Review Intestinal Absorption of Drugs Mediated by Drug Transporters: Mechanisms and Regulation

Drug Metab. Pharmacokin. 18 (1): 1–15 (2003).

Toshiya KATSURA and Ken-ichi INUI Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto, Japan

Summary: The absorption of drugs from the gastrointestinal tract is one of the important determinants for oral bioavailability. Development of in vitro experimental techniques such as isolated membrane vesicles and cell culture systems has allowed us to elucidate the transport mechanisms of various drugs across the plasma membrane. Recent introduction of molecular biological techniques resulted in the suc- cessful identiˆcation of drug transporters responsible for the intestinal absorption of a wide variety of drugs. Each transporter exhibits its own substrate speciˆcity, though it usually shows broad substrate speciˆcity. In this review, we ˆrst summarize the recent advances in the characterization of drug transporters in the small intestine, classiˆed into peptide transporters, organic cation transporters and organic anion transporters. In particular, peptide transporter (PEPT1) is the best-characterized drug transporter in the small intestine, and therefore its utilization to improve the oral absorption of poorly absorbed drugs is brie‰y described. In addition, regulation of the activity and expression levels of drug transporters seems to be an important aspect, because alterations in the functional characteristics andWor expression levels of drug transporters in the small intestine could be responsible for the intra- and interindividual variability of oral bioavailability of drugs. As an example, regulation of the activity and expression of PEPT1 is summarized.

Key words: intestinal absorption; transporter; brush-border membrane; basolateral membrane; Caco-2

transepithelial transport properties of various com- Introduction pounds. This cell line forms con‰uent monolayers of The absorption of drugs from the gastrointestinal well-diŠerentiated enterocyte-like cells with functional tract is one of the important determinants for oral properties of transporting epithelia4) and has extensively bioavailability. It has long been considered that intesti- been used to characterize intestinal transport mechan- nal absorption of drugs after oral administration is isms of various drugs.5–8) Using isolated intestinal brush- mediated by a simple diŠusion process, which depends border membrane vesicles or Caco-2 cells, transport on physicochemical properties of drugs such as characteristics of various drugs have been investigated, hydrophobicity and ionizing state. However, there have and the following criteria were usually used to deˆne the been numerous drugs exhibiting higher absorption rates transporter-mediated permeation across the plasma after oral administration than expected from their phys- membrane: saturability, temperature-dependence, icochemical properties. In the 1980's, development of in energy-dependence, cis-inhibition and trans-stimulation vitro experimental techniques such as isolated mem- eŠects by related compounds. Modifying reagents for brane vesicles and cell culture systems has allowed us to amino acid residues of the membrane proteins were elucidate the transport mechanisms of various drugs as sometimes used to see whether transport process was well as nutrients across the plasma membrane of small mediated by transport proteins (transporters). Extensive intestine and renal proximal tubules. The development surveys concerning the intestinal absorption mechan- of methods isolating the brush-border and basolateral isms for various ionic drugs revealed that drug transmembrane vesicles from these epithelial cells permitted porters were mainly classiˆed into three systems; organ- to characterize the detailed mechanisms involved in the ic cation transport systems, organic anion transport transport of various compounds across each mem- systems and peptide transport systems. However, except brane.1–3) In addition, the human colon adenocarcinoma for a few drugs, less attention had been paid to trans- cell line Caco-2 has been introduced to characterize the porter-mediated drug absorption in drug discovery and

Received; January 9, 2003, Accepted; March 6, 2003 To whom correspondence should be addressed: Prof. Ken-ichi INUI,Ph.D.,Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. Tel. ＋81-75-751-3577, Fax. ＋81-75-751-4207, E-mail: inui＠kuhp.kyoto-u.ac.jp

1 2ToshiyaK2 ATSURA and Ken-ichi INUI

Table 1. Major transporter families involved in drug absorption and disposition

Transporter Family HGNCa) Typical Substrates

ABC transporter MDR family (MDR1 etc.) ABCB hydrophobic compounds, anticancer agents, digoxin, immunosuppressants MRP family (MRP2, MRP3 etc.) ABCC anionic conjugates, anticancer agents, methotrexate, pravastatin

Peptide transporter PEPT family (PEPT1, PEPT2) SLC15 diWtripeptides, b-lactam antibiotics, bestatin, valacyclovir

Monocarboxylic acid transporter MCT family (MCT1 etc.) SLC16 lactic acid, salicylic acid

Organic anion transporter OATPWoatp family (OATP-C etc.) SLC21 taurocholic acid, estradiol 17b-glucuronide, sulfobromophthalein, thyroxin, pravastatin Organic ion transporter SLC22 OAT family (OAT1, OAT3 etc.) p-aminohippuric acid, b-lactam antibiotics, estrone-3-sulfate, methotrexate, cimetidine OCT family (OCT1, OCT2 etc.) tetraethylammonium, choline, dopamine, 1-methyl-4-phenylpyridinium, cimetidine OCTN family (OCTN1, OCTN2 etc.) L-carnitine, tetraethylammonium

