Figure 6 Topological models of the human SLC22; (a) organic cation transporters (OCT), organic anion transporters (OAT), and SLCO; (b) the organic anion polypeptide transporter (OATP). Features common to all members of the OCT, OAT, OATP transporter family include 12 transmembrane-spanning domains (TMDs) with intracellular amino and carboxyl termini. Transmembrane a-helical domains (TMD) indicated by red rectangular bars; N-glycosylation sites (indicated by branches ) are present on extracellular protein loops. Cytoplasmic (in) and extracellular (out) orientations are indicated.

more than 30-fold lower than in wild-type mice. The metformin-induced blood lactate concentration is also decreased in the knockout mice, suggesting that Octl is involved in the hepatotoxicity of biguanides.90

The OCTNs differ markedly in their mode of action, unlike the OCTs, which have a common energy-supply mechanism (Table 3). OCTN1 supports electroneutral OC/H + exchange, OCTN2 supports both Na + -dependent co-transport (e.g., carnitine) and electrogenic-facilitated diffusion (e.g., TEA and type 1 OCs), and OCTN3 mediates the electrogenic transport of carnitine. OCTN3 and CT2 are present only in the testes of mice and humans,91 where transported carnitine improves sperm quality and fertility. OCTN1 and OCTN2 were originally cloned from human fetal liver and placenta.92'93 OCTN1 is most abundant in the kidney (at the apical membrane of the tubular cells), small intestine, bone marrow, and fetal liver (but not in the adult liver).92 OCTN2 is mainly found in the heart, placenta, skeletal muscle, kidney, and pancreas.93 Both OCTN1 and OCTN2 have a low affinity for MPP +, cimetidine, and TEA, and OCTN2 plays a major role in carnitine homeostasis.85,93 OAT (SLC22) transporters

As their name implies, small organic anions (300-500 Da) possess a net negative charge at physiological pH and their transepithelial transport into the negatively charged environment of the cell requires energy. This is largely to the OATs (SLC22 family) that are found mainly in cells playing a critical role in the excretion and detoxification of xenobiotics. The OAT family contains six members (OAT1, OAT2, OAT3, OAT4, OAT5, and URAT1), present mainly in the liver, kidney, placenta, brain capillaries, and choroid plexus (Table 4). Their topological structures are very similar to those of the OCTs (Figure 6), but they have more complex energy requirements.94 The members of the OAT1, OAT2, and OAT3 group form a tertiary active system, with the first driving element being a Na + gradient furnished by the Na +,

K+-ATPase pump. The inward movement of Na+ drives the uptake of dicarboxylate a-ketoglutarate (a-KG) by a second transport protein, the Na+ /dicarboxylate co-transporter 3 (NaDC3). This, in conjunction with mitochondrial a-KG production, maintains an outward a-KG gradient.95 Finally, the third transporter in the chain is one of the three OATs; it functions like an a-KG/organic anion exchanger for translocation of organic anion substrates into the cell. This differs slightly from the mechanism depicted in Figure 3. As a result, a negatively charged molecule can enter the cell against its chemical concentration gradient and the negative electrical potential of the cell. While these three OATs are present at the basolateral membrane and function mainly as influx systems, OAT4 and URAT1 are found at the cell apex and may function as either Na+ -independent organic anion exchangers or membrane potential-facilitators allowing the bidirectional transport of organic anions.96

The first OAT, rat and mouse Oat1, was cloned by several groups in 1997, and its orthologs were subsequently isolated from the kidneys of rabbits, humans, and pigs.97 OAT1 is mainly a kidney-specific transporter, found at the basolateral membrane of the renal proximal tubular cells. OAT1 was earlier known as the ^-aminohippurate (PAH) transporter. As PAH is almost completely extracted during a single pass through the kidney, it has been used as the prototypical substrate for the renal organic anion transport system and as a biological marker of renal plasma flow, which is equal to the renal clearance of PAH.98

