Drug Transporters: Molecular Characterization and Role in Drug Disposition

This new edition overviews drug transporters and presents the principles of drug transport and associated techniques, featuring new chapters on multidrug and toxin extrusion proteins, placental transport,  in silico approaches in drug discovery, and regulatory guidance for drug transport studies in drug development.

• Describes drug transporter families, mechanisms, and clinical implications along with experimental methods for studying and characterizing drug transporters
• Includes new chapters on multidrug and toxin extrusion proteins, placental transport and in silico approaches in drug discovery
• Has a new chapter covering regulatory guidance for the evaluation of drug transport in drug development with global criteria used for drug transporters in clinical trials
• Arranges material to go from fundamental mechanisms to clinical outcomes, making the book useful for novice and expert readers

• Language: English
• Publisher: Wiley
• Release date: Jul 7, 2014
• ISBN: 9781118704981

Book preview

1

Overview of Drug Transporter Families

Guofeng You¹ and Marilyn E. Morris²

¹ Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ, USA

² Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA

1.1 What Are Drug Transporters?

Transporters are membrane proteins whose primary function is to facilitate the flux of molecules into and out of cells. Drug transporters did not evolve to transport specific drugs. Instead, their primary functions are to transport nutrients or endogenous substrates, such as sugars, amino acids, nucleotides, and vitamins, or to protect the body from dietary and environmental toxins. However, the specificity of these transporters is not strictly restricted to their physiological substrates. Drugs that bear significant structural similarity to the physiological substrates have the potential to be recognized and transported by these transporters. As a consequence, these transporters also play significant roles in determining the bioavailability, therapeutic efficacy, and pharmacokinetics of a variety of drugs. Nevertheless, because drugs may compete with the physiological substrates of these transporters, they are also likely to interfere with the transport of endogenous substrates and consequently produce deleterious effects on body homeostasis.

1.2 Structure and Model of Drug Transporters

Because of the involvement of transporters in all facets of drug absorption, tissue distribution, excretion, and efficacy/toxicity, characterization of transporter structure can provide a scientific basis for understanding drug delivery and disposition, as well as the molecular mechanisms of drug interaction and interindividual/interspecies differences. However, compared to soluble proteins, the atomic resolution crystal structures of membrane transporters have been extremely difficult to obtain for several reasons: first is the amphipathic nature of the surface of the transporters, with a hydrophobic area in contact with membrane phospholipids and polar surface areas in contact with the aqueous phases on both sides of the membrane; second is the low abundance of many transporters in the membrane, making it impossible to overexpress them, a prerequisite for structural studies; and third is the inherent conformational flexibility of the transporters, making it difficult to obtain stable crystals.

Due to these difficulties, high-resolution three-dimensional structures have been obtained for only a limited number of transporters. For other transporters, the three-dimensional structures have been achieved through homology modeling. In this approach, similar folding patterns between any protein and one for which the crystal structure is known enable the construction of a fairly accurate three-dimensional protein model of the unknown structure using the related crystal structure as a template and modern computational techniques. Three-dimensional structures have revealed that transporters have alpha-helical structures of the membrane-spanning domains, and some of the helices have irregular shapes with kinks and bends. Certain transporters undergo substantial movements during the substrate translocation process. Construction of three-dimensional transporter models has provided insight into functional mechanisms and molecular structures and enabled formulation of new hypotheses regarding transporter structure and function, which may be experimentally validated.

1.3 Transport Mechanisms

Not only different transporters reside in the membrane with different three-dimensional structures, but also they transport their substrates through different transport mechanisms. According to their transport mechanisms, transporters can be divided into passive and active transporters: passive transporters, also called facilitated transporters, allow molecules to move across the cell membrane down their electrochemical gradients. Such a spontaneous process decreases free energy, and increases entropy in a system, and therefore does not consume any chemical energy. In contrast to facilitated transporters, active transporters typically move molecules against their electrochemical gradients; such process is entropically unfavorable and therefore needs the coupling of the hydrolysis of ATP as an energy source. This coupling can be either primary or secondary. In primary active transport, transporters that move molecules against their electrical/chemical gradient hydrolyze ATP. In the secondary active transport, transporters utilize ion gradients, such as sodium or proton gradients, across the membrane produced by the primary active transporters and transport substrates against an electrochemical difference.

1.4 Polarized Expression of Drug Transporters in Barrier Epithelium

Most drug transporters are expressed in tissues with barrier functions such as the liver, kidney, intestine, placenta, and brain. Cells at the border of these barriers are usually polarized. For example, enterocytes of the intestine and proximal tubule cells of the kidney have an apical domain facing the lumen and a basolateral domain facing the blood circulation; hepatocytes polarize into a canalicular membrane facing the bile duct and sinusoidal membrane facing the blood circulation; syncytiotrophoblasts of placenta have an apical domain facing maternal blood and a basolateral domain facing the fetus. Brain capillary endothelial cells, which function as the blood–brain barrier, also polarize into apical and basolateral membranes. In most cases, the expression of drug transporters is highly restricted to one side (i.e., apical or basolateral domain) of polarized cells. Such polarized expression of the transporters is essential for the concerted transport of drugs in the same direction. One of the most well-studied examples of concerted transport is the kidney. Kidney proximal tubule cells play a critical role in the body clearance of drugs. These drugs are first taken up from the blood into the proximal tubule cells by transporters at the basolateral membrane. Once inside the cells, these drugs are then transported out of the cells into the tubule lumen by transporters at the apical membrane and subsequently eliminated in the urine. The alliance between transporters at both the basolateral membrane and the apical membrane of the kidney proximal tubule cells ensures the clearance of the drugs from the body.