Nucleoside transporter CNT family (CNT1, CNT2 etc.) SLC28 purineWprimidine nucleoside, nucleoside derivatives ENT family (ENT1, ENT2) SLC29 purineWprimidine nucleoside, nucleoside derivatives a) Gene family nomenclature classiˆed by the Human Gene Nomenclature Committee (HGCN). See its homepage (http://www.gene.ucl.ac.ukW nomenclatureWgenefamily.shtml) for details. development stage and in clinical situations such as disposition. drug-drug interactions. In this review, we describe the recent studies concern- During the last decade, molecular biological tech- ing the intestinal absorption of drugs mediated by drug niques have been employed to identify drug transporters transporters. Although the fundamental characteristics responsible for drug absorption from the intestinal of each transporter and the classiˆcation of each trans- lumen. In 1987, Hediger et al.9) ˆrst succeeded in clon- porter family (nomenclature) are not described in detail, ing of the Na＋ Wglucose cotransporter (SGLT1) from several excellent reviews covering such information have rabbit small intestine by means of a functional expres- been published in the last few years.14–26) sion cloning strategy using Xenopus laevis oocytes.10) Transporters Involved in Drug Absorption Thereafter, various transporters for nutrients, neurotransmitters and other endogenous compounds have Peptide Transporters: Peptide transporters mediate been cloned using this technique, and PCR analyses H＋-coupled active transport of di- or tripeptides across allowed us to identify homologous genes. Since 1994, the brush-border membranes of the small intestine and when drug transporters such as peptide transporter the renal proximal tubules. The acidic luminal pH PEPT1,11) organic cation transporter OCT112) and or- generated by the Na＋ WH＋ exchanger (NHE3) expressed ganic anion transporting polypeptide oatp113) were ˆrst in the brush-border membrane serves as the driving isolated, many drug transporters have been cloned and force for the transport of small peptides (Fig. 1). Using characterized. It has been demonstrated that drug trans- intact small intestinal preparations, isolated intestinal porters are selectively expressed in pharmacokinetically brush-border membrane vesicles and Caco-2 cells, it has important tissues such as small intestine, liver, kidney been demonstrated that intestinal absorption of peptide- and brain capillary endothelial cells. Therefore, it is like drugs such as orally active b-lactam antibiotics is now well accepted that drug transporters play an im- mediated by the H＋ Wpeptide cotransport system. In portant role in drug absorption and disposition. Table 1 1994, intestinal peptide transporter was ˆrst cloned summarizes the major transporter families considered from rabbit small intestine and designated PEPT1.11) to be important determinants for drug absorption and Thereafter, PEPT1 was cloned and characterized from Intestinal Absorption of Drugs by Drug Transporters 3