OAT2 was originally isolated from the rat liver as a novel liver-specific transporter (NLT) labeling the sinusoidal membrane of rat hepatocytes; it was later found in the kidney and choroid plexus.95 The concentration of Oat2 is higher in the kidney than in the liver of female rats, but the concentrations are reversed in male rats. OAT3 gene expression has been detected in mammalian kidneys and in the apical membrane of the choroid plexus and basolateral membrane of the endothelial cells at the BBB from rodents. No orthologs of human OAT4 have been identified. OAT4 is present in the kidney, liver, and placenta.96 Oat5 is the first rodent Oat to be detected in the kidney, but not in the choroid plexus, in marked contrast to Oat1, Oat2, and Oat3. URAT1 is expressed in the luminal membrane of the kidney proximal tubules, and was originally identified in the mouse as a renal specific transporter (RST). URAT1 is involved in renal reabsorbtion of urate and helps to maintain blood levels of uric acid. It is the in vivo target for the uricosuric acid drugs probenicid and benzbromarone and the antiuricosuric acid drug pyrazinamide.96

The OAT proteins play a critical role in the excretion and detoxification of a wide variety of drugs, toxins, hormones, and neurotransmitter metabolites. Uremic toxins, which accumulate in the blood during chronic renal failure, inhibit renal transport, hepatic drug metabolism and serum protein binding. Several uremic toxins (indoxyl sulfate, indoleacetic acid, hippuric acid, etc.) are substrates or inhibitors of both rat and human OAT1 and OAT3; this could explain changes in the pharmacokinetics of other OAT substrates, including drugs.96 Several acidic metabolites of neurotransmitters are excreted into the CSF via OAT transporters, which is consistent with the presence of OATs in the choroid plexus. A number of common nonsteroidal anti-inflammatory drugs (NSAIDs), including acetyl salicylate and salicylate, acetaminophen, diclofenac, ibuprofen, ketoprofen, indomethacin, and naproxen, are substrates of one or more OAT isoforms, so that there can be significant interactions between NSAIDs and other drugs. The b-lactam antibiotics (penicillins, cephalosporins, and penems) and the antiviral nucleosides adefovir, cidofovir, aciclovir, and AZT are also substrates of one or more OAT isoforms and are actively excreted in the urine.98 Toxins like chlorinated phenoxyacetic acid herbicides, mercuric conjugates, cadmium, and ochratoxin A are also transported either into renal tubular or hepatocyte cells via the OAT network, and this predisposes these tissues to nephrotoxicity or hepatotoxicity.95 PEPT1 (SLC15A1) and PEPT2 (SLC15A2) transporters