1.5 Classifications of Drug Transporters

Drug transporters can be classified in a number of different manners, including as efflux transporters versus influx transporters, secretory transporters versus absorptive transporters, and ATP-binding cassette (ABC) transporters versus solute carrier (SLC) transporters.

1.5.1 Definition of Efflux and Influx Transporters

Drug transporters can be categorized as efflux or influx transporters according to the direction they transport substrate across the cell membranes. This classification is often observed in the literature where drug transport studies are performed at the cellular level. With this definition, transporters that pump the substrates out of the cells are called efflux transporters, whereas transporters that transfer substrates into cells are called influx transporters.

1.5.2 Definition of Absorptive and Secretory Transporters

The other way of classifying drug transporters is from a pharmacodynamic or pharmacokinetic point of view. In such a classification, the transporter that transfers its substrates into the systemic blood circulation is called an absorptive transporter, whereas the transporter that excretes its substrates from the blood circulation into the bile, urine, or gut lumen is known as a secretory transporter. However, when absorptive or secretory transporters in the blood–brain barrier and placenta are discussed, the definition needs to be modified. The brain and fetus have been traditionally considered as two isolated compartments in the human body. In drug therapy, many strategies have been utilized to achieve either enhanced or reduced penetration of drugs into these two compartments. Conventionally, the transporters facilitating drug penetration into the brain or fetus are referred to as absorptive transporters.

1.5.3 Relationship between Influx/Efflux and Absorptive/Secretory Transporters

An absorptive transporter does not necessarily mean that it influxes a substrate. Similarly, a secretory transporter does not have to be an efflux pump. For example, organic anion transporter (OAT) OAT1, present at the basolateral membrane of the kidney proximal tubule, is an influx transporter based on its role of taking up drugs from the blood into the proximal tubule cells for their subsequent exit across the apical membrane into the urine for elimination. However, considering its overall role of removing drugs out of the blood circulation into the urine, OAT1 is a secretory transporter. Intestinally expressed organic anion-transporting polypeptide 1A2 (OATP1A2) is localized on the apical domain of enterocytes. It can take up (i.e., influx) orally administered drugs into the enterocytes for their subsequent exit across the basolateral membrane into the bloodstream, so OATP1A2 is considered an absorptive transporter. Therefore, influx transporters can function as either absorptive or secretory transporters depending on the tissue and on the membrane domain where they are expressed.

1.5.4 ABC Transporters and SLC Transporters

Most of the drug transporters can also be molecularly and mechanistically classified as a member of the ABC transporter family or the SLC transporter family (Table 1.1).

Table 1.1 Classifications of representative drug transporters

a References where the human chromosome locus can be found.

ABC transporters are a family of membrane transport proteins that require ATP hydrolysis for the transport of substrates across membranes. Therefore, ABC transporters are primary active transporters. The protein family derives its name from the ATP-binding domain found on the protein. The best studied drug transporters that are classified as ABC transporters are multidrug resistance protein (MDR), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP).

Some of the SLC transporters utilize an electrochemical potential difference of the transported substrate and are therefore classified as facilitated transporters; other SLC transporters utilize an ion gradient, such as sodium and proton gradients across the membrane produced by the primary active transporters, and transport substrates against an electrochemical difference. These transporters are classified as secondary active transporters. In contrast to ABC transporters, SLC transporters do not possess ATP-binding sites. Most drug transporters belong to the SLC transporter family.

1.6 Regulation of Drug Transporters

Given the importance of drug transporters in the absorption, distribution, and excretion of a diverse array of environmental toxins and clinically important drugs, alteration in the function of these transporters plays a critical role in intra- and interindividual variability of the therapeutic efficacy and the toxicity of the drugs. As a result, the activity of drug transporters must be under tight regulation so as to carry out their normal duties. Key players involved in the regulation of transporters are hormones, protein kinases, nuclear receptors, scaffolding proteins, and disease conditions. These players may affect transporter activity at multiple levels, including (i) when and how often a gene encoding a given transporter is transcribed (transcriptional control), (ii) how the primary RNA transcript is spliced or processed (RNA processing control), (iii) which mRNA in the cytoplasm is translated by ribosomes (translational control), (iv) which mRNA is destabilized in the cytoplasm (mRNA degradation control), and (v) how a transporter is modified and assembled after it has been made (posttranslational control). Posttranslational modification may alter physical and chemical properties of the transporters, their folding, conformation, distribution, stability, and their activity. Because of such loops and layers of regulation, the functional diversity of these transporters often far exceeds the considerable molecular diversity of the transporter genes, which may help in utilizing identical transporter proteins for different cellular functions in different cell types.

Regulation of transporter activity at the gene level usually occurs within hours and days and is therefore classified as long-term or chronic regulation. Long-term regulation usually occurs when the body undergoes massive change, such as during development or the occurrence of disease. Regulation at the posttranslational level usually occurs within minutes or hours and is therefore classified as short-term or acute regulation. Short-term regulation usually occurs when the body has to deal with rapidly changing amounts of substances as a consequence of variable intake of drugs, fluids, or meals, as well as metabolic activity.