L-dopa and its metabolite, dopamine, on the basolateral side was much greater than after addition of a parental compound, L-dopa. In addition, peptidic prodrugs of a novel aminomethyl tetrahydrofuranyl-1 b-methylcar- bapenem (CL 191,121) with L-amino acids such as alanine, valine, isoleucine and phenylalanine have been shown to improve the e‹cacies after oral administration, though PEPT1-mediated transport was not directly demonstrated.44) Another possible approach is to conjugate a dipeptide to parental drugs, thus produc- ing peptidyl prodrugs. Ezra et al. synthesized dipeptidyl-bisphosphonates, Pro-Phe-pamidronate and Pro- Phe-alendronate, and successfully demonstrated the improvement of oral absorption of these dipeptidyl- bisphosphonates.45) After oral administration, intestinal absorption of Pro-Phe-pamidronate and Pro-Phe- alendronate was increased 3-fold. In addition, the apical-to-basolateral transport of these prodrugs across Fig. 1. Peptide transporters in the small intestine. DiWtripeptides Caco-2 cell monolayers was much greater than that of and peptide-like drugs are absorbed across the brush-border membrane (BBM) by H＋-coupled peptide transporter (PEPT1). Intracellu- the parent drug. These in vivo and in vitro studies indi- lar peptides and peptide-like drugs exit from cells across the basolater- cated that PEPT1-mediated transport of dipeptidyl- al membrane (BLM) by an unidentiêd peptide transporter. bisphosphonates was involved in the improvement of oral absorption of bisphosphonates. However, it should be noted that this approach appeared not to be applica- human,27) rat,28) mouse,29) ovine30) and chicken.31) In all ble to any compounds. Friedrichsen et al. developed a species, PEPT1 is expressed predominantly, but not ‰exible synthetic pathway for attaching compounds to exclusively, in the small intestine. So far, PEPT1 is the dipeptides through ester or amide bonds.46) Glucose-6- best-characterized transporter mediating the drug phosphatase inhibitor was chosen as a model drug, absorption in the small intestine. PEPT1-mediated however, none of the peptide-coupled compounds transport of peptide-like drugs such as orally active b- seemed to be transported by PEPT1 in Caco-2 cells. lactam antibiotics, an anti-cancer agent bestatin, Since the low aqueous solubility of the parent com- angiotensin converting enzyme inhibitors, renin inhibi- pounds was not increased after attachment to a dipep- tors and thrombin inhibitors has been shown directly us- tide, they suggested that only compounds with a certain ing PEPT1 expressing Xenopus oocytes or cultured intrinsic aqueous solubility should be targeted to cells, or indirectly using isolated intestinal brush-border PEPT1 by attachment to a dipeptide. Recently, Nielsen membrane vesicles or Caco-2 cells. Structural features et al. reported that 4-toluenesulfonylureido-carnosine, and several parameters for PEPT1-mediated transport an anticancer agent synthesized by linking a dipeptide of these drugs were summarized in previous review arti- L-carnosine to the parent drug via an amide bond, was cles,32–38) and therefore will not be described in detail. not transported by PEPT1 in Caco-2 cells, though it Readers should also refer to the most recent reviews to inhibited the PEPT1-mediated uptake of glycylsarco- understand the recent advances in peptide transporter sine.47) research.39,40) ThesearchforthesubstrateofPEPT1is These strategies utilized dipeptidyl moiety introduced still continuing, and PD 15847341) and sulpiride,42) an into the parental drug to be recognized by PEPT1. Since NMDA receptor antagonist and an antipsychotic agent, endogenous substrates of PEPT1 are di- or tripeptides, respectively, were suggested to be transported by it was initially considered that the substrate of PEPT1 PEPT1. should possess a peptide bond and free amino andWor Due to the broad substrate speciˆcity of PEPT1, it carboxyl terminal in its structure. However, the struc- has been considered that utilization of PEPT1 is a ture-transport relationships for the interactions of vari- promising strategy for oral drug delivery. One success- ous compounds with PEPT1 revealed that a peptide ful approach to be a substrate for PEPT1 is peptidyl bond is not an absolute structural requirement to be derivation of parent compounds. It was demonstrated recognized by PEPT1. Temple et al. ˆrst demonstrated that a dipeptide-mimetic derivative of L-dopa, L-dopa- that 4-aminophenylacetic acid, a peptide mimic lacking L-Phe, was e‹ciently transported by PEPT1.43) Using a peptide bond, was transported by PEPT1 and the Caco-2 cell monolayers, it was shown that after addition presence of a peptide bond was not a requirement for of L-dopa-L-Phe on the apical side, the appearance of rapid translocation.48) Thereafter, delta-aminolevulinic 44ToshiyaKATSURA and Ken-ichi INUI acid,49) v-amino fatty acids50) and amino acid aryl protein was novel with no obvious similarity to PEPT1. amides,51) all of which lack a peptide bond, were shown Molecular identiˆcation of this protein will allow us to to be substrates for PEPT1. Almost at the same time, it understand the detailed mechanisms of the transport of was demonstrated that valacyclovir, a prodrug of an peptide-like drugs across the basolateral membrane. antiviral agent acyclovir, interacted with PEPT152) and Organic Cation Transporters: Transport mechan- was directly shown to be a good substrate for isms of various cationic drugs in the small intestine have PEPT1.53,54) Valacyclovir is the L-valyl ester of acyclovir been examined using techniques such as everted intesti- without a peptide bond and has initially been developed nal sacs, isolated intestinal mucosa mounted on Ussing to improve the poor oral bioavailability of acyclovir. chambers, isolated brush-border membrane vesicles and The ˆnding that transport of valacyclovir is mediated by Caco-2 cells. Organic cation transport systems charac- PEPT1 leads to another idea that 5?