Peptide transporters 1 and 2 (PEPT1 and PEPT2, SLC15A1 and SLC15A2, respectively) are H + -coupled oligopeptide symporters (Figure 3) whose predicted membrane topology anticipates the 12 TMDs of the SLC family (Figure 6). More recently, two new type 1 (PHT1, SLC15A4) and type 2 (PHT2, SLC15A3) peptide/histidine transporters have been identified in mammals. PEPT1 and PEPT2 translocate dipeptides and tripeptides produced by protein catabolism. Considering all possible combinations, they can transport 400 dipeptides and 8000 tripeptides derived from the 20 L-a amino acids present in proteins.99 Their pharmacological importance results from their ability to transport a wide variety of peptide-mimetic drugs, such as b-lactam antibiotics of the cephalosporin and penicillin classes and drugs like captopril, enalapril, and fosinopril. Other drugs include the dopamine D2 receptor antagonist sulpiride and the peptidase inhibitor bestatin.99 PHT1 and PHT2 transport histidine and certain di- and tripeptides, but their location on the cell or lysosomal membranes remains as questionable as their implication in pharmacotherapy.100 PEPT1 and PEPT2 differ in their transport properties and tissue distributions (Table 5). PEPT1 is the low-affinity (millimolar range), high-capacity transporter that is mainly found in the apical membranes of enterocytes in the small intestine, in renal proximal tubular cells of the S1 segment, and in bile duct epithelial cells. In contrast, PEPT2 is a high-affinity (micromolar range), low-capacity transporter that is more widely distributed in the apical membranes of kidney tubular cells of the S2 and S3 segments, brain astrocytes, and epithelial cells of the choroid plexus. They are involved in the uptake of their substrates, leaving a basal transporter(s) to account for the exit. This basal transporter could be PHT1 and/or PHT2, or the amino acid transporters of the SLC1 and SLC7 families. Both PEPT1 and PEPT2 can mediate the renal reabsorption of the filtered compounds in kidney tubules, whereas PEPT2 may be responsible for the removal of brain-derived peptide substrates from the CSF via the choroid plexus.101 Recent studies on ppt2-deficient mice (which are viable and have no kidney or brain abnormalities) have confirmed that Pept2 is involved in the uptake role of peptides into the cells of the choroid plexus and the proximal tubule.102 The pharmaceutical relevance of these peptide transporters is closely linked to the design of drug delivery strategies mediated by the intestinal PEPT1. One successful approach has been to produce peptide derivatives of parent compounds as substrates for PEPT1. The pharmacophoric pattern for the transporter includes the rules that the peptide bond is not a prerequisite for a substrate and that 5'amino acid esterification, mostly using L-valine or L-alanine, markedly improves recognition by PEPT1.103 This prodrug strategy was used to improve the bioavaibility of oral enalapril from 3-12% to 60-70% for the ester enalaprilat, which resembles the structure of the tripeptide Phe-Ala-Pro. The oral bioavaibility of the nucleoside antiviral aciclovir (22%) was similarly improved by adding a valine residue to give valaciclovir (70%).103 Current studies on the regulation of PEPT1 and PEPT2 synthesis in inflammatory intestinal diseases may provide helpful information on the variations in bioavaibility of oral PEPT1 drug substrates.99 CNT (SLC28) and ENT (SLC29) transporters

The members of the human SLC28 and 29 families catalyze mainly the transport of purine and pyrimidine nucleosides.104 The hydrophilic nucleosides, like the purine adenosine, are important signaling molecules that control both neurotransmission and cardiovascular activity. They are also precursors of nucleotides, the constitutive elements of DNA and RNA, and are the basic elements of a variety of antineoplastic and antiviral drugs. The SLC28 proteins in the apical membranes of polarized cells work in tandem with the SLC29 proteins found in the basolateral membrane. Thus, the members of these two families are critical for the transepithelial transport of nucleosides - they form a coordinated transport system.

The SLC28 family consists of three dependent concentrative nucleoside transporters, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively). They co-transport Na + and the substrate in a symporter mode and are considered to be a high-affinity, low-capacity transporter.105 The members of the SLC28 family have 13 TMDs, unlike the 12 TMDs of most of the SLC, with the Na+ and substrate recognition sites in the carboxyl half of the protein. The three subtypes differ in their substrate specificities. CNT1 transports naturally occurring pyrimidine nucleosides plus the purine adenosine. Several antiviral analogues, like AZT, lamivudine (3TC), and ddc, are substrates of CNT1. The cytotoxic cytidine analogues, cytarabine (AraC) and gemcitabine (dFdc) are also transported by CNT1.106 CNT1 is primarily found at the apical membrane in epithelial tissues including the small intestine, kidney, and liver (Table 6).

