Review Transporter-Mediated Drug Interactions

Drug Metabol. Pharmacokin. 17 (4): 253–274 (2002).

Review Transporter-mediated Drug Interactions

Akira TSUJI Laboratory of Innovating Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

Summary: Since 1994, researchers have isolated various genes encoding transporter proteins involved in drug uptake into and eŒux from tissues that play key roles in the absorption, distribution and secretion of drugs in animals and humans. The pharmacokinetic characteristics of drugs that are substrates for these transporters are expected to be in‰uenced by coadministered drugs that work as inhibitors or enhancers of the transporter function. This review deals with recent progress in molecular and functional research on drug transporters, and then with transporter-mediated drug interactions in absorption and secretion from the intestine, secretion from the kidney and liver, and transport across the blood-brain barrier in humans. Although the participation of the particular transporters in observed drug-drug interactions can be di‹cult to conˆrm in humans, this review focuses mainly on pharmacokinetic interactions of clinically important drugs.

Key words: transporter; uptake; eŒux; intestine; kidney; liver; blood-brain barrier

in vivo pharmacokinetic proˆle of drugs from in vitro Introduction data. Construction of in vitro expression systems, using Processes involved in metabolic biotransformations, human transporter cDNA clones, may provide as a use- especially those mediated by the cytochrome P-450 ful models for the prediction of transporter-mediated monooxygenases, have clinically signiˆcant eŠects on drug-drug interactions. Fig. 1 and Table 1 summarize the disposition of many drugs. Now, it is increasingly the transporters so far identiêd and characterized as recognized that other processes, such as transport via drug transporters in human intestine, liver, kidney, and membrane-bound systems, may also substantially in- brain capillary endothelial cells (which form the blood- ‰uence the absorption of administered drugs from the brain barrier; BBB). These transporters are classiêd as intestine, as well as distribution into various tissues, and solute transporters, for which gene symbols are preˆxed organs and secretion into urine, bile and intestinal lu- with SLC, and ATP binding cassette (ABC) transport- men. Although membrane transport of synthesitic drugs ers, for which gene symbols are preˆxed with ABC. had long been believed to proceed by simple diŠusion Modulation of the function of such membrane trans- depending on the lipophilicity of the drug molecules, the porters may potentially be responsible for certain drug- role and importance of active carrier systems in the drug interactions. This review describes several exam- transport of drugs across biological membranes is now ples of pharmacokinetic drug interaction, which are well recognized. able to be predicted from in vitro data or animal studies Recent biochemical and molecular cloning methodol- as being transporter-mediated. Although the participa- ogies have identiêd a number of transporter proteins tion of an individual transporter in an observed drug- that are expressed in organs important for drug disposi- drug interaction can be di‹cult to conˆrm in humans, tion. However, it is only in the past several years that the identiˆcation of mutations resulting in deˆcient speciˆc transporter gene cDNAs have been cloned. The transport function may provide valuable information functions of the transporter proteins have been charac- about the role of the transporter as a pharmacokinetic terized by several in vitro and in vivo techniques using determinant. Since studies on polymorphisms of human cells expressing the gene(s) or using gene-knockout drug transporters have only recently been initiated, the animals. Species diŠerences in substrate selectivity, tis- present review is limited to cases which can be clearly sue distribution, and levels of expression of the drug judged as transporter-mediated drug interaction in a transporters need to be explored in order to predict the specialized tissue in humans. In this review, the trans-

Received; June 14, 2002, Accepted; September 2, 2002 To whom correspondence should be addressed :Prof.AkiraTSUJI, Ph.D., Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanzawa 920-0934, Japan. Tel. +81-76-234-4479, Fax. +81-76-234-4477, E-mail: tsuji＠kenroku.kanazawa-u.ac.jp

253 254 Akira TSUJI

Fig. 1. Human membrane transporters expressed in intestine, kidney, liver, blood-brain barrier. porter preˆxes are indicated as m: mouse, r: rat, rb: rab- zymatic breakdown of dietary or endogenous proteins.1) bit, h: human. In 1994, a rabbit cDNA was ˆrst isolated that encodes a 707-amino-acid peptide transporter, rbPEPT1 Transporter-Mediated Drug Interactions (SLC15A1), with twelve putative membrane-spanning in Intestine regions and an unusually large hydrophilic loop having Intestinal transport of drugs by simple diŠusion several N-glycosylation sites.2) Saito et al.3) and our deˆnitely occurs. However, participation of transpor- group4) have also cloned a homologue of the rbPEPT1 ter-mediated intestinal absorption has also been demon- gene from a rat intestinal or renal cDNA library. A strated both directly and indirectly for several drugs.1) human homologue, hPEPT1, which shares 81z amino The primary function of the intestinal transporters is to acid sequence identity with rbPEPT1, was identiˆed in absorb molecules that are produced during the digestion the intestine.5) Human and rat PEPT2 (SLC15A2), of food. In addition, the intestinal epithelium is an im- cloned as a homologue of rbPEPT1, is expressed only in portant site of secretion of many substrates, including the kidney,6) while PEPT1 is expressed in the apical drugs, via eŒux transporters. Solute transporters and membrane of both intestinal and renal epithelial ABC transporters expressed in the human intestine are cells.2,3,7) Immunohistochemical detection of r, listed in Table 1. Currently available information on rbPEPT1 showed that PEPT1 is expressed only in the their membrane localization in human epithelial cells is brush-border membrane, but not the basolateral mem- summarized in Fig. 1. Relatively little is yet known brane, in both species.3,5) hPEPT1, like rPEPT1,3–5) about the substrates (including drugs) transported by transports several b-lactam antibiotics, ACE inhibi- these molecules. tors,7–9) and a non-peptide prodrug, valacyclovir.10) Table 2 shows typical results obtained using hPEPT1 Oligopeptide-transporters expressed Xenopus laevis oocytes in our laboratory; The physiological role of the H+ Woligopeptide co- b-lactam antibiotics (cefaclor, cefadroxil, cefdinir, transporters lies in the absorption of di- and tripeptides ceˆxime, FK089 and ceftibuten), ACE inhibitors from the intestinal tract after their formation by en- (enalapril, temocapril and temocaprilate) are taken up, Transporter-mediated Drug Interaction 255

Table 1. ATP-binding cassette (ABC) transporters and solute transporters expressed in human tissues for organic anions and organic cations

ABC Transporters Solute Transporters

OrganWTissue Organic Anion Organic Cation Transporters Transporters Small Intestine MDR1WP-Glycoprotein (ABCB1) MCT1 (SLC16A1) OCT1 (SLC22A1) MDR3 (ABCB4) MCT4 (SLC16A4) OCTN1 (SLC22A4) MRP1 (ABCC1) MCT5 (SLC16A5) OCTN2 (SLC22A5) MRP2WCMOAT (ABCC2) MCT8 (SLC16A8) MRP3 (ABCC3) OATP-B (SLC21A9) MRP4WMOATB (ABCC4) OATP-D (SLC21A11) MRP5 (ABCC5) OATP-E (SLC21A12) MRP6WARA (ABCC6) PEPT1 (SLC15A1) BCRPWABCP (ABCG2) PGT (SLC21A2) ASBTWNTCP2 (SLC10A2) AE2 (SLC4A2)