-amino acid esteriˆ- terized earlier were summarized in previous review arti- cation of poorly absorbed compounds may improve cles,63,64) and we will not discuss previously described their intestinal absorption. Indeed, transport of valyl transport systems in detail. So far, as compared with the esters of AZT54) and ganciclovir55) has been shown to be kidney and liver, molecular mechanisms of organic mediated by PEPT1. Sawada et al. demonstrated that cation transport in the small intestine remain largely to L-valyl ester compound showed higher a‹nity for be elucidated. Most organic cation transporters PEPT1 than other amino acid esters.56) Taken together, described are not identiêd yet. these ˆndings suggest that L-valyl esteriˆcation of Organic cation transporter 1 (OCT1) is the ˆrst mem- poorly absorbed drugs may be a useful and promising ber of the OCT family cloned from rat kidney in 1994.12) approach for improving their oral bioavailability. Subsequently, members of a family of organic cation In addition to the above mentioned approaches to transporters (OCT1–3), as well as the more distantly utilize the PEPT1 as the target for oral drug delivery, it related proteins that transport carnitine and organic should be understood how PEPT1 recognizes its sub- cations (OCTN1–3) and organic anion transporters strate. Bailey et al. provided a model for the orientation (OAT1–4), have been cloned and characterized. Due to of the key binding features of substrates for PEPT1.57) their signiˆcant homology, these transporters turned They showed that this model allowed us to predict the out to be members of the organic ion transporter super- relative a‹nity of substrates for PEPT1. Although crys- family (SLC22). Examination of tissue distribution of tal structure of PEPT1 is still not known, a complete the OCT family indicated that OCT1 and OCT3 are understanding of the molecular features recognized by expressed in the small intestine of rats. OCT1 is PEPT1 would be important for rational design of drug expressed much less in the human small intestine, while molecules which can be well absorbed from the small expression of OCT3 in the human small intestine is not intestine. conˆrmed. Although OCT2 is predominantly expressed Peptide-like drugs taken up by PEPT1 from the intes- in the rat kidney,65) expression of OCT2 in the human tinal lumen must then exit from the cells across the small intestine was detected by RT-PCR.66) On the anal- basolateral membrane of intestinal epithelium. Another ogy of their localization in the renal proximal tubules, peptide transporter localized at the basolateral mem- localization of OCTs in the small intestine is supposed brane should mediate this step to achieve the e‹cient to be in the basolateral membrane (Fig. 2). In addition, transepithelial transport (Fig. 1). The basolateral pep- recent studies using Oct1 knockout mouse suggested its tide transporter expressed in Caco-2 cells has been basolateral localization in the small intestine (see characterized.58,59) It was demonstrated that the below). However, direct immunolocalization data are basolateral peptide transporter is less sensitive to not available so far. Functional characterization of changes in extracellular pH than the apical PEPT1.58,60) these transporters using various heterologous expression Glycylsarcosine uptake from the basolateral side was systems revealed that these transporters showed broad shown to be mediated by a low-a‹nity, facilitated trans- substrate speciˆcity. OCTs are likely to function port system,58) whereas concentrative uptake of primarily in the elimination of cationic drugs and other fosinopril was demonstrated.60) In addition, substrate xenobiotics in tissues such as kidney, small intestine and speciˆcity of the basolateral peptide transporter is some- liver. However, the role of OCTs in the intestinal trans- what diŠerent from that of PEPT1.61) Most recently, a port of cationic drugs is poorly understood. candidate protein for the basolateral peptide transporter Recently, Jonker et al. generated mice with a targeted was identiêd from the rat small intestine.62) By pho- disruption of the organic cation transporter 1 (Oct1W toa‹nity labeling using [4-azide-3,5-3H-D-Phe]-L-Ala as Slc22a1) gene.67) In Oct1-W- mice, intestinal secretion of a probe, a single protein band of 112 kDa was identiêd tetraethylammonium was reduced about two-fold after in the basolateral membrane of rat small intestine. intravenous administration (Table 2), indicating that MALDI-TOF analysis of tryptic digests of the protein Oct1 plays an important role in the secretion of organic followed by database searches established that this cations into the small intestinal lumen. In contrast, Intestinal Absorption of Drugs by Drug Transporters 5

