta is already being pursued. It is thus of interest to review the existing knowledge of human placental transporters. Transporters in all groups which take part in drug transport are found in human placenta. Especially, ATP-binding cassette transporters ABCG2/breast cancer resistance protein, ABCB1/P-glycoprotein and ABCC2/MRP2 are all expressed at the apical surface of syncytiotrophoblast facing maternal blood and are putatively important protective proteins both for placental tissue and the fetus, because they are efflux transporters and their substrates include many drugs and also environmental chemicals. Such protective effect has been shown in animals, but these results cannot be directly extrapolated to humans due to interspecies differences in placental structure and function. Experimental models utilizing human placental tissue, especially human placental perfusion, offer valuable possibilities, which have been insufficiently studied so far.     

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.     

In vitro evaluation of potential transporter‐mediated drug interactions of evogliptin

To date, little is known about the transporter‐mediated drug–drug interaction (DDI) potential of evogliptin, a novel DPP‐4 inhibitor. The objective of this study was to evaluate the DDI potential of evogliptin using various in vitro assays in transporter‐expressing cell lines. After incubating evogliptin with cells overexpressing OAT1, OAT3, OCT2, OATP1B1 and OATP1B3, there was no notable cellular accumulation of evogliptin (fold accumulation, 0.41–1.86). In bidirectional transport assays using a Caco‐2 cell monolayer, a high efflux ratio (ER, 522) of evogliptin was observed, which was significantly decreased (97.96%) in the presence of a potent P‐gp inhibitor. In assays using MDCKII‐BCRP cell monolayers, by contrast, a low net ER (1.16–1.26) was found. In similar cellular uptake and bidirectional studies with probe substrates of P‐gp, BCRP, OAT1, OAT3, OCT2, OATP1B1 and OATP1B3, the active transport of the substrates was not significantly suppressed by evogliptin. These results suggest that evogliptin may be a substrate of P‐gp, but not a substrate of BCRP, OAT1B1, OAT1B3, OAT1, OAT3 or OCT2, and not an inhibitor of any of these transporters. Therefore, it could be concluded that evogliptin has some DDI potential involving P‐gp, but it has low potential of DDI mediated by the other transporters.     

Decreased Expression of Na+/K+-ATPase, NHE3, NBC1, AQP1 and OAT in Gentamicin-induced Nephropathy

The present study was aimed to determine whether there is an altered regulation of tubular transporters in gentamicin-induced nephropathy. Sprague-Dawley male rats (200~250 g) were subcutaneously injected with gentamicin (100 mg/kg per day) for 7 days, and the expression of tubular transporters was determined by immunoblotting and immunohistochemistry. The mRNA and protein expression of OAT was also determined. Gentamicin-treated rats exhibited significantly decreased creatinine clearance along with increased plasma creatinine levels. Accordingly, the fractional excretion of sodium increased. Urine volume was increased, while urine osmolality and free water reabsorption were decreased. Immunoblotting and immunohistochemistry revealed decreased expression of Na⁺/K⁺-ATPase, NHE3, NBC1, and AQP1 in the kidney of gentamicin-treated rats. The expression of OAT1 and OAT3 was also decreased. Gentamicin-induced nephropathy may at least in part be causally related with a decreased expression of Na⁺/K⁺-ATPase, NHE3, NBC1, AQP1 and OAT.     

Transport mechanisms at the pulmonary mucosa: implications for drug delivery

ABSTRACT

Introduction: Over the past years, a significant number of papers have substantiated earlier findings proposing a role for drug transporter proteins in pulmonary drug disposition. Whilst the majority of reports present data from in vitro models, a growing number of publications advance the field by introducing sophisticated ex vivo and in vivo techniques. In a few cases, evidence from clinical studies in human volunteers is complementing the picture.

Areas covered: In this review, recent advances in pulmonary drug transporter research are critically evaluated. Transporter expression data in tissues and cell-based in vitro models is summarized and information on transport activity assessed. Novel techniques allowing for better quantification of transporter-related effects following pulmonary delivery are also described.