Kidney MDR1WP-Glycoprotein (ABCB1) OAT1 (SLC22A6) OCT1 (SLC22A1) MRP1 (ABCC1) OAT3 (SLC22A8) OCT2 (SLC22A2) MRP2WCMOAT (ABCC2) OAT4 (SLC22A9) OCT3 (SLC22A3) NPT1 (SLC17A1) OCTN1 (SLC22A4) PEPT1 (SLC15A1) OCTN2 (SLC22A5) PEPT2 (SLC15A2)

Liver MDR1WP-Glycoprotein (ABCB1) OCT1 (SLC22A1) MDR3 (ABCB4) OCTN2 (SLC22A5) MRP2WCMOAT (ABCC2) MRP3WCMOAT2 (ABCC3) OATP-B (SLC21A9) MRP5 (ABCC5) OATP-CWLST-1WOATP-2 (SLC21A6) SPGPWBSEP (ABCB11) OATP-8 (SLC21A8) NTCP (SLC10A1) NPT1 (SLC17A1)

Blood-Brain Barrier MDR1WP-Glycoprotein (ABCB1) MCT1 (SLC16A1) OCT2 (SLC22A1) MRP1 (ABCC1) MCT2 (SLC16A2) OCT3 (SLC22A3) OAT1 (SLC22A6) OCTN2 (SLC22A5) OAT3 (SLC22A8) OATP-AWOATP (SLC21A3)

As shown in Fig. 2,plasmapeaktimeandtheareaun- der the curve (AUC) normalized with respect to the dose after oral administration of 5 mgWkg cefadroxil (Fig. 3A), a b-lactam antibiotic, in humans were signiˆcantly delayed and decreased, respectively, by co-administration of 15 mgWkg cephalexin (Fig. 3B),11) presumably due to competitive inhibition by cephalexin of the intestinal hPEPT1-mediated transport of cefadroxil. On the other hand, 70z-increased absorption rate and 25z increase in the bioavailability of 1 g of amoxicillin upon co-administration of 20 mg of nifedipine in humans are considered to be attributable to an increase of the proton concentration at the apical surface of epithelial cells by nifedipine thereby increasing the driving force for Fig. 2. Mean plasma concentrations of cefadroxil in healthy volun- 12) teers after the oral administration of 5 (——), 15 (——), and 30 amoxicillin transport via hPEPT1. Thechangeofthe (——) mg・kg„1 of cefadroxil and after 5 mg・kg„1 of cefadroxil surface pH by nifedipine may be consequence of the takenwith45mg・kg„1 of cephalexin (——). (From Ref. 11) decreased concentration of intracellular Ca++.12)

Monocarboxylate transporters but orally eŠective lisinopril and faropenem, a penem To date, nine isoforms of the proton-coupled antibiotic, are not (unpublished observations). monocarboxylate transporter (MCTs) have been identi- 256 Akira TSUJI

Table 2. Uptake of various b-lactam antibiotics and ACE inhibitors by Xenopus laevis oocytes injected with human PEPT1 cRNA.

Cell-to-medium Ratio (nlWoocyte) Compounds PEPT1 cRNA-injected Water-injected

[ 14C]Gly-Sar 378±42.1* 71.6±5.23 [ 3H]Carnosine 222±36.5* 43.2±5.62 Cefaclor 28.9±7.22* N.D. Cefadroxil 48.3±6.32* 3.62±1.26 Cefdinir 16.8±4.03* 5.21±0.49 Ceftizoxime 3.88±0.90 3.40±0.86 [ 14C]Faropenem 10.2±1.21 9.83±0.89 [ 14C]PCG 51.1±1.85* 38.5±3.02 Ceˆxime 41.8±11.4* N.D. FK089 23.8±4.66* N.D. Ceftibuten 22.6±3.20* N.D. [ 14C]Temocapril 519±87.9* 280±33.1 [ 3H]Temocaprilat 50.0±2.85* 44.0±1.06 Enalapril 52.7±8.05* 20.0±5.14 Enalaprilat 8.49±1.15* 4.52±0.79 Lisinopril 12.3±1.30 11.4±1.09

Uptakes of b-lactam antibiotics and ACE inhibitors were measured for 2 hr in Xenopus laevis oocytes injected with human PEPT1 cRNA or water alone. Each datum represents the mean±S.E. of three to eight experiments.

Fig. 3. Chemical structures of cefadroxil (A) and cephalexin (B). ˆed in humans, each having a diŠerent tissue distribution.13,14) At least four MCT isoforms, hMCT1 (SLC16A1), hMCT4 (SLC16A4), hMCT5 (SLC16A5) and hMCT8 (SLC16A8), are present in the human small ylate drugs. intestine, colon 13,14) and Caco-2 cells.15) Among them hMCT1 the most abundant isoform.15) Cationic drug transporters Monocarboxylates such as acetic acid, benzoic acid, Most drugs in therapeutic use (antihistamines, salicylic acid, and valproic acid are transported via the skeletal muscle relaxants, antiarrhymics, and b-adreno- proton-coupled monocarboxylate transporter, rMCT1 receptor blocking agents) and endogenous bioactive (SLC16A1)16,17) andWor anion-exchange transporter amines (catecholamines, 5-hydroxytryptamine, hista- mAE2 (SLC4A2).18) The short-chain fatty acid mine, choline and N-methylnicotinamide) are organic butyrate15) and monocarboxylates such as benzoic acid, cations. Because many of these organic cations are polar salicylate, (S)W(R)-mandelic acid and (S)W(R)- and positively charged at physiological pH, membrane- ibuprofen19–21) are rapidly taken up by Caco-2 cells with bound transporters are anticipated to participate in the saturable kinetics, pH dependency, Na+-independent absorption, distribution and secretion of these com- transport, and stereospeciˆcity. Transient transfection pounds. However, compared with kidney and liver, of an antisense oligonucleotide of hMCT1 resulted in there are few reports concerning organic cation trans- signiˆcant inhibition of butyrate uptake by Caco-2 cells, porters in the intestine.24–28) Transporter-mediated intes- demonstrating that the hMCT1 transporter plays a tinal absorption has been proposed for the cationic major role in the transport of butyrate into Caco-2 cells. drugs 1-methyl-4-phenyl-pyridinum, MPP+,24) azase- HMG Co-A reductase inhibitors such as pravastatin 22) tron,28) celiprolol,29) and morphine.30) Although MDR1W and atrovastatin23) are also transported in the absorp- P-gp (ABCB1)-mediated active secretion of organic tion direction via proton coupled monocarboxylate cations into the intestinal lumen has recently been exten- transporter(s) in Caco-2 cells, although atrovastatin is sively investigated, as described later, secretion via or- eŒuxed in the secretion direction via P-glycoprotein (P- ganic cation transporters has been also suggested to oc- gp), the MDR1 (ABCB1) gene product.23) Whether cur. A H+ Wguanidine antiporter was characterized in intestinal absorption of monocarboxylates via MCT1 or intestinal brush-border membrane vesicles.31) It was sug- other MCT isomers occurs under physiological condi- gested that the H+ Wguanidine exchanger functions as an tions is not known at present. eŒux system for organic cations. Katsura et al.27) There is no report of any drug interaction that demonstrated that another type of H+ Wtertiary amine reduces or increases the bioavailability of monocarbox- antiport system, diŠerent from H+ Wguanidine antiport- Transporter-mediated Drug Interaction 257

