biliary excretion mechanism of cpt-11 and its …...because biliary excretion is one of the...

[CANCER RESEARCH 58. 5137-514.1. November 15. 1998)

Biliary Excretion Mechanism of CPT-11 and Its Metabolites in Humans:Involvement of Primary Active Transporters1

Xiao-Van Chu, Yukio Kato, Kaoru Ueda, Hiroshi Suzuki, Kayoko Niinuma, Charles A. Tyson, Valorie Weizer,Jack E. Dabbs, Ritchie Froehlich, Carol E. Green, and Yuichi Sugiyama2

Graduate School nf Pharmaceutical Sciences, University of Tokyo, Hongo. Bunk\o-ku, Tokyo Â¡13-0033. Japan Â¡X-Y.C, Y. K., K. U.. H. S., K. N., Y.S.I, and ToxicologyLaboratory. SRI International. Menlo Park. California V4Ã“2S-3943Â¡C.A. T.. V. W.. J. E. D.. R. F.. C. E. G.I

ABSTRACT

After administration of CPT-11, a camptothecin derivative exhibiting awide spectrum of antitumor activity, dose-limiting gastrointestinal toxicity

with great interpatient variability is observed. Because the biliary excretion is a major elimination pathway for CPT-11 and its metabolites [anactive metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), and its glu-curonide, SN38-Glu], several hypotheses for the toxicity involve biliary

excretion. Here, we investigated whether primary active transport isinvolved in the biliary excretion of anionic forms of CPT-11 and its

metabolites in humans using bile canalicular membrane vesicles (cMVs).Uptake of the carboxylate form of CPT-11 and the carboxylate andlactone forms of SN38-Glu by cMVs prepared from five human liversamples was ATP dependent. The concentration dependence of the ATP-dependent uptake of the carboxylate form of CPT-11 and SN38-Glu

suggests the involvement of at least two saturable transport components,both with lower affinity and higher capacity than in rats. The ATP-dependent uptake of the carboxylate form of SN-38 showed a single

saturable component but was detectable only in one human cMV sample.Both carboxylate and lactone forms of SN38-Glu uptake also showed a

large intersample variability, although the variability was less than thatobserved for the carboxylate form of SN-38. On the other hand, thecarboxylate form of CPT-11 exhibited much less variability. The carboxylate forms of SN38-G1U and SN-38 almost completely inhibited theATP-dependent uptake of leukotriene (',. a well-known substrate of

canalicular multispecific organic aniÃ³n transporter, whereas the inhibition by the carboxylate form of CPT-11 was not as marked. Thus, multiple

primary active transport systems are responsible for the biliary excretionof CPT-11 and its metabolites, and the major transport system for CPT-11

differs from that for the other two compounds. A greater degree ofinter-cMV variability in the uptake of SN-38 and SN38-Glu may imply

that interindividual variability in biliary excretion of these metabolitesmight contribute to interpatient variability in the toxicity caused byCPT-11.

INTRODUCTION

Irinotecan, CPT-11,3 is a novel, water-soluble, semisynthetic de

rivative of camptothecin, which exhibits a wide spectrum of antitumoractivity by inhibiting mammalian DNA topoisomerase I (1-3).CPT-11 acts as a prodrug in vivo and is converted to a primary activemetabolite, SN-38, by the enzyme carboxylesterase (4, 5). SN-38 has

been shown to undergo glucuronic acid conjugation to form thecorresponding glucuronide (SN38-Glu), which is mainly eliminated

Received 5/11/98; accepted 9/16/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by a Grant-in Aid for Scientific Research provided

by the Ministry of Education. Science. Sports and Culture of Japan and in part by a Grantfor Cancer Research from the Ministry of Health and Welfare of Japan.

2 To whom requests for reprints should be addressed, at Graduate School of Pharma

ceutical Sciences. University of Tokyo. Hongo. Bunkyo-ku. Tokyo 113-0033. Japan.3 The abbreviations used are: CPT-11. 7-elhyl-10-[4-(l-piperidino)-l piperidino] car-

bonyloxycamptothecin: SN-38. 7-ethyl-10-hydroxy-camplothecin; SN38-Glu. SN-38 glucuronide; AUC. area under the plasma concentration-time curves; LTCj. leukotriene C4;

cMV. canalicular membrane vesicle; cMOAT, canalicular multispecific organic aniÃ³ntransporter; MRP. multidrug resistance-associated protein; P-gp, p-glycoprotein; AIC,Akaike's Information Criterion; HPLC. high-performance liquid chromatography; ALP.

alkaline phosphatase; LAP. leucine amino peptidase.