Expert opinion: Different tissue and cell populations of the lung have distinct transporter expression patterns. Whether these patterns are affected by disease, gender and smoking habits requires further clarification. Transporters have been found to have an impact on drug absorption processes, at least in vitro. Recent ex vivo experiments using isolated, perfused lung models, however, suggest that mainly efflux pumps have significant effects on absorption into the pulmonary circulation. Whether these rodent-based ex vivo models predict the human situation is basis for further research.     

The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation

Introduction: Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug ‘exporters’ and the Solute Carrier transporters, with various transport directions and mechanisms.

Areas covered: In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges.

Expert opinion: The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of ‘stemness’ and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation.     

MicroRNA对药物转运体调控作用的研究进展

MicroRNA作为一类单链非编码小分子RNA,通过与靶基因mRNA 3＇端非编码区（3＇-untranslated region,3＇-UTR）的互补结合来调节靶基因的表达,在生物发育、细胞分化、增殖和凋亡中发挥重要作用,与人类多种疾病如恶性肿瘤、糖尿病等的发生发展密切相关。MicroRNA表达的变化可影响药物代谢酶和转运体的表达,进而影响药物效应。目前有关药物代谢酶的研究较为深入,而对转运体的研究则相对较少。本文通过总结近几年国内外相关研究,从MicroRNA的作用机制及其对药物转运体的调控作用方面进行综述,以期为指导临床个体化用药提供理论依据。     

Mechanisms of renal anionic drug transport

By utilizing filtration, active secretion and reabsorption processes, the kidney can conserve essential nutrients, and eliminate drugs and potentially toxic compounds. Active uptake of organic anions and cations across the basolateral membrane, and their extrusion into the urine across the brush border membrane mainly takes place in the renal proximal tubule cells, and is facilitated via a range of substrate-specific tubular transporters. Many drugs and their phase II conjugates are anionic compounds, and therefore renal organic anion transporters are important determinants of their distribution and elimination. Competition for renal excretory transporters may cause drugs to accumulate in the body leading to toxicity, which is a potential hazard of concomitant drug administration. Here, we present a brief update on the most prominent human proximal tubule organic anion transporters, which either belong to the ATP-binding cassette (ABC) or the solute carrier transporter (SLC) families. We focus on the participation of the individual transporters in renal anionic drug elimination, in an attempt to understand their overall biological and pharmacological significance, hoping to inspire further studies in the renal transporters field.     

Monocarboxylate Transporter (MCT) Mediates the Transport of γ-Hydroxybutyrate in Human Kidney HK-2 cells

Purpose Previous studies in our laboratory have suggested that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCT). The objectives of this study were to characterize the renal transport of GHB using HK-2 cells and the role of MCT in the renal transport of GHB. Materials and Methods Western blot was used to detect the protein expression of MCT1, 2, and 4. Cellular uptake and directional flux studies were conducted to investigate the transport of GHB and L-lactate. RNA interference assay was used to investigate the involvement of MCT isoforms in the transport of GHB. Results MCT1, 2 and 4 were present in HK-2 cells. The cellular uptake of L-lactate and GHB exhibited pH- and concentration-dependence (L-lactate: K _m of 6.5 ± 1.1 mM and V _max of 340 ± 60 nmol mg⁻¹min⁻¹; GHB: K _m of 2.07 ± 0.79 mM, V _max of 27.6 ± 9.3 nmol mg⁻¹min⁻¹, and a diffusional clearance of 0.54 ± 0.15 μl mg⁻¹min⁻¹), but not sodium-dependence. α-Cyano-4-hydroxycinnamate (CHC) competitively inhibited the uptake of GHB and L-lactate with inhibition constants (K _i) of 0.28 ± 0.1 mM, and 0.19 ± 0.03 mM, respectively. Using small-interference RNA (siRNA) for MCT1, the protein expression of MCT1 and the uptake of L-lactate and GHB were significantly decreased. The siRNA treatment of MCT2 in HK-2 cells inhibited the uptake of GHB by 17%, and the siRNA treatment of MCT4 demonstrated no inhibition of GHB uptake. GHB exhibited a directional flux across HK-2 monolayer from apical to basal chambers in the presence of a pH gradient of pH 6.0 to pH 7.4. Conclusion These data suggest that MCT1 represents an important transporter for GHB transport in renal tubule cells, responsible for the reabsorption of GHB in the kidney.     

Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: A resource for investigations into drug disposition

Carrier-mediated transporters play a critical role in xenobiotic disposition and transporter research is complicated by species differences and their selective tissue expression. The purpose of this study was to generate a comprehensive data set of xenobiotic transporter gene expression profiles in humans and the pre-clinical species mouse, rat, beagle dog and cynomolgus monkey. mRNA expression profiles of 50 genes from the ABC, SLC and SLCO transporter superfamilies were examined in 40 human tissues by microarray analyses. Transporter genes that were identified as enriched in the liver or kidney, or that were selected for their known roles in xenobiotic disposition, were then compared in 22 tissues across the five species. Finally, as clinical variability in drug response and adverse reactions may be the result of variability in transporter gene expression, variability in the expression of selected transporter genes in 75 human liver donors were examined and compared with the highly variable drug metabolizing enzyme CYP3A4.     

寡肽转运体在生理、药物转运的重要作用及临床相关性

寡肽转运体（PEPTs）属于溶质转运体（SLC）大家族,以H⁺梯度为驱动力,包括PEPT1和PEPT2。PEPT1是低亲和力、高容量转运蛋白,主要表达于小肠;而PEPT2是高亲和力、低容量的转运蛋白,主要在肾脏、脑和肺中表达,在生物体中分布较广。PEPTs除重吸收二肽和三肽以及维持脑中神经肽的稳态作用外,还能够吸收和处置许多重要的化合物,如一些氨基头孢菌素、血管紧张素转化酶抑制剂、抗病毒前药等,而且PEPTs也与一些肠道疾病和癌症相关。因此综述了PEPTs在生理、药物转运中的重要作用及临床相关性。     

Isolation,identification and excretion profile of the principal urinary metabolite of the recently banned designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rats

The metabolism of 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a recently banned designer drug, in rats was studied by analysing its urinary metabolites. p-Hydroxy-TFMPP (p-OH-TFMPP) was isolated and identified as the main metabolite by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). The time-course excretion profiles of TFMPP and p-OH-TFMPP in rats were investigated following a single intraperitoneal dosing of 5?mg?kg^?1 TFMPP by using an optimized analytical procedure that combined solid-phase extraction and LC-ESI MS techniques. The cumulative amount of p-OH-TFMPP excreted within the first 48?h reached approximately 64% of the dose, of which 70% was the glucuronide conjugated form. The cumulative amount of parent TFMPP excreted was less than 0.7% of the dose. The results suggest that p-OH-TFMPP would be the most relevant metabolite to be detected for TFMPP exposure in the forensic and clinical analysis of human urine.     

Comparison of uptake transporter functions in hepatocytes in different species to determine the optimal model for evaluating drug transporter activities in humans

1. A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data.

2. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared.

3. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis–Menten constant (K_m,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17β-glucuronide and metformin. The K_m,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PS_inf,act) while no difference in %PS_inf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug–drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin.

4. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

     

Glucuronidation: driving factors and their impact on glucuronide disposition

Glucuronidation is a well-recognized phase II metabolic pathway for a variety of chemicals including drugs and endogenous substances. Although it is usually the secondary metabolic pathway for a compound preceded by phase I hydroxylation, glucuronidation alone could serve as the dominant metabolic pathway for many compounds, including some with high aqueous solubility. Glucuronidation involves the metabolism of parent compound by UDP-glucuronosyltransferases (UGTs) into hydrophilic and negatively charged glucuronides that cannot exit the cell without the aid of efflux transporters. Therefore, elimination of parent compound via glucuronidation in a metabolic active cell is controlled by two driving forces: the formation of glucuronides by UGT enzymes and the (polarized) excretion of these glucuronides by efflux transporters located on the cell surfaces in various drug disposition organs. Contrary to the common assumption that the glucuronides reaching the systemic circulation were destined for urinary excretion, recent evidences suggest that hepatocytes are capable of highly efficient biliary clearance of the gut-generated glucuronides. Furthermore, the biliary- and enteric-eliminated glucuronides participate into recycling schemes involving intestinal microbes, which often prolong their local and systemic exposure, albeit at low systemic concentrations. Taken together, these recent research advances indicate that although UGT determines the rate and extent of glucuronide generation, the efflux and uptake transporters determine the distribution of these glucuronides into blood and then to various organs for elimination. Recycling schemes impact the apparent plasma half-life of parent compounds and their glucuronides that reach intestinal lumen, in addition to prolonging their gut and colon exposure.     