Fig. 4. Chemical structures of digoxin (A) and rifampin (B). er, in the intestinal brush-border membrane participates refampin treatment.38) The eŠect was much less in the active secretion of procainamide.27) Martel et al.24) pronounced after intravenous administration of digox- reported that hOCT1 (SLC22A1) may be involved in or- in. These results are shown in Fig. 5. The oral bioavaila- ganic cation absorption in Caco-2 cells on the basis of bility of digoxin decreased by 30z during rifampin the fact that Caco-2 cells express hOCT1 mRNA. therapy. When duodenal biopsies from volunteers before and after administration of rifampin were ana- ABC transporters lyzed, rifampin treatment increased the content of intes- The intestinal secretion of several lipophilic drugs, tinal MDR1WP-gp, 3.5-fold, which was in good agree- amphipathic drugs and several organic anions occurs via ment with the decrease in AUC after oral, but not ATP-binding cassette (ABC) transporters.32–36) Several intravenous digoxin.38) Since rifampin is a known in- ABC transporter family members, working as drug ducer of cytochrome P450 enzymes (e.g., CYP3A4),39) eŒux pumps, such as MDR1WP-gp (ABCB1), as well as MDR1WP-gp,40) the induction of intestinal MDR3 (ABCB4), MRP1 (ABCC1), MRP2WCMOAT CYP3A4 protein may increase intestinal metabolism, (ABCC2), MRP3 (ABCC3), MRP4WMOATB which would lead one to expect a decrease of plasma (ABCC4), MRP5 (ABCC5), MRP6WARA (ABCC6) digoxin level after rifampin treatment. However, and breast cancer resistance protein, BCRPWABCP although the CYP3A4 in duodenal samples was in- (ABCG2), are expressed in the healthy human intestine. creased 4.4-fold, there was no correlation between the BCRP and MRP2 are more extensively expressed than AUC of digoxin and CYP3A4 expression of individual MDR1. The transcript levels of these nine ABC trans- volunteers.38) These data strongly indicate that MDR1W porters in healthy human jejunum and in Caco-2 cells P-gp modulates intestinal digoxin absorption, leading were correlated well, although BCRP exhibited a 100- to low drug concentrations in individuals with high fold lower transcript level in Caco-2 cells compared with MDR1WP-gp expression and high concentrations in jejunum.37) These results indicate that the Caco-2 cell those with low MDR1WP-gp expression. Accordingly, line can be a useful model of drug eŒux transport in hu- rifampin-mediated MDR1WP-gp induction is associated man jejunum, if the low expression of BCRP is taken with a reduction of plasma digoxin. account. Very similar drug-rifampin interactions have been A number of clinically important drug interactions reported for fexofenadine41) and talinolone42) in hu- with rifampin have been reported that are caused by its mans. Fexofenadine (Fig. 6A), a nonsedating antihista- powerful induction of intestinal cytochrome P450 3A4. mine and an active metabolite of terfenadine (Fig. 6B), Recent ˆndings, however, indicated that concomitant has recently been approved for the symptomatic relief administration of rifampin (Fig. 4A)atadoseof of seasonal allergy. The relatively poor bioavailability 600 mgWday for 10 days in healthy volunteers reduced of fexofenadine, estimated to be about 30z,43) may in- digoxin plasma concentrations substantially after an volve MDR1WP-gp-mediated eŒux in the small intes- oral single-dose (1 mg) administration of digoxin tine, as occurs with a number of other drugs. When an (Fig. 4B) compared with the plasma-time proˆle without oral dose of 60 mg of fexofenadine was administered af- 258 Akira TSUJI ter treatment with 600 mg of oral rifampin for 6 days in P-gp. twelve young (age range, 33 to 35 years) and twelve Talinolol is a b1-blocker antagonist without apprecia- elderly (age range, 65 to 76 years) volunteers, the peak ble metabolic disposition but with intestinal secretion in serum concentration of fexofenadine was signiˆcantly humans. Westphal et al.42) determined the pharmacoki- decreased, and oral secretion clearance was signiˆcantly netics of talinolol (Fig. 7A) in 8 male healthy volunteers. increased in comparison with those before rifampin Duringrifampintreatment(600mgperdayfor9days), treatment. This eŠect was independent of age.41) the expression of duodenal MDR1WP-gp was increased Hamman et al.41) concluded that the fexofenadine- 4.2-fold, resulting in a the signiˆcant reduction of the rifampin interaction is due to reduced bioavailability of AUC of intravenous (30 mg) or oral (100 mg) talinolol. fexofenadine caused by induction of intestinal MDR1W These results indicate that rifampin induces MDR1WP- gp-mediated excretion of talinolol predominantly at the gut wall, and the increased clearance from blood into the lumen of the gastrointestinal tract should be predict- able from the level of intestinal MDR1WP-gp expression, in individuals. Digoxin is absorbed from the intestine to the extent of ca. 70z and eliminated from the body mainly by glomerular ˆltration and MDR1WP-gp-mediated tubular secretion of the unchanged drug; its metabolism is negligible. Indeed, digoxin has been identiˆed by experiments with in vitro MDR1WP-gp-expressing cultured cells and mdr1-a knockout mice as a substrate of MDR1 WP-gp expressed in renal, intestinal and blood-brain barrier cells.34,44–47) Direct evidence for a role of intestinal MDR1WP-gp in determining the oral bioavailability of digoxin was obtained in volunteers with a silent polymorphism in exon 26 (G 3435T) of MDR1. Individuals homozygous for this polymorphism showed signiˆcantly lower duodenal MDR1WP-gp expression and high digoxin plasma levels and AUC.48) Intravenously administered talinolol is actively secreted into the human small intestine. This secretion was reduced by intraluminal administration of the MDR1W P-gp-modulating drug, R-verapamil (Fig. 7B). This fact Fig. 5. (A) Mean (n＝8) plasma concentration (mean±SD) time gives ˆrm support to the concept of MDR1WP-gp- curves of orally administered digoxin (1 mg) before (open circles) and mediated intestinal drug secretion as a cause of poor during (ˆilled circles) coadministration of rifampin (600 mg). (B) Mean (n＝8) plasma concentration (mean±SD)timecurvesofin- oral bioavailability, and provides an explanation for 49) travenously administered digoxin (1 mg) before (open circles) and drug interaction during intestinal absorption. during (ˆlled circles) coadministration of rifampin (600 mg). (From A patient treated with tacrolimus (Fig. 7C)8mgtwice Ref. 38) a day followed by coadministration with diltiazem

Fig. 6. Chemical structures of fexofenadine (A) and terfenadine (B). Transporter-mediated Drug Interaction 259

Fig. 7. Chemical structures of taninolol (A), verapamil (B), tacrolimus (C) and diltiazem (D).