via biliary excretion (6). SN38-Glu is reported to be deconjugated bythe intestinal microflora to give SN-38 (7, 8). Recently. 7-ethyl-10-[4-A/-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptoth-ecin, a product of the cytochrome P-450-mediated metabolism ofCPT-11, was also identified in human plasma (9, 10). After administration of CPT-11, dose-limiting gastrointestinal toxicity. diarrhea, is

observed in both humans and rats, and there is great interpatientvariability in the severity of this side effect in clinical trials (11, 12).Clarification of the mechanism behind this CPT-11-induced diarrhea

has received a great deal of attention lately. It has been suggested thataccumulation of SN-38 in the intestine is responsible for the diarrheaattributed to CPT-11 administration in nude mice (13). Because biliary excretion of CPT-11 and its metabolites is a major elimination

pathway, accounting for about 60% of the administered dose (6),several hypotheses for the toxicity of CPT-11 directly involve the

biliary excretion of these compounds. One of the proposed hypothesesfor CPT-11-induced diarrhea involves the biliary excretion of itsmetabolite, SN38-Glu. which may be deconjugated to give SN-38 in

the gastrointestinal tract, producing a toxic effect on intestinal epithelial cells (14, 15). In addition, excessive biliary excretion of SN-38,

because of low glucuronidation activity in the liver, may also contribute to the diarrhea (16, 17). Moreover, CPT-11 accumulated in the

intestine, from either the sinusoidal or the brush border side, followingits biliary excretion may be converted into SN-38 by intestinal car

boxylesterase, leading to diarrhea (6). To evaluate the above hypotheses, it is important to clarify the biliary excretion mechanism ofCPT-11 and its metabolites. Our previous studies demonstrated that

multiple transport systems are involved in the biliary excretion ofCPT-11 and its metabolites (anionic form) in rats (18, 19). However,

their biliary excretion mechanisms in humans are still unknown.The pharmacokinetics of CPT-11 and its metabolites in humans has

been studied (20-22). Whatever the CPT-11 schedule, its toxicitydepends on the AUC of SN-38; this metabolite has been given only

sporadically in patients with diarrhea in a few studies (23), but toxicitywas confirmed when several Phase I studies were pooled (24). Because of the difficulty in obtaining human bile samples, study of thebiliary excretion of these compounds in humans has been limited.Rothenberg et al. (25) reported that biliary concentrations of CPT-11and SN-38 in one patient were 10- to 60-fold and 2- to 9-fold higher,

respectively, than the corresponding plasma concentrations. In another study, mean bilerplasma concentration ratios for CPT-11 werereported to be 70 and 135. whereas those of SN-38 were 29 and 57during the 1st and 3rd weeks of CPT-11 administration, respectively(23). A recent study in two patients treated with CPT-11 who had a

percutaneous biliary catheter for extrahepatic biliary obstruction indicated that the cumulative biliary excretion levels of CPT-11 and of

its metabolites up to 48 h were 26 and 53%, respectively (26). Theabove findings suggest that the biliary excretion of CPT-11 and its

metabolites in humans might also be mediated by active transportsystems and show large interindividual differences. Because we havealready shown that primary active transporters are responsible for thebiliary excretion of CPT-11 and its metabolites (anionic form) in rats

5137

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BILIARY EXCRETION OF CPT-II AND ITS METABOLITES

(18, 19), we now attempted to determine whether this is the case inhumans.

Thus far, several kinds of primary active transport systems havebeen identified on the rat bile canalicular membrane for xenobioticsand endogenous substrates: mdr-1 (27), which excretes amphipathiccompounds; bile acid transporter (28, 29); cMOAT (30-33); andmdr-2, which transports phospholipids (34). In human MRP, whichhas a common ATP-binding cassette region and similar substrate

specificity to that of rat cMOAT, is reported to be overexpressed in amultidrug-resistant human lung cancer cell line (35). It has been

shown to transport cysteinyl leukotriene, LTC4, and several otherglutathione 5-conjugates (36-39), as well as estradici-17/3-o-glucu-ronide, a glucuronide conjugate (40), in an ATP-dependent manner.

Recently, the complete cDNA of human cMOAT has been isolated(41). Human cMOAT is highly homologous to rat cMOAT, a homologue of the human MRP gene (41, 42). A mutation in this gene is thecause of human Dubin-Johnson syndrome, an autosomal recessive

liver disorder characterized by chronic conjugated hyperbilirubinemia(43, 44).

Because biliary excretion is one of the important pathways involvedin the detoxification of xenobiotics, many transport studies involvingthe biliary excretion of drugs have been carried out using cMVsisolated from rat liver (45-47). Unfortunately, biliary excretion stud

ies in humans are limited because of the restricted availability ofhuman cMVs. Nevertheless, prediction of biliary excretion in humansfrom in vitro cMV uptake studies is important for the development ofdrugs and their subsequent clinical application. Recently, Wolters etal. (48) developed a method to simultaneously isolate cMVs andsinusoidal membrane vesicles from human liver and investigatedtaurocholate transport in cMVs and sinusoidal membrane vesicles(49). Access to human cMVs would provide valuable information onthe biliary excretion mechanism of drugs in humans. In this study, weinvestigated the uptake of CPT-11 and its metabolites with anionic

charges by cMVs prepared from five human livers to confirm whethera primary active transport system is involved in the biliary excretionof these compounds.

MATERIALS AND METHODS

Materials. CPT-11. SN-38, and SN38-Glu were obtained from Daiichi

Pharmaceutical Co. Lid. (Tokyo, Japan) and Yakult Honsha Co. Ltd. (Tokyo,Japan). The lactone and carboxylate forms of CPT-11, SN-38, and SN38-Glu

were produced by dissolving them in 50 mM phosphate buffer at pH 3.0 or 9.0and leaving them overnight. Conversion of the lactones into carboxylates orcarboxylates into lactones was virtually complete (>99%), as determined byHPLC. Camptothecin. used as an internal standard, was obtained from SigmaChemical Co. (St. Louis, MO). [3H1LTC4 (52.0 /liCi/nmol) was purchased

from New England Nuclear (Boston. MA). ATP, AMP. creatine phosphate,creatine phosphokinase, and acivicin were purchased from Sigma. All otherchemicals were commercial products and of analytical grade.