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2

Organic Cation and Zwitterion Transporters (OCTs, OCTNs)

Hermann Koepsell

Department of Molecular Plant Physiology and Biophysics of the Julius-von-Sachs-Institute, University of Würzburg, Würzburg, Germany

2.1 Introduction

The SLC22 protein family is a member of the major facilitator superfamily (MFS), which is assigned 2.A.1 in the transporter classification system of Milton Saier (see http://www.tcdb.org/). The SLC22 family contains three well-characterized subgroups. These are the organic cation transporters (OCTs) (OCT1 (SLC22A1), OCT2 (SLC22A2), OCT3 (SLC22A3)), the zwitterion/organic cation transporters (OCTNs) (OCTN1 (SLC22A4), OCTN2 (SLC22A5)), and the organic anion transporters (OATs (SLC22A6-13,20)). Most transporters of the SLC22 family are polyspecific; they interact with many structurally different compounds, which may be substrates or nontransported inhibitors. All members of the SLC22 family have the same predicted membrane topology consisting of 12 α-helical transmembrane domains (TMDs), a large extracellular loop between TMDs 1 and 2 and a large intracellular loop between TMDs 6 and 7. The current review focuses on OCT1, OCT2, OCT3, OCTN1, OCTN2, and OCT6 also called CT2 (SLC22A16) (Table 2.1). All these transporters are capable of translocating cationic drugs. OCTN1, OCTN2, and OCT6 are also able to translocate zwitterions including l-carnitine. Due to structural and functional similarities of all SLC22 family members, some considerations concerning substrate recognition and translocation obtained from studies with OCT1–3 may be also valid for OCTN1, OCTN2, OCT6, and OATs. Several reviews on OCTs and/or OCTNs have been published [1–5]. Some of these reviews provide detailed information about functional properties and physiological and pathophysiological functions of these transporters including detailed descriptions of expression sites and localizations in different species [2], substrate and inhibitor affinities [2, 3], and polymorphisms in humans [3]. The aim of the present review is to provide a comprehensible updated overview in which the current knowledge concerning functional properties, structure function relationships, physiological functions in different organs, roles in drug handling, as well as biomedical implications of OCT1–3, OCTN1, OCTN2, and OCT6 is summarized. Specific emphasis is put on aspects concerning the understanding of how these polyspecific transporters recognize and translocate structurally different drugs and how these drugs may interact at individual transporters. Quotation has been restricted to most relevant earlier findings and to new findings that have not been quoted in the previous reviews.

Table 2.1 Human cation and zwitterion transporters of the SLC22 transporter family

2.2 hOCT1 (SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3)

One subgroup of the human SLC22 transporter family comprises the organic cation transporters hOCT1 (SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3). In 1994, rat Oct1 (rOct1) was cloned and characterized [6]. rOCT1 was the first identified transporter of the SLC22 family. In 1996, rat OCT2 (rOct2) was cloned [2]. In 1998, rat Oct3 (rOct3) and human OCT3 (hOCT3) were cloned [2]. Cloning of human OCT1 (hOCT1) and human OCT2 (hOCT2) was reported in 1997 [2]. Although many differences concerning tissue distribution, substrate selectivity, and regulation between the OCT1–3 and between the individual subtypes in different species were detected, no differences concerning the basic transport mechanism became apparent. Thus, assuming that OCT1–3 operate in the same way, a basic transport function and mechanism of OCT1–3 is proposed on the basis of results that were mostly obtained from studies performed with rOct1 and rOct2.

2.2.1 Basic Functional Properties of OCT1–3

Functional properties of OCT1–3 have been investigated using different heterologous expression systems and employing proteoliposomes containing purified protein. In various epithelial cell lines, overexpressing OCT1–3 cellular uptake was measured using radioactively labeled or fluorescent substrates or measuring intracellular concentrations of unlabelled substrates by mass spectrometry. Functions of OCT1 and OCT2 were also characterized by measuring transcellular flux through polarized epithelial cells in which OCT1 or OCT2 was expressed in the basolateral membrane and the efflux transporter MATE1 or MDR1 were expressed in the luminal membrane [7–9]. In oocytes of Xenopus laevis and in proteoliposomes, uptake and efflux measurements with radioactively labeled substrates were performed [2]. Because unidirectional cation transport via OCT1–3 is electrogenic, transport could be also measured electrically employing voltage-clamp procedures [2]. The possibility to analyze transport properties of OCT1–3 by electrical measurements enabled a detailed investigation of different functions because it allowed the analysis of influx and efflux in intact oocytes at clamped membrane potentials as well as cation flux in the absence of a substrate gradient at different membrane potentials in giant membrane patches of oocytes [2, 4]. Employing these methods on rOct1 and rOct2 and some of them on rOct3 and hOCT2, the following basic functional properties of OCTs have been defined.

First, the OCTs are transporters that perform a set of conformational transitions during translocation of an individual substrate rather than channels that allow passage of many compounds in the open state. The evidence that OCT1–3 are transporters has been described in detail [4]. It is based on the observations (i) that trans-stimulation by substrates was observed under voltage-clamp conditions [4], (ii) that rOct2-mediated voltage-dependent currents in the presence of equal substrate concentrations on both sides of the plasma membrane were decreased—rather than increased as expected for a channel—when the substrate concentration on both sides of the plasma membrane was increased [4], and (iii) that the activation energies for rOct2-mediated uptake of tetramethylammonium+ and Cs+ were in the range observed for transporters [4].