(Fig. 7D) 30 mg orally every eight hours showed a dra- renal tubular secretion of organic anions and cations matic increase of blood tacrolimus concentration from have been studied in detail and reviewed else- 12.9 ngWmL to 55 ngWmL, which resulted in tacrolimus where.25,51–55) The process of secreting organic anions toxicity symptoms, such as delirium, confusion and agi- and cations through the proximal tubular cells is tation.50) Since tacrolimus and diltiazem are known to achieved via transcellular transport, involving the up- be substrates andWor inhibitor for MDR1WP-gp and take of organic anions into the cells from the blood CYP3A, drug interaction due to hepatic andWor intesti- across the basolateral membrane, followed by transport nal CYP3A inhibition andWor intestinal MDR1WP-gp in- across the brush border membrane into urine.56–58) hibition is not surprising. Careful monitoring of tacrolimus concentration, together with tacrolimus dose Organic anion transporters reduction, is recommended for transplant patients if dil- Several renal organic anion transporters have recently tiazem co-therapy cannot be avoided. been cloned and characterized. The organic anion transporter rOAT1 (SLC22A6) in rat kidney was cloned59–61) Transporter-Mediated Drug Interactions in Kidney and shown to be located in the basolateral membrane of Molecular mechanisms of transport involved in the the proximal tubules.62) Three mammalian OAT 260 Akira TSUJI isoforms, m, t, hOAT1 from mice, rats and humans, r, diuretics, sulfobromophthalein, benzylpenicilllin, and hOAT2 (SLC22A7) from rats63,64) and humans,64) and r, bile salt.71) PAH and glutaric acid, high-a‹nity sub- hOAT3 (SLC22A8) from rats65) and humans,66) have strates of hOAT1, showed no or little inhibitory activity been identiêd. Variants of hOAT1 have also been iden- on hOAT4-mediated estrone sulfate transport. The role tiêd. A variant termed hOAT1-167) and two splice vari- of hOAT4 in the secretion of organic anions from tubu- ants termed hOAT1-3 and hOAT1-468) have also been lar cells remains unclear. rOAT-K1, rOAT-K2 and identiêd in the human kidney, but their functions roatp1 (SLC21A1), have been shown to be expressed at remain to be determined. Most recently, hOAT4 the apical membrane and to transport substrates with a (SLC22A9)70) has been identiêd to localize to human steroid moiety (estradiol-17 b-D-glucuronide) and kidney. hOATs except hOAT4 are expressed in the methotrexate. However, PAH interacts only weakly basolateral membrane, whereas hOAT4 is localized in with these organic anion transporters.53) In addition, the the apical membrane of the proximal tubules.71) Among counterparts of hOATPs corresponding to rOAT-K1, these organic anion transporters, hOAT1 and hOAT3 rOAT-K2, roatp1 and roatp2 (SLC21A5) remain un- have been precisely localized to the basolateral mem- clear.71) brane of proximal tubules and, thus, is expected to par- We have characterized an Na+-dependent phosphate ticipate in active excretion of organic anions from blood transporters, m, hNPT1 (SLC17A1), that are localized into the tubular lumen.72) at the apical membrane of proximal tubular cells, and Mammalian OAT1 was proved to be an organic mediates the transport of PAH as well as organic an- anionWdicarboxylate exchanger, and mediates transport ions, including anionic b-lactam antibiotics. Although of a number of endogenous organic anions such as rbNPT1 transports benzylpenicillin well and PAH poor- p-aminohippurate (PAH), cyclic AMP, cGMP, ly, m, hNPT1 transports both PAH and benzylpenicillin prostaglandin E2, urate, and a-ketoglutarate.59–61) well in a manner such that outside chloride ion inhibits hOAT1 and hOAT3 transport clinically important the PAH uptake. We suggest that m, hNPT1 works as drugs such as b-lactam antibiotics, antiviral nucleotide an apical organic anion transporter, secreting into urine analogs, antitumor drugs, histamine H2-receptor an- the intracellular organic anions imported via basolateral tagonist, prostaglandins, diuretics, nonpeptidic an- OATs.82) giotensin inhibitors, and nonsteroidal anti-in‰ammato- Probenecid, allopurinol, non-steroidal in‰ammatory ry drugs.67,72–77) Some diŠerences in characteristics exist drugs (NSAIDs), penicillins and salicylates are consi- between hOAT1 and hOAT3, such as substrate speciˆci- dered to work as inhibitors for the secretion of several ty and localization; hOAT1 is localized on the basolater- organic anionic drugs via basolateral andWor brush- al side of the S2 segment of the proximal tubule,67) border membrane-bound organic anion transport- whereas hOAT3 is localized in the ˆrst, second, and ers.83–86) third segments (S1, S2 and S3) of the proximal tubule.72) To identify the renal secretion pathway for a particu- In addition, hOAT1, but not hOAT3, exhibits exchan- lar drug, in vivo pharmacokinetic drug interaction stu- ge-type transport properties.67,72) Variants of OAT1, r, dies are usually conducted with probe drugs such as hOAT1-1, show a‹nity for a number of endogenous or- probenecid and cimetidine. It has been well recognized ganic anions and anionic drugs and is therefore consi- that probenecid inhibits renal secretion of other anionic dered to be the multispeciˆc organic anion transporter drugs transported via organic anion transport system(s), responsible for the excretion of anionic drugs from the resulting in decreased drug renal excretion. Probenecid kidney,73) whereas another variant, r, hOAT1-2 is a potent inhibitor of hOAT1.87) In contrast, cimeti- (PATH), was suggested to show narrow substrate dine, an e‹cient inhibitor of hOCT1 and hOCT2,88) is speciˆcity, since prostaglandins and methotrexate were known to compete for active tubular secretion primarily not transported.78) hOAT2 mediates the transport of or- with cationic drugs. In addition, cimetidine has recently ganic anions including salicylate and prostaglandin been identiêd as a potent inhibitor of the hOAT3- F2a.64) mediated transport of organic anions.72) Coadministra- On the other hand, in the apical membrane of the tion of two drugs interacting with the same renal trans- tubular cells, several transporters have been identiêd porter may lead to the inhibition of active secretion of as organic anion transporters, such as rOAT-K1 one or both drugs. (SLC21A4),79) rOAT-K2,80) phosphate transporter The renal clearance of furosemide, an organic acid hNPT1 (SLC17A1)81) and hOAT4,71) and oligopeptide and a diuretic, after iv administration of a 40 mg dose transporters PEPT1 (SLC15A1) and PEPT2 was decreased from 1.04 to 0.29 mLWkg after 1 g (SLC15A2).2,7) hOAT4 is exclusively expressed in the probenecid treatment in humans.89) Acyclovir, a anti- placenta and kidney. In the kidney, hOAT4 is localized herpesvirus agent, is eliminated predominantly by renal in the apical membrane of the tubular cells and mediates excretion via glomerular ˆltration and active tubular the transport of nonsteroidal anti-in‰ammatory agents, secretion. After oral probenecid treatment (1 g), the Transporter-mediated Drug Interaction 261 mean terminal plasma half life of acyclovir after transporter is a physiologically important Na+ Wcarni- 5mgWkg intravenous infusion increased by 18z (from tine transporter.97) We analyzed the mOCTN2 gene and 2.3 to 2.7 h), and the area under the plasma concen- found a missense mutation in juvenile visceral steatosis tration-time curve from zero to inˆnity increased by (jvs) mice, with primary systemic carnitine deˆciency 40z. After probenecid administration the mean 25-h (SCD). Biochemical analysis revealed that this mutation acyclovir urinary excretion declined 12.4z from 79.0 to abrogates carnitine and TEA transport. Analysis of the 69.2z of the dose, resulting in a decrease of renal clear- hOCTN2 gene in SCD patients revealed mutations of ance from 248 to 168 mLWminW1.73 m2.90) In the cases of hOCTN2 in all patients, providing evidence that loss of panipenem and betamipron, and imipenem and silasta- hOCTN2 function causes SCD.100) Interestingly, these tin, inhibition of the renal tubular basolateral transport mutations of OCTN2 resulted in loss of the ability to of the penem antibiotics via organic anion transporters transport carnitine, but some of the mutants retain (probably hOATs) by betamipron and silastatin, respec- transport activity for TEA.101) The results suggest that tively, is utilized to prevent nephrotoxity caused by in- the binding sites of carnitine and TEA are distinct. We tracellular penem antibiotics. observed increased eŒux of intracellular TEA during Na+-dependent carnitine uptake by HEK293 cells trans- Organic cation transporters fected with the hOCTN2 gene, indicating that hOCTN2 Three human organic cation transporters, hOCT1 participates in the reabsorption of carnitine and simul- (SLC22A1), hOCT2 (SLC22A2) and hOCT3 taneously in the active secretion of intracellular organic (SLC22A3), have been reported.51,52,56,91,92) hOCT2 is cations transported via basolateral OCTs in the kid- highly expressed in the kidney with no detectable ney.97) The tubular secretion clearance of [14C]TEA, transcript in other tissues in both rats and humans.57,91) when correlated with the glomerular ˆltration, was Within the kidney, hOCT2 has been shown to be more than 40z decreased in jvs mice in comparison localized on the basolateral side of the proximal tub- with that in normal mice and accumulation of [3H]car- ules.67,72,93) In rats, the highest rOCT1 mRNA expression nitine in the kidney was 2.5-fold increased in jvs mice. is in the kidney, with detectable expression in liver and This in vivo result in jvs mice indicates strongly that intestine,56) whereas hOCT1 mRNA expression in hu- mOCTN2 plays an important role in the for urinary ex- mans is localized only in liver.57,94) Although the expres- cretion of organic cations.101) sion pattern of mOCT3 is similar to that of rOCT3, Cephaloridine (Fig. 8)isab-lactam antibiotic that which is expressed highly in placenta and also in the kid- contains a quaternary nitrogen and exists as a zwitter ney, intestine, heart and brain,95) hOCT3 has a much ion under physiological conditions. It shows a signiˆ- broader tissue distribution, being highly expressed in the cant structural similarity to carnitine. Since cephalori- aorta, liver, prostate, salivary glands, adrenal glands, dine induces severe nephrotoxicity, this antibiotic is not skeletal muscle, and fetal lung.92,95) Intracellular locali- currently used in humans. Cephaloridine increases the zation of OCT1 and OCT3 has not yet been determined fractional renal excretion of carnitine, suggesting that it in any species. may interfere with reabsorption of carnitine in the In humans, hOCT2, which is expressed in the kidney.102,103) Ganapathy et al.104) clearly demonstrated, basolateral membrane, is the most important organic using a human cell line expressing hOCTN2 (SLC22A5) cation transporter for the ˆrst step in renal tubular or hPEPT1 (SLC15A1) and hPEPT2 (SLC15A2), that secretion. hOCT2 is pH-independent, electrogenic, and cephaloridine, cefoselis, cefepime and ce‰uprenam polyspeciˆc, and is responsible for the ˆrst step in the (Fig. 8) were recognized by hOCTN2, but did not inter- renal tubular secretion of tetraethylammonium (TEA)91) act with hPEPT1 or hPEPT2, whereas many of the b- and many organic cations such as procainamide, and lactam antibiotics that were not recognized by hOCTN2 desipramine.56,57) were good substrates for both hPEPT1 and hPEPT2. On the other hand, in the apical membrane of the The interaction of cephaloridine with hOCTN2 is tubular cells, organic cation transporters such as competitive with respect to carnitine, indicating that OCTN1 (SLC22A4)96) and organic cationWcarnitine cephaloridine-induced carnitine deˆciency is likely to be transporter OCTN2 (SLC22A5)97–99) have been identiêd due to inhibition of carnitine reabsorption in the kid- from mouse and human. Both OCTN1 and OCTN2 ney.104) In the similar way, it is expected that cefoselis have only about 30z homology with OCTs and trans- and cefepime also interfere with the renal reabsorption portinanNa+-independent manner organic cations of carnitine, but the interference may be much smaller such as TEA.96,97) Transport activity of mOCTN1 and in magnitude compared with that in the case of hOCTN1 for TEA is higher than that of OCTN2 from cephaloridine due to the diŠerence in their a‹nities for both species, and OCTN1 may work as an H+ Wantiport- hOCTN2.104) er.96,97) Interestingly, m, hOCTN2 transports L-carnitine Pilsicainide hydrochloride (Fig. 9A) is a new antiar- e‹ciently in a completely Na+-dependent manner. This rhythmic agent that was developed in Japan. In hu- 262 Akira TSUJI