WATER CHANNELS AND UREA TRANSPORTERS

1. It now appears that when water crosses an endothelium which is not fenestrated, or an epithelium with tight junctions, it does so rapidly, and with low energy cost, only if the cell membrane contains an adequate number of specific water channels, encoded by one of at least six different genes. 2. The water channel genes so far cloned encode a series of integral membrane proteins called aquaporins, all of approximately 30kDa (265–282 amino acids), in the unglycosylated state. All but one (AQP₃) are specific water channels and all but one (AQP₄) are inactivated by mercurial compounds. 3. Aquaporin 0 is the major (60%) intrinsic protein (MIP) of lens fibre cells of the eye. Mutations in this gene are associated with cataract formation in mice. 4. Aquaporin 1, also called CHIP-28, exists in the membrane as a homotetramer, and is present in red blood cells, the choroid plexus, the proximal tubule and descending limb of the loop of Henle in the kidney as well as in many other sites. Surprisingly, no pathological consequence is known in patients lacking a functional AQP₁ gene. 5. Aquaporin 2, also called WCH-CD, is the water channel of the principal cell of the cortical and medullary collecting duct, and is located in cytoplasmic vesicles unless arginine vasopressin is acting, when it is translocated to the apical membrane by synaptobrevins or vesicle associated membrane protein 2 (VAMP₂). Lack of a functional AQP₂ gene leads to a rare form of nephrogenic diabetes insipidus. 6. Aquaporins 3, 4, and 5 are located in many tissues—AQP₃ and AQP₄ being in the basolateral membrane of the renal cortical and medullary principal cell, as well as in the gastrointestinal tract (AQP₃) and the brain (AQP₄). 7. Four sequences are known for urea transporters HUT₁₁—the urea transporter of the human red cell membrane, and HUT₂, rUT₂, rbUT₂—the arginine vasopressin inducible urea transporters of the human, rat and rabbit kidney. They are specifically permeable to urea, not to water, and are claimed to be inhibited by phloretin. 8. The water channel proteins contain six membrane-spanning regions, whilst the urea transporters are thought to contain at least 10 membrane spanning segments. 9. Very little work has examined the ontogeny of these proteins, except in the rat, and virtually nothing is known of the expression of these genes in pregnancy or in any disorder of fluid balance in the mother or foetus.     

Cytochrome P450 isozymes responsible for the metabolism of toluene and styrene in human liver microsomes

1. Cytochrome P450 isozymes from Asian (31 Chinese subjects) and Caucasian (14 Finnish subjects) livers were examined for their roles in the metabolism of toluene (rates of benzyl alcohol, o- and p-cresol formation) and styrene (rates of styrene glycol formation). 2. For toluene, the overall rate of metabolism was higher in samples from Finnish than from Chinese subjects. At 0·20 mM toluene, the rate of o-cresol formation was significantly higher in Finnish microsomes than in Chinese ones. The formation rates of benzyl alcohol and p-cresol in Finnish samples were also higher than those of Chinese samples, but only at a high substrate concentration (5·0 mM). For styrene metabolism, the Chinese liver microsomes showed higher metabolic rates than the Finnish ones at 0·085 mM styrene, but not at the higher substrate concentration. 3. Mean expression levels of immunochemically detected CYP1A2 1 and CYP2B6 were almost 3-fold higher in Finnish microsomes, whereas CYP2E1 was 1 7-fold higher in Chinese samples. 4. Correlation analysis showed that CYP2E1 (benzyl alcohol formation) and CYP1A2/1 (o-cresol formation) contributed to the metabolism of toluene at the low substrate concentration, whereas CYP2C8 was the form more actively involved at the higher toluene concentrations. At the higher concentration (1·8 mM) of styrene, CYP2B6 was most active isozyme to catalyse the formation of styrene oxide from styrene. 5. These results suggest that CYP2E1 and CYP1A2/1 are the main isoforms responsible for the metabolism of toluene at low substrate concentrations in human liver microsomes, CYP2E1 at low styrene concentration, and CYP2C8 and CYP2B6 at high concentrations of toluene and styrene respectively.