Isolation of cMVs from Human and Rat Liver. Fresh and fro/en humanliver samples from five people were obtained: HI, a Hispanic male age 34years who died from anoxic brain injury: H2, a white female age 21 years whodied from head trauma; H4. a Hispanic female age 47 years who died fromclosed head trauma: H5. a white male age 35 years who died from head trauma;and H6, a white female age 45 years who died from head trauma: only onesubject smoked, and none drank alcohol. Male Sprague-Dawley rats (250-300

g body weight) were obtained from Charles River Japan, Inc. (Kanagawa,Japan). Human and rat cMVs were prepared as described previously.4 One

human cMV preparation came from one human liver, whereas one rat cMVpreparation came from the livers of five or six Sprague-Dawley rats. After

suspension in 50 mM Tris buffer (pH 7.4) containing 250 mM sucrose, thecMVs were frozen in liquid N, and stored at -100Â°C until use.

The purity of the prepared cMVs was checked by determining the activityof ALP, LAP, and 7-glutamyl transpeptidase as described previously.4 The

ALP enrichment factors were 48.9 Â±10.7 (mean Â±SE of five cMV preparations) and 68.9 Â±6.3 (mean Â±SE of three cMV preparations) in cMVs fromhumans and Sprague-Dawley rats, respectively, compared with the corre

sponding activity in liver homogenate. The LAP enrichment factors were26.1 Â±5.8 and 48.5 Â±9.2 in humans and Sprague-Dawley rats, respectively.The y-glutamyl transpeptidase enrichment factors were 22.3 Â± 4.2 and109 Â±17 in humans and Sprague-Dawley rats, respectively. Protein concen

trations were determined as described previously (50), using the Bio-Rad

protein assay kit with BSA as a standard.

Uptake Study by cMVs. The uptake study using the carboxylate andlactone forms of CPT-11 and its metabolites was performed as reportedpreviously ( 18). The ATP-independent uptake of the substrate was determined in the presence of 5 mM AMP and the ATP-regenerating system.

Because the uptake of the carboxylate forms of CPT-11, SN-38, andSN38-Glu in the presence of 5 mM ATP was linear up to 2 min, as shown

in Fig. 1, the initial rates for the uptake of these compounds in humans wereestimated by determining the uptake for 2 min after the start of the reaction.The initial uptake of CPT-11 and its metabolites at concentrations of 5 and50 /Â¿Mby cMVs from Sprague-Dawley rat livers was also determined using

an uptake time of 2 min, because the uptake of these compounds in thepresence of ATP was linear over the first 2 min, as we showed in ourprevious reports (18, 19). After the uptake study, the pH of the incubationmedium was also checked and proved to be 7.4. For the uptake of [ 'H]LTC4

in humans, membrane vesicles were pretreated with 1 mM acivicin at 25Â°Cfor 30 min to inhibit the activity of y-glutamyl transpeptidase.4 Transport

was terminated after incubation at 37Â°C for 3 min. The radioactivity

retained on the filter and in the reaction mixture was combined with ascintillation mixture (Clear-sol I; Nacalai Tesque, Tokyo, Japan) and

measured in a liquid scintillation counter (LS 6000SE; Beckman Instruments, Fullerton, CA). The ATP-dependent uptake was obtained by sub

tracting the uptake in the absence of ATP from that in the presence of ATP.HPLC Analysis. Determination of the carboxylate forms of CPT-11 and its

metabolites, and the lactone form of SN38-Glu present on Filters and in the

medium was accomplished by HPLC as described previously (18). In theuptake study, conversion of the lactone and carboxylate forms of the drug onthe filter and in the medium during the experiment was less than 5%, asdetermined by HPLC.

150 50 100Time (Sec)

150

50 100Time (Sec)

150

4 K. Niinuma, Y. Kato, H. Suzuki, C. A. Tyson. V. Weizer. J. E. Dabbs. R. Froehlich.

C. E. Green, and Y. Sugiyama. Primary active transport of organic anions on bilecanalicular membrane in humans, submitted for publication.

Fig. I. ATP-dependent uptake of the carboxylale forms of CPT- !!(/Â»), SN-38 (ÃŸ).andSN38-GIU (O by human cMVs. cMVs (H5 for CPT-11 and H2 for SN38-GÃŒUand SN-38)were incubated in the presence of substrate (50 (J.Mfor CPT-11 and SN38-Glu and 100 ftMfor SN-38) with (â€¢)or without (D) ATP (5 HIM)and the ATP-regenerating system. Dataare means (bars in A, SE) from three experiments for uptake in the presence of ATP: dataare means from two experiments for uptake in the absence of ATP.