Second, OCT1–3 are polyspecific transporters. They translocate cations with unrelated structures and are inhibited by many additional compounds that are not transported. Most transported cations are monovalent organic cations [1–3, 10]. However, individual divalent organic cations, noncharged compounds, monovalent inorganic cations, and divalent inorganic cations may be also transported [1, 3, 11]. Compared to substrates, a much larger number of compounds inhibit OCTs and are not transported themselves. The nontransported inhibitors include organic cations, which are often larger in size compared to the substrates. Some of them have a higher affinity compared to high-affinity substrates [3, 10]. The inhibitors include noncharged compounds such as corticosterone and organic anions [10].

Third, OCT1–3 are able to translocate organic cations in both directions across the plasma membrane dependent on the driving force that may be the concentration gradient of the substrate and/or an electrical membrane potential [4]. They are able to translocate an organic cation when no substrate is present on the other side (trans-side) of the plasma membrane (uniporter function). In this situation, cation translocation is electrogenic. When the concentration of organic cations on both sides of the plasma membrane is identical, the membrane potential is the only driving force.

Fourth, organic cation transport by OCT1–3 is not influenced by transmembrane gradients of sodium, potassium, or chloride [4, 10]. It is also not influenced by proton gradients provided the membrane potential is kept constant.

2.2.2 Structure and Proposed Transport Mechanism of OCT1–3

OCT1, OCT2, and OCT3 from different species contain 50–70% identical amino acids and reveal the same predicted membrane topology as demonstrated for rOct1 (Fig. 2.1a). They contain 12 predicted α-helical TMDs, one large extracellular loop between TMD1 and TMD2 and one larger intracellular loop between TMD6 and TMD7. The large extracellular loop is glycosylated [2]. It contains six cysteine residues, which form three disulfide bridges (shown for rOct1, T. Keller and H. Koepsell, unpublished data) that are supposed to stabilize the tertiary structure of the loop. The disulfide bridge-stabilized tertiary structure of the extracellular loop mediates homo-oligomerization [1]. Oligomerization is required for membrane targeting but does apparently not influence transport function, which is accomplished on the level of monomers [1]. The large intracellular loop contains several predicted sites for protein kinase C (PKC)-dependent phosphorylation [2]. Phosphorylation of these sites changes substrate selectivity [2]. Homology models of inward-facing and outward-facing tertiary structures of OCTs were generated [4]. The OCT model with the inward-facing substrate-binding cleft was calculated in analogy to solved crystal structures of the inward-facing conformations of the lactose permease LacY and the glycerol-3-phosphate transporter GlpT from Escherichia coli (Fig. 2.1c). The model with the outward-facing substrate-binding cleft was generated by employing a rearrangement mechanism for LacY that was proposed on the basis of biochemical data [4] (Fig. 2.1b).

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Figure 2.1 Structure models of rOct1 with amino acids that are critical for substrate affinity.

Reprinted with permission from Ref. 4

Modeling of the outward- and inward-facing conformation was performed using tertiary structures of LacY in the outward- and inward-facing conformations. Mutagenesis experiments showed that the indicated amino acids are critical for affinity and/or selectivity of the substrates TEA and MPP and suggest that extracellular and intracellular corticosterone interacts with F160, W218, R440, L447, and D475. (a) Predicted membrane topology of rOct1. (b) Modeled tertiary structure of rOct1 in the outward-facing conformation (side view). (c) Modeled tertiary structure of the inward-facing conformation (side view).

A hypothesis about how OCT1–3 may bind and translocate structurally different compounds and how transport may be inhibited has been developed. It is based on current knowledge concerning structure function relationships of other transporters, on the modeled outward-facing and inward-facing tertiary structures of OCT1–3, and on results obtained using the following six methods. First, affinities of nontransported inhibitors were determined that were added to the extracellular or intracellular side of the plasma membrane [4]. Second, extensive site-directed mutagenesis was performed with rOCT1 including detailed functional characterizations of the mutants [4]. In particular, effects of point mutations on the affinity of different ligands were determined. Third, substrate- and inhibitor-dependent structural changes that include movements of charges within the transporter were analyzed by measuring ligand-induced capacitance changes [4]. Fourth, substrate- and inhibitor-dependent movements at defined positions were determined by employing voltage-clamp fluorometry [4, 12]. Fifth, high-affinity ligand-binding sites were identified employing voltage-clamp fluorometry [13]. Sixth, stoichiometries between different transported organic cations and translocated positive charge were determined [4].

