Intestinal drug transporters: An overview

ADR-12405; No of Pages 17 Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

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Intestinal drug transporters: An overview☆ Margarida Estudante a, b, José G. Morais a, Graça Soveral c, d, Leslie Z. Benet b,⁎ a

Pharmacological Sciences Unit, iMed.UL, University of Lisbon, Faculty of Pharmacy, Portugal Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, USA c REQUIMTE, Dep. Química, FCT-UNL, 2829-516 Caparica, Portugal d Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal b

a r t i c l e

i n f o

Article history: Received 24 May 2012 Accepted 24 September 2012 Available online xxxx Keywords: Intestinal drug transport Transporter–enzyme interplay P-gp MRP BCRP PEPT OCT OCTN OATP MCT BDDCS

a b s t r a c t The importance of drug transporters as one of the determinants of pharmacokinetics has become increasingly evident [1]. While much research has been conducted focusing the role of drug transporters in the liver [2–5] and kidney [2,6,7] less is known about the importance of uptake and efflux transporters identified in the intestine [8]. Over the past years the effects of intestinal transporters have been studied using in vivo models, in situ organ perfusions, in vitro tissue preparations and cell lines. This review aims to describe up to date findings regarding the importance of intestinal transporters on drug absorption and bioavailability, highlighting areas in need of further research. Wu and Benet [9] proposed a Biopharmaceutics Drug Disposition Classification System (BDDCS) that allows the prediction of transporter effects on the drug disposition of orally administered drugs. This review also discusses BDDCS predictions with respect to the role of intestinal transporters and intestinal transporter-metabolizing enzyme interplay on oral drug pharmacokinetics. © 2012 Published by Elsevier B.V.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intestinal drug transporters . . . . . . . . . . . . . . . . . . . . . . . 2.1. ATP binding cassette transporters (ABC) . . . . . . . . . . . . . . 2.1.1. MDR1 drug-transporting P-glycoprotein (P-gp; ABCB1) . . . 2.1.2. Multidrug resistance-associated protein family (MRP; ABCC) 2.1.3. Breast cancer resistance protein (BCRP; ABCG2) . . . . . . 2.2. Solute carrier transporters (SLC; SLCO) . . . . . . . . . . . . . . . 2.2.1. Oligopeptide transporters (PEPT; SLC15A) . . . . . . . . . 2.2.2. Organic anion transporters (OAT; SLC22A) . . . . . . . . . 2.2.3. Organic cation transporter (OCT, OCTN; SLC22A) . . . . . . 2.2.4. Plasma membrane monoamine transporter (PMAT; SLC29) . 2.2.5. Organic anion transporter polypeptide (OATP; SLCO) . . . . 2.2.6. Monocarboxylate transporters (MCT1; SLC16A) . . . . . . Synergistic action of CYP3A4 and P-glycoprotein . . . . . . . . . . . . . BDDCS prediction of transporter effects . . . . . . . . . . . . . . . . . . 4.1. Class 1 compounds . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Class 2 compounds . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Class 3 and Class 4 compounds . . . . . . . . . . . . . . . . . . Intestinal permeability assessment . . . . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Editor's Choice 2013”. ⁎ Corresponding author at: University of California San Francisco, 533 Parnassus Avenue, Room U-68, San Francisco, CA 94143-0912, USA. E-mail address: [email protected] (L.Z. Benet). 0169-409X/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.addr.2012.09.042

Please cite this article as: M. Estudante, et al., Intestinal drug transporters: An overview, Adv. Drug Deliv. Rev. (2012), http://dx.doi.org/ 10.1016/j.addr.2012.09.042

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6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Membrane transporters can be major determinants of the pharmacokinetics, safety and efficacy profiles of drugs [1]. Transporters are the gatekeepers for cells and organelles, controlling uptake and efflux of crucial compounds such as sugars, amino acids, nucleotides, inorganic ions and drugs [10]. Specific membrane transporters are expressed in the luminal and/or basolateral membranes of enterocytes, hepatocytes, renal tubular epithelial cells and other important barrier tissues, including the blood–brain barrier, blood–testis barrier and the placental barrier [11]. Transporter expression in the intestine and/or liver, the two major sites affecting how much of a drug will get into the systemic circulation after an oral dose, suggests that factors affecting their function will be important determinants of oral drug pharmacokinetics. Regulatory elements controlling protein levels, genetic polymorphisms leading to increased or reduced function and coadministration with inhibitors are all important avenues by which a transporter's ability to transport substrates is altered [12]. While much research has been conducted focusing on the role of drug transporters in the liver [2–5] and kidney [2,6,7] less emphasis has been placed on the importance of uptake and efflux transporters identified in the intestine [8]. In this review we will focus on the role of uptake and efflux transporters identified in the apical and basolateral membranes of the enterocytes with respect to substrate and inhibitor interactions. Detailed characterization, pharmacogenetics and mechanisms of transport can be found in several reviews [12–23]. 2. Intestinal drug transporters Orally administered drugs must pass through the gut wall mucosa before reaching the capillaries that lead to the portal vein. This mucosal barrier consists of polarized enterocytes that are closely linked

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together by means of tight junctions. In these cells several drug transporters have been identified, but only a few are known to be involved in intestinal drug absorption (Fig. 1) (Tables 1, 2). Thus, intestinal drug transporters are of increasing interest as evidence emerges about the importance of transporters in affecting pharmacokinetics [16]. In Tables 1 and 2 we catalog the substrates, inhibitors, inducers and potential for polymorphic changes that have been identified in the literature for the various efflux (Table 1) and uptake (Table 2) transporters. With such an extensive listing we are not suggesting that each of the effects/interactions/pharmacogenetics is clinically relevant. Rather the tables provide readers with information indicating which of the hundreds of drugs and interactions are available in the literature. Furthermore, it is well recognized that the specificity of particular substrates for individual transporters is poor (much less than has been found for enzymes). Therefore, Tables 1 and 2 should not be presumed to infer substrate specificity. Drug molecules may pass through membranes via passive diffusion and/or via transporter processes. Membrane transport can be divided into facilitated and active mechanisms. Facilitated transport is performed through transporters that allow the passage of solutes (e.g., glucose, amino acids, urea) across membranes down their electrochemical gradients and without energy expense. Active transport, in contrast, utilizes diverse energy-coupling mechanisms and is performed by active transport systems that create ion/solute gradients across membranes. These latter mechanisms are classified as primary or secondary active transporters if the energy used results from ATP hydrolysis or from the dissipation of the ion gradients previously generated by active transporters such as ion pumps. According to the guidelines of the HUGO Gene Nomenclature Committee, membrane transporters have been classified into the solute carrier (SLC) and the ATP-binding cassette (ABC) transporter superfamilies [10]. More than 400 membrane transporters in the two major superfamilies have been annotated in the human genome so far [1]. In general, the SLC family transporters consist of proteins with a molecular mass in the range of 40–90 kDa containing 300–800 amino acid residues, while the ABC family transporters have masses of 140–180 kDa and contain 1200–1500 residues [11]. Transporter isoforms are denoted as rodent (lowercase) and/or human (uppercase). 2.1. ATP binding cassette transporters (ABC)

Fig. 1. Diagram of major drug transporters proteins expressed at the intestinal epithelia including intestinal uptake (yellow) and efflux (light blue) transporters. Multidrug resistance protein (MDR1, P-glycoprotein), multidrug resistance associated protein (MRP), breast cancer resistance protein (BCRP), monocarboxylate transporter protein (MCT), peptide transporter protein (PEPT), organic anion transporting polypeptide (OATP), organic cation transporter (OCT), carnitine/organic cation transporter (OCTN), and plasma membrane monoamine transporter (PMAT). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The most investigated transporters at the intestine are the ABC family of efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP 2) and breast cancer resistance protein (BCRP). These transporters are highly abundant at the apical (luminal) membrane of enterocytes thereby limiting the intestinal absorption of many clinically important and frequently prescribed drugs such as statins, antibiotics, HIV protease inhibitors, immunosuppressants, anticancer and cardiac drugs, which were shown to be substrates of such efflux carriers. The expression levels for these transporters at the human intestine were recently quantified [17]. Members of this superfamily use ATP as an energy source, allowing them to pump substrates against a concentration gradient. Drugs can be simultaneously substrates and/or inhibitors of more than one efflux transporter, suggesting that ABC transporters exert a combined role in detoxification at the intestine. Moreover, the simultaneous down-regulation [18] and induction [19] of these transporters can occur following administration of a single compound. Down-regulation or inhibition of ABC efflux transporters at the

Please cite this article as: M. Estudante, et al., Intestinal drug transporters: An overview, Adv. Drug Deliv. Rev. (2012), http://dx.doi.org/ 10.1016/j.addr.2012.09.042

M. Estudante et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

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Table 1 Drug efflux transporters identified in the intestine. Drug Gene transporter

Intestinal Substrate specificity localization

Substrates

Inhibitors

Inducers

MDR1/ P-gp

ABCB1

Apical

a

Verapamil, immunosuppressive agents, SDZ PSC 833, LY335979, GF120918 (GG918), grapefruit juice, cyclodextrin, PEG 400, Tween 80 and Cremophor EL

St. John's wort, Yes rifampicin

BCRP/MXR

ABCG2 Apical

b

MRP1

ABCC1

Basal

Estrone, 17-β-estradiol, GG918, flavonoids, herb Efavirenz extracts, gefitinib, imatinib, tamoxifen, novobiocin, nelfinavir, ritonavir, dipyridamole, fumitremorgin C (FTC), Ko143, cyclosporine MK571, LTC4, sulfinpyrazone, benzbromarone, probenecid

MRP2

ABCC1

Apical

Broad subtrate specificity; preference for hydrophobic, amphipathic or cationic molecules Broad substrate specificity Acids and drug conjugates Hydrophobic drugs, conjugates to glutathione, glucuronic acid or sulfate Glutathione, glucuronide, sulfate and heavy metals conjugates Unconjugated organic anions

Vinca alkaloids, anthracyclines, etoposide, teniposide, mitoxantrone, methotrexate c