the apical-to-basolateral transport, indicating the net secretion of MPP＋. However, addition of tetraethylammonium or decynium-22, inhibitors of OCTs, had no eŠect on the basolateral-to-apical transport of MPP＋, suggesting the lack of involvement of OCT1 and OCT2 in the secretion of MPP＋. They also indicated the Na＋- dependent MPP＋ uptake from the apical side of Caco-2 cells grown on permeable supports. Although the nature of this novel Na＋-dependent transporter is not clear, the results suggest the existence of a novel Na＋-coupled organic cation transporter in the intestinal brush-border membrane. In contrast, Martel et al.72) have suggested that apical uptake of MPP＋ by Caco-2 cells is mediated by Na＋-independent transporters, probably OCT1 and OCT3 (they termed EMT as Extraneuronal Monoamine Transporter). However, they evaluated the uptake of MPP＋ by Caco-2 cells grown on plastic dishes and considered the amount of MPP＋ accumulated as the apical Fig. 2. Organic cation and anion transporters in the small intestine. uptake. Since the accumulation of substrates by Characteristics of each transporter are described in the text. In some epithelial cells grown on plastic dishes does not neces- cases, membrane localization and preferential transport direction are sarily re‰ect the apical uptake, and OCTs are considered not determined. BBM, brush-border membrane; BLM, basolateral membrane. to be located on the basolateral membrane, it seems un- likely that OCT1 and OCT3 mediate the apical uptake of organic cations.

