[CANCER RESEARCH 50, 1715-1720. March 15. 1990]

Metabolism and Pharmacokinetics of the Camptothecin Analogue CPT-11

in the MouseNorimasa Kaneda,1 Hiroshi Nagata, Tornio I-unita, and Teruo Yokokura

Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186, Japan

ABSTRACT

A new water-soluble derivative of camptothecin, 7-ethyl-10-[4-(l-pi-peridino)-l-piperidino|carbonyloxycamptothecin (CPT-11), did not exhibit potent antitumor activity in vitro against experimental tumor cells.The 50% effective doses of CPT-11 against KB and LI210 cells were1100 and 5500 ng/ml, respectively. These values were markedly higherthan those of camptothecin (CPT, 0.98 and 3.7 ng/ml) or 7-ethyl-10-hydroxycamptothecin (SN-38, 0.37 and 3.6 ng/ml).

CPT-11 was found to be converted into SN-38 in mouse serum. Invitro incubation of CPT-11 in mouse serum or tissue homogenate enhanced the growth-inhibitory activity much more than that expected fromthe concentration of CPT-11. This enhancement of the activity coincidedwith that expected from the SN-38 concentration in incubated serum orhomogenate, though the contribution of CPT-11 could not be refuted.SN-38 is considered to play a major role in the antitumor activity whenCPT-11 is incubated in serum or homogenate.

The plasma CPT-11 concentration decreased biexponentially after i.v.administration of CPT-11 into mice with a biological half-life of 0.8 to1.1 li. The area under the plasma CPT-11 concentration-time curveshowed dose dependency. The SN-38 concentration decreased for thefirst 30 min after administration and was then maintained for a few hoursat about 0.1 fig/ml after i.v. administration of 20 and 40 mg/kg of CPT-11 followed by the log-linear terminal phase with a half-life of about 2 hwhich was independent of the dose.

It is suggested that the maintenance of plasma SN-38 concentrationmight be necessary for it to exhibit antitumor activity in vivo.

INTRODUCTION

CPT2 (Fig. 1) is an alkaloid extracted from Camptotheca

acuminata (1). Although CPT shows strong antitumor activityin vitro (2, 3) and in experimental animal tumor systems (1,4),it has not demonstrated encouraging clinical results because ofthe therapeutic responses and severe toxicity (5-9). Currently,CPT is not used clinically.

The other important reason that makes the clinical use ofCPT impractical is that it is only slightly soluble in water. Forclinical use, CPT is dissolved in NaOH and administered as thesodium salt which has an open lactone ring structure. Butopening the lactone ring results in lower antitumor activity (10,11). Solubilizing CPT for i.v. administration inevitably diminishes its antitumor activity.

In this institute, various CPT derivatives have been semisyn-thesized (12, 13), aiming at two characteristics. One is reinforcement of the antitumor activity and a decrease in toxicity, andthe other is increased water solubility without opening of thelactone ring. 7-Ethyl-10-[4-(l-piperidino)-l-piperidino]carbon-yloxycamptothecin (CPT-11; Fig. 1) (14, 15) was synthesizedas a water-soluble derivative of CPT without an open lactone

Received 7/20/89; revised 11/28/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.2The abbreviations used are: CPT, camptothecin: ED50, 50% effective dose;

T/C, relative survival rate; HPTLC. high-performance thin-layer chromatogra-phy; HPLC, high-performance liquid chromatography; rrt, half-life; AUC, areaunder the plasma concentration-time curve; CLp. plasma clearance; F„„.volumeof distribution at steady state; AUMC. first moment of the plasma concentration-time curve; ECPT, 7-ethylcamptothecin; ED,,, 95% effective dose.

ring. CPT-11 has been reported to have higher activity and lesstoxicity than CPT, ECPT, and 7-ethyl-10-hydroxycamptothe-cin (SN-38; Fig. 1) against murine tumors (16, 17) and higheractivity against pleiotropic drug-resistant tumors in vitro andin vivo (18). While studying the conditions for determiningCPT-11, we found that CPT-11 is metabolized to SN-38 inmouse serum. In this paper we present the in vitro activation ofCPT-11 in mouse serum and tissue homogenates, its plasmapharmacokinetics in mice, and a discussion of the antitumoractivity in vitro and in vivo.

MATERIALS AND METHODS

Chemicals. All the chemicals used were of analytical grade. CPT-11(hydrochloride trihydate, M, 677), SN-38 (monohydrate, M, 410), andCPT (M, 348) were semisynthesized at our institute. CPT-11 wasdissolved in saline solution by sonication and warming. SN-38 andCPT were used as sodium salts for animal administration.

Animals. Female BALB/c x DBA/2 F, (hereafter called CD2F,)mice were purchased from Charles River Japan (Atsugi, Japan). Allexperiments were performed on 7-wk-old animals.

Preparation of Tissue Homogenate. Mice were killed by exsanguina-tion after overnight fasting. The liver was perfused with ice-cold 0.15M KC1, and the small intestinal mucosa was scraped with a glass slideto collect epithelium. Tissue (liver or scraped epithelium) was homogenized with 4 volumes of ice-cold 0.15 M KC1 with a Teflon hornoge-nizer. The supernatant, after centrifugation at 9000 x g for 20 min at2°C,was used as tissue homogenate.

Incubation of CPT-11 in Serum and Tissue Homogenate. CPT-11 wasadded to mouse serum or tissue homogenate at 100 fig/ml, and it wasincubated for various periods at 37°C.Then the concentrations of CPT-

11 and SN-38 were determined by HPLC as described later. Forbioassay, the incubated samples were fixed with methanol hydrochloride (100/1, v/v) and then centrifuged. The supernatant was dried inair, resuspended, and diluted with culture medium to the appropriateconcentrations.

Bioassay System. KB and 1.1210 cells were cultured in Eagle's

minimal essential medium and RPMI 1640 medium, respectively,containing 10% fetal bovine serum under a humidified atmospherecontaining 5% CO2 at 37°C.About 1.5 x IO4cells per ml were exposed

to medium containing test materials at various concentrations for 3days. The treated cells were counted with a Coulter Counter. Thegrowth inhibition rate and ED50 were determined as described previously (11). Under these conditions extracts of mouse serum and tissuehomogenates without drug were not toxic to cell growth.

Antitumor Activity in Vivo. CD2F, mice were inoculated i.p. withLI210 cells (1.0 x IO5cells/animal) on Day 0 and were given SN-38

or CPT i.p. on Days 1, 5, and 9 to give a total dose of 1.5 to 400 mg/kg. The relative survival rate for the animals was calculated as follows.

median no. of survival days of drug-treated miceT/C (%) = —¿� - ; —¿�—- —¿�- ; x 100.

median no. ot survival days ol untreated mice

Identification of SN-38. CPT-11 was incubated with serum fromCD2F, mice at the initial concentration of about 10 jig/ml at 37°C

overnight. The fluorescence emission spectrum at the excitation wavelength of 300 nm in the samples at the beginning of and after overnightincubation in mouse serum and SN-38 in mouse serum was monitoredwith a Model RF-510 spectrofluorophotometer (Shimadzu Seisakusho,Kyoto, Japan).

Mouse serum was incubated with CPT-11