LTC4, MK571, phenolphthalein glucuronide, fluorescein methotrexate, probenecid, furosemide, indomethacin, grapefruit juice

Polymorphisms

Yes

Yes Rifampin, 1,25(OH)2D3, spironalactone

a Steroid hormones, bile salts, glycocholate, tauroursodeoxycholate, doxorubicin, daunorubicin, reserpine, vincristine, vinblastine, valinomycin, cyclosporine, tacrolimus, tandutinib, aldosterone, hydrocortisone, dibucaine, talinolol, digoxin, TPP, ivermectin, paclitaxel, grepafloxacin, indinavir, nelfinavir, saquinavir, grepafloxacin, colchicine, darunavir, Rhei Rhizoma extract, flavonoids, glyburide, imatinib, methotrexate, mitoxantrone, prazosin, temocapril and SN-38. b Topotecan, irinotecan and its active analog SN-38, mitoxantrone, doxorubicin, daunorubicin, imatinib, gefitinib, tandutinib, statins, prazosin, glyburide, dipyridamole, quercetin, temocapril, sulfate conjugates, porphyrins, nitrofurantoin, fluoroquinolones, zidovudine, lamivudine, efavirenz, ciprofloxacin, rifampicin, sulfasalazine, quercetin, flavonoids, phytoestrogens, porphyrins, estrone 3-sulfate, PhIP, resveratrol conjugates, naringenin glucuronides, methotrexate, 7-hydroxymethotrexate, ezetimibe, gefitinib, 9-aminocamptothecin, diflomotecan, rosuvastatin, atorvastatin, fluvastatine, simvastatin lactone. c Leukotrienes glutathione, 2,4-dinitrophenyl-S-glutathione, bromosulfophthalein, conjugates of bile salts and heavy metals, resveratrol conjugates, naringenin glucuronides, vinblastine, reduced folates, irinotecan and its metabolite SN-38, pravastatin, ceftriaxone, ampicillin, grepafloxacin, sulfasalazine, fexofenadine, lopinavir, fosinopril, ochratoxin A, epicatechin, PhIP, phenols, colchicine, darunavir, Rhei Rhizoma extract, flavonoids, methotrexate, 7-hydroxymethotrexate, ezetimibe.

intestine can be used as a strategy to improve oral drug bioavailability of known substrates [18], as these transporters prevent drug molecules from being absorbed.

2.1.1. MDR1 drug-transporting P-glycoprotein (P-gp; ABCB1) The multidrug resistance 1 (MDR1) gene encodes for P-glycoprotein (P-gp), the most studied efflux transporter in the gut. In rodents, the

Table 2 Drug uptake transporters identified in the intestine. Drug Gene transporter

Intestinal Substrate localization specificity

PEPT1

SLC15A Apical

Di–tri-peptides

OCTN1

SLC22A Apical

OCTN2

SLC22A Apical

Carnitine and organic cations Carnitine and organic cations

OCT1/ OCT2

SLC22A Basal

PMAT

SLC29

Apical

Low molecular weight organic cations Organic cations

OATP2B1

SLCO

Apical

Organic anions

OATP1A2

SLCO

Apical

Organic anions

MCT1

SLC16A Apical

Substrates

Inhibitors

Cephalosporins, penicillins, enalapril, alacepril, Gly-Sar, zinc ions, lisinopril, oseltamivir, renin inhibitors, thrombin inhibitors, JBP485 betastin, L-alpha-methyldopa-phenylalanine, D-phenylglycine-L-alpha-methyldopa, L-val-acyclovir, 5′-O-L-valyl didanosine TEA, quinidine, verapamil, ergothioneine, pyrilamine Levofloxacin, grepafloxacin TEA, quinidine, verapamil, ergothioneine, pyrilamine, Levofloxacin, grepafloxacin cephaloridine, imatinib, ipatropium, valproic acid, spironolactone. TEA, metformin, acyclovir, zalcitabine, memantine, ranitidine 1-Methyl-4-phenylpyridinium (MPP+), tetraethylammonium, serotonin, dopamine, epinephrine, norepinephrine, guanidine, histamine, metformin Estrone-3-sulfate, BSP, pravastatin, DHEAS, rosuvastatin, atorvastatin, pitavastatin, fexofenadine, mesalazine, glyburide, taurocholate, aliskiren

Bile salts, BSP, steroid sulfates, thyroid hormones, prostaglandin E2, fexofenadine, opioid peptides, N-methylquinine, N-methylquinidine, ouabain, BQ-123, talinolol, CRC-220, celiprolol, atenolol, ciprofloxacin Acetate, pyruvate, lactate, acetoacetate, Unbranched β-hydroxybutyrate, p-aminohippuric acid, benzoic aliphatic and acid, foscarnet, mevolonic acid, salicylic acid, substituted monocarboxylates carbenicillin indanyl sodium, phenethicillin, propicillin.

Decynium-22, GBR12935, fluoxetine, desipramine, cimetidine, quinidine, quinine, verapamil, rhodamine123 Grapefruit juice, green tea, sirolimus, everolimus, budesonide, cyclosporine, rifampin. Grapefruit, orange and apple juices, green tea, sirolimus, everolimus, budesonide, cyclosporine, rifampin.

Inducers

Polymorphisms

Oxidized fats Oxidized fats, clofibrate

Yes

Yes

Please cite this article as: M. Estudante, et al., Intestinal drug transporters: An overview, Adv. Drug Deliv. Rev. (2012), http://dx.doi.org/ 10.1016/j.addr.2012.09.042

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Table 3 Digoxin P-glycoprotein (P-gp) interactions described in the intestine. Affected drug

Inhibitor

Impact on affected drug

Experimental model

Species

Reference

Digoxin

Ritonavir Drodenarone Ranolazine Verapamil Quinidine

Talinolol

86% increase in AUC 2.5-Fold increase in steady-state level 1.46-Fold increase in AUC and 1.60-fold increase in Cmax Increase in the absorption rate 2-Fold increase in plasma concentration levels Decrease in net flux ratio Decrease in net flux ratio Decrease in net flux ratio Decrease in net flux ratio 20% increase in AUC Decrease in net flux ratio 1.7-Fold increase in AUC Decrease in net flux ratio 15% increase in AUC and 20% increase in Cmax 23% increase in AUC and 45% increase in Cmax

Clinical study Clinical study Clinical study Single pass intestinal perfusion Clinical study MDR1-MDCKII Caco-2 Isolated ileal tissues Caco-2 Clinical study Caco-2 Clinical study Caco-2 Clinical study Clinical study

Human Human Human Rat Human Transfected cell line Human Rat Human Human Human Human Human Human Human

[207] [208] [209] [210] [211] [23] [212] [213] [214] [214] [215] [216] [217] [217] [218]

Inducer

Impact on affected drug

Experimental model

Species

Reference

Rifampicin St. John's wort

30% decrease in bioavailability and 58% decrease in Cmax 18% decrease in AUC

Clinical study Clinical study

Human Human

[38] [219]

Dipyridamole Clarithromycin Atorvastatin

gene responsible for the primary MDR isoforms' expression is depicted by lower case letters as mdr1 (a and b) and mdr2 [20]. Initially discovered as a result of its interaction with multiple anticancer drugs [1], numerous studies have demonstrated that P-gp possesses broad subtrate specificity, with a preference for hydrophobic, amphipathic or cationic molecules containing a planar ring system ranging in size from 200 to 1900 Da [1]. P-gp is also involved in the transport of neutral compounds such as digoxin and cyclosporine, negatively charged carboxyl groups such as those found in atorvastatin and fexofenadine, and hydrophilic drugs such as methotrexate [20]. The degree of hydrogen bonding and partitioning into the lipid membrane has been determined to be a rate-limiting step for substrate interactions with P-gp. The polarized, apical membrane localization of P-gp within the intestine (Fig. 1) suggests a normal excretory or barrier role for P-gp in mediating the efflux of xenobiotics and toxins into the intestinal lumen [21]. P-gp is therefore ideally positioned to limit the oral absorption of compounds by driving efflux of substrates back into the lumen. Furthermore, intestinal P-gp may contribute to the systemic clearance of intravenously administrated drugs by active secretion into the intestinal lumen [22]. The expression of P-gp increases from proximal to distal regions of the small intestine [22]. Net intestinal secretion of paclitaxel and digoxin in excised mdr1a (+/+) mice tissues followed the order ileum> distal colon >distal jejunum ~ proximal colon. Similar profiles were also reported for the P-glycoprotein substrates etoposide in rabbit and rhodamine 123 in mouse and rat [23], revealing that P-gp secretion varies across different segments of the intestine. The first evidence indicating that P-gp acts as a secretory detoxifying system to limit drug absorption came from studies in human

intestinal epithelial cell lines Caco-2, HT29 and T84. Polarized, apical P-gp expression in these cells was accompanied by secretory (basal-to-apical; blood-to-lumen) transport of the cytotoxic anticancer drug, vinblastine, which was reduced in the presence of MRK16, an inhibitory monoclonal antibody directed against P-gp, and the P-gp inhibitors/substrates verapamil and nifedipine [24]. Competitive binding studies using colchicine and actinomycin D revealed a lack of competition for the vinblastine-binding site, suggesting that P-gp has multiple drug binding domains [20]. Shapiro et al. [25] demonstrated that progesterone was not effluxed by P-gp, although it was shown to bind P-gp and block the efflux of other substrates. Mutiple binding sites for substrates and inhibitors on P-gp have been identified using site-directed mutagenesis [1]. Naturally occuring substrates for P-gp (Table 1) include biologically active compounds found in a normal diet, such as plant chemicals [26], and endogenous compounds like steroid hormones [27] and bile salts [28–30] A number of clinically important drugs are P-gp substrates, which are as diverse as anthracyclines (doxorubicin, daunorubicin), alkaloids (reserpine, vincristine, vinblastine), specific peptides (valinomycin, cyclosporine), steroid hormones (aldosterone, hydrocortisone), local anesthetics (dibucaine), immunosuppressive agents (cyclosporine, tacrolimus), talinolol, digoxin, tandutinib [16,31] and the cancer imaging agent tetraphenylphosphonium (TPP) [32]. Examples of intravenous administered drugs that undergo intestinal excretion mediated by P-gp include digoxin, cyclosporine, ivermectin, paclitaxel, vinblastine, grepafloxacin, indinavir, nelfinavir and saquinavir [23]. P-gp inhibitors (Table 1) include several calcium channel blockers, immunosuppressive agents and other well-characterized compounds