14 - - Other organic cation transporters yet to be identiêd Table 2. Excretion of [ C]tetraethylammonium in Oct1 W and wild- type (wt) mice (From ref. 67) should exist in the small intestine. As mentioned above, transport mechanisms of various cationic drugs have Mean excretion (z) - - been investigated using a variety of techniques. Tissue Ratio (Oct1 WWwt) wt Oct1- W- Mizuuchi et al. examined the transport characteristics of diphenhydramine, an antihistamine, in Caco-2 cells.74) Bile 0.35±0.09 0.14±0.01* 0.41 Small intestine 1.31±0.19 0.67±0.09** 0.51 Diphenhydramine uptake by Caco-2 cells was tempera- Cecum 0.12±0.02 0.09±0.04* 0.72 ture-dependent, saturable and pH-dependent. Chlor- Colon 0.03±0.01 0.04±0.01 1.23 pheniramine, another antihistamine, competitively Urine 53.3±16.8 80.0±15.6* 1.50 inhibited the diphenhydramine uptake, whereas 14 tetraethylammonium and cimetidine, typical substrates After i.v. administration of [ C]tetraethylammonium (0.2 mgWkg), bile was collected every 10 min for 1 h and organs, feces, and urine of OCTs, had no eŠect. In addition, biological amines were collected after 1 hr. Each value represents the mean±S.D. of 4 and neurotransmitters, such as histamine and choline, mice. *Pº0.05, **Pº0.01. did not inhibit the diphenhydramine accumulation. These results suggested that transport of diphenhydramine is mediated by a pH-dependent speciˆc transport small intestinal distribution of 1-methyl-4-phenyl- system in Caco-2 cells. Detailed investigation of the sub- pyridinium (MPP＋)inOrct3 (mouse homologue of strate speciˆcity of this transport system indicated the OCT3)-null mice was not diŠerent from wild-type existence of a novel pH-dependent tertiary amine trans- mice.68) Using the Oct1 knockout mice, Wang et al. port system that recognizes N, N-dimethyl or N, N- clearly demonstrated the involvement of Oct1 in hepatic diethyl moieties.75) Although pH-dependent transport of and intestinal distribution of a biguanide, metformin.69) diphenhydramine suggested the involvement of H＋ W These results indicate that OCT1 mediates the basolater- organic cation antiport system in diphenhydramine al uptake of organic cations from the blood in the small uptake, it is possible that diphenhydramine transport is intestine as well as in the liver. regulated not by H＋ gradient but by pH itself. There- In Caco-2 cells, the expression of OCT1, OCT2 and fore, to determine the driving force of the tertiary amine OCT3 has been demonstrated.70–73) Bleasby et al.investi- transport system, the transport of procainamide, gated the transport characteristics of organic cations another tertiary amine with an N, N-diethyl moiety, by across Caco-2 cell monolayers using MPP＋ as a typical rabbit intestinal brush-border membrane vesicles was substrate.71) It was demonstrated that the basolateral-to- examined.76) The data showed that an outward H＋ apical transcellular transport of MPP＋ was greater than gradient serves as the driving force for procainamide 66ToshiyaKATSURA and Ken-ichi INUI uptake by intestinal brush-border membrane vesicles. pH-dependent, saturable transport of anionic drugs. This H＋ gradient-dependent transport system speciˆcal- Direct demonstration of MCT-mediated transport using ly recognizes tertiary amines and is diŠerent from previ- expression systems is necessary to conclude the MCT- ously characterized H＋ Wguanidine antiport system77) mediated absorption of anionic drugs. Indeed, and H＋ Wthiamine antiport system.78) Taken together, Okamura et al. recently demonstrated that the uptake of these ˆndings clearly indicate that organic cations with nateglinide, an oral hypoglycemic agent, by Caco-2 cells N, N-dimethyl or N, N-diethyl moieties such as diphen- was saturable, pH-dependent and inhibited by various hydramine and procainamide are transported by a novel monocarboxylic acids.87) Although nateglinide inhibited H＋ Wtertiary amine antiport system in the intestinal the MCT1-mediated uptake of L-lactic acid in MCT1- brush-border membrane. Judging from its substrate expressing Xenopus oocytes, nateglinide itself was not speciˆcity, it seems likely that characteristics of this transported by MCT1.87) transporter are diŠerent from those of the OCT family Other organic anion transporters have been charac- of organic cation transporters. This transport system terized extensively in the kidney and liver. Organic seems to play an important role in intestinal absorption anion transporters in these tissues are classiêd into two andWor secretion of tertiary amine compounds. families: organic anion transporter (OAT) family and Other cationic drugs such as azasetron79) and nico- organic anion transporting polypeptide (OATPWoatp) tine80) have been reported to be transported by speciˆc family.15,17,18,20–24) OAT1 is the ˆrst member of the OAT transport systems. It was reported that azasetron uptake family identiêd in 1997,88) and it is almost exclusively by Caco-2 cells is saturable, Na＋-independent, and expressed in the kidney. So far, members of a family of inhibited by various organic cations such as imipramine, OAT identiêd were not expressed in the small intestine, desipramine and serotonin.79) This transport system and therefore the role of OAT family members in the seemed to be diŠerent from the Na＋ and Cl－-dependent intestinal absorption of drugs seems to be negligible. serotonin transporter. It was demonstrated that trans- OATP (human)Woatp (rodents) family members are port of nicotine in Caco-2 cells is mediated by a pH- Na＋-independent, multispeciˆc organic anion trans- dependent speciˆc transport system distinct from the porters that mediate the transport of various am- H＋ Wtertiary amine antiport system.80) Although the sub- phipathic organic anions as well as conjugated and strate speciˆcity and molecular nature of these transport unconjugated bile salts.23) Among the members of systems are still unknown, there might be multiple OATPWoatp family, their tissue distribution and sub- transporters responsible for the absorption of cationic strate speciˆcity is diŠerent from each other. The ˆrst drugs in the small intestine. member of this family, oatp1, was cloned from rat liver Organic Anion Transporters: Intestinal absorption in 1994.13) Thereafter, other members were identiêd mechanisms of anionic drugs have been mainly from rat, mouse and human. Comparison of tissue explained by the passive diŠusion of nonionized com- distribution and substrate speciˆcity of these members pounds according to the pH-partition theory, because revealed that there were species diŠerences in the expres- intestinal lumen is more acidic than the intracellular pH. sion and function of each transporter. However, several studies have suggested the involve- It was demonstrated that rat oatp3 mRNA is ment of speciˆc transporters in intestinal absorption of expressed at similar levels down the length of the small weak acids, especially short-chain fatty acids such as intestine.89) Oatp3 is localized to the brush-border acetic acid and butyric acid (Fig. 2). Monocarboxylic membrane of rat jejunum by immuno‰uorescence and acid transporter (MCT) family, originally cloned from mediates the transport of various bile acids, suggesting Chinese hamster ovary cells as a lactate transporter that rat oatp3 is the facilitative transporter responsible MCT1,81) mediates the transport of various short-chain for the jejunal absorption of bile acids as well as anionic fatty acids.82) Although it was clearly demonstrated that drugs (Fig. 2). It was also demonstrated that the mouse members of MCT family transport various short-chain oatp3 gene was localized to a region of chromosome 6 fatty acids, their role in the intestinal absorption of syntenic with human chromosome 12p12, where human anionic drugs remains to be elucidated. HMG-CoA OATP-A gene was mapped. Walters et al. therefore reductase inhibitors such as pravastatin and atorvastatin suggested that rodent oatp3 is orthologous to human have been suggested to be transported by MCT1, OATP-A.89) However, OATP-A was not expressed in though no direct evidence for MCT1-mediated trans- the human small intestine but expressed predominantly port of these drugs has been shown.83,84) Since transport in the brain. Tamai et al. examined the expression of weak acids such as salicylic acid could be explained proˆles of human members of the OATP family by RT- not only by a MCT1-mediated mechanism85) but also by PCR and demonstrated the expression of OATP-B, -D a physical process of nonionic diŠusion of protonated and -E in the human small intestine, but not OATP-A compound and ion trapping of deprotonated form,86) and -C.90) Kullak-Ublick et al. reported the expression care must be taken to interpret the data that indicate the of OATP-B in the small intestine by Northern blot Intestinal Absorption of Drugs by Drug Transporters 7