Fig. 8. Chemical structures of cephalosporins which interact via OCTN2 transporter. mans, about 75 to 86z of orally administered pil- forms of herpes simplex infections, cytomegalovirus in- sicainide is recovered as the unchanged form in urine. fections and HIV infections, respectively. Approximate- The elimination half-life of pilsicainide has been report- ly 83z of ACV, 90z of GCV and 80z of AZT are ex- ed to be prolonged in patients with renal impairment creted unchanged in urine through by the renal tubular and in elderly patients with decreased creatinine secretion.90,106,107) Although these drugs have no typical clearance. Concominant administration of cimetidine anionic moiety, the renal excretion of ACV, GCV (Fig. 9B) signiˆcantly increased the AUC of pilsicainide and AZT is reduced by co-administration of pro- by 33z on average, prolonged the elimination half-life benecid,90,108,109) suggesting that the renal organic anion by 24z (from 5.0 to 6.2 h), reduced apparent oral clear- transporters are responsible for the tubular secretion of ance by 26z (from 14.7 to 10.8 LWh and reduced renal these drugs. Takeda et al.110) demonstrated by using cells clearance by 28z (from 196.8 to 141 mLWmin). The net stably expressing organic cation transporters (hOAT1, renal clearance by tubular secretion was signiˆcantly hOAT2, hOAT3, hOAT4) and organic cation transport- reduced by 38z on avarage, from 151.4 to 93.0 mLW ers (hOCT1, hOCT2) that hOAT1 and hOCT1 mediate min. However, coadministration of probenecid did not renal ACV and GCV transport, whereas hOATs, but cause any change in plasma concentration, oral clear- not hOCTs, mediate renal AZT transport. Probenecid ance or net renal clearance of pilsicainide.105) From these exerted only a weak inhibitory eŠect the hOAT1- results, pilsicainide, an organic cation with a pKa value mediated ACV transport, i.e., to about 80z of the con- of ca. 10, appeared to be secreted by organic cation trol at 1 mM probenecid,110) indicating that hOAT1 is transporters (s) in the tubules. not responsible for the drug interaction between ACV Acyclovir (ACV), ganciclovir (GCV) and zidovudine and probenecid. (AZT) (Fig. 10) are used in the treatment of various Transporter-mediated Drug Interaction 263

Fig. 9. Chemical structures of pilsicainide (A) and cimetidine (B).