Table 4 Other drug P-glycoprotein (P-gp) interactions described in the intestine. Affected drug

Inhibitor

Impact on affected drug

Experimental model

Species

Reference

Ivermectin Paclitaxel Cyclosporine Talinolol

Verapamil GG918 Grapefruit juice Verapamil Grapefruit juice R101933 Cyclosporine

Reduced intestinal luminal accumulation Increase in AUC and Cmax 55% increase in AUC and 35% in Cmax Reduced intestinal luminal accumulation Increase in AUC and Cmax 8.47% decrease in dose excreted in the feces 8-Fold increase in AUC

In vivo In vivo Clinical study In vivo In vivo Clinical study Clinical study

Rat Rat Human Rat Rat Human Human

[220] [221] [222] [223] [35] [224] [225]

Docetaxel Paclitaxel Affected drug

Inducer

Impact on affected drug

Experimental model

Species

Reference

Talinolol Cyclosporine Fexofenadine

Rifampicin St. John's Wort St. John's Wort

35% decrease in AUC 1.9-Fold decrease in AUC 1.6-Fold decrease in AUC

Clinical study Clinical study Clinical study

Human Human Human

[39] [226] [226]

Please cite this article as: M. Estudante, et al., Intestinal drug transporters: An overview, Adv. Drug Deliv. Rev. (2012), http://dx.doi.org/ 10.1016/j.addr.2012.09.042

M. Estudante et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

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Table 5 BCRP and MRP2 interactions described in the intestine. Affected drug

Inhibitor

Impact on affected drug

Experimental model

Species

Reference

Topotecan

GG918a Novobiocina Indomethacinb

2.43-Fold increase in systemic exposure 50% decrease in intestinal secretion Increase in permeability Increase in plasma quercetin levels

Human Rat Human Rat Mice

[80] [227] [69]

FCTa

Clinical study Ileal perfusion Caco-2 Jejunal perfusion In situ intestinal perfusion

Sulfasalazine Quercetin glucuronides a b

[75]

BCRP inhibitors. MRP2 inhibitor.

such as SDZ PSC 833, LY335979 and GF120918 (GG918) [20]. Additionally, common pharmaceutical excipients such as hydrophilic cyclodextrin, cosolvents (PEG 400) and surfactants (Tween 80, Cremophor EL) have been shown to inhibit P-gp activity. Surfactants or cosolvents have also been shown to indirectly influence P-gp by inducing changes in cellular membrane fluidity. The flavonoids and other ingredients present in fruits, vegetables and herbs have been found to modulate the activity of P-gp function and may cause detrimental effects on pharmacokinetics [20,26]. One of the most recognized interactions is the ingestion of grapefruit juice that was initially believed to change drug absorption only by inhibiting CYP3A enzymes [33], but then was hypothesized to change the pharmacokinetic profile of cyclosporine also by inhibition of P-gp mediated transcellular intestinal absorption [34]. The oral coadministration of grapefruit juice with talinolol, a nonmetabolized drug, resulted in an increase of talinolol Cmax, AUC and a reduced Tmax without significantly affecting the terminal talinolol half-life [35]. This study confirmed that grapefruit juice could inhibit intestinal P-gp mediated efflux resulting in enhanced talinolol bioavailability. Therefore concomitant intake of herbal extracts or fruits/foods and nutraceuticals may modulate the pharmacokinetic profile with impact on the therapeutic index of drugs, in particular for those agents with a narrow therapeutic index, resulting in an altered clinical response. More recently, Custodio et al. [36] demonstrated in MDCK and MDR1-MDCK cell lines the inhibitory effect of monoglycerides on the P-gp efflux of vinblastine. These results suggest a potential effect of monoglycerides, breakdown products from a high fat meal, on increasing drug bioavailability. P-gp expression can be induced by various factors such as xenobiotics, environmental stress, differentiating agents, and hormones under cell culture conditions (Table 1). St John's Wort results in a change of the pharmacokinetic profile of digoxin due to induction of P-gp mediated efflux [37]. Concomitant rifampin therapy (600 mg/ day for 10 days, p.o.) has also resulted in significant reduction in the area under the plasma concentration time curve (AUC) of oral digoxin (single-dose, 1 mg oral and 1 mg intravenous), a known P-gp substrate. These results were confirmed by measuring intestinal P-gp levels, which revealed a threefold increase in this efflux transporter expression [38]. Similarly, rifampicin decreased talinolol oral exposure, consistent with an ~ 4 fold increase in duodenal P-gp expression[39]. In conclusion, two major mechanisms have been described regarding P-gp drug–drug interactions: inhibition and induction. Interactions described for P-gp are summarized in Tables 3 and 4. It is important to note that substrate specificity of P-gp may also vary across populations due to genetic polymorphisms. The three most frequent single nucleotide polymorphisms in the ABCB1 gene are C1236T in exon 12, G2677T/A in exon 21 and C3435T in exon 26. These SNPs could affect the pharmacokinetics and pharmacodynamics of drugs that are P-gp substrates. For example, oral exposure of levosulpiride in ABCB1 2677TT and 3435TT subjects was significantly higher than that of subjects with at least one wild-type allele [16]. Kurata et al. [40] demonstrated a significant difference in oral digoxin bioavailability between two allelically diverged MDR1

populations, which resulted in population specific absorption and/ or distribution outcomes. Nevertheless, findings from many studies on the effect of ABCB1 polymorphisms on P-gp substrates have not been consistently reproduced; therefore, routine application of ABCB1 polymorphism analysis to clinical studies is not warranted at this time. Studies with a larger number of subjects may be needed to clarify the role of ABCB1 polymorphisms in pharmacokinetics and pharmacodynamics [1]. Information on the extent of interaction of P-gp with drug molecules as they move along the length of the small and large intestine is crucial to our understanding of drug absorption. The FDA [41] and the Drug Transporter Consortium [1] recommend the use of a decision tree for P-gp substrate interactions. A new molecular entity (NME) is considered to be a potential P-gp substrate if the efflux ratio (Eq. (1)) is equal or higher than 2 in an epithelial cell system that expresses the transporter. Efflux ratio ¼ BasaltoApical Permiability ðBA PappÞ=

ð1Þ

ApicaltoBasal Permiability ðAB PappÞ: Nevertheless, a high efflux ratio in a cellular system does not always translate into poor oral absorption. The involvement of P-gp is more pronounced if the drug has a poor apparent permeability coefficient (BDDCS Classes 3 and 4 drugs), or in cases in which there is interplay between metabolism and efflux (BDDCS Class 2 drug). The pharmacokinetics of a NME that has high solubility, high permeability and/or is highly metabolized (BDDCS Class 1 drugs) are less likely to be affected by co-administered drug that is a P-gp inhibitor [1,42,43]. The very recent finding of Broccatelli et al. [44] that the relevant P-gp efflux ratio of 2 is only valid for studies using the “Borst” cell line, which corresponds to a higher cut-off ratio of 8.5 for studies in the NIH cell line, must be considered in reviewing the published results. 2.1.2. Multidrug resistance-associated protein family (MRP; ABCC) To date, nine members within the MRP family have been identified, although only MRPs 1 to 5 have been demonstrated to have a well-defined role in the transport of drugs [45–47]. MRP, ABCC, transporters are all able to transport organic anions, such as drugs conjugated to glutathione, sulfate or glucuronide and a variety of endogenous compounds, such as leukotriene, bilirubin glucuronides prostaglandins E1 and E2, cGMP and several glucuronosyl-, or sulfatidyl steroids [48]. MRP2 is expressed at the apical side of the enterocyte and acts as a barrier to drug absorption. MRP1, MRP3, MRP4 and MRP5 are localized at the basolateral membrane of the enterocyte and facilitate drug entrance into the circulation [49,50]. Here, we will review MRP1 and MRP2 in further detail. 2.1.2.1. MRP1; ABCC1. MRP1 was first cloned by Cole et al. [51] from a multidrug-resistant human lung cancer cell line. MRP1 is highly expressed in the small and large intestine, where it is localized in the basolateral membrane of enterocytes (Fig. 1) [52]. Unlike MRP2 and P-glycoprotein, MRP1 transports drugs from the cell into the

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Table 6 OATP interactions described in the intestine. Affected drug

Inhibitor

Impact on affected drug

Experimental model

Species

Reference

Aliskiren

Grapefruit juice

Clinical study

Human

[143]

Fexofenadine Estrone-3-sulfate

Grapefruit juice Sirolimus and everolimus Green tea

61% reduction in AUC 81% reduction in Cmax 63% reduction in AUC Uptake inhibition Uptake inhibition