Fig. 3. Mean plasma fexofenadine concentration-time proˆles for persons (n＝10) orally administered fexofenadine (120 mg) with 300 mL water, grapefruit juice at 25z of regular strength (25z GFJ), grapefruit juice (GFJ), orange juice (OJ), or apple juice (AJ) followed by 150 mL of the same ‰uid every 0.5 to 3 hours (total volume, 1.2 L). (From ref. 92)

analysis, though OATP-B did not transport bile salts.91) Membrane localization of OATPs in the small intestine Fig. 4. ATP-dependent eŒux transporters in the small intestine. P- has not been reported (Fig. 2). glycoprotein (P-gp), MRP2 and BCRP are expressed in the brush- Recently, Kim and colleagues reported that con- border membrane (BBM), whereas MRP3 is located at the basolateral comitant intake of fruit juices such as grapefruit, membrane (BLM). orange and apple juices decreased the fexofenadine AUC (Fig. 3).92) Since the metabolism of fexofenadine is negligible in human, it seems that such food-drug several drugs such as talinolol, digoxin and tacrolimus. interaction may occur at the transporter level in the MRP2 located at the brush-border membrane is consi- small intestine. Using a recombinant vaccinia virus dered to mediate the intestinal secretion of anionic expression system, they have demonstrated that human drugs as well as conjugated metabolites. In contrast, OATP-A and rat oatp1–3 mediate fexofenadine MRP3 is expressed in the basolateral membrane and uptake.92,93) These in vivo and in vitro results suggested reported to mediate the transfer of bile acids to the that fruit juices inhibited the OATP-mediated fexofena- blood. The role of MRP3 in the intestinal absorption of dine absorption, thereby reducing the oral bioavailabil- drugs needs to further be clariˆed. ity. However, as described above, OATP-A is not Although we should pay attention to eŒux transport- expressedinthehumansmallintestine.Identiˆcationof ers to elucidate the oral bioavailability of drugs, we do the human OATP family member(s) responsible for the not describe the detailed characteristics of eŒux trans- intestinal absorption of anionic drugs will allow us to porters in this article. Recent reviews focusing on the understand the food-drug interaction. eŒux transporters in the intestinal absorption process EŒux Transporters: It is now well recognized that have been published elsewhere.94–96) eŒux transporters function as an absorptive barrier and Other Transporters: Other nutrient transporters are limit the oral bioavailability of various drugs. EŒux expressed in the small intestine and have been character- transporters include ABC transporters such as P- ized extensively. Those include Na＋ Wglucose cotran- glycoprotein, breast cancer resistance protein (BCRP) sporter, amino acid transporters and nucleoside and multidrug resistance associated protein (MRP), and transporters. Interaction of various drugs with these organic ion transporters as described above. Figure 4 transporters has been examined, though such studies shows the localization of ABC transporters expressed in mainly showed the inhibitory eŠect of drugs on the the small intestine. P-glycoprotein and BCRP are transport of typical substrates. Thus, the role of expressed in the brush-border membrane and mediate nutrient transporters in the intestinal absorption of the secretion of various hydrophobic drugs including drugs needs to be clariˆed in detail. Among them, anticancer agents into the lumen. Recent studies have nucleoside transporters have relatively been well-charac- revealed that expression levels of P-glycoprotein in the terized and shown to mediate the intestinal absorption human small intestine correlate the absorption proˆle of of nucleoside derivatives used as anticancer and anti- 88ToshiyaKATSURA and Ken-ichi INUI