ABC transporters Multidrug resistance-associated protein, MRP1 (ABCC1) belongs to the ABC transporter superfamily and is located in the basolateral membrane of the tubular cells. Most substrates for MRP1 are anionic conjugates of hydrophobic substances with glutathione as well as unmodiêd anticancer drugs.111,112) ABC transporters such as MDR1WP-gp (ABCB1)113) and MRP2WCMOAT (ABCC2)114) are expressed in the apical membrane of the tubular cells and play an important role in cellular detoxiˆcation. MDR1WP-gp Fig. 10. Chemical structures of acyclovir (A), ganciclovir (B) and pumps various hydrophobic xenobiotics into the lumen zidovudine (C). from the tubular cells. MRP2WCMOAT mediates the secretion of endogenous and xenobiotic anionic compounds mostly in the form of conjugates. in the levels of both proteins was maximal at 2 days The ˆnding that digoxin is actively secreted by the after cisplatin treatment and lasted for 8 days.117) These renal proximal tubules via mammalian MDR1WP-gp is results suggest that therapies using cisplatin as a single of particular importance for transporter-mediated inter- chemotherapeutic agent or in combination with other actions of clinically used drugs. Decreased renal clear- drugs must be carefully designed. ance andWor increased serum concentration of digoxin Transporter-Mediated Drug Interactions in Liver when co-administered with quinidine, verapamil, itraconazole or clarithromycin are attributed to inhibi- Numerous endogenous and xenobiotic compounds in- tion of the eŒux transport of digoxin via mammalian cluding non bile salt organic anions and drugs are trans- MDR1WP-gp andWor MRP.44,115,116) A rapid increase of ported from the blood into intracellular space through MDR1WP-gp expression by À200–300z was observed the sinusoidal membrane of hepatocytes by carrier- in rat liver, kidney and intestine after administration of mediated processes rather than by simple diŠusion. Fol- cisplatin. Similarly, MRP2WCMOAT expression in- lowing hepatocellular uptake, many of these com- creased À10-fold in the apical membrane of tubular pounds are biotransformed in two phases. Phase I cells after cisplatin administration in rats. The increase metabolism is mediated by cytochrome P450 enzymes 264 Akira TSUJI and prepares the drug for conjugation by creating polar hOATP-E showed much broader expression proˆles.127) groups. Phase II metabolism is the conjugation of drugs Strong expression of hOATP-A is observed in both with glucuronate, sulfate, or glycine, and represents a adult and fetal brain, with lower levels of expression in detoxiˆcation step. The conjugates are then excreted via fetal lung and adult liver, testis and prostate.127) transporters into the bile through the bile canalicular hOATP-C 122,127) and hOATP8 123) are exclusively expres- membrane or into urine through renal tubular epithelial sed at the basolateral membrane of hepatocytes. Com- cells after eŒux transport from hepatocytes into the pared with hOATP-A and hOATP-C, the expression blood stream. This ˆnal secretory process is called proˆles of hOATP-B, hOATP-D and hOATP-E are Phase III detoxiˆcation. much broader. In adults, hOATP-B is expressed in pancreas, liver, lung, gut, ovary, testes, and spleen. Organic anion transporters hOATP-D is expressed strongly in leukocytes and Hepatocellular uptake of endogenous compounds spleen, but hOATP-E is not. In skeletal muscle, and xenobiotics is mediated by organic anion transport- hOATP-E is the only transporter detected among ing polypeptides, hOATPs. roatp1 (SLC21A1) was ˆrst hOATP members.127) molecularly identiêd in rats as a multispeciˆc and sodi- Although the one-to-one correspondence generally um ion-independent transporter for various organic an- found between rat and human gene counterparts is not ions, including bile acids, and conjugated metabo- clear in the case of hOATPs, the rat liver oatp4 is 64z lites.118) Subsequently, roatp2 (SLC21A5) and roatp3 and 66z identical with hOATP-C and hOATP8, (SLC21A7) were isolated as homologues of roatp1 and respectively, and the substrate speciˆcities of hOATP-C were shown to be present in various tissues and to trans- and hOATP8 and roatp4 are very similar.69,128) port anionic and neutral compounds.119) As they are The hOATP family of transporters plays a central structurally very closely related, they are considered to role in hepatocellular organic anion and drug clearance. form a single protein family—the OATP family. In hu- hOATP-B, -C, -D and -E exhibit transport activity for mans, hOATP was cloned from a human liver-derived estrone-3-sulfate as a common substrate.127) hOATP-A cDNA library as a homologue of rat oatp1.69) However, transports bile salts, BSP, estrone-3-sulfate, de- subsequent analysis showed that hOATP-AWhOATP hydroepiandrosterone sulfate (DHEAS),129) the opioid (SLC21A3) is localized mainly in the brain and is not receptor agonists DPDPE and deltorphin O,130) the anti- functional in the hepatic membranes.120) Although there histamine fexofenadine,43) and the amphipathic organic are many functional similarities between roatp1 and cations APD-ajmalinium, rocuronium, and N-methyl- hOATP-A, it was suggested that hOATP-A is not a quinine.131) Interestingly, hOATP-A also transports am- functional counterpart of rat oatp1, due to the obvious phipathic organic cations. hOATP-B exhibits a limited diŠerences in substrate speciˆcity, a‹nity and tissue dis- substrate speciˆcity for the organic anions BSP, tribution.120,121) hOATP-A also shows high similarity to estrone-3-sulfate and DHEAS. Transport substrates of roatp2, roatp3, rOAT-K1 (SLC21A4), and rOAT-K2, hOATP-C include taurocholate,122,125) bilirubin mono- but it is not yet clear whether it is the human counter- glucuronide, DHEAS, estradiol-17 b-D-glucuronide,132) part of any of these. Recently, a novel homologue of estrone-3-sulfate, prostaglandin E2, thromboxane B2, hOATP family termed hLST-1 (also called hOATP-C leukotriene C4, leukotriene E4, T3, T4,122) pravasta- and hOATP2) was identiêd.122) hLST-1WhOATP-CW tin 125) and BSP.132) hOATP8 exhibits a closely overlap- hOATP2 (SLC21A6) is exclusively expressed in liver ping substrate speciˆcity with hOATP-C, but addition- and is localized at the basolateral (sinusoidal) mem- ally transports the cardiac glycoside digoxin and is brane of hepatic parenchymal cells.123) Subsequent stu- particularly e‹cient in transporting the oligopeptides dies on the hOATP family have identiêd at least six BQ-123 (endothelin receptor antagonist), DPDPE hOATPs, which are assigned the gene symbol SLC21A (opioid receptor agonist) and cholecystokinin.132,133) and include the prostaglandin transporter hPGT Although rOAT2 (SLC22A6) is expressed in the (SLC21A2).124) They are called hOATP-A (originally basolateral membrane of rat liver and kidney, expres- termed hOATP, SLC21A3), hOATP-B (SLC21A9), sion of hOAT2 was reported in human liver.55) hOATP-C (also called hOATP2 and hLST-1, Intrahepatocellular compounds can be secreted back SLC21A6), hOATP-D (SLC21A11), hOATP-E into the sinusoidal space. m, rb, hNPT1 (NaPi-1, (SLC21A12) and hOATP8 (SLC21A8).122,125–127) SLC17A1), which has been cloned as the renal inorganic hOATP-A, hOATP-B, hOATP-C, hOATP-D, and phosphate transporter, is present in both kidney and hOATP-E consist of 670, 709, 691, 710, and 722 amino liver, and rbNPT1 was shown to transport organic an- acids, respectively, and all of the hOATP members have ions such as benzylpenicillin, probenecid and phenol twelve potential transmembrane regions.127) red.81) We have previously demonstrated that b-lactam hOATP-A and hOATP-C are expressed in restricted antibiotics are taken up by hepatocytes and excreted ranges of tissues, whereas hOATP-B, hOATP-D and into bile via multiple organic anion transporters.134–138) Transporter-mediated Drug Interaction 265