Clinical study Transfected HEK293T cells Transfected cells

Human Human Human

[147,148] [156] [155]

interstitial fluid (body), rather than moving them out into the intestinal lumen. As a result, MRP1 acts as an absorptive transporter and protects enterocytes from the accumulation of toxic chemicals [23]. Substrate anticancer drugs include substances like vinca alkaloids, anthracyclines, etoposide, teniposide, mitoxantrone and methotrexate (Table 1). Unlike P-gp, MRP1 does not confer high levels of resistance to paclitaxel or bisantrene in cells. It can transport hydrophobic drugs or other compounds that are conjugated or complexed to the anionic tripeptide glutathione (GSH), to glucuronic acid, or to sulfate. Knockout mice lacking Mrp1 are viable and fertile, but they do show deficiencies in leukotriene (LTC4) mediated inflammatory reactions, suggesting that secretion of LTC4 is an important physiological function of MRP1 [53]. Other studies demonstrated that a combined deficiency of Mdr1a/Mdr1b P-gps and Mrp1 in knockout mice resulted in a markedly increased toxicity to intraperitoneally administered vincristine (up to 128-fold), but also to etoposide (3.5 fold), whereas a P-gp deficiency alone resulted in a 16- and 1.75-increase in toxicity to these drugs, respectively [54]. Up to now, it has been more difficult to find good small molecule inhibitors for MRP1 than for P-gp, especially ones that function in intact cells. This probably has to do with the preference of MRP1 for anionic compounds as substrates and inhibitors: most anionic compounds enter cells poorly, so it may be difficult to obtain sufficient intracellular concentrations of the inhibitor for efficacious inhibition. MRP inhibitors (Table 1) include the LTC4 analog MK571, LTC4 itself, sulfinprazone, benzbromarone and probenecid. Nevertheless, for specific in vivo inhibition of MRP1 these compounds are also not appropriate as they extensively affect organic anion uptake systems as well. For the specific aim of the study of in vivo MRP1 inhibition, inhibitors will have to be developed, with reasonable specificity and cellular penetration properties [53]. 2.1.2.2. MRP2; ABCC2. MRP2 is localized on the apical side of enterocytes (Fig. 1) and transports leukotrienes C4, D4 and E4 and various glutathione conjugates, including oxidized glutathione, 2,4-dinitrophenylS-glutathione, bromosulfophthalein glutathione, as well as conjugates of heavy metals including arsenic and cadmium [45]. Additionally, MRP2 also transports glucuronide and sulfate conjugates of several bile salts, a range of unconjugated organic anions such as methotrexate, vinblastine, reduced folates, irinotecan and its metabolite SN-38, pravastatin, ceftriaxone and ampicillin [20,55]. Other substrates include antihyperlipidemics, angiotensin-converting enzyme inhibitors, fexofenadine [56], lopinavir [57], fosinopril [58], as well as many toxins and their conjugates, such as naphthylisothiocyanate, heavy metals, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and some dietary compounds, such as ochratoxin A, epicatechin, PhIP [59] and phenols in St John's Wort supplements [60] (Table 1). MRP2 is important clinically as it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by certain compounds, toxins, health care supplements and disease states [18,55,59]. Alterations in MRP2 expression appear to be modulated during cholestasis and carcinogenesis and in response to various hormones and chemically unrelated compounds [55]. Shibayama et al. [18] showed that cholestasis induced by 5-FU treatment decreased intestinal MRP2 expression levels in vitro. In a clinical study, rifampin treatment of normal human subjects increased MRP2 mRNA and

protein in the duodenum [61]. In recent studies rat intestinal PepT1 and Mrp2, but not P-gp, were functionally induced by 1,25(OH)2D3 treatment [19]. Spironalactone was reported to induce Mrp2 in rats [62] (Table 1). In the rat small intestine, Mrp2 expression is concentrated at the tip of the villus, with the highest concentrations seen in the proximal jejunum, with little Mrp2 protein detected in the distal ileum [63]. A similar distribution of the phase II conjugating enzymes, UDPglucuronosyltransferase and glutathione S-transferase, suggests that metabolism and subsequent efflux of the organic anion conjugates act coordinately to decrease the intestinal absorption of food contaminants and drugs that enter the enterocytes via the digestive tract [59]. Mrp2 has been shown to mediate the transport of the abundant foodderived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats [64]. Unlike MRP1, the overlapping substrate specificity of MRP2 with P-gp, coupled with their intestinal and cellular colocalization to the apical membrane of duodenum and jejunum (Fig. 1), suggests a concerted function between these two transporters that would comprise a significant barrier to the intestinal absorption of many xenobiotics. For example, the uptake of grepafloxacin [31], colchicine [62], darunavir [65] and Rhei Rhizoma extract [66] were observed to be directly influenced by the combined effect of P-gp and MRP2. Flavonoids, such as quercetin and naringenin, were secreted via MRP2 and MRP2 + P-gp, respectively, in a Caco-2 cell model [67]. Grapefruit juice has been reported as an inhibitor of both P-gp and MRP2 [68]. As with MRP1, small molecule inhibitors of MRP2 that can be used in intact cells are quite limited. Obviously, many of the anionic transported substrates of MRP2 will readily serve as competitive inhibitors when applied in in vitro systems where MRP2 is present in an inside-out (vesicle) orientation. Some examples (Table 1) are LTC4, MK571, phenolphthalein glucuronide, fluorescein methotrexate, probenecid, furosemide and indomethacin [21,69]. MRP2 has clinically relevant genetic polymorphisms [1] and the pharmacokinetics of fexofenadine was affected by ABCC2 C-24T polymorphism [56]. The gene is fully deficient in two mutant rat strains (TR −/GY and EHBR) and in patients suffering from Dubin–Johnson syndrome [53,63]. These latter patients suffer from a recessively inherited conjugated hyperbilirubinemia that can result in jaundice. 2.1.3. Breast cancer resistance protein (BCRP; ABCG2) Breast cancer resistance protein (BCRP, MXR, ABCG2) similar to P-glycoprotein and MRPs is a multidrug resistance protein and a member of the ABC family of secretory transporters. BCRP was first cloned based on its overexpression in a highly doxorubicin-resistant MCF7 breast cancer cell line (MCF-7/AdrVp). BCRP sequences were also cloned by Miyake et al. [70] and Allikmets et al. [71], who called the gene MXR (for mitoxantrone resistance) and ABCP (for placental ABC protein), respectively. Since structural and sequence homology revealed that BCRP belongs to the ABCG gene subfamily, the Human Genome Nomenclature Committee conferred the official designation as ABCG2 [20]. BCRP is expressed at the apical membrane of the small and large intestine (Fig. 1) where it has a role in limiting oral bioavailability. Unlike P-gp, the expression of BCRP does not vary significantly along the length of the small intestine [72] and the BCRP mRNA

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level is higher than other efflux transporters in the human intestine [73]. BCRP transports a highly diverse range of substrates that has been rapidly expanding since its discovery (Table 1). BCRP is demonstrated to offer resistance to intestinal absorption of anti-cancer agents including topotecan, irinotecan and its active analog SN-38, mitoxantrone, methotrexate, doxorubicin, daunorubicin, tyrosine kinase inhibitors imatinib, gefitinib and tandutinib [31], non-chemotherapy drugs such as statins, prazosin, glyburide, dipyridamole [74], quercetin[75], temocapril, an ester-type prodrug of temocaprilat [76], sulfate conjugates, porphyrins[77], nitrofurantoin, some fluoroquinolones [1], antivirals like zidovudine, lamivudine and efavirenz [78], ciprofloxacin [79], rifampicin, sulfasalazine, quercetin and nontherapeutic compounds such as dietary flavonoids, phytoestrogens such as genistein, daidzein and coumestrol, porphyrins, estrone 3-sulfate and the carcinogen PhIP [16]. Topotecan and sulfasalazine had a 10-fold to 110-fold increase in relative AUCs when administered to Bcrp (−/−) knockout mice [1]. Topotecan was also tested in patients with solid tumors. The apparent oral bioavailability of topotecan increased by nearly 2.5-fold and the mean Cmax increased by nearly 3-fold in the presence of GF120918, a dual BCRP/P-gp inhibitor [80] (Table 5). Taken together, these reports reveal that BCRP appears to play a vital role in limiting substrate drug absorption across the intestine in conjunction with P-glycoprotein, since topotecan is a weak P-glycoprotein substrate as well [81] and GF120918 is a good inhibitor of both transporters. Of the BCRP substrates, glyburide, imatinib, tandutinib, methotrexate, mitoxantrone, prazosin, temocapril and SN-38 are also P-gp substrates, although methotrexate is a weak substrate of P-gp [76,82,83]. Notably, in addition to hydrophobic substrates such as mitoxantrone, BCRP can also transport hydrophilic conjugated organic anions like the sulfate conjugates, whereas P-gp generally transports hydrophobic compounds [74]. As with P-gp, there is also an overlap in substrate specificity between BCRP and MRP2 that can lead to a synergistic effect of the transporters in limiting drug penetration across tissue barriers like the intestine. Following oral administration, sulfasalazine is characterized by poor intestinal absorption [84] that essentially enables its colonic targeting and therapeutic action in inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. The reason for sulfasalazine's low oral bioavailability was attributed initially to poor solubility and permeability characteristics [46]; however, sulfasalazine has a calculated log P (cLog P) value of 3.88, suggesting that it should be a high permeability compound [85]. It has been recently shown that efflux transport mediated by MRP2 and BCRP is responsible for the poor permeability of sulfasalazine in the small intestine [86]. This effect was partially reversed by the MRP2 inhibitor indomethacin in the Caco-2 cell line and in an in situ rat jejunal perfusion model [69] (Table 5). In conclusion, efflux transport mediated by MRP2 and BCRP shifts sulfasalazine permeability from high to low, thereby enabling its therapeutic action at the distal GI. This is a demonstration of intestinal efflux acting in favor of oral drug efficacy. Other dual BCRP and MRP2 substrates include resveratrol glucuronide and sulfate conjugates [87], naringenin glucuronides [88], methotrexate and its metabolite 7-hydroxymethotrexate [89], as well as ezetimibe [90]. Known BCRP inhibitors (Table 1) include estrone, 17-β-estradiol, dietary flavonoids such as chrysin and biochanin A, herb extracts, GG918, gefitinib, imatinib, tamoxifen, novobiocin, dipyridamole [88] and the anti-HIV protease inhibitors nelfinavir and ritonavir [91–93]. Fumitremorgin C (FTC), an extract of Aspergillus fumigatus, and the FTC analog Ko143 selectively inhibit BCRP with no overlapping affinity for P-gp or MRP1 [94–96]. Drugs like cyclosporine and anti-HIV protease inhibitors seem to be general inhibitors of ABC transporters [74]. BCRP induction has also been reported. Peroni et al. [78] found that a five-day oral treatment with 20 mg/kg efavirenz promotes the over-expression of BCRP in the rat intestine, with a decline in