5138



BILIARY EXCRETION OF CPT-1] AND ITS METABOLITES

-50Ã´o 40

a 10atI 0

50

I 40

!;3Â°:

| 20

0 1000 2000 3000 4000Vo (pmol/min/mg protein)

0 2000 4000 6000 8000Vo (pmol min mg protein)

-400

2300

|200

-100

I 0 1000 2000 3000Vo (pmol/min/mg protein)

Fig. 2. Eadie-Hofslee plots for the ATP-dependcnt uptake of the carhoxylate forms ofCPT-ll (A}. SN-38 (fl). and SN38-Glu (O by human cMVs. Human cMVs <H5 forCRT-11 and H2 for SN38-Glu and SN-38) were incubated in the presence of substratewith or without ATP or the ATP-regenerating system for 2 min. ATP-dependent uptakewas obtained hy subtracting the uptake in the absence of ATP from that in its presence.Kinetic parameters were obtained by fitting using Eqs. A and B. and these parameters arelisted in Table I. Data are means (bars. SE) from three experiments. Insets, linearrepresentations of the data.

Data Analysis. The kinetic parameters for ATP-dependent uptake, as

sessed by subtraction of the uptake in the absence of ATP from that in itspresence, were estimated from the following equations:

Va = Vâ€ž,â€žX S/(Km + S) (A)

Va = Vmâ€ž,X S/(KmÂ¡+SÃŒ+ Vmâ€ž,X S/(Km, + S) (B)

where Vâ€žis the initial uptake rate of substrate (pmol/min/mg protein). S is thesubstrate concentration in the medium (JXM). Kn, is the Michaelis constant(/Â¿M).and Vmax is the maximum uptake rate (pmol/min/mg protein). Theuptake data sets were fitted to the above equations by an iterative nonlinearleast-squares method using a MULTI program (50) to obtain estimates of the

kinetic parameters. The input data were weighted as the reciprocal of thesquare of the observed values, and the algorithm used for fitting was theDamping Gauss Newton Method (51). The decision whether to assume one(Eq. A) or two (Eq. B) components was based on the AIC value (52).Theoretically, the equation with the minimum AIC is regarded as the bestrepresentation of the experimental data (52).

RESULTS

Uptake of Carboxylate Forms of CPT-ll, SN-38, and SN38-Gluby Human cMVs. The uptake of carboxylate forms of CPT-11 (Fig.\A), SN-38 (Fig. IB), and SN38-Glu (Fig. 1C) by human cMVs was

stimulated by ATP and was essentially linear for at least 2 min. Theinitial rate of uptake of the carboxylate form of CPT-11 into human

cMVs (H5) was examined in the presence and absence of ATP. TheATP-dependent uptake, obtained by subtracting the uptake in the

absence of ATP from that in its presence, shows saturation (Fig. 2A).

By fitting the data to either Eq. A or Eq. B, the goodness of the tit wasassessed by means of the AIC value, and the presence of two saturabletransport components for the ATP-dependent uptake was demon

strated, because the AIC was lower when two components wereassumed (Table I ). Concentration dependence was also investigatedfor the uptake of the carboxylate forms of SN-38 (Fig. IB) andSN38-G1U (Fig. 2O by human cMVs (H2). The ATP-dependentuptake of the carboxylate form of SN38-Glu exhibited nonlinearity

with two saturable transport components (Fig. 2C), whereas only asingle transport component was involved in the ATP-dependent uptake of the carboxylate form of SN-38 (Fig. 2ÃŸ).as judged from the

AIC values (Table 1). The obtained kinetic parameters are shown inTable 1.

Transport Activity of CPT-ll and Its Metabolites by cMVsfrom Humans and Sprague-Dawley Rats. The initial uptake velocity of the carboxylate forms of CPT-11 and SN-38, and the carboxylate and lactone forms of SN38-Glu at substrate concentrations of 5

and 50 /J.M,was determined using cMVs from five human and threeSprague-Dawley rat preparations (Tables 2 and 3). For the carboxylateforms of CPT-11 and the carboxylate and lactone forms of SN38-Glu.an ATP-dependent uptake was observed in all cMVs, from bothhumans and Sprague-Dawley rats. The average value of the ATP-dependent and ATP-independent uptake in humans was not muchdifferent from that in Sprague-Dawley rats for these compounds

(Tables 2 and 3). The difference was at most twice. In the case of thecarboxylate form of SN-38. however, uptake at a substrate concen

tration of 5 /AMwas only detectable in one human cMV sample (H2);uptake by the other samples was below the detection limit for SN-38

(2 /xl/min/mg protein; Table 2). At a substrate concentration of 5 /LIM,the ATP-dependent uptake of both the carboxylate and lactone formsof SN38-Glu in H2 shows a much higher transport activity than thatof the other human cMVs. For these forms of SN38-Glu at 5 JU.M,thetransport activity in HI was approximately 2-fold lower than that of

H2. The transport activities in H4, H5, and H6 were comparable andapproximately 5- to 6-fold lower than that in H2 (Table 3). On theother hand, compared with the uptake of SN-38 and SN38-Glu,intersample variability in the ATP-dependent uptake of the carboxylate form of CPT-11 at 5 /J.Mwas minimal: the highest ATP-dependent

uptake was found in H5. and the intersample variability was less than2-fold (Table 3). At a substrate concentration of 50 /MM,the intersample variability in the ATP-dependent uptake of the carboxylateform of SN-38 was 41-fold, much higher than that in the ATP-dependent uptake of the carboxylate form of CPT-11 (2.2-fold), thecarboxylate form of SN38-Glu (6.3-fold), and the lactone form ofSN38-GIU (5.0-fold) in human cMVs (Tables 2 and 3).