The tissue distribution and organic anion-transporting which mediates ATP-dependent canalicular secretion of function of m, hNPT1 suggest that it may participate in phosphatidylcholine; 3) MRP2WCMOAT (ABCC2), a the hepatic transport of b-lactam antibiotics. mNPT1 multispeciˆc organic anion transporter, which primarily was demonstrated to be localized in the sinusoidal mem- transports divalent organic anions such as GSSG, brane of mouse liver, and the transport of benzylpenicil- leukotrienes, bilirubin diglucuronide, S-(2,4-dinitro- lin by mNPT1 examined in Xenopus laevis oocytes was pheny)lglutathione, BSP-glutathione conjugate, and p- Na+-independent and was inhibited by several organic nitrophenylglucuronide, and many glucuronide andWor anions.139) However, since its transport activity was sulfate conjugates of drugs; 4) BSEPWSPGP (ABCB11), reduced by a high concentration of chloride ion, NPT1 a bile salt transporter which mediates transport of may function for the eŒux of organic anions such as b- monovalent bile acids. lactam antibiotics from hepatocytes to blood across the Fexofenadine (Fig. 6A), an active metabolite of ter- sinusoidal membrane.139) fenazine (Fig. 6B), does not undergo signiˆcant biotran- sformation in humans because 95z of the dose is ex- Organic cation transporters creted unchanged in urine or feces after biliary excre- The ˆrst organic cation transporter to be isolated, tion.149) When fexofenadine was coadministered with called rOCT1 (SLC22A1), was cloned from rat kid- the cytochrome P-450 3A inhibitor erythromycin ney.56) It is expressed at the basolateral membrane of (500 mg every 8 hr) and ketoconazole (400 mg once dai- hepatocytes, small intestinal enterocytes and cells of the ly) in humans, the steady-state plasma levels of fex- renal proximal tubule S1 segment.25) Jonker et al.140) ofenadine increased by 109z and 164z, respectively.43) generated mOCT1-knockout mice and proved by meas- Because fexofenadine is not signiˆcantly metabolized, it uring the pharmacokinetics of tetraethylammonium, a is likely that uptake and eŒux transporters could be model organic cation, in wild-type and knockout mice predominantly involved in such drug interactions. Cvet- that mOCT1 plays an important role in the uptake of kovic et al.43) demonstrated in a recombinant vaccinia small organic cations such as tetraethylammonium, N- expression system that members of the hOATP family methylnicotinamide, and dopamine into the liver and in and roatp1 (SLC21A1) and roatp2 (SLC21A5) mediate their direct excretion into the lumen of the small intes- [ 14C]fexofenadine cellular uptake, whereas the bile acid tine.26,91) transporter human sodium taurocholate cotransporting polypeptide, NTCP (SLC10A1) and rOCT1 (SLC22A1) ABC transporters at sinusoidal and bile canalicular do not exhibit transport activity. P-gp was identiêd as membranes a fexofenadine eŒux transporter, using the P-gp- The sinusoidal (basolateral) membrane of hepato- overexpressing LLC-PK1 cell line. In addition, in- cytes also contains several members of MRP. MRP1 travenous administration of [14C]fexofenadine to mice (ABCC1) mediates the ATP-dependent eŒux of lacking mdr1a-encoded P-gp resulted in 5- and 9-fold glutathione S-conjugates, leukotriene C4, steroid increases in the drug's plasma and brain levels, respec- conjugates such as estradiol-17-E-D-glucuronide and tively, compared with wild-type mice. A number of drug glucuronidated or sulfated bile salts.141) MRP3 (ABCC3) inhibitors of MDR1WP-gp (ABCB1) were found to be is expressed at the basolateral hepatocyte membrane 142) eŠective inhibitors of hOATP.43) These results strongly and mediates basolateral eŒux of the organic anions indicate that drug-drug interaction involving fexofena- estradiol-17 b-D-glucuronide and S-(2,4-dinitro- dine can be attributed to the combined inhibition of phenyl)glutathione, the anticancer drugs methotrexate both drug uptake and eŒux transporters. Because and etoposide143,144) and even of monovalent bile hOATP transporters and MDR1WP-gp are colocalized salts.145) MRP5 (ABCC5) appears to be an anion trans- in organs of importance for drug disposition, such as porter, though its expression level in the adult liver is the liver, modulation of their activity provides a plausi- very low.146) MRP6 (ABCC6) is localized at the ble explanation for changes in the disposition of other basolateral membrane of hepatocytes and transports xenobiotics and for drug-drug interactions. the cyclic pentapeptide and endothelin antagonist Although the blood level of doxorubicin was not BQ-123.143,144,147) changed after co-administration of cyclosporin A in Unlike the sinusoidal organic anion transport sys- rats, the cellular concentrations of doxorubicin in sever- tems, most canalicular transport systems belong to the al tissues highly expressing MDR1WP-gp, such as liver, ABC transporter superfamily. Four ATP-dependent or- adrenals, kidney and intestine, were dramatically in- ganic substrate transporters have so far been identiêd creased, owing to the inhibitory eŠect of cyclosporin A at the bile-canalicular front of rat hepatocytes:148) 1) on the eŒux transport of intracellular doxorubicin via MDR1WP-gp (ABCB1) which mediates canalicular MDR1WP-gp.150) Even if there is no signiˆcant change of excretion of hydrophobic organic cations such as blood concentration of an anticancer drug after co-ad- daunomycin, vinblastine, and verapamil; 2) MDR3 ministration of another drug(s), there is a possibility of 266 Akira TSUJI signiˆcant intracellular accumulation of the anticancer tions of the P-gp substrates can be explained in terms of drug, which might result in severe side-eŠects. the drug-drug interaction via P-gp at the BBB. Transporter-Mediated Drug Interactions A Draft of Japanese Guidance for Drug Interactions: at the Blood-Brain Barrier In Vitro and In Vivo Assessment Brain capillary endothelial cells (BCECs) mediate the The draft of the guidance for predicting clinically sig- exchange of solute between the blood and brain intersti- niˆcant drug interactions and for evaluating their occur- tial ‰uid. Monocarboxylate transporters, rMCT1 renceinhumanswascompletedattheendof2000and (SLC16A1) and rMCT2 (SLC16A2), are expressed at submitted it to the Ministry of Health, Labor and Wel- both the luminal and abluminal membranes of fare (MHLW) MHLW sent notiˆcation June 4, 2001 BCECs.16) rMCT1 expressed at the BBB transports lac- that it should be used as a reference under the name tic acid, short-chain fatty acids, benzoic acid and other ``Methods for Drug Interaction Studies.'' This docu- monocarboxylates.151) Data on the BBB transport of or- ment describes in some parts the guidance for assessing ganic cations suggest that there are at least two diŠerent the likelihood of the transporter-mediated drug interac- carrier-mediated transport systems for choline and tion in gastrointestinal absorption, tissue distribution lipophilic basic drugs.47,152) and elimination into urine and bile. The following MDR1WP-gp (ABCB1) works as an eŒux pump for points are cited in part from the guidance: various lipophilic compounds such as digoxin, quini- (1) Transporters expressed on the plasma membrane dine, immunosuppressants and new quinolone antibac- of intestinal epithelial cells participate in the absorption terial agents. Although Goden and Pardridge160) report- process of some drugs, and therefore competitive inhibi- ed that in the human brain MDR1WP-gp is localized at tion of absorption may occur when the same transporter the astrocyte foot processes but not at the brain capilla- participates in the absorption of plural drugs or food ry endothelial cells, this ABC transporter has been wide- components. Also, some transporters, such as P- ly recognized to be expressed at the luminal membrane glycoprotein, expressed in the intestinal wall, contribute of the brain capillary endothelial cells, thereby prevent- to the eŒux of drugs taken up by epithelial cells from ing their entry into the brain.45–47,152) Brain distribution the luminal side before going to the basal side (or into of MDR1WP-gp substrates increased dramatically in the portal vein). Inhibition of this process may lead to mdr1a-knockout mice. Recent in vivo and in vitro stu- increased absorption. When active transport is suspect- dies on BBB transport have provided evidence that ed to contribute to the absorption and eŒux processes in unidentiêd eŒux transporters other than P-gp actively the gastrointestinal tract, it may be useful to examine exclude a variety of drugs from the brain.45,46) the extent of this contribution using in vitro test sys-