The mutagenesis data indicate that transported organic cations bind to amino acids in the innermost cavity of the outward-open binding cleft. The binding sites for different transported organic cations are overlapping so that exchange of one amino acid in this innermost cleft may change the affinity for one substrate but not for another one [4]. Binding of substrates to this innermost cleft induces structural changes during which the respective substrate is occluded within the transporter. These structural changes include bending of the 11th transmembrane α-helix that contains two juxtaposed glycine residues. The glycine residues are framed by two amino acids that are involved in substrate binding [12]. The intermediate occluded state is required to prevent slippage of small ions during translocation of organic cations; however, at zero membrane potential, small ions may be translocated together with transported organic cations [4]. Following substrate occlusion, the transporter switches to the inward-open conformation, and the substrate can be released at the intracellular side of the plasma membrane. Consistent with the tertiary structural models, mutagenesis combined with inhibition experiments with a nontransported inhibitor applied from either side of the plasma membrane indicated that some of the amino acids forming the innermost cavity of the outward-open cleft also participate in the innermost cavity of the inward-open cleft [4]. After the organic substrate has been dissociated, the empty transporter switches back to the outward-open conformation.

Transport may be inhibited by other substrates in a competitive or noncompetitive way [14] dependent on the degree of overlap between the respective substrate-binding sites. Nontransported inhibitors may also inhibit organic cation transport competitively or noncompetitively [14]. They may bind at more peripheral regions of the binding cleft. Measuring ligand-induced conformational change by voltage-clamp fluorometry, high-affinity binding sites of substrates and the nontransported inhibitor tetrabutylammonium were identified. The functional role of these high-affinity binding sites is not fully understood. Importantly, it was observed that binding of nontransported inhibitors to high-affinity inhibitor sites can partially inhibit transport of organic cation substrates [4] when the concentration of the applied substrate was far below their Michaelis–Menten Km values. Comparing IC50 values for inhibition of uptake of different substrates by hOCT1, hOCT2, or hOCT3, Nies and coworkers suggested that the affinity of inhibitors to a specific OCT subtype may be dependent on the employed substrate [3]. Recently, this hypothesis was verified in side-by-side experiments [15]. We observed that the inhibitor selectivity of hOCT2 was completely different when three different substrates were employed for uptake measurements using substrate concentrations that were far below the respective Km values. These data implicate that nontransported inhibitors can inhibit translocation after the substrate has bound to the transporter and that binding of different substrates induces short-term allosteric effects on the inhibitor-binding sites.

2.2.3 Comparison of Substrate and Inhibitor Selectivities of hOCT1–3

hOCT1, hOCT2, and hOCT3 have broadly overlapping substrate and inhibitor selectivities (Table 2.2, Table 2.3, and Table 2.4). There is also an overlap in substrate selectivity between hOCTN1, hOCTN2, and OCT6/CT2, but only few compounds are common substrates of hOCT1, hOCT2, and/or hOCT3 and hOCTN1, hOCTN2, and/or OCT6 (see acetylcholine and oxaliplatin in Table 2.3). So far, identified common substrates of hOCT1, hOCT2, and hOCT3 are the model cations tetraethylammonium (TEA), 1-methyl-4-phenylpyridimium (MPP); the neuromodulators histidyl-proline diketopiperazine (cyclo(His-Pro)) and salsolinol, and agmatine; the antidiabetic metformin; the antiviral drug lamivudine; and the cytostatic drug oxaliplatin [3] (Table 2.2 and Table 2.3). Similar affinities for the three transporters were observed for metformin and lamivudine. Other substrates exhibited different rank orders of affinities to hOCT1, hOCT2, and hOCT3. For example, the affinity rank order for cyclo(His-Pro) and agmatine was hOCT2 > hOCT3 > hOCT1. For TEA and MPP, the rank order was hOCT2 > hOCT1 > hOCT3 (Table 2.2). Some compounds are transported by two and some only by one of the three transporters. For example, epinephrine and norepinephrine are transported by hOCT2 and hOCT3 but not by hOCT1, dopamine and serotonin are only transported by hOCT2, and histamine is only transported by hOCT3.

Table 2.2 Model substrates for analysis of transport activity

Fluorescent compounds are given in italics.

Table 2.3 Substrates of hOCT1, hOCT2, and hOCT3

K m values are indicated in parentheses. If more than two different Km values have been reported, the mean value is presented and indicated by ~. Compounds that are either transported by hOCT1 plus hOCT2 plus hOCT3 or by hOCTN1 plus hOCTN2 plus OCT6 are printed in bold. Substrates of two of the six indicated transporters of the SLC22 family are printed in italics.

Table 2.4 High-affinity inhibitors of hOCT1-3

The data indicated by (a) were obtained during the thesis of Sonja Utner in the laboratory of H. Koepsell submitted to the University of Würzburg in December 2010. The title of the thesis is Interaktion von Psychopharmaka mit humanen organischen Kationentransportern. The measurements were performed using stably transfected Chinese hamster ovary cells that were grown to confluence and then dissociated by removal of Ca²+ and Mg²+.

In Table 2.4 inhibitors are compiled, which exhibit at least for one of the hOCT1–3 transporters with one substrate an IC50 value below 5 μM. The measurements were performed in transfected epithelial cell lines. Most uptake measurements were performed using confluent monolayers. Some measurements were performed with cells that had been dissociated from confluent cell layers by removal of Ca²+ and Mg²+.

The Km values for transport of different substrates by hOCT1–3 differ by a factor more than 30,000 (rhodamine uptake by hOCT1; Km = 0.54 μM, agmatine uptake by hOCT1; Km = 18 mM). The Vmax values obtained for different substrates may show less variation compared to be Km values. For example, the Km value of hOCT1-mediated metformin uptake is more than 200-fold lower compared to the Km value of hOCT1-mediated MPP uptake, whereas the Vmax value for metformin uptake is 1.4 times higher compared to MPP uptake [40].

