Roles of Transporters in Pharmacokinetics

Transporters are now recognized to be as important as the metabolizing enzymes in the modulation of the main steps controlling the fate and action of xenobiotics in the body. They affect all the main pharmacokinetic events, such as the oral bioavailability, distribution, and clearance of any substrates. As most drugs, toxicants, and carcinogens are taken orally, we focus here on their impact on the enterocytes, as these are the main intestinal cells involved in the passage of compounds from the intestine to the blood. The absorbed compounds are then cleared by the liver, where the hepatocytes may produce, like the enterocytes, coordinated transporter-enzyme systems that influence the overall availability of compounds taken via the mouth before they can be distributed throughout the body. Tissue distribution depends on many factors, such as the degree to which a drug is bound to plasma proteins, the organ perfusion rate, and the rate at which the drug permeates biological membranes. Here too, transporter-mediated effects are particularly important at the so-called barriers between the blood and specific organs, like the BBB, the blood-CSF barrier at the choroid plexus, and the blood-testis, blood-prostate, and blood-placenta barriers. All these barriers protect tissues from external agents, and transporters are now considered to be the predominant actors in this defense system. They are frequently described as the gatekeepers of these physiological barriers between the exterior and interior environments. Finally, the last step concerns the elimination of parent compounds and their metabolites. The two most frequent routes for the removal of xenobiotics that do not require their metabolism are excretion in the bile or urine. Figure 7 illustrates all these steps from the absorption to the elimination of a xenobiotic, including the key organs in drug disposition and the networks of transporters at the apical or basolateral poles of their cells.

5.04.6.1 Intestinal Absorption

Drugs can be absorbed from the intestine by passive transcellular, paracellular, and carrier-mediated transport. The development of various in vitro experimental techniques in the 1980s, such as the human colon adenocarcinoma cell line Caco-2, which forms confluent monolayers of well-differentiated enterocyte-like cells, facilitated the characterization of intestinal transepithelial transport. Numerous SLC transporters translocating a wide range of natural and essential nutrients were found on both the intestinal brush border and the basolateral membrane.124 Some of them, like the amino acid (SLC7) and monocarboxylic acid transporters (MCT, SLC16), may also transport drugs and xenobiotics.125 Both influx and efflux transporters modulating drug absorption are present in the epithelium of the various segments of the intestine.126 PEPT1, OATP2B1, OATP3A1, and OATP4A1 are all on the apical membrane and mostly import substrates from the lumen to the circulation (Figure 8). PEPT1 is the best-characterized drug transporter in the small intestine of mammals, and is widely used to improve the absorption of poorly absorbed oral drugs using a prodrug strategy (see Section 5.04.4.2.4). ABC transporters, including MDR1, MRP2, and BCRP, are also found on the apical membrane, where they either limit the intestinal uptake of their substrates or contribute to the active secretion of drugs from the blood to the intestinal lumen.127 For example, the antineoplastic agent, paclitaxel, a P-gp substrate, is poorly absorbed when taken orally in humans (only 5% is bioavailable), but when it is administered with the P-gp competitor cyclosporin A, its bioavailability is increased to 50%.127 The roles of basolateral transporters are much less well known. Oct1 is present on the basolateral sides of cells, and studies using Oct1 knockout mice indicate that Oct1 is important for the secretion of OCs into the lumen of the small intestine. The intestinal distribution of human OCT1 and OCT3 is still poorly documented, whereas the functional activity of OCT1, 2, and 3 has been demonstrated using Caco-2 cells.128 The network of OCTs in the small intestine remains to be identified.

Brain

Efflux Uptake

Brain

Efflux Uptake

Body And Drug Absorption Excretion
Figure 7 Drug absorption, distribution, and elimination: the ABC and/or SLC transporter network mediate vectorial transport in each of the key body organs, with transporters at the basolateral or apical membranes of the organ-specific cells indicated in brackets.

Systemic blood

Systemic blood

Abc Slc Transporters

oatp4a1 Intestinal lumen

Figure 8 Distribution of the main drug ABC (blue) and SLC (red) transporters on the apical and basolateral membranes of intestinal enterocytes. All apical transporters (except MRP1) lie at the top of the villi.

oatp4a1 Intestinal lumen

Figure 8 Distribution of the main drug ABC (blue) and SLC (red) transporters on the apical and basolateral membranes of intestinal enterocytes. All apical transporters (except MRP1) lie at the top of the villi.