First-generation H1 receptor antagonists, which are tems, such as cells in which transporters are expressed. used to treat allergic disorders, are often associated with (2) The involvement of transporters in distribution adverse CNS eŠects such as sedation. These are cationic to the liver, kidneys, and brain has been reported for drugs with high lipophilicity and are distributed well to certain drugs. In particular, interactions involving an the brain via organic cation transport system(s) which active transport process may change the unbound drug 153,154) transport the H1-antagonist mepyramine. The or- concentration in tissue and thereby alter the eŠects and ganic cation transporter responsible for mepyramine side eŠects of the drug. When an interaction occurs in a transport across the blood-brain barrier showed lower major organ of distribution and elimination, such as the a‹nity for zwitter ionic H1-antagonists such as fex- liver or kidneys, the volume of distribution of the drug ofenadine and cetirizine than for cationic derivatives, and its systemic clearance may also be aŠected. such as cyproheptadine and terfenadine.155) (3) Many highly polar drugs are excreted into the The transport of amino-acid-mimetic drugs such as L- renal tubular ‰uid or actively reabsorbed by the renal dopa, baclofen and melphalan (Fig. 11) into the brain tubules via transporters. Inhibitory interactions may oc- was signiˆcantly reduced by leucine or phenylalanine, cur in these processes among acidic drugs or among bas- and this was attributed to the competitive inhibitory ic drugs. Caution is therefore required. There are more eŠects of neutral amino acids on the BBB transport of reports of drug interactions with acidic drugs than with these amino-acid-like drugs via aneutralaminoacid basic drugs. Some drugs interact with concomitantly ad- transporter (probably LAT1).156–158) Rhodamine-123 ministered drugs through their metabolites. It is neces- accumulation in the brain was increased 3- to 4-fold by sary to consider the possibility of drug interactions in intravenous infusion of cyclosporin A.159) Similarly, the process of urinary excretion for a drug that is exten- intravenous bolus administration of SDZ PSC833 sively secreted into the renal tubular ‰uid and whose (Fig. 11), a non-immunosuppressive cyclosporin A ana- renal clearance is high. logue, enhanced 16-fold the BBB permeability clearance Patients with renal disease or of advanced age often of quinidine in rats.159) The increased brain concentra- show low drug excretion rates, and thus blood concen- Transporter-mediated Drug Interaction 267

Fig. 11. Chemical structures of L-dopa (A), baclofen (B), melphalan (C) and SDZ PSC833 (D). trations of renal clearance-dependent drugs tend to be to select drug candidates that do not require drug inter- high. Therefore, in such patients, it is necessary to con- action studies in humans. In vitro studies using human sider the possibility of side eŠects due to drug interac- tissue-derived samples, human enzyme expression sys- tions particularly in the urinary excretion process. tems, and animal studies conducted when necessary (4) Drugs are often conjugated and excreted in bile. should contribute to this selection. The results of studies Some drugs are excreted in bile without biotransforma- with related drugs andWor other drugs are useful for the tion. For example, in humans, most water-soluble drugs extrapolation of in vitro data to the in vivo situation and and metabolites of relatively high molecular weight of animal data to humans. (more than about 450) are excreted largely in the bile. Conclusion This excretion is mainly via transporters, and the possibility exists of interaction with concomitantly ad- Although only a small proportion of drug interac- ministered drugs. Conjugates such as glucuronides are tions mediated via transporters, compared with those often excreted in bile and then deconjugated in the intes- mediated via metabolizing enzymes, is clinically sig- tinal tract and reabsorbed (enterohepatic circulation). niˆcant, serious adverse reactions sometimes occur. In Drug interactions in the process of biliary excretion may particular, interactions involving drugs having a narrow aŠect the residence time and AUC of the unchanged therapeutic range may have serious adverse conse- drug in plasma. quences. Therefore, in the evaluation and clinical appli- To predict interactions in the processes of urinary and cation of drugs, appropriate eŠorts should be made to biliary excretion, in vitro inhibition studies using human predict the nature and extent of drug interactions so that tissue-derived samples, cells expressing transporters, patients will not be adversely aŠected. and membrane vesicles may be useful. Molecular cloning and the functional characterization (5) Clinical studies must be both ethical and scien- of drug transporters expressed in various tissues and tiˆcally rational. It is desirable to avoid conducting un- utilization of transporter-gene-knockout or deˆcient necessary studies in humans. Therefore, it is important animals will be of great help in identifying the therapeu- 268 Akira TSUJI tically signiˆcant drug-drug interactions in drug trans- Kitada, H., Leibach, L. H., Tsuji, A. and Sinko, P. J.: port processes. 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A.: Blood-brain barrier transport of H1-antagonist