Human CNT2 is widely distributed in the kidney, liver, heart, brain, intestine, skeletal muscle, pancreas, and placenta. CNT2 transports purine nucleosides and uridine. Pharmaceutical substrates include the antiviral didanosine (ddI) and ribavirin.107

Human CNT3 has, like CNT2, a wide tissue distribution with high concentrations in the pancreas, bone marrow, and mammary gland. CNT3 is broadly selective and transports both purine and pyrimidine nucleosides in a 2:1 Na+ /nucleoside coupling ratio, in contrast to the 1:1 ratio employed by CNT1 and CNT2. CNT3 transports several anticancer nucleoside analogues, including cladrabine, dFdc, fludarabine, and zebularine.108

A number of coding region SNPs have been reported for human CNT1 and CNT2,108a and the determination of their function is an important goal for future investigations.105

The human SLC 29 family contains four members. These equilibrative nucleoside transporters (ENTs) include the well-characterized, low-affinity facilitators ENT1 (SLC29A1) and ENT2 (SLC29A2), but the cation-dependence, and the tissue and subcellular distribution of ENT3 have not yet been fully evaluated. The ENT proteins have 11 TMDs, in which the amino-terminus is in the cytoplasm and the carboxyl-terminus is extracellular. ENT1 and ENT2 can both transport adenosine, but differ in their abilities to transport other nucleosides and nucleobases.109 ENT1 is almost ubiquitously distributed in human and rodent tissues and transports purine and pyrimidine nucleosides with Km values of 50 mM (adenoside) to 680 mM (cytidine). The antiviral drugs ddC and ddI are also poorly transported.110 ENT2 is present in a wide range of tissues, including the brain, heart, pancreas, prostate, and kidney, and is particularly abundant in skeletal muscle. ENT2 differs from ENT1 in that it can also transport nucleobases like hypoxanthine and AZT.111 ENTs also mediate the uptake and efflux of several nucleoside drugs because of their bidirectional transport property. Selective inhibition of ENTs may be a strategy for improving therapy with nucleoside drugs. For example, the vasodilator draflazine increases and prolongs the cardiovascular effects of adenosine by inhibiting nucleoside uptake into endothelial cells. Dipyridamole, dilazep, and imatinib all selectively inhibit ENTs and modulate the toxic or therapeutic effects of the endogenous nucleosides.104

5.04.5 Expression and Function Properties of Transporters in Tissues

About 40 transporters belonging to the ABC and SLC superfamilies are presently known to influence the pharmacokinetics, pharmacodynamics, and toxicity of drugs and xenobiotics. While there are still many gaps in our knowledge of the function of transporters, it is possible to outline their main properties. Transporters at the Plasma Membrane and Within Cells

There are about 200 different types of cells in human tissues, and all their plasma membranes and the membranes of their organelles contain transporters. As outlined above, they can import or export endogenous and exogenous compounds, thereby regulating intracellular ions and essential nutrients and controlling the traffic of exogenous compounds into, from, and within cells. They are thus the fundamental effectors of physiological and pharmacological processes. The drug transporters at the organelles may well become most important in future studies. This was recently documented during a dramatic phase II trial in which the nucleoside antiviral fialuridine (FIAU) caused the death of subjects as a result of severe toxicity, including hepatotoxicity, pancreatis, neuropathy, or myopathy.112 These toxic events were clearly linked to mitochondrial damage due to the inactivation of the mitochodrial DNA polymerase g by FIAU or its triphosphorylated metabolite. Before this can happen, FIAU must be transported into the cytosol, and then it, or its phosphorylated metabolite, must be transported into the mitochondria. Molecular and imaging techniques were used to show that FIAU is transported by ENT1, which is located in both the plasma and mitochondria membranes, but not in the nuclear envelope, endosomes, lysosomes or Golgi complex. But the mitochondrial membranes of rodents do not contain Ent1, and they are not affected by this toxic action. This variation in FIAU toxicity and ENT1 expression between species dramatically highlights the inability of preclinical studies on rodents to predict the toxicity of nucleoside analogs for human mitochondria. It also shows how the toxicity of antiviral drugs like AZT, stavudine, and ddI for mitochondria is linked to the role of a transporter.112 Tissue Specificity