The ABC transporter MRP3 is concentrated in the basolateral membranes, where it mediates the transfer of bile acids to the blood; its role in the intestinal absorption of drugs needs further clarification. The mRNAs of MRP4 and MRP5 were recently detected in the human intestine, suggesting that they are involved in the transport of nucleosides.127 The intestinal transporters are not identically distributed along the crypt-villus axis. Many of those implicated in the absorption of drugs, like PEPT1, MDR1, BCRP, MRP2, and MRP3, are villus-specific.113 This restriction of transporters to the villus is also correlated with the presence of CYP3A in intestinal cells, suggesting coordinated phase 0 and 1 activities of MDR1 and CYP3A4 in the so-called intestinal first-pass effect.115 By contrast, MRP1 is predominantly found on the membranes of crypt cells in the small intestine, the site of enterocyte renewal, where it can protect these cells from toxins. The function and interplay of all these intestinal transporters must be very carefully considered when looking at their roles in drug absorption. A major concern is the way in which their densities vary along the gastrointestinal tract. For example, MRP3 is the most abundant ABC protein throughout the human intestine, except for the terminal ileum where MDR1 is most abundant. Similarly, the concentration of MDR1 increases from the duodenum to the colon, whereas BCRP is found throughout the small intestine and colon, and MRP2 is most prevalent in the duodenum and becomes undetectable toward the terminal ileum and colon.45,113 MCT1 has also been detected throughout the intestinal tract from the stomach to the large intestine, but it is most abundant in more proximal regions of the duodenum-ileum.129 These diverse densities of the intestinal transporters may have dramatic pharmaceutical consequences. The pharmaceutical form of an oral drug can vary from a simple solution to a solid, controlled-release complex, and this can influence the gastrointestinal site (stomach, duodenum, jejunum, ileum, or colon) at which the active compound is released. Such differences may also influence the efficacy of the carrier-mediated transporters, as these may vary from one region of the intestine to another.

This raises the question of how useful in vitro Caco-2 cells are. They are representative only of the colon, whereas the majority of drugs are absorbed more proximally. Caco-2 cells can also bear some OCTs, as indicated above, although these have not yet been identified in vivo, but they lack BCRP and CYP3A4, which are now known to be important for effluxing drugs. The great risk of saturating active transport can also affect the kinetics of drug absorption. This can occur when a large amount of drug is rapidly dissolved in the intestinal lumen and ready to be absorbed by a relatively small area of intestine. Active transport can be saturated by a relatively high concentration of substrate (Figure 4a and b), so shifting absorption toward diffusion. Here, too, the properties of the oral preparation, like its rate of dissolution, may influence the contribution of active transport to drug absorption.

In summary, the intestinal drug transporters play two major roles. First, they take part in drug influx - the absorption of drugs such as PEPT1 may be used to develop drug-delivery strategies for poorly absorbed drugs. Second, they are very important for drug efflux, either limiting the intestinal uptake of xenobiotics or mediating the secretion into the intestine of substrates circulating in the blood.

5.04.6.2 Liver and Hepatic Clearance

The liver is the most important drug-metabolizing organ in the body and acts an apparently 'homogeneous' pool of enzymatic activity. Its parallel sinusoids lined with fenestrated endothelial cells form a freely accessible extracellular space surrounded by plates of hepatocytes. The hepatocyte plasma membrane is the only barrier to the entry of drugs into the hepatocytes. Hepatocytes are polarized cells with basolateral (sinusoidal and lateral) and apical (canilicular) membranes (Figure 9). Molecules may be excreted from the hepatocytes across their canalicular membrane into the

Hepatocyte Sinusoidal Membrane Apical
Figure 9 Distribution of the main drug ABC (blue) and SLC (red) transporters on the basolateral (sinusoid) and apical (bile canaliculus) membranes of hepatocytes.

bile, or across the basolateral membrane into sinusoidal blood, from which they are subsequently removed by other organs (e.g., the kidney). Hepatic clearance is a combination of metabolic (phase I and II) and biliary clearance. As previously indicated, hepatocytes can take up drugs by diffusion or active transport (phase 0).