Effect of CPT-ll and Its Metabolites on the Uptake of[3H]LTC4 by Human cMVs. The effect of the carboxylate forms of

CPT-ll, SN-38, and SN38-Glu on the ATP-dependent uptake of[3H]LTC4 is shown in Fig. 3. LTC4 uptake was significantly inhibited

by the carboxylate form of SN38-Glu at a concentration of 50 /LIMandby the carboxylate form of SN-38 at a concentration of 500 JAM(Fig.

3). This inhibition was almost complete (Fig. 3). On the other hand,the inhibitory effect of the carboxylate form of CPT-ll was notmarked, even at 500 /UM(Fig. 3). The carboxylate form of CPT-11 at

Table I Kinetic parameters for the ATP-dependent cMV uptake of carboxylate farms of CPT-II. SN-38. and SNJK-Clu in humans"

OnecomponentCPT-llSN-38

SN38-GIU*â€ž48.9

Â±180 Â±

5.70 Â±t20.4191.13vr

max1456

Â±3646873 Â±5971919 Â±189AIC-3.04

-17.6-9.77ffml18.9

Â±10.6140 Â±30

3.56 Â±0.60Vâ€ž659522613X7laxlÂ±302

Â±865Â±142Two

components*m24019

Â±21374509 Â±1882

747 Â±578vâ€ž12519108463268iax2Â±

2095Â±3491Â±1181AIC-h.5X

-12.6-23.4

' Data are represented as mean Â±calculated SD. Kms are given in /IM; V'ma,,tsare given in pmol/min/mg protein.

5139




Table 2 Transpon activity of the carboxylale forms of CPT-II and SN-38 by cMVs isolated from human and normal rats"

cMV

5 /J.M

ATPI + J* ATP(-)rf ATP(dep)' ATP(-)'' ATP(dep)'

CPT-11HumansAverageRatsAverageSN-38HumansAverageRatsAverage"

Data are givenHIH2H4HJH6RlR:R3HIH2H4HSHi,RlR2R3in/il/min/mg protein.37.4

8.8569.513.547.75.353.516.556.57.053.05.554.0440.16.1562.08.452.06.4<246.0

Â±4.8<2<2<238.8

Â±1.849.4Â±0.440.9e43.0

Â±3.212.9e29.3

Â±8.225.1e13.9'33.4e22.9

4.120.22.716.21.9515.56.417.31.5<211.3Â±

1.2<2<2<28.45

Â±1.2015.9Â±0.222.7e15.7

Â±4.124.5

Â±8.937.0Â±7.922.6

5.339.716.323.07.229.43.734.04.723.86.4546.510.634.86.6<234.4

Â±4.2<2<2<230.3

Â±2.233.5Â±0.418.2e27.3

Â±4.718.0

Â±3.735.0Â±10.811.2Â±1.825.8Â±3.440.4Â±7.526.1Â±5.421.

4Â±2.818.5Â±1.225.9Â±3.321.9Â±2.211.6

Â±0.830.8Â±2.93.80Â±0.203.55Â±0.358.60Â±0.5511.7Â±5.029.0Â±1.631.4

Â±4.014.9Â±0.625.1Â±5.15.95e17.3

Â±9.82.15e5.60e24.4e11.1

Â±4.26.38Â±1.999.35Â±3.6011.1

Â±0.98.94Â±1.385.75'7.80

Â±1.603.20e1.75e2.30e4.16

Â±1.146.80Â±0.5010.4Â±0.84.77

Â±0.197.32Â±1.6512.114.29.0520.216.114.315.09.2014.913.05.8022.40.5501.756.357.3522.221.110.13.73.91.803.47.451.93.43.792.81.90.801.800.2000.350.553.901.604.00.617.8Â±3.9''

Uptake in the presence of ATP and the ATP-regeneralingsystem.'Data are meanvalues of two differentexperiments. Other datashown here wereobtained fromdifferentexperiments.dUptake in the absence ofATP.'ATP-dependentuptake.

50 H.Mstimulated the uptake of LTC4 (Fig. 3). Additional studies areneeded to clarity the mechanism for such stimulation.

DISCUSSION

Although CPT-11 has been widely used as a potent anticancer drug,

its gastrointestinal toxicity, manifested as diarrhea, greatly limits itsclinical application. The pharmacokinetics of CPT-11 and its activemetabolite SN-38 exhibit large, interpatient variability (22, 53). The

cause of the great interindividual variability in its toxicity as well asin its human pharmacokinetics is currently unknown. Although thebiliary excretion of CPT-11 and/or its metabolites is believed to be

responsible for the toxicity of CPT-11, the mechanism involved in its

biliary excretion in humans is still unknown. The present study wasdesigned to clarify the involvement of primary active transport in thebiliary excretion of CPT-11 and its metabolites in humans by means

of an in vitro uptake study using human cMVs. For the carboxylateform of CPT-11, the uptake by all cMVs from human subjects was