7

the intestinal permeability of this antiretroviral. BCRP expression normalizes within 24 h after the last drug administration together with the ability of efavirenz to permeate through the small intestine wall. As for P-gp, the Drug Transporter Consortium [1] recommends the use of a decision tree for assessment of BCRP substrate interactions. A new molecular entity (NME) is considered to be a potential BCRP substrate if the efflux ratio (Eq. (1)) is equal or higher than 2 in an epithelial cell system that expresses the transporter. BCRP presents clinically relevant genetic polymorphisms [1,74]. Recent studies have demonstrated that individuals with reduced BCRP expression levels (Q141K variant) are at increased risk for gefitinib-induced diarrhea and altered pharmacokinetics of 9aminocamptothecin, diflomotecan, irinotecan, rosuvastatin, sulfasalazine and topotecan [1]. BCRP polymorphisms significantly affect the pharmacokinetics of several HMG-CoA reductase inhibitors including atorvastatin, rosuvastatin, fluvastatin and simvastatin lactone, but have no significant effect on pravastatin or simvastatin acid [16]. 2.2. Solute carrier transporters (SLC; SLCO) The major uptake transporters responsible for xenobiotic transport belong to the two solute carriers (SLC and SLCO) superfamilies [12]. The members of these superfamilies are involved in the transport of a wide range of substrates including amino acids, peptides, sugars, vitamins, bile acids, neurotransmitters, and xenobiotics. The SLC superfamily includes many pharmacokinetically important transporters such as proton dependent oligopeptide transporters (PEPT1, PEPT2, PHT1; SLC15A family), organic anion transporters (OAT; SLC22A), organic cation transporters (OCT; SLC22A family), nucleoside transporters (CNT and ENT; SLC28A and 29A family) [20], plasma membrane monoamine transporter (PMAT; SLC29 family) and the monocarboxylate transporters (MCT; SLC16A family) [97]. The SLCO family is made up of the organic anion transporting polypeptides (OATP; SLC21A). These uptake transporters use a variety of porter mechanisms (i.e. uniporter, antiporter, symporter), not all of which have been fully elucidated for each specific transporter and use chemiosmotic gradients created by translocations of ions across the membrane [12]. Solute carriers known to play a relevant role in drug transport at the intestine include PEPTs, OCTNs, OCTs, PMAT, OATPs and MCT. 2.2.1. Oligopeptide transporters (PEPT; SLC15A) The currently known peptide transporters include the peptide transporters 1 and 2, PEPT1 and PEPT2, and the peptide/histidine transporters 1 and 2, PHT1 and PHT2 [20]. PEPT1 protein expression has been demonstrated in the human small intestine and was localized at the apical plasma membrane of enterocytes (Fig. 1) in rat, mouse, rabbit and sheep, exhibiting high homology between species [98]. It was first cloned in the intestine and the expression level of this transporter increases from duodenum to ileum. Among PEPT1 substrates (Table 2) are cephalosporins, penicillins, ACE inhibitors, antivirals like oseltamivir [99], renin inhibitors, thrombin inhibitors and the peptide-like antineoplastic drug betastin [23,100] that present different degrees of affinity and capacity. It is well known that beta-lactam antibiotics are hydrophilic weak acids containing a peptide bond and that these ionize at the intestinal pH. Therefore, on the basis of their physicochemical characteristics, these antibiotics should exhibit relatively poor oral absorption. However, pharmacokinetic studies have clearly demonstrated that beta-lactam antibiotics are fairly well absorbed in the intestine [101]. Beta-lactam antibiotics possess at least one peptide bond and a free terminal carboxyl group, a minimum requirement to be transported by PEPT1. The majority of beta-lactam antibiotic drugs including ampicillin, amoxicillin, cyclacillin, cefaclor, cefadroxil, cefatrizine, cefixime, cephalexin and cephradine demonstrate PEPT carrier mediated transport characteristics [23,102].

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Peptidomimetic drugs are increasingly utilized as therapeutic agents for the treatment of numerous disorders including AIDS, hypertension and cancer [103–105]. Understanding the nature of PEPT1 and the structural requirements of drug transport by PEPT1 led to a strategic development of prodrugs [106]. Generally PEPT1 has a low affinity [16] but a high transport capacity, which makes it highly attractive as a drug target [20,107]. For instance, the dipeptidyl derivative L-αmethyldopa-phenylalanine of L-α-methyldopa, which otherwise is a poorly absorbed antihypertensive agent, displayed at least 20-fold enhanced intestinal permeability [108]. The 5′-O-L-valyl ester prodrug of didanosine (DDI) demonstrated a 5-fold increase in the oral bioavailability of DDI [106]. Another prodrug D-phenylglycine-L-α-methyldopa showed 3.5-fold increased permeability in in situ single pass rat jejunum perfusion studies [109]. Additionally, cefixime and cefdinir prodrugs of beta-lactam antibiotics, the L-valyl-acyclovir prodrug of acyclovir and prodrugs for ACE inhibitors such as enalapril and alacepril were found to be good substrates for the di–tri-peptide transporters, although some controversy remains in the literature regarding ACEnhibitors as PEPT1 substrates [76]. Lisinopril, enalapril, quinapril, benazepril [110] and temocapril [76] were reported to be poor and non-PEPT1 substrates. Potential drug–food interactions via PEPT1 have been investigated [103]. A recent clinical study confirmed that intestinal absorption of oseltamivir (Tamiflu), a prodrug of the influenza virus neuraminidase inhibitor Ro 64-0802 and a substrate of PEPT1, is markedly inhibited by administration with milk in rats [104]. Milk proteins are rapidly broken down to free amino acids and small peptides in the stomach and duodenum, competing for absorption with PEPT1 substrates. In humans, although the initial rate of absorption of oseltamivir decreases with milk, the total extent of absorption was unchanged [104]. Glycylsarcosine (Gly-Sar) has been reported as a typical PEPT1 inhibitor [104–106] (Table 2). It was recently demonstrated in rat and in HeLa-hPEPT1 cells that zinc ions can also inhibit the transport activity of PEPT1, while basal intestinal Pept1 expression does not change [111]. Other authors [93,105] reported lisinopril and JBP485 (cyclotrans-4-L-hydroxyprolyl-L-serine, a dipeptide with anti-hepatitis activity) to be competitive inhibitors for PEPT1 in HeLa cells transfected with human peptide cotransporter 1 (PEPT1), in everted rat intestinal sacs and in rat jejunal perfusions. The influence of the fed-fasted state on Pept1 expression was studied in rat. It was reported that 16 h of fasting can cause significant upregulation of Pept1 protein expression in the small intestine [112]. Other studies showed that Pept1 activity and expression were markedly reduced in animal models of type 2 diabetes/obesity [113]. During chronic inflammation such as that associated with inflammatory bowel disease, Pept1 expression is upregulated in the colon where it is normally minimally expressed. Several recent studies have shown that Pept1 binds to and transports various bacterial di/ tripeptides into colon cells, leading to activation of a downstream proinflammatory response [114].

2.2.2. Organic anion transporters (OAT; SLC22A) To date, five structurally related isoforms of organic anion transporters (OAT1-5) have been identified. OAT1 and OAT3 are mainly expressed in the kidney and localized on the basolateral membrane of the proximal tubules. OAT2 transports relatively small and hydrophilic organic anions, such as indomethacin and salicylate and may be involved in the hepatic uptake of these drugs [115]. While OAT isoforms have broad substrate specificity, members of the OAT family have not been identified in the human intestine. The intestinal expression of OAT members is limited, with only one report suggesting the presence of Oat2 mRNA in mouse fetal intestine. Therefore, the role of the OAT family in the intestinal absorption of drugs is considered negligible [20].

2.2.3. Organic cation transporter (OCT, OCTN; SLC22A) The SLC22 family is a member of the major facilitator superfamily that comprises transporters from bacteria, plants, animals and humans in 18 transporter families. This group of transporters consists of the electrogenic cation transporters OCT1-3, the organic cation/ carnitine transporters (OCTN1, OCTN2) and the multidrug and toxin extrusion H +/cation antiporters MATE1, MATE2-K and MATE2-B. Relevant SLC22A members in the intestine include OCT [15] and OCTN. OCTN1 and OCTN2 are expressed at the enterocyte apical membrane (Fig. 1) and transport carnitine and other organic cations. Known substrates for both OCTN1 and OCTN2 (Table 2) include tetraethylammonium, quinidine, verapamil, ergothioneine and pyrilamine [14,16]. OCTN2 also transports cephaloridine, imatinib, ipatropium [15], valproic acid and spironolactone [14] (Table 2). Gene expression of OCTN1 and OCTN2 is downregulated in patients with ulcerative colitis [116] and intestinal mRNA levels of other SLC family members are deregulated in inflammatory bowel disease patients [117]. Fasting was reported to increase mRNA concentrations of OCTN2 in the small intestine of rats and pigs [118]. The lipid lowering agent clofibrate caused upregulation of OCTN 2 in the small intestine of rats [119] and pigs [120]. Oxidized fats also seem to be involved in upregulation of OCTN in the rat small intestine [121]. Zwitterionic drugs like levofloxacin and grepafloxacin inhibited L-carnitine transport by OCTN in Caco-2 cells [122]. Organic cation transporter 1 (OCT1) was the first member of the OCT family identified and was cloned from rat kidney [123]. OCT1 and OCT2 are expressed in epithelial cells of the intestine and are localized to the basolateral membranes of the enterocytes, mediating basolateral uptake into enterocytes [15] (Fig. 1). Substrates of OCT transporters have relatively low molecular weight and are hydrophilic organic cations exhibiting diverse molecular structures [14] (Table 2). The pharmacological and physiologic role of Oct1 has been investigated using Oct1 knockout (Oct1 −/−) showing that Oct1 also mediates basolateral uptake of TEA into enterocytes (Table 2). Pharmaceuticals such as metformin, acyclovir, zalcitabine, memantine and ranitidine have been identified as Oct/OCT substrates [14]. Rat Oct3 mRNA has been found to be most abundant in the placenta, with a moderate presence in the intestine, heart and brain [124]. Most of the literature dealing with the function of OCT transporters has been conducted in the liver and kidney, while few papers discuss the role of OCT transporters in the gastrointestinal tract [20,125]. 2.2.4. Plasma membrane monoamine transporter (PMAT; SLC29) A novel organic cation transporter, the plasma membrane monoamine transporter (PMAT), has been recently identified. It belongs to the equilibrative nucleoside transporter (ENT) family (SLC29) [126]. Unlike the existing ENT members that mainly transport nucleosides and their structural analogs, PMAT (also known as ENT4) specifically transports monoamines, such as the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), tetraethylammonium, serotonin, dopamine, epinephrine, norepinephrine, guanidine and histamine [126,127] (Table 2). The substrate and inhibitor specificity of PMAT largely overlaps with that of the OCTs [127]. Known inhibitors of the dopamine transporter (DAT), the serotonin transporter (SERT), the norepinephrine transporter (NET) and the organic cation transporters (OCTs) such as decynium-22, GBR12935, fluoxetine, desipramine, cimetidine, quinidine, quinine, verapamil, and rhodamine123 were also reported to inhibit PMAT [126,127]. PMAT is highly expressed in the human and rat [128] brain, where it was first identified as a biogenic monoamine transporter, but also in a number of other human tissues, including skeletal muscle, kidney, liver, and heart [126]. More recently Barnes et al. [129] revealed a high level of PMAT mRNA expression in various segments of the human intestinal tract, localized at the apical membrane of polarized epithelial cells [130] (Fig. 1). Zhou et al. [97] demonstrated that