The basolateral membrane OATs include OAT2, OAT4, OATP1A2, -1B1, -1B3, and -2B1, the organic cation transporter OCT1 and the Na-taurocholate co-transporting polypeptide NTCP (SLC10A1).130 They are responsible for the uptake of a wide variety of drugs by the liver because of their broad, overlapping substrate specificities. Phase III, which follows phases 0, I, and II, results in the elimination of the intact drug and/or metabolite(s) via efflux transporters on the apical and basolateral membranes. The hepatobiliary transporters include several ABC proteins (MDR1, MDR3, MRP2, BSEP, and BCRP) that are the main mediators of the excretion of numerous endogenous conjugated and unconjugated bile salts and drugs via the bile. Phase III also includes the efflux of compounds from hepatocytes back into the systemic circulation via basolateral membrane efflux transporters. Some of the OATPs, OATs, and OCT1 are bidirectional and may facilitate efflux, but the main exporters are the ABC proteins, which transport a wide range of glucuronides, and sulfated and GSH conjugates. The main ones are MRP1 and MRP3, the synthesis of which is readily induced, and the cyclic nucleoside transporters MRP4 and MRP5.113'130

This huge network of hepatobiliary transporters can give rise to variations in drug disposition between individuals by modulating the uptake or the exit of drugs and their metabolites from hepatocytes. A change in hepatic uptake may have clinical consequences. It may modulate the pharmacological activity of drugs that act via the intrahepatocellular transduction pathways, it may cause hepatotoxicity, or give rise to drug-drug interactions. The concentration of the cholesterol-lowering HMGCoA inhibitors in hepatocytes must be adequate for their pharmacological activity, and most of the statins (e.g., pravastatin, simvastatin, lovastatin, cerivastatin, and pitavastatin) enter hepatocytes via OATP1B1, and to a lesser degree via OAT1B3.131 Recently identified genetic polymorphisms like the SLCO1B3 haplotype *17 are associated with reduced statin clearance by the liver and lower concentrations in hepatocytes; they thus have less effect on cholesterol synthesis.132 Large-scale clinical studies are needed to confirm the impact of OATP1B1 polymorphisms on the considerable variation between individuals to therapy with hypolipidemic agents. The clinical efficacy and adverse effects of oral antidiabetic drugs similarly vary greatly between individuals. Polymorphisms of CYP2C8/2C9, the main enzymes catalyzing the transformation of sulfonylureas, and the meglitinide-class drugs, such as repaglinide and nateglinide, have been advanced to explain these variations, but one SLCO1B1 genotype was recently shown to affect markedly the pharmacokinetics of repaglinide and its effect on blood glucose.133 The clinical efficacy of the antidiabetic drug metformin, which is not significantly metabolized, mainly depends on its hepatic uptake by OCT1 (see Section 5.04.4.2.2), which can also be affected by several SNPs.134

Transporters can also mediate hepatotoxicity. For example, the sulfate conjugate of the antidiabetic troglitazone can cause troglitazone hepatotoxicity by inhibiting OATP1B1 and OATP1B3.135 Phalloidin, the major toxin of the mushroom Amanitaphalloides, enters hepatocytes via OATP1B1 and -1B3, and cyclosporin A is reported to be the most potent competitive inhibitor of OATP1B1-mediated phalloidin transport in the liver.136 Several other drugs also inhibit the basolateral OATPs. The fibrate gemfibrozil interacts with statins by inhibiting OATP1B1. Thus drug-drug interactions do not concern only the inhibition of metabolic enzyme, but may involve the first line of hepatocyte transporters. These hepatic impacts of the basolateral transporters have their counterpart at the apical pole. The multiple ABC transporters may also be responsible for variable drug disposition. For example, giving patients receiving digoxin the P-gp inhibitor verapamil decreases the biliary clearance of digoxin by 43% and increases its plasma concentration by 44%.138 Furthermore, each of the apical ABC proteins contains several genetic polymorphisms and is very sensitive to liver diseases.139 Both the influx and efflux hepatic transporters are thus critical influences on drug efficacy and toxicity. They are also very important for selecting the appropriate in vitro system for evaluating hepatic clearance and predicting hepatic clearance in vivo. The absence of transporters from the hepatic microsomal assays widely used to assess the metabolism of xenobiotics means that both suspensions and sandwich-cultures of primary hepatocytes are presently the most relevant in vitro models for studying hepatic uptake, biliary excretion, and drug-drug interactions, because they integrate the entire hepatobiliary transporter network.130