ATP dependent (Table 2). Kinetic analysis of human cMVs revealedthat the ATP-dependent uptake was saturable and consisted of bothhigh-affinity and low-affinity transport components (Fig. 2/4; Table1). Similarly, in cMVs from Sprague-Dawley rats, the ATP-dependentuptake also showed two saturable transport components: one high-

affinity component (Km, 3.4 JLIM;Vma%,115 pmol/min/mg protein) and

Table 3 Transport activity of the carboxylate and laclarteform of SN38-CÃŒUby cMVs isolated from human and normal rats"

SN38-G1U(carboxylaleform)HumansAverageRatsAverageSN38-G1U

(lactoneform)HumansAverageRatsAveragecMVHIH:H4asHCRlR2R3HIH2H4HSHhRlR:RÃŽATP(

+)*90.0

Â±9.0149Â±1434.6Â±2.922.7Â±0.426.4Â±1.4564.5Â±24.567.5Â±2.062.5Â±9.376.6Â±8.768.9

Â±4.152.5

Â±3.0112Â±726.3

Â±1.612.3Â±3.417.6Â±I.I44.1Â±18.337.1Â±2.835.8Â±1.931.2e34.7

Â±1.795

/J.MATP(-)''0.650'1.15

Â±0.100.300'0.400'0.550'0.610

Â±0.1501.55Â±0.122.55

Â±0.253.70Â±0.152.60Â±0.620.600'2.30

Â±0.141.050'0.500'0.700e1.03

Â±0.332.21Â±0.463.60

Â±0.154.55e3.45

Â±0.68ATP(dep)'89.5

Â±9.0148Â±1434.3Â±2.922.3Â±0.425.8Â±1.564.0Â±24.565.5Â±2.059.9Â±9.373.0Â±8.766.1Â±3.852.0

Â±3.21IOÂ±725.2

Â±1.611.8Â±3.416.9Â±1.143.1

Â±18.034.9Â±2.932.2Â±1.9226.7e31.3

Â±2.4ATP(

+)fc27.7

Â±0.938.0Â±3.68.80Â±0.556.30Â±0.258.50Â±1.1517.9Â±6.358.75Â±0.8511.3Â±1.88.35Â±0.559.47Â±0.9215.6

Â±1.829.9Â±3.46.35

Â±0.108.85Â±0.557.90Â±0.4513.7Â±4.46.95Â±1.059.50Â±0.856.21

Â±0.247.55Â±0.1050

Â»IMATP(-)rf1.20'1.23

Â±0.250.500'0.500'1.70'1.03

Â±0.231.50Â±0.022.25

Â±0.151.23Â±0.131.66Â±0.310.550r1.23

Â±0.150.550'0.250r1.15'0.745

Â±0.1901.30Â±0.242.80

Â±0.100.947Â±0.0731.68Â±0.57ATPfdep)'26.4

Â±0.936.7Â±3.58.30Â±0.555.80Â±0.256.80Â±1.1516.8Â±6.257.25Â±0.859.00Â±1.797.12

Â±0.677.79Â±0.6115.0

Â±1.828.6Â±3.35.75Â±0.108.60Â±0.556.75Â±0.4513.0

Â±4.35.65Â±1.106.75Â±0.855.26Â±0.255.89Â±0.45

" Data arc given in /il/min/mg protein.'' Uptake in the presence of ATP and ATP-regenerating system.' Data are mean values of two different experiments. Other data shown here were obtained from three different experiments.'' Uptake in the absence of ATP.' ATP-dependent uptake.

5140




Control

SN38-GIU carboxylate form SuM

SOuM

CPT-11 carboxylate term SO|iM

500(1M

SN-38 carboxylate term SOuM

SOOpM

10 20 30 40 50 60Uptake (pl/min/mg protein)

Fig. 3. The effect of the carboxylate forms of CPT-ll and its metabolites on theATP-dependent uptake of ['H]LTC4 (O.Ol /J.M)by human cMVs. Human cMVs (H2) werepreincubated with acivicin (I HIM)at 25Â°Cfor 30 min. The uptake study was performed

in the presence of substrate and inhibitors with or without ATP and the ATP-regeneratingsystem at 37Â°Cfor 3 min. ATP-dependent uptake was obtained by subtracting the uptake

in the absence of ATP from that in its presence. Data are means (bars, SE) from threeexperiments. *, P < 0.05. significantly different from control. **, P < O.Ol. significantly

different from control.

one low-affinity component (Km 240 piM, Vmax 1990 pmolAnin/mg

protein), according to our previous report (19). As far as the carboxylate form of SN38-Glu is concerned, its uptake by human cMVs was

ATP dependent (Fig. 1C; Table 3), with two saturable transportcomponents (Fig. 2C; Table 1). Similarly, in cMVs from Sprague-Dawley rats, the ATP-dependent uptake of the carboxylate form ofSN38-Glu also showed two saturable transport components: one high-

affinity component (Km, 1 piM; Vmax,250 pmol/min/mg protein) andone low-affinity component (Km, 75 JU.M;Vmax, 360 pmol/min/mgprotein; Ref. 19). Unlike CPT-11 and SN38-Glu, the ATP-dependenttransport of the carboxylate form of SN-38 by human cMVs (H2; Fig.