resulted in an increased expression of PEPT1 mRNA in the rat small intestine.102) In contrast, the expression of PEPT1 protein was decreased when fasted rats were allowed to drink an amino acid mixture.103) It is still not clear how dietary components such as amino acids and small peptides regulate the expression of PEPT1. Besides the high protein diets and dipeptide sup- plementation, the expression of PEPT1 mRNA and protein in the rat small intestine was also increased after a brief fast.103–105) The mechanism underlying the up- regulation of PEPT1 expression after a brief fast was considered to be an adaptive response to e‹ciently absorb small peptides from the intestinal lumen. Ihara Fig. 5. Correlation between the eŠective intestinal permeability et al. also demonstrated that the expression of PEPT1 (PeŠ) of cephalexin and the PEPT1 expression in rat jejunum. The results were obtained from 15 rats with jejunum in situ single pass per- was increased in rats given total parental nutrition as 105) fusion of cephalexin. The amount of PEPT1 was expressed as the ra- well as in starved rats. They speculated that tio of optical density of PEPT1 to villin in rat jejunum mucosal sam- insu‹cient nutritional supply to the whole body might ples. (From ref. 98) cause an increase in PEPT1 expression. Physiological signals aŠecting the PEPT1 expression in dietary regulation should further be determined. Recently, Pan et al. viral agents.97) It is likely that utilization of these trans- demonstrated that the intestinal PEPT1 underwent porters as targets for oral drug delivery is a promising diurnal regulation in its activity and expression.106) It is strategy, and therefore several studies have been at- possible that this diurnal rhythm of PEPT1 relates to tempted to improve the intestinal absorption of drugs. food content and feeding schedule of rats, because rodents show a nocturnal feeding behavior. Regulation of Function and Expression of Drug The expression of PEPT1 along with functional Transporters properties was also aŠected by various endogenous The function and expression levels of drug transport- and exogenous factors. Increased expression of PEPT1 ers should be under the control of various physiological was demonstrated in Caco-2 cells treated with s-recep- and exogenous stimuli. Alterations in the functional tor ligand pentazocine107) and in streptozotocin-induced characteristics andWor expression levels of drug trans- diabetic rats.108) In contrast, several factors were report- porters in the small intestine could be responsible for ed to down-regulate the expression of PEPT1. Nielsen the intra- and interindividual variability of oral et al. reported that treatment of Caco-2 cells with bioavailability of drugs often observed in the clinical epidermal growth factor resulted in a decrease in situations. As already described above, the peptide PEPT1 expression after long-term treatment.109) It transporter PEPT1 is the best-characterized transporter was also demonstrated that the expression of PEPT1 in the small intestine in relation to drug absorption. mRNA and protein was decreased in Caco-2 cells treat- Therefore, we focus on the regulation of PEPT1 activity ed with thyroid hormone.110) Moreover, Shu et al. and expression in the present review. Since a very sig- showed that lipopolysaccharide treatment in rats niˆcant correlation between expression levels of PEPT1 caused a decreased expression of PEPT1 in the small and cephalexin permeability in the rat small intestine intestine and this decrease was attenuated by the treat- was observed (Fig. 5),98) variation of PEPT1 expression ment with dexamethasone.111) It was suggested that in the small intestine could be correlated with absorp- lipopolysaccharide-induced increase in tumor necrosis tion permeability variation of peptide-like drugs. Earlier factor-a and interleukin-1 b levels seemed to mediate the works concerning the regulation of PEPT1 have been decrease in PEPT1 expression. Overall, these factors are summarized in a previous review.38) Therefore, updated likely to regulate, in coordination, the PEPT1 expres- information is mainly discussed below. sion that may vary under several disease states. The Dietary regulation of peptide transporter in the small mechanisms underlying the regulation of PEPT1 intestine had been studied. It was demonstrated that expression seemed to be at the transcriptional level andW peptide transport activity was increased in response to or post-transcriptional level (i.e. changes in mRNA high protein diets.32,34) The mechanism of this response stability). turned out to be increased expression of PEPT1 mRNA Another mechanism of the regulation of PEPT1 after and protein.99–102) Shiraga et al. reported that sup- short-term treatment has also been reported. plementation not only with casein or glycyl-phenylala- Thamotharan et al. demonstrated that short-term treat- nine but with phenylalanine in the protein-free diet ment of Caco-2 cells with insulin stimulated the activity Intestinal Absorption of Drugs by Drug Transporters 9 of PEPT1 without aŠecting the expression of PEPT1 mRNA.112) The mechanism was suggested to increase translocation of PEPT1 from a preformed cytoplasmic pool to the apical membrane, because the stimulatory eŠect of insulin was abolished by colchicine, a microtubule disrupting agent. Subsequently, stimulation of PEPT1 translocation from the intracellular com- partment was observed when Caco-2 cells were treated with a2-adrenergic receptor agonists, clonidine and UK14304113) or leptin.114) In both cases, stimulatory eŠect on PEPT1 was also abolished by colchicine. In addition, leptin treatment resulted in an increased membrane expression of PEPT1 protein and decreased intracellular PEPT1 content, without changes in PEPT1 mRNA level (Fig. 6). However, intracellular compart- ment where PEPT1 is present is not demonstrated ultrastructurally and signals mediating the translocation of PEPT1 are not known at this stage. From a pathophysiological point of view, PEPT1 was reported to play an important role in several disease states. Merlin et al. reported that PEPT1 mediated the transport of formyl-Met-Leu-Phe (fMLP), a bacteria- derived major peptide neutrophil chemotactic factor and that PEPT1-mediated fMLP uptake induced neutrophil-transepithelial migration in Caco2-BBE cells, suggesting that PEPT1 mediates intestinal in‰amma- tion115) They later demonstrated that expression of PEPT1 was observed in the human colon from patients with in‰ammatory bowel disease such as chronic ulcera- tive colitis and Crohn's disease, but not in the normal Fig. 6. Leptin modiˆes the amount of PEPT1 protein in Caco-2 cell colon.116) In addition, it was shown that PEPT1-mediat- monolayers. (a) Representative immunoblots of PEPT1 protein in ed fMLP uptake enhanced major histocompatibility membranes of leptin-treated cells (left) and in intracellular extracts complex class I surface expression. These data suggested from leptin-treated cells (right). (b) Densitometric analysis of immunoblots of PEPT1 protein. The changes are expressed as mean± the link between PEPT1 and immune eŠector status in S.E. of three to four analyses. *Pº0.05 and **Pº0.01 vs. vehicle. (c) the epithelial surface of the intestine. Colonic expres- EŠect of leptin on abundance of mRNA encoding PEPT1. Total sion of PEPT1 was also found in patients with short- mRNA was extracted from control and leptin-treated cells and sub- bowel syndrome and this up-regulation of PEPT1 ex- jected to Northern blot analysis. (From ref. 114) pression was considered to be an adaptive response in patients independent of changes in the mucosal surface area.117) In contrast to the adaptive response after mas- that ‰avonoids with epidermal growth factor-receptor sive enterectomy, it was reported that midgut resection tyrosine kinase inhibitory activity such as quercetin and caused a decreased expression of PEPT1 in rabbit prox- genistein enhance the uptake of ceˆxime, a PEPT1 sub- imal jejunum but had no eŠect on the expression in the strate.122) It is possible that inhibition of tyrosine kinase distal ileum.118) In addition, it was demonstrated that by ‰avonoids activates the apical Na＋ WH＋ exchanger expression of PEPT1 was decreased after allogeneic rat (NHE3), thereby PEPT1 activity is stimulated by main- intestinal transplantation even with tacrolimus treat- taining the transmembrane H＋ gradient. They also ment.119) reported that reduction of intracellular free Ca2＋ con- The function of PEPT1 can be altered by modulating centration by the treatment of Ca 2＋ channel blockers its driving force, H＋ gradient across the brush-border such as nifedipine caused an increased uptake of membrane. Acidic luminal pH is generated by the Na＋ W ceˆxime by Caco-2 cells, whereas ceˆxime uptake was H＋ exchanger 3 (NHE3) located at the brush-border reduced by increased intracellular free Ca 2＋ concentra- membrane of intestinal epithelium. It was demonstrated tion by Ca 2＋ ionophores.123) It was already demonstrat- that regulation of NHE3 activity by a speciˆc inhibitor ed that nifedipine administration enhanced the intesti- or cAMP indirectly modulates the PEPT1 activity in nal absorption of b-lactam antibiotics in humans.124,125) Caco-2 cells.120,121) In addition, Wenzel et al.reported Wenzel et al. suggested that alterations in intracellular 10 Toshiya KATSURA and Ken-ichi INUI free Ca 2＋ concentration aŠected the pH regulatory sys- Pharm. Sci., 87: 403–410 (1998). tems, presumably NHE3, which in turn aŠected the 8) Artursson, P., Palm, K. and Luthman, K.: Caco-2 PEPT1 activity.123) Although previous studies have sug- monolayers in experimental and theoretical predictions gested that the eŠect of nifedipine on the absorption of of drug transport. Adv. Drug Deliv. 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