2.2.4 Distribution of hOCT1

In humans, OCT1 is most strongly expressed in the liver where it is located in the sinusoidal membrane of the hepatocytes [3] (Fig. 2.2b). Strong expression in the liver and location in the sinusoidal membrane were also observed in rodents [2]. The expression and location of OCT1 in the kidney are different between human and rodents. Whereas in rodents Oct1 is also strongly expressed in the kidney and located in basolateral membranes of S1 and S2 segments of proximal tubules [2], only small amounts of SLC22A1 mRNA were detected in human kidney, and hOCT1 was localized to luminal membranes of the proximal and distal tubules [3] (Fig. 2.2c). Human OCT1 has been also detected in many other organs such as the small intestine, lung, heart, skeletal muscle, brain, placenta, mammary gland, adrenal gland, and eye; adipose tissue; immune cells; and in various tumors [1, 3, 45]. In the small intestine, hOCT1 is probably located at the brush-border membrane of enterocytes (Fig. 2.2a). Similarly, hOCT1 has been localized to the luminal membrane of bronchial epithelial cells (Fig. 2.2d) [1, 2]. In the brain, hOCT1 has been detected in endothelial cells of microvessels [1] (Fig. 2.2f).

c2-fig-0002c2-fig-0002c2-fig-0002c2-fig-0002c2-fig-0002c2-fig-0002c2-fig-0002c2-fig-0002

Figure 2.2 Location of human organic cation and zwitterion/organic cation transporters in enterocytes (a), hepatocytes (b), renal proximal tubule cells (c), airway epithelial cells (d), syncytiotrophoblast of the placenta (e), endothelial cells of the blood–barrier (f), brain neurons (g), and immune cells. Transporters of the SLC22 family are prepresented by filled circles, whereas transporters of other families that transport organic cations are presented by open symbols. Abbreviated names of transporters that do not belong to the SLC22 family are the following: MDR1 (ABCB1), MATE1 (SLC47A1), and MATE2-K (isoform of SLC47A2, NM_001099646). NC, noncharged compound; OC+, organic cation; ZI, zwitterion.

2.2.5 Regulation of hOCT1

Transcriptional and posttranscriptional regulation of OCT1 shows large differences between species [2]. Here, only some findings concerning regulation of hOCT1 are summarized notwithstanding that regulation must be known in much more detail to understand physiological phenomena and biomedical responses. For example, the observation that the expression of hOCT1 protein in the liver varies dramatically in patients with the same genetic background is due to transcriptional and/or posttranscriptional regulation [1, 3]. Relevant issues are regulation during development [46], tissue-specific regulation, gender-dependent regulation, regulation during diseases [47, 48], regulation in response to different metabolic states, regulation in response to drug treatment [49], and regulation during tumor development [45, 50].

Transcription of hOCT1 in the liver is activated by the hepatic nuclear transcription factor HNF4α, which interacts with the promoter of hOCT1 [1, 2]. Chenodeoxycholic acid reduces the transcription of hOCT1 in the liver by inhibiting transcriptional activation by HNF4α via a component of the bile acid-inducible transcription repressor. In chronic myeloid leukemia cell lines, hOCT1 expression is upregulated by the peroxisome proliferator-activated receptor α (PPARα) [51]. Transcription of hOCT1 in the liver is regulated by methylation of the SLC22A1 gene [50]. So far, posttranscriptional regulation of hOCT1 has been only investigated after overexpression of hOCT1. The available data suggest posttranscriptional modification of hOCT1 changes function of hOCT1 as well as the amount of hOCT1 in the plasma membrane. The mechanisms for posttranscriptional regulations involve protein kinase A (PKA)-dependent phosphorylation and a Ca²+/calmodulin-stimulated pathway [1, 2]. It has been shown that the Ca²+/calmodulin pathway, which modulates phosphorylation of hOCT1, has an influence on the affinity of TEA.

2.2.6 Physiological and Biomedical Roles of hOCT1

hOCT1 in the luminal membrane of enterocytes participates in small intestinal absorption of food compounds such as cyclo(His-Pro) and salsolinol and of cationic drugs like metformin and lamivudine (Fig. 2.2a). Being the most abundant OCT in the sinusoidal membrane of hepatocytes (Fig. 2.2b), hOCT1 plays a pivotal role for hepatic secretion of organic cations. This has been demonstrated experimentally in Oct1 knockout mice [2]. hOCT1 in the brush-border membrane of renal proximal tubules (Fig. 2.2c) participates in the reabsorption of ultrafiltrated cationic drugs from the primary urine [1, 3]. In the lung, hOCT1 participates in the absorption of drugs from the bronchi (Fig. 2.2d). It is also supposed to participate in the absorption of organic cationic drugs across the blood–brain barrier (Fig. 2.2f). In CD4 immune cells (Fig. 2.2h), hOCT1 facilitates uptake of endogenous substrates and accelerates uptake of antiviral drugs [52, 53].