IÃŸ)showed only a single saturable transport component (Fig. 2B;Table 1). ATP-dependent saturable uptake by cMVs from Sprague-

Dawley rats also exhibited a single saturable component (Km, 70 Â¿AM;Vmax, 2900 pmol/min/mg protein; Ref. 19). Thus, the number oftransport components for the carboxylate form of CPT-11 and its

metabolites is the same in humans and rats. By comparing the kineticparameters for humans and rats, it was found that the primary activetransport systems, for the carboxylate forms of CPT-11, SN-38, andSN38-Glu in humans, show lower affinity and higher capacity thanthose in Sprague-Dawley rats. Nevertheless, the intrinsic clearanceunder linear conditions (V/maxand Km) was comparable in humans and

rats (Table 1).To gain a deeper understanding of species differences in the pri

mary active transport of CPT-11 and its metabolites, we carried out anuptake study using cMVs from five human livers and three Sprague-Dawley rat preparations. For the carboxylate form of CPT-11 and thecarboxylate and lactone forms of SN38-Glu, ATP-dependent uptake

was observed in all five human cMV samples (Tables 2 and 3). Wepreviously compared the transport activity for several anionic compounds using cMVs from humans and rats and found that the uptakeof several glucuronide conjugates [6-hydroxy-5,7-dimethyl-2-methyl-amino-4-(3-pyridylmethyl) benzothiazole-glucuronide, estradiol-17ÃŸ-

glucuronide, and grepafloxacin glucuronide] was similar in humansand rats, whereas the uptake of glutathione conjugates [S-(2,4-dini-trophenyl)-glutathione, LTC4, and sulfobromophthalein glutathione]and other types of organic anions [pravastatin, sodium cyclo-(o-Trp-D-Asp-L-Pro-D-Val-L-Leu), and methotrexate] in humans was approximately 5-fold lower than in rats.4 Our present observations, that the

lactone and carboxylate forms of SN38-Glu exhibit similar transport

activity in humans and rats (Table 3), are in accordance with thesefindings. This implies that glucuronides may undergo relatively similar transport activity in humans and rats, compared with other organicanions. Interestingly, the carboxylate form of CPT-11 also showed a

minimal species difference, although it has no glucuronide moiety.To obtain more detailed information about the transport mechanism

for CPT-ll and its metabolites, LTC4, a typical substrate for rat

cMOAT (37) and human MRP (38), was used in the inhibition study.As shown in Fig. 3, ATP-dependent uptake of LTC4 in humans can bealmost completely inhibited by the carboxylate form of SN38-Glu andSN-38. For the carboxylate form of SN38-Glu, almost complete

inhibition was observed at a concentration of 50 /J.M,whereas minimalinhibition was found at 5 JUM(Fig. 3). Therefore, its approximate K,value should be within 5-50 /UM,which is not very different from itsown high-affinity Km (3.56 /XM).On the other hand, the K-tvalue forthe carboxylate form of SN-38 should be within 50-500 /J.Mbased on

the result shown in Fig. 3, which is near its own Km (180 ^M). Theseresults are reasonable if the carboxylate forms of SN38-Glu andSN-38 share the same transporter as LTC4 in humans. On the otherhand, the carboxylate form of CPT-11 has only a slight inhibitoryeffect on the ATP-dependent uptake of LTC4, even at 500 piM. whichis much higher than the Km (18.9 JU.M)for the high-affinity uptake ofthe carboxylate form of CPT-11 (Fig. 3). This suggests that the majortransporter for the carboxylate form of CPT-11 may be different from

the LTC4 transporter in humans. Thus, the major transporter forCPT-ll and its metabolites (SN-38 and SN38-Glu) is unlikely to be

the same. In our previous reports (18, 19), the carboxylate form ofSN38-G1U and SN-38 was found to be mainly excreted by cMOAT inrats, whereas the high-affinity site for the carboxylate form of CPT-11

is not cMOAT, but the transporter that is also present in EHBR, amutant rat strain with a genetic cMOAT deficiency. Therefore, thisdiscrepancy in transport between CPT-11 and its metabolites occurs

both in humans and rats.The present finding may also give us an insight into the interindi

vidual variability in biliary excretion in humans. The ATP-dependentuptake of the carboxylate form of SN-38 at a substrate concentration

of 5 IJM was only detectable in one human cMV sample (H2, 34.4Â¿il/min/mg protein; Table 2). Considering the detection limit forSN-38, the intersample variability in the transport activity of thecarboxylate form of SN-38 is at least 17-fold. The intersample variability in the enrichment of marker enzymes (ALP, LAP, and y-glu-tamyl transpeptidase) in human cMV samples was at most 3-fold.

Therefore, such large intersample variability in the transport activityof the carboxylate form of SN-38 cannot be attributed to the purity of

the membrane preparations. Thus, the present findings seem to indicate that the biliary excretion of the carboxylate form of SN-38 inhumans exhibits much larger interindividual variability. The ATP-dependent uptake for the carboxylate form of CPT-ll shows littleintersample variability (Table 2). As for the lactone form of SN38-Glu, there was a 5- to~ 10-fold intersample variability, whereas, forthe carboxylate form of SN38-Glu, the intersample variability was 6-to~ 7-fold (Tables 2 and 3). In these human cMV samples, H2 shows

particularly high transport activity for not only the carboxylate formof SN-38, but also for the lactone and carboxylate forms of SN38-Glu.