The distribution of certain transporters may be restricted to a limited range of cells or tissues. Thus, MRP2, and MRP3, OCT1, and OATPs are mainly found in the liver, while OAT1 is restricted to the kidney. In contrast others, like MRP1, the CNTs, and ENTs, are ubiquitous, being present throughout the human body. This indicates that the transporters with a limited distribution are more specialized. For example, MRP2 and MRP3 are concerned with the export of conjugated metabolites from hepatocytes, while the ubiquitous transporters are implicated in essential physiological functions such as the export of LTC4 and GSH (MRP1) and uptake of nucleosides (CNTs and ENTs), and their influence on pharmacokinetics may be less organ-specific.35,104 Polarized Cellular Distribution

The cellular location of transporters at the plasma membrane is a critical issue because most of the cells involved in the A, D, and E pharmacokinetic processes are polarized. Hence their apical (luminal) and basolateral (abluminal) membranes do not have the same populations of transporters. The same transporter is found at both the apical and basolateral membranes in only a few cases. Good examples are the glucose transporter GLUT1 (SLC2) and the monocarboxylic transporter MCT1 (SLC7), and they ensure the efficient transepithelial transport of their substrates.8 However, most of the ABC and SLC transporters are found at either the apical or the basolateral epithelial membranes, and their location helps define the direction of substrate transport and the resulting pharmacokinetic event. Efflux transporters are usually found in the apical membrane of epithelial cells, where their main function is to reduce the uptake of substrates. Most of the ABC efflux transporters (P-gp, BCRP, MRP2, and MRP4) lie in the apical membrane, although MRP1, MRP3, and MRP5 are more usually found in basolateral membranes.55 In contrast, influx transporters are more equally distributed on both apical and basolateral membranes. Although the great majority of transporters are specific to the apical or basolateral sides, there are exceptions. One is the rat Oatp1a1, which is found on the basolateral membrane of hepatocytes, where it takes up substrates from the blood, and on the apical membrane of renal tubular cells, where it reabsorbs substrates from the urine.113 Another is MRP4, which is localized in the basolateral membrane in prostatic glandular cells and in apical membrane in rat kidney tubule cells.114 The basolateral segment of the epithelial cells of the small intestine and the kidney renal tubules face the blood, while the apical sides are in contact with the gut lumen or urine. However, the blood (luminal) side of the BBB is in contact with the apical side of the brain endothelial cells, while their basolateral sides face the brain. Thus, each tissue must be carefully examined to determine the apical/basolateral distributions of transporters and the correlation between this and the cell's environment (blood, CSF, extracellular fluid, urine, etc.). The mode of transport, such as bidirectional facilitated diffusion or one-directional active transport, must also be considered before assigning a direction to the transport of the substrate. Coordinated and Vectorial Transports

The net pharmacokinetic effect of active transport processes is frequently due to the involvement of several transporters that may not always belong to the same superfamily or subfamily. For example, the OATPs (SLCO) on the sinusoidal (basolateral) membrane of hepatocytes take up organic anions, while the MRP2 (ABC) on the apical bile canicular membrane excrete them. The combined activities of these two transporters thus results in the vectorial transport of drugs from the blood to the bile.113 Similarly, the basolateral transporters OCT1, OCT2, and OCT3 of the kidney tubular cells act in a coordinated, vectorial manner with the apical transporters OCTN1 and OCTN2 to secrete OCs from the blood to the urine, whereas the basolateral OAT1, OAT2, and OAT3 are coupled to the apical OAT4, and URAT1 for transporting organic anions in the same tissue.85'95 Unfortunately, the apical or basolateral partners of several coordinated systems have not yet been identified. Two examples are the apical peptide transporters PEPT1 and PEPT2; their basolateral partners have not yet been found.99 Coordination between Transporters and Enzymes