2.2.7 Pathological Implications of hOCT1 and Therapeutical Aspects

Due to genetic and epigenetic factors and to posttranscriptional regulation, large interindividual differences in small intestinal absorption, tissue distribution, and hepatic excretion of cationic drugs transported by hOCT1 are expected. In the livers of healthy individuals, largely different levels of hepatic hOCT1 expression were observed [3]. Many synonymous and nonsynonymous single nucleotide polymorphisms (SNPs) have been identified that alter expression, intracellular trafficking, and/or transporter functions [3, 54]. Different ethnic groups show significant differences in frequencies of individual SNPs in the SLC22A1 gene. Six nonsynonymous SNPs have been identified that alter substrate selectivity [3]. During diseases, expression of hOCT1 may be altered. For example, expression of hOCT1 in the liver is reduced during cholestasis [3], hepatitis C virus-related cirrhosis [48], and inflammation [47]. In human hepatocellular carcinoma, the expression of OCT1 is downregulated compared to nontransformed hepatocytes [50]. This downregulation is mediated by hypermethylation of the SLC22A1 gene [50].

The pharmacokinetics of drugs that are transported by hOCT1 are influenced by the level of hOCT1 expression provided hOCT1 is critically involved in their small intestinal absorption and/or hepatic excretion. Intracellular effects of hOCT1 substrates may be influenced by the level of hOCT1 expression in the target cells. Therapeutic efficacy of hOCT1 substrates may be also influenced by comedication of cationic drugs, which inhibit transported drugs at the applied therapeutic concentrations. Toxic side effects of drugs transported by hOCT1 may be dependent on hOCT1 expression or activity in the effected cells and may be reduced by comedication of drugs that inhibit uptake of the toxic compounds.

The antiemetic drug tropisetron is taken up into hepatocytes via hOCT1 where it undergoes metabolic inactivation via cytochrome P450. In patients with loss-of-function mutations of hOCT1, the plasma concentrations and the efficacy of tropisetron are increased [18]. In hepatocytes and fat cells where the antidiabetic drug metformin is effective, hOCT1-mediated metformin transport is essential for the therapeutic effect [1, 3]. Loss-of-function mutations in hOCT1 and comedication with proton pump inhibitors and tyrosine kinase inhibitors that block hOCT1 may blunt the antidiabetic effect of metformin [1]. It has been demonstrated that expression of hOCT1 in chronic myeloid leukemia cells is correlated with the therapeutic effect of the cytostatic drug imatinib [1, 3, 51, 55]. Clear experimental evidence has been provided that imatinib is a high-affinity inhibitor of hOCT1; however, so far, no conclusive demonstration of imatinib transport via hOCT1 has been reported [1, 55, 56]. Thus, either very low hOCT1-mediated imatinib uptake into chronic myeloid leukemia cells is effective or the investigated hOCT1 mutants are linked to another transporter for imatinib such as hOCTN1 [57] or imatinib blocks the uptake of an endogenous compound that modulates cell viability [58].

2.2.8 Distribution of hOCT2

hOCT2 is most strongly expressed in the kidney but also in the small intestine, lung, placenta, thymus, brain, and inner ear [1, 2]. In the kidney, hOCT2 is located at the basolateral membrane of renal proximal tubules (Fig. 2.2c), whereas it is located at the luminal membrane of tracheal epithelial cells (Fig. 2.2d) [2]. In the human small intestine, the location of hOCT2 has not been determined. In the brain, hOCT2 has been detected in the choroid plexus, in neurons (Fig. 2.2g) [10], and in microvessels where it has been localized to the luminal membrane of the endothelial cells (Fig. 2.2f) [1].

2.2.9 Regulation of hOCT2

Similar to hOCT1, transcription of hOCT2 is regulated by genetic imprinting. For example, in the liver, transcription of hOCT2 is suppressed by methylation of the hOCT2 promoter [1]. Methylation of the promoter inhibits binding of the upstream stimulating factor (USF). In the placenta, transcription of hOCT2 is inhibited by methylation of lysine 9 of histone H3 [59]. Different mechanisms for posttranscriptional regulation of hOCT2 have been described involving regulation of hOCT2 trafficking by effects on endocytosis and exocytosis and changes of transport properties that are probably mediated by phosphorylation of hOCT2. Regulation of endocytosis may involve lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) [1], whereas the exocytotic pathway is downregulated by regulatory protein RS1 (RS1A1), which blocks the release of transporter containing vesicles from the trans-Golgi network [60, 61]. It has been shown that PKC, PKA, phosphatidylinositol-3-kinase (PI3K), and calmodulin participate in posttranscriptional regulation of hOCT2 [1, 2]; however, the individual pathways responsible for different types of posttranscriptional regulation have not been identified.

2.2.10 Physiological and Biomedical Roles of hOCT2

Knowledge concerning the physiological roles of hOCT2 is limited. hOCT2 in the basolateral membrane of renal proximal tubules is critically involved in renal excretion of incorporated toxins (Fig. 2.2c). hOCT2 in the kidney is also involved in the secretion of creatinine [23, 24]. It participates in secretion of the other endogenous compounds, which are substrates such as food components, monoamine neurotransmitters, and/or histamine (see Table 2.3). Because hOCT2 is also capable to mediate efflux of organic cations provided the driving forces are appropriate, it may be also involved in the reabsorption of compounds in the proximal tubule by mediating release across the basolateral membrane of proximal tubular cells. This may play a role for reabsorption of choline and dopamine [1]. The release of acetylcholine across the luminal membrane of tracheal epithelial cells may be an important physiological function of hOCT2 in the lung. Thus, extraneuronal cholinergic stimulation may be mediated independent of exocytosis (Fig. 2.2d). Because extracellular acetylcholine is rapidly degraded by acetylcholine esterase, the driving force for hOCT2-mediated acetylcholine release, which is defined by the fraction of intracellular/extracellular acetylcholine, may be high enough to overcome the membrane potential. The expression of hOCT2 in the blood–brain barrier (Fig. 2.2f), the choroid plexus, and the neurons (Fig. 2.2g) implicates that hOCT2 is involved in the regulation of brain interstitial concentrations of epinephrine, norepinephrine, acetylcholine, serotonin, and/or histamine.