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metformin is a PMAT substrate and suggest that it may be responsible for the intestinal uptake of this biguanide. Therefore, PMAT may be further involved in the intestinal uptake of other organic cationic drugs. Currently there are insufficient data on potential genetic polymorphisms for PMAT [131]. 2.2.5. Organic anion transporter polypeptide (OATP; SLCO) The SLCO family is made up of the organic anion transporting polypeptides (OATPs) [12]. These transporters mediate the sodiumindependent transport of a diverse range of amphiphilic organic compounds with relatively high molecular weight, including bile acids, steroid conjugates, thyroid hormones, anionic peptides, numerous drugs and other xenobiotics. Interestingly, OATP family members are poorly conserved evolutionarily and orthologs for human OATPs may not exist in rodents [1]. In rats, the main Oatps are Oatp1a1 (formerly known as oatp1) expressed in the liver and kidney, the widely expressed Oatp2a1 (formerly known as rPGT), Oatp1a4 (formerly known as oatp2) expressed in the retina, liver and brain and Oatp1a5 (formerly known as oatp3) expressed in retina, brain, liver, kidney and the intestine [132]. Additional studies are needed to more fully validate the utility of rodent models to predict human OATP-mediated drug disposition [1]. OATP1A2 (formerly termed OATP-A) was described in 1995 and followed by the discovery of the second member OATP1B1 (formerly termed OATP2 or OATP-C). Today the OATP superfamily consists of 39 members [16], including 10 OATPs and the prostaglandin transporter OATP2A1 (formerly known as PGT). While most of the OATP proteins are expressed in multiple tissues, OATP1B1 and OATP1B3 are predominantly if not exclusively expressed in the liver. Almost all OATP family members are localized to the basolateral membrane of polarized cells. Interestingly, in addition to its basolateral localization in liver and placenta, OATP2B1 and OATP1A2 have been detected in the apical membrane of enterocytes in the human small intestine (Fig. 1) [133] and have a role in intestinal absorption of drugs [134]. It was shown in vitro that OATP2B1 was responsible for the pH dependent transport of estrone-3-sulfate, sulfobromophthalein (BSP) [135] and pravastatin [136,137]. Extracellular acidification, consistent with the acidic microenvironment of intestinal mucosa, promoted solute uptake. Further studies using rabbit intestinal apical membrane vesicles [138] and a clinical study [139] suggest that OATP2B1 may play a role in the oral absorption of pravastatin. Other substrates (Table 2) of OATP2B1 are dehydroepiandrosterone sulfate (DHEAS), rosuvastatin, atorvastatin, pitavastatin [140], mesalazine [141], and the anti-diabetic glyburide, although the clinical relevance of intestinal uptake for the BDDCS Class 2 drugs has not been shown. At a lower pH taurocholate is an OATP2B1 substrate [16] (Table 2). OATP2B1 presents a narrow substrate specificity compared with other OATPs [142]. Aliskiren has been described as a substrate of intestinal CYP3A4, P-gp and OATP2B1. Grapefruit juice has been reported as an inhibitor of intestinal CYP3A4, P-gp and OATP2B1 [22]. In a study in 11 healthy volunteers Tapaninen et al. [143] observed a reduction of aliskiren AUC and Cmax by 61 and 81% respectively, with concomitant administration of grapefruit juice. Inhibition of CYP3A4 and P-gp would be expected to reduce presystemic aliskiren extraction and thus increase aliskiren bioavailability. The opposite effect was observed, and the authors propose that inhibition of intestinal uptake is the major mechanism in the interaction between grapefruit juice and aliskiren (Table 6). Aliskiren is a BDDCS Class 3 drug (although incorrectly categorized in reference 183) and would be expected to require an intestinal uptake transporter. The physiological and pharmacological role played by OATP2B1 in intestinal absorption may also vary between individuals. A single nucleotide polymorphism (SNP) (found in 31% of the Japanese population) is associated with a greater than 50% reduction in transport capacity [144].

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Another OATP transporter that enhances the intestinal absorption of xenobiotics is OATP1A2, which possesses a broad spectrum of solutes (Table 1) including bile salts, bromosulfophthalein (BSP), steroid sulfates, thyroid hormones, prostaglandin E2, fexofenadine, opioid peptides, N-methylquinine and N-methylquinidine, ouabain, the endothelin receptor antagonist BQ-123, talinolol and the thrombin inhibitor CRC-220 [16]. Dresser et al. [145] have shown in vitro that grapefruit, orange and apple juices at b 5% concentration were good inhibitors of OATP mediated uptake of fexofenadine (Table 6). Fexofenadine is also a substrate of P-gp [146]; interestingly, the effect of these same juices on P-glycoprotein mediated secretion of fexofenadine was detected only at >20% concentration. In the clinic, the oral plasma exposure of fexofenadine was decreased by 63% with concomitant intake of grapefruit juice [147,148]. These results are likely to be mediated by inhibition of intestinal absorption via OATP1A2. A very recent study reports that, when the extracellular pH is acidic, OATP2B1 may mediate uptake of fexofenadine, and this uptake is inhibited by apple juice in vitro[149]. It is therefore possible that the inhibition of OATP2B1 by fruit juices also contribute to the food–fexofenadine interactions at the level of the small intestine. Similar results were reported in a study that evaluated the effect of grapefruit ingestion on the oral plasma exposure of talinolol in humans. The decrease in the oral plasma exposure of talinolol (44%) was attributed to OATP1A2 inhibition [150]. Grapefruit and/or orange juice also decreased the AUC and Cmax of other orally substrates of OATP1A2 such as celiprolol [151,152], atenolol [153] and ciprofloxacin[154]. A recent study describes the inhibitory effect of green tea (Camellia sinensis) catechins on the function of both OATP1A2 and OATP2B1 [155]. Other reported inhibitors of intestinal OATPs include sirolimus, everolimus [156] budesonide, cyclosporine and rifampin [141] (Table 2). Similar to OATP2B1, genetic variations have been reported in OATP1A2. Lee et al. [157] identified six non-synonymous SNPs in the coding region of SLCO1A2. The c.516A>C variant had markedly reduced uptake capacity for the OATP1A2 substrates estrone3-sulfate and the d-opioid receptor agonists in vitro[157]. 2.2.6. Monocarboxylate transporters (MCT1; SLC16A) The bi-directional movement of monocarboxilic acids across the plasma membrane is catalyzed by a family of proton-linked monocarboxylate transporters (MCTs). MCTs are encoded by the SLC16A gene family, of which there are 14 known members. Only MCTs 1–4 have been shown to catalyze the proton-coupled transport of metabolically important monocarboxylates such as lactate and pyruvate. The first member of the MCT family, MCT1 (SLC16A), is well characterized and known to play a role in intestinal drug absorption. MCT1 is highly expressed at the apical side of the small and large intestine (Fig. 1), where it is responsible for the absorption of short chain fatty acids such as acetate, propionate and butyrate, produced from microbial fermentation and dietary fiber (Table 2) [16]. MCT1 is a low affinity, high capacity transporter that has been shown to transport unbranched aliphatic monocarboxylates such as acetate and propionate and substituted monocarboxylated pyruvate, lactate, acetoacetate and β-hydroxybutyrate. MCT1 substrates also include exogenous acids p-aminohippuric acid, benzoic acid, foscarnet, mevolonic acid and salicylic acid. MCT1 is thought to be responsible for the intestinal absorption of β-lactam antibiotics such as carbenicillin indanyl sodium as well as phenethicillin and propicillin [16]. 3. Synergistic action of CYP3A4 and P-glycoprotein CYP3A4 is the most prominent oxidative cytochrome P450 enzyme present in human enterocytes. Despite the lower CYP3A4 content in the intestine relative to the liver, first-pass metabolism in the intestine by CYP3A affects a large number of drugs. CYP3A4 has

Please cite this article as: M. Estudante, et al., Intestinal drug transporters: An overview, Adv. Drug Deliv. Rev. (2012), http://dx.doi.org/ 10.1016/j.addr.2012.09.042

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Fig. 2. Cartoon depicting CYP3A4 and P-glycoprotein interplay in the enterocytes. In Mol Pharm. 2009 [34].