5.04.6.3 Blood Barriers and Tissue Distribution

The tissue distribution of a drug can be affected by transporters because they lie on either the luminal or abluminal membranes of the endothelial cells of the tissue blood vessels, or on the membranes of the specific cells of the underlying organ. The transporters on the membranes of the blood vessels may be several key physiological components of the blood barriers throughout the human body if tight junctions seal adjacent cells and prevent the paracellular exchange of solutes. In contrast, solutes can freely communicate between extracellular spaces when blood vessels are fenestrated, as in the liver sinusoids, and transporters on the plasma membranes of the tissue cells (e.g., the

Luminal Blood
Figure 10 Distribution of the main drug ABC (blue) and SLC (red) transporters on the abluminal (facing brain extracellular fluid) and luminal membranes of the brain microcapillary endothelial cells constituting the BBB.

hepatocyte membranes) become the first barrier regulating the import and export of solutes. Several organs, including the brain, nose, eyes, testes, prostate, and placenta, are protected by endothelial barriers that contain extensive i r 79 140-143

networks of transporters.

Figure 10 illustrates the luminal and abluminal distributions of several transporters at the BBB. The two ABC proteins MDR1 and BCRP are most abundant on the luminal side of the endothelial cells and are most important for protecting the brain from numerous xenobiotics (see Sections 5.04.4.1.1 and 5.04.4.1.3).144 Most of the MRPs have been also identified in the brain microvessel endothelial cells, but their luminal or abluminal location remains questionable and no important functional effects on drug transport across the BBB have been documented, except in a few cases for MRP2 and MRP4 (see Section 5.04.4.1.2). Few SLCs have been characterized in the rat BBB, except for the important network of SLC transporters that allows the blood-brain exchange of amino acids and sugars. Rat Oatp1a4 is found on both the luminal and abluminal membranes of the brain capillaries.145 The human isoform OATP1A2 is also present, but its membrane location has not been determined. Both OATPs can mediate uptake or efflux transport because of their bidirectional transport characteristic (see Section 5.04.4.2.1). The members of the SLC22, OAT3, OCTN2, and URAT1, have been found in the BBB. OAT3 is abluminal and effluxes benzylpenicillin, cimetidine, PAH, and several acidic metabolite of neurotransmitters from the brain to the blood. The luminal position of URAT1 enables this vectorial transport of the OAT3 substrates. OCTN2, which is believed to be luminal, can simultaneously transport carnitine into the brain and efflux OCs from the brain to the blood.

The other BBB, the blood-CSF barrier (BCSFB), which regulates the solute exchanges between the blood and the CSF is a monolayer of epithelial cells at the choroid plexus floating in the brain ventricles. The apical and basolateral locations of ABC and SLC transporters makes this second barrier an important interface for brain homeostasis and drug disposition.147 Here, too, drug transporters on the membranes of physiological barriers or on specific membranes of the tissue cells can affect drug distribution and, consequently, the fraction of the drug available for binding to intracellular receptors or other biological targets.

5.04.6.4 Kidney and Renal Clearance

The presence of a drug in the urine is the net result of filtration, secretion, and reabsorption. Filtration occurs by passive glomerular filtration of unbound plasma solutes, whereas secretion and reabsorption are generally carrier mediated. They can occur in the proximal tubule, which has three segments (S1, S2, and S3): the loop of Henle, the distal tubule, and the collecting tubule. These specific anatomical and functional regions of the kidney must be carefully considered, just like the regions of the intestine, because region-specific distributions of transporters define their action in renal clearance. Most renal transporters lie on the apical and basolateral membranes of the proximal tubule cells, with fewer on the epithelial membrane of the other components of the nephron. The resulting vectorial transport from the peritubular capillaries to the tubule lumen, or vice versa, can produce either secretion or reabsorption. Figure 11 shows the locations of the major drug transporters in the human proximal renal tubule cells. Multiple SLC transporters at their basolateral membrane (close to peritubular capillaries) mediate drug uptake into the tubule cells. Although by nature bidirectional, the direction of the transmembrane driving gradients favors tubular uptake rather than the efflux of organic anions and cations. Organic anions enter these cells via OAT1, OAT2, and OAT3 - and probably via OATP1A2 and OATP4C1, which was recently identified, and transports digoxin and

Basolateral membrane

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