The above findings can be explained if we assume that the primaryactive transport system for SN-38, which also mediates the transportof SN38-G1U at the high-affinity site and LTC4 in cMVs. might

exhibit considerable interindividual variability, whereas the majortransporter for the carboxylate form of CPT-11 and that for the lowaffinity site of SN38-Glu show only minimal interindividual variabil

ity.Considerable interpatient variability in the pharmacokinetics of

CPT-11 and its metabolites has been reported (22, 53). A pharmaco-

kinetic and pharmacodynamic analysis in 36 patients demonstratedthat episodes of diarrhea correlated more closely with the AUC ofSN-38 than that of CPT-11 itself (22). The variability of SN-38 AUC

is affected by many factors, including its biliary and urinary excretionand glucuronide conjugation. In addition, enterohepatic recycling ofSN-38 followed by biliary excretion and deconjugation of SN38-Glu

5141




by intestinal microflora, as well as the conversion of CPT-11 to SN-38

by intestinal carboxylesterase, may also contribute to the interindividual variation of SN-38 AUG. Gupta and colleagues (16) reportedthat the AUC of SN38-Glu in patients also greatly contributed to the"biliary index," the product of the relative area ratio of SN-38:SN38-

Glu multiplied by the AUC of CPT-11, which has been shown to be

correlated with the severity of diarrhea in humans. This interpatientvariability is possibly due to a difference in the metabolism of thesecompounds, e.g.. the content or activity of the converting enzyme,such as carboxylesterase and UDP-glucuronosyltransferase, or in the

transport systems involved in their biliary excretion. Our presentobservation indicates that there is considerable intersample variabilityin the ATP-dependent uptake of the carboxylate form of SN-38, aswell as the lactone and carboxylate forms of SN38-Glu. Therefore,

such variability in biliary excretion activity may also contribute to theinterpatient variability observed in the pharmacokinetics of CPT-11

and its metabolites. Because of the limit of detection in the HPLCassay, the concentration of SN-38 used in the transport study (5 and

50 /MM)was much higher than the plasma levels seen in patients (20,21, 23). Therefore, caution is needed in extrapolating the present datato clinical situations. We should also note that the compound showingthe most marked interindividual variability is the active metaboliteSN-38. Gupta and colleagues (16) suggested that excessive biliaryexcretion of SN-38, because of reduced glucuronidation in patients,may be responsible for the CPT-11 -induced diarrhea. Therefore, in-

terindividual variability in biliary excretion, as well as glucuronidation of the active metabolite SN-38, might contribute to the interpa-tient variability seen in the toxicity of CPT-11. On the other hand, the

intersample variability in the transport activity of the carboxylate formof CPT-11 was relatively lower (Table 2). This may imply that theinterpatient variability in the toxicity of CPT-11 is not related to the

biliary excretion of the parent compound. To gain a better understanding of the biliary excretion of CPT-11 and its metabolites in humans

and the correlation with diarrhea, additional studies using largernumbers of human cMV samples and more human biliary excretiondata are needed.

Although the present findings suggest that the major transporterdiffers between CPT-11 and other metabolites, the identification of

each transporter has to be performed by additional studies. Our recentstudy demonstrated that the high-affinity transporter for the carboxylate form of CPT-11 can be inhibited by several substrates orinhibitors of P-gp (verapamil, cyclosporin A, and PSC-333) in cMVsfrom Sprague-Dawley rats.5 In addition, its ATP-dependent uptake

can be observed by membrane vesicles isolated from human KB-C2cell lines, which show high expression of P-gp/' These previous

results suggest that P-gp is one possible candidate responsible for thebiliary excretion of the carboxylate form of CPT-11 in humans. Ourrecent study also indicated that there was ATP-dependent saturabletransport of the carboxylate form of SN-38 and SN38-Glu in membrane vesicles from CA-500 cell lines, which are human KB-derivedcells with high expression of MRP.6 This observation agrees with our

finding that both the carboxylate forms of SN-38 and SN38-Glu can

inhibit the uptake of LTC4 (Fig. 3). Considering the similar substratespecificity of MRP and cMOAT, it is possible that cMOAT or acMOAT-related transporter may be involved in the ATP-dependenttransport of the carboxylate form of SN-38 and SN38-Glu in humans.

This hypothesis can be examined if the transport activity of thesecompounds were compared in cMVs from normal subjects and pa-

5 X-Y. Chu. Y. Kalo, and Y. Sugiyama. Possible involvement of P-glycoprolein in

biliary excretion of CPT-11 in rats, submitted for publication.6 X-Y. Chu, H. Suzuki. K. Ueda, Y. Kalo, S. Akiyama, and Y. Sugiyama. Active efflux

of CPT-11 and its metabolites in human KB-derived cell lines, submitted for publication.

tients with Dubin-Johnson syndrome, a hereditary deficiency in hu

man cMOAT. Additional studies are needed to clarify the transportsystems for each compound.

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1998;58:5137-5143. Cancer Res Xiao-Yan Chu, Yukio Kato, Kaoru Ueda, et al. Humans: Involvement of Primary Active TransportersBiliary Excretion Mechanism of CPT-11 and Its Metabolites in

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