conclusively been shown to be important in the disposition of midazolam [158] and cyclosporine [159] from studies in anhepatic patients. Drug interaction studies performed with grapefruit juice (inhibitor of intestinal CYP3A) have also shown significant increases in the oral bioavailability of many CYP3A4 substrates including felodipine [160]. As discussed above, drug absorption can also be impaired by efflux transporters in the intestine [161]. The synergistic action of CYP3A and P-glycoprotein in limiting oral drug delivery is suggested by their joint presence in small intestine enterocytes, the significant overlap in their substrate specificities and the poor oral bioavailability of joint substrates for CYP3A and P-glycoprotein [162]. These proteins are induced or inhibited by many of the same compounds [163,164]. In the enterocyte, the spatial separation of P-gp, located on the apical plasma membrane, and that of CYP3A4, located in the endoplasmatic reticulum, supports the idea that P-gp may control the access of drugs to intracellular metabolism by CYP3A4. Drugs absorbed into the intestinal epithelium can interact with P-gp and be actively extruded back into the intestinal lumen. If this process of diffusion and active transport occurred repeatedly, the circulation of the drug from the lumen to the intracellular compartment would potentially prolong the intracellular residence time of the drug, decrease the rate of absorption, and result in increased drug metabolism by CYP3A4 relative to the parent drug crossing the intestine (Fig. 2). Support for this hypothesis was obtained from a simulation model [165] as well as experimental studies examining the metabolism and transport of indinavir in vitamin D3-induced Caco-2 cells [166]. Further studies by Cummins et al. [161] were designed to identify the contribution of P-gp in determining the extent of CYP3A4 drug metabolism using selective CYP3A4/P-gp substrates and inhibitors in CYP3A4-transfected Caco-2 monolayers. Two compounds were tested: one a dual P-gp and CYP3A4 substrate (K77: N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl) and the other only a CYP3A4 substrate (felodipine). The substrates were administered with the inhibitors cyclosporine (dual inhibitor of P-gp and CYP3A4) or GG918 (inhibitor of P-gp and not CYP3A4). When P-gp alone was inhibited, the extent of metabolism of the dual CYP3A4 and P-gp substrate was decreased, but there was no change in the extent of metabolism for the exclusive CYP3A4 substrate. These data indicate that P-gp, when active, can work in concert

with CYP3A4 to increase metabolism. Studies using the in vivo rat single pass intestinal perfusion model were performed to determine whether a similar drug metabolism–efflux alliance was present in vivo[167]. The results obtained were the first to show the specific interaction of P-gp with CYP3A in this isolated organ and support the proposed interplay between P-gp and CYP3A in the intestine [167]. As stated above, in the intestine, inhibition of the efflux transporter P-gp with no effect on the enzyme will decrease metabolism. Thus, when both the enzyme and the efflux transporter are inhibited, a significant decrease in metabolism will be observed with the achievement of higher AUCs. These results revealed that metabolism can be altered by changes that occur only in drug transport. Incorporating efflux, as well as uptake processes, affecting drug absorption and disposition should lead to better predictions of drug clearances from in vitro systems [168]. While the interplay between P-gp and CYP3A4 has been extensively studied, this phenomenon may also occur with other enterocytic drug metabolizing enzymes (CYP3A4, CYP2C9, CYP2C19, CYP2C8, CYP2D6,

Fig. 3. The Biopharmaceutics Drug Disposition Classification System (BDDCS) after Wu and Benet. In Pharm Res. 2005 [9].

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M. Estudante et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

esterases, epoxide hydrolases, UGT1A1, UGT1A7-10, SULT1E1, SULT2A1, SULT1A3, N-acetyltransferases, glutathione-S-transferases) and other apical efflux transporters (BCRP, MRP2) [12,75].

4. BDDCS prediction of transporter effects Amidon et al. [169] devised a Biopharmaceutics Classification System (BCS) that categorized drugs into four classes based on aqueous solubility and intestinal permeability, fundamental parameters controlling the rate and extent of drug absorption from immediate release (IR) solid oral dosage forms. In 2005, Wu and Benet [9] recognized that the overwhelming majority of Classes 1 and 2 high permeability drugs were extensively metabolized, while for the great majority of Classes 3 and 4 compounds elimination was predominantly via biliary and/or renal excretion of unchanged drug. They suggested that changing the permeability component to a route of elimination component in a Biopharmaceutics Drug Disposition Classification System (BDDCS) (Fig. 3) may be useful in predicting overall drug disposition for new molecular entities, including routes of drug elimination, potential for drug–drug interactions and the effects of efflux and absorptive transporters on oral drug-absorption [9]. Both uptake and efflux transporters in the intestine [161,167,168] and liver [170–173] are important in determining oral drug disposition by controlling absorption and bioavailability. As described above, some drugs may fulfill the BCS 90% permeability criteria because of the activity of uptake transporters in the intestine, rather than just due to high passive permeability [174]. Some of these compounds, including D-glucose [175], levodopa, L-leucine and phenylalanine [176], are known to be absorbed via an endogenous carrier mediated mechanism. Thus, some drugs listed as highly permeable according to BCS may show marked changes in bioavailability when intestinal uptake transporters are inhibited. Moreover, it was demonstrated that interplay between transporters and intestinal metabolic enzymes may control the access of drug molecules to the enzymes and changes in transporter function can modulate intestinal metabolism without changes of enzyme activity [34,168]. There is an ongoing debate among the scientific community regarding the role of transporters in drug intestinal bioavailability and intestinal bioavailability predictions from in vitro systems [9,12,36,177–179]. The body of data evaluating the role of transporters and transporter– enzyme interplay led Wu and Benet [9] to the generalizations regarding transporter effects following oral dosing depicted in Fig. 4.

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4.1. Class 1 compounds The gut lumen of the gastrointestinal tract is sufficiently leaky so that small molecular weight, soluble, non-polar compounds (i.e. Class 1 compounds) readily pass through the membrane. Alternatively, the high permeability/high solubility of Class 1 drugs allows high concentrations in the gut lumen and enterocytes to saturate any transporter, both efflux and absorptive [43]. That is, Class 1 compounds may be substrates for both uptake and efflux transporters in vitro in cellular systems under the right conditions (for example, midazolam [180] and verapamil [181] are reported to be substrates for P-glycoprotein), but transporter effects will not be important clinically because their role is insignificant compared to passive diffusion. Cao et al. [182] demonstrated this in situ with an intestinal rat perfusion model in which they determined verapamil permeabilities and showed that they were independent of P-gp expression level, which increased 6-fold along the intestinal tract from the duodenum to the ileum. Very recently, Broccatelli et al. [44] demonstrated that this lack of clinically relevant transporter effects for BDDCS Class 1 drugs also held for the brain. 4.2. Class 2 compounds For Class 2 drugs, BDDCS predicts that the high permeability will allow ready access into the gut membranes making intestinal uptake transporters generally unimportant due to the rapid permeation of the drug molecule into the enterocytes. That is, absorption of Class 2 compounds is primarily passive and a function of lipophilicity. However, the low solubility of these compounds will limit the concentrations coming into the enterocytes, thereby preventing saturation of the efflux transporters. Consequently, efflux transporters will affect the extent of oral bioavailability and the rate of absorption of Class 2 drugs. Sulfasalazine has been classified as a BCS Class 4 drug, due to its low intestinal permeability but in BDDCS it is a Class 2 drug [183]. Consistent with this BDDCS class, the MRP2 inhibitor indomethacin was found to significantly increase sulfasalazine permeability [69]. Sulfasalazine is an example of a BDDCS Class 2 drug affected by efflux transporters in the gut. Moreover, there will be little opportunity to saturate intestinal enzymes such as CYP3A4 and UDP-glucuronosyltransferases (UGTs) due to the low drug solubility. Thus, changes in transporter expression, and inhibition or induction of efflux transporters will cause changes in intestinal metabolism of drugs that are substrates for the intestinal enzymes. Many Class 2 compounds, particularly the immunosuppressants cyclosporine [184] and tacrolimus [170] (Table 1), are primarily substrates for CYP3A as well as substrates or inhibitors of the efflux transporter P-glycoprotein as previously reviewed by Shugarts and Benet [12,34]. 4.3. Class 3 and Class 4 compounds

Fig. 4. Transporter effects, following oral dosing, by BDDCS class. In Pharm Res. 2005 [9].

For Class 3 compounds, sufficient drug will be available in the gut lumen due to good solubility, but an absorptive transporter will be necessary to overcome the poor permeability characteristics of these compounds. However, intestinal apical efflux transporters may also significantly decrease the absorption of such compounds when sufficient enterocyte penetration is achieved via an uptake transporter. That is, since influx of Class 3 compounds will generally be rate limited by an absorptive transporter, the counter effects of efflux transporters will not be saturated and can also be important. In general, these principles also hold for Class 4 compounds, although Class 4 drugs may achieve sufficient solubility in the natural surfactant containing gut contents so that they act like Class 3 compounds (Fig. 3). Uptake transporters effects on Class 3 drugs are demonstrated by the role of OATP2A1 on fexofenadine (Class 3 drug) absorption [145,147,148]. Also the Class 3 compounds famotidine and cimetidine

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showed a P-gp dependent permeability determined in rat in situ intestinal perfusion studies [185]. BDDCS Class 3 compounds digoxin and talinolol are well-known substrates for P-glycoprotein and drug– drug interactions are well characterized for these two drugs (Tables 3 and 4).