Tissue locations and the capability of hOCT2 to transport various cationic drugs implicate important biomedical functions. Participation in the renal excretion of various drugs may be most important. Thus, hOCT2 mediates uptake of organic drugs such as the antiviral drug lamivudine, the antiallergic drug cimetidine, and the antineoplastic drugs cisplatin and oxaliplatin across the basolateral membrane. The excretion of the drugs into the urine is performed by a proton cation antiporter (MATE1 (SLC47A1), MATE2-K (SLC47A2)), by the ATPase MDR1 (ABCB1), by the carnitine/cation transporters OCTN1 (SLC22A4), or by the carnitine cation transporter OCTN2 (SLC22A5) (Fig. 2.2c). In the brain, hOCT2 may be important to facilitate transport of drugs such as the anti-Parkinson drugs amantadine and memantine across the blood–brain barrier or may transport drugs into neurons. In the lung, drugs applied as aerosols may enter tracheal epithelial cells via hOCT2 or may block cholinergic extraneuronal regulation by inhibiting the release of acetylcholine.

2.2.11 Pathological Implications of hOCT2 and Therapeutical Aspects

The hOCT2 gene contains one frequent nonsynonymous SNP and various rare nonsynonymous SNPs, some of which decrease transport activity [3]. Decreased expression of hOCT2 or impaired transporter function alters pharmacokinetics of drugs by decreasing renal drug excretion or altering drug uptake and distribution in the brain [1]. Decreased expression and function may be due to mutations in the hOCT2 gene or in proteins that are involved in the regulation of hOCT2 expression or function. Decreased expression in the kidney or brain may be observed in the course of diseases and/or due to short-term or long-term effects of drugs. Changes in the expression of hOCT2 in the brain may have an effect on the clearance of released neurotransmitters such as norepinephrine or serotonin. This may lead to changes of mood-related behavior [62].

Blood and tissue concentrations of drugs that are excreted by the kidney and transported by hOCT2 can be increased by comedication of other hOCT2 substrates or hOCT2 inhibitors slowing down renal excretion [1]. Thus, the doses for medication can be decreased and a potential nephrotoxicity can be reduced. For example, nephrotoxicity of cisplatin, which may be correlated with the expression and activity of hOCT2 in the kidney, may be reduced by coadministration of drugs that inhibit hOCT2 [1]. Because MATE1 in the brush-border membrane of renal proximal tubular cells mediates the efflux of cisplatin into the tubular lumen [25, 63], an inhibitor of hOCT2 must be selected that does not block MATE1.

2.2.12 Distribution of hOCT3

hOCT3 is expressed in many organs such as the heart, skeletal muscle, brain, small intestine, liver, lung, kidneys, urinary bladder, mammary gland, cornea, skin, and blood vessels [2, 3]. hOCT3 is also expressed in tumors [27, 45, 64]. In the brain, hOCT3 is located in epithelial cells of the choroid plexus, in neurons, and in glial cells. Similar to hOCT1 and hOCT2, the subcellular distribution of hOCT3 is organ specific. In the liver, hOCT3 is located in the sinusoidal membrane of hepatocytes; in the placenta, it is located in the basolateral membranes of the placental epithelium [1, 65]; and in the lung, it is located in the luminal membrane of bronchial epithelial cells [2, 3].

2.2.13 Regulation of hOCT3

Expression of OCT3 is regulated under various conditions, for example, in the placenta during pregnancy [66, 67]. Epigenetic regulation of hOCT3 expression by methylation of the basal promoter region has been described [68]. Posttranscriptional short-term regulation of hOCT3-mediated cation uptake by the Ca²+/calmodulin pathway has been observed that is supposed to include changes of function and trafficking [2]; however, the involved mechanisms have not been elucidated. Since the postsynaptic density 95/disk-large/ZO-1 (PDZ) domain of protein IKEPP binds to the C-terminus of hOCT3 [2], it may be speculated that IKEPP is involved in the regulation of intracellular trafficking.

2.2.14 Physiological and Biomedical Roles of hOCT3

Some interesting observations concerning the potential role of hOCT3 in the brain have been reported; however, information concerning physiological roles of hOCT3 in other organs is missing. Experimental data in mice suggest that hOCT3 contributes to the regulation of interstitial concentrations of monoamine neurotransmitters in the brain and may thereby modulate neuronal activities and behavior [1]. Speculations on additional physiological and pathophysiological functions of hOCT3 may help to design future research. For example, hOCT3-mediated uptake of food components like cyclo(His-Pro) and salsolinol and uptake of putrescine may influence cellular metabolism and thereby influence, for example, the response to antineoplastic drugs. Removal of histamine by hOCT3 from the interstitial fluid may help to terminate allergic reactions. This could be relevant in