Drug metabolism was considered to be one of the main processes of removing xenobiotics prior to the emergence of transporters. The CYP isoenzymes catalyze the first step of biotransformation and this function was called phase I metabolism. The subsequent conjugation step was called phase II metabolism. We now know that these two phases occur in specialized cells like the hepatocytes and enterocytes, and that they are preceded and followed by two other phases controlled by transporters. Efflux or influx transporters reduce or increase the uptake of substrates, and these actions help regulate the amounts of xenobiotics reaching the enzyme binding sites or the rate at which the metabolites produced are eliminated. The first step has been called 'phase O' and the second 'phase III,' indicating a close relationship between transporters and enzymes. They provide the cell with a suite of events and operate both in parallel and in series. This integrated biological function of combined transport and metabolic processes is strongly supported by the presence of common regulation pathways that act via similar nuclear receptors, such as PXR, RXR, and others, to induce or repress the genes encoding enzymes and transporters.45,115 Polyspecific Transport and Inhibition

The substrate specificities of transporters are often very broad, as indicated by the many overlaps of substrates and inhibitors, much like the specificity of the drug metabolism enzymes. Thus, probenecid was initially known to produce many drug interactions by blocking the secretion by the kidney of many drugs, including the penicillins and the antiviral Tamiflu.116 Probenicid is today known to be a polyspecific inhibitor of several MRPs, OATs, OATPs, and even OCTs.117 Similarly, MRP1, MRP2, and MRP3 have broad, overlapping substrate specificities, while OCT1, OCT2, and OCT3 transport a wide range of similar OCs. P-gp interacts with a multitude of xenobiotics, many of which are metabolized by CYP3A4/5, and some of them are also substrates of MRP1 and BCRP.21 The tyrosine kinase inhibitor imatinib is effluxed by both P-gp and BCRP, and imported by OCT1.118 Thus all ionized chemicals, peptides, and nucleosides that cannot diffuse freely across membranes are very likely to interact with one or more transporters. A review of the entry and export of 14 antiretroviral nucleoside agents into and from cells found that 14 distinct transporters of the ABC and SLC superfamilies were involved in translocating these drugs, illustrating this unique property of polyspecificity.104 Variations in Transport Kinetics

As each transporter has a limited capacity, it can be saturated by substrate concentrations greater than its Km. The Km of transporters can vary from nanomolar to millimolar values and the risk of saturating transport will depend on the amount of substrate in the transporter environment. It was mentioned above that the efflux of P-gp at the BBB can be saturated with a taxane derivative, leading to its nonlinear penetration into the brain parenchyma.119

Transport can also be inhibited in a competitive or noncompetitive manner, in the same way as the drug-metabolizing enzymes, so that transporters can promote drug-drug interactions that were initially thought to be due to the drug-metabolizing enzymes alone. The calcium channel blocker mibefradil, which was first believed to be only a CYP3A4 inhibitor, is now known to inhibit P-gp.120 Similarly, the effect of grapefruit juice on the disposition of fexofenadine, which was attributed to the inhibition of CYP3A4, is now known to result from its ability to inhibit the OATP-mediated uptake of fexofenadine.121 In vitro transporter assays are increasingly being used to assess the potential risks of drug-drug interactions mediated by transporters. The in vitro inhibition constant (Ki) can be measured and used to predict changes in the clearance or systemic exposure by measuring the area under the curve (AUC).122 Transport kinetics may also depend on the amount of transporter, which will depend on the actions of drugs, nutrients, and disease states on the nuclear receptor pathways mentioned above. The most recent area of variation concerns the presence of genetic polymorphisms; these have been described for MDR1, BCRP, and nucleoside transporters. However, studies on the pharmacogenetics of most drug transporters have only recently begun. SNPs have been identified in drug-uptake transporters such as OCT1 and OATP1B1, and those in OATP1B1 may have an impact on the therapeutic efficacy and toxicity of HMG-CoA reductase inhibitors such as pravastatin.123

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