5. Intestinal permeability assessment Due to the multivariate processes involved in the intestinal absorption of drugs it is often difficult to accurately predict the in vivo permeability characteristics of a compound. Several physicochemical parameters, in vitro and in vivo models can be used to estimate intestinal permeability [186]. Important physicochemical parameters are size, charge, lipophilicity and hydrogen-bonding potential as well as other molecular descriptors [187,188]. In vitro methods include artificial lipid membranes such as parallel artificial membrane permeability assays (PAMPA), cell based systems such as Caco-2 cells, Mardin– Darby canine kidney cells (MDCK) [36] and tissue based assays like the intestinal everted sac [189,190], intestinal membrane vesicles [191] and the Ussing chamber [192,193]. In situ methods consist of intestinal single pass perfusion [194,195] and Thiry–Vella loops [196]. In vivo methods involve whole animal absorption studies [197,198], including the use of knock out animals for the transporter of interest [199,200]. In vitro techniques for assessment of permeability are less laborious and cost intensive compared to in vivo animal studies. However, the effect of physiological factors such as gastric emptying rate and GI transit rate are not taken into account, and thus not incorporated into the data interpretation. One or more of these methods can be used as a screening tool for the assessment of GI permeability [197]. Another important limiting feature is that healthy intestinal epithelial permanent cell lines are not commercially available. Despite efforts to maintain them in culture, major problems such as low viability and short life-times are still not overcome. Therefore, immortalized (tumor) cell lines that grow rapidly into confluent monolayers have been developed and currently enjoy widespread popularity. These cells exhibit several characteristics of differentiated epithelial cells providing an ideal system for the rapid assessment of the intestinal permeability of drugs [198]. Transfected cell lines are widelly used to study a particular transporter [173,201] or group of transporters [202]. Examples of cell models that are used routinely for permeability screening are listed in Table 7. CYP3A4 is the most prominent oxidative cytochrome P450 enzyme present in the intestine and plays a significant role in first-pass metabolism [168]. One issue with the use of cell lines is that they do not express CYP3A4 and thus the transporter–enzyme interaction cannot be ruled out. Although Caco-2 cell based models are known to include adequate amounts of hydrolase, esterase and brush border enzymes [198], they fail to simulate a complete in vivo intestinal environment because they do not express adequate quantities of CYP3A4, the main CYP present in human epithelial cells. Efforts have been made to develop Caco-2 cells expressing high levels of cDNA-derived CYP3A4, either by modification of the growth media or using a transfection strategy [198]. Cummins and colleagues [161,203] successfully used CYP3A4-transfected Caco-2 cells as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption of sirolimus, K77, felodipine and midazolam. Because in vivo studies performed with humans and laboratory animals are expensive, time consuming and often even unethical, in vitro methods, as accurate as possible, are needed for screening new drug candidates [204]. Computational or virtual screening has received much attention in the last few years since in silico predictive models would minimize extremely time consuming steps of synthesis as well as experimental studies [197,205,206].

6. Conclusions One area of increasing interest is that of membrane transporters localized in the intestine. Enterocytes express several transporters belonging to the adenosine triphosphate (ATP) binding cassette (ABC) superfamily and the solute carrier (SLC) superfamilies, on the apical and basolateral membranes for the influx or efflux of endogenous substances and xenobiotics. Although a variety of transporters are expressed in the enterocyte, only a few have been investigated and are known to play a key role in the intestinal absorption of drugs. ABC transporters expressed in the intestine include P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance proteins (MRP1 and MRP2). SLC transporters suggested as relevant at the intestinal apical surface of epithelial cells include peptide transporter (PEPT1), organic anion polypeptide transporters (OATP1A2 and OATP2B1), monocarboxylate transporters (MCT1), carnitine/organic cation transporters (OCTN1 and OCTN2) and the plasma membrane monoamine transporter (PMAT). Several other SLC transporters including organic anion or cation transporters (OATs or OCTs) have also been identified in the intestine, but seem to be of less importance in oral drug absorption [16]. The current information on intestinal transporter proteins should be considered in rational design process. BDDCS predicts that drug transporter effects would not be relevant for Class 1 drugs, while efflux transporter effects and transporter–enzyme interplay can be expected for Class 2 compounds. Enhanced effect of uptake transporters can be a very beneficial strategy for compounds that have low permeability, i.e. compounds in BDDCS Classes 3 and 4. Further work is needed to characterize the effect of gut apical transporters on Class 3 and 4 drugs. Also, the influence of intestinal basolateral uptake and efflux transporters has not yet been examined in depth. Another aspect that has been underestimated is the possible interaction between intestinal enzymes and efflux and uptake transporters in the intestine. To date only the intestinal CYP3A4–P-gp interaction has been described in detail. While there is a large body of knowledge regarding the effect of intestinal efflux proteins on oral drug absorption, there is an Table 7 Cell culture models used for drug permeability assessment. Cell line Species of origin

Special characteristics

Caco-2

Human colon adenocarcinoma

MDCK

Mardin–Darby canine kidney cells

Most well-established cell model. Differentiates and expresses some relevant efflux transporters. Expression of uptake transporters is variable. Polarized cells with low intrinsic expression of ABC transporters. Ideal for transfections. (e.g. MDR1-MDCK). Polarized cells with low intrinsic transporter expression. Ideal for transfections.

LLC-PK1 Pig kidney epithelial cells Lewis lung carcinoma-porcine kidney 2/4/A1 Rat fetal intestinal epithelial cells

TC-7 T84

Caco-2 subclone Human carcinoma

HT-29 IEC-18

Human colon Rat small intestinal cell line Human embryonic kidney Cervical cancer cells

HEK HeLa

Temperature-sensitive. Ideal for paracellularly absorbed compounds because of leakier pores with 9.0 ± 0.2 Å, similar to human small intestine pores. TEER of 50 Ω·cm2. Contains brush-border enzymes as well as transporter proteins. Similar to Caco-2. Have receptors for many peptide hormones and neurotransmitors; maintains vectorial electrolyte transport. Contains mucus-production goblet cells. Provides a size-selective barrier for paracellularly transported compounds. Used for transfections. Wide use in research.

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opportunity to explore the role of intestinal uptake transporters. Some interactions thought to be P-gp or CYP3A4 related, like interactions with grapefruit juice, have been recently revealed to be mediated by inhibition of intestinal uptake transporters, like OATPs as well. Also, PMAT has now been suggested to be the basis for the saturable intestinal absorption of metformin. A major issue remaining in the study of drug transporters effects is that many drugs are substrates of multiple membrane transporters and drug metabolizing enzymes, and that inhibitors frequently affect both metabolism and transport of a specific drug. Future work is needed to clarify these complex interactions. There is a need to find specific transporter substrates and inhibitors that allow a detailed characterization of the individual transporters. It is necessary to determine the contribution of each transporter to the overall transport process because such information enables the prediction of changes in membrane permeability when the functions of transporters are altered, e.g., by genetic polymorphism, disease state, or drug–drug interactions. It is therefore very difficult to quantitatively predict drug–drug interactions from in vitro data [22]. Human pharmacokinetic studies and human in vivo intestinal perfusion studies are expensive and time consuming. The use of representative animal models is less expensive and time consuming but there are species differences in expression and substrate profiles for transporters and metabolic enzymes that can lead to discrepancy between human and animal results. The use of human cell lines and cell lines transfected with human transporters has been widely used but care must be taken as the expression levels of transporters and enzymes are not representative of those seen in vivo. Improved in vitro methods and models that allow the reliable prediction of the situations in humans are needed. References [1] K.M. Giacomini, S.M. Huang, D.J. Tweedie, L.Z. Benet, K.L. Brouwer, X. Chu, A. Dahlin, R. Evers, V. Fischer, K.M. Hillgren, K.A. Hoffmaster, T. Ishikawa, D. Keppler, R.B. Kim, C.A. Lee, M. Niemi, J.W. Polli, Y. Sugiyama, P.W. Swaan, J.A. Ware, S.H. Wright, S.W. Yee, M.J. Zamek-Gliszczynski, L. Zhang, Membrane transporters in drug development, Nat. Rev. Drug Discov. 9 (2010) 215–236. [2] Y. Shitara, T. Horie, Y. Sugiyama, Transporters as a determinant of drug clearance and tissue distribution, Eur. J. Pharm. Sci. 27 (2006) 425–446. [3] M.K. DeGorter, R.B. Kim, Hepatic drug transporters, old and new: pharmacogenomics, drug response, and clinical relevance, Hepatology 50 (2009) 1014–1016. [4] P. Li, G.J. Wang, T.A. Robertson, M.S. Roberts, Liver transporters in hepatic drug disposition: an update, Curr. Drug Metab. 10 (2009) 482–498. [5] K.N. Faber, M. Muller, P.L. Jansen, Drug transport proteins in the liver, Adv. Drug Deliv. Rev. 55 (2003) 107–124. [6] J.E. van Montfoort, B. Hagenbuch, G.M. Groothuis, H. Koepsell, P.J. Meier, D.K. Meijer, Drug uptake systems in liver and kidney, Curr. Drug Metab. 4 (2003) 185–211. [7] R. Kikuchi, S. Yagi, H. Kusuhara, S. Imai, Y. Sugiyama, K. Shiota, Genome-wide analysis of epigenetic signatures for kidney-specific transporters, Kidney Int. 78 (2010) 569–577. [8] S. Oswald, M. Grube, W. Siegmund, H.K. Kroemer, Transporter-mediated uptake into cellular compartments, Xenobiotica 37 (2007) 1171–1195. [9] C.Y. Wu, L.Z. Benet, Predicting drug disposition via application of BCS: transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system, Pharm. Res. 22 (2005) 11–23. [10] M.A. Hediger, M.F. Romero, J.B. Peng, A. Rolfs, H. Takanaga, E.A. Bruford, The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction, Pflugers Arch. 447 (2004) 465–468. [11] Y. Sai, Biochemical and molecular pharmacological aspects of transporters as determinants of drug disposition, Drug Metab. Pharmacokinet. 20 (2005) 91–99. [12] S. Shugarts, L.Z. Benet, The role of transporters in the pharmacokinetics of orally administered drugs, Pharm. Res. 26 (2009) 2039–2054. [13] R.M. Franke, E.R. Gardner, A. Sparreboom, Pharmacogenetics of drug transporters, Curr. Pharm. Des. 16 (2010) 220–230. [14] C.D. Klaassen, L.M. Aleksunes, Xenobiotic, bile acid, and cholesterol transporters: function and regulation, Pharmacol. Rev. 62 (2010) 1–96. [15] Y. Shu, Research progress in the organic cation transporters, J. Cent. South Univ. (MedSci) 36 (2011) 913–926. [16] M.V. Varma, C.M. Ambler, M. Ullah, C.J. Rotter, H. Sun, J. Litchfield, K.S. Fenner, A.F. El-Kattan, Targeting intestinal transporters for optimizing oral drug absorption, Curr. Drug Metab. 11 (2010) 730–742. [17] T.G. Tucker, A.M. Milne, S. Fournel-Gigleux, K.S. Fenner, M.W. Coughtrie, Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast

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Please cite this article as: M. Estudante, et al., Intestinal drug transporters: An overview, Adv. Drug Deliv. Rev. (2012), http://dx.doi.org/ 10.1016/j.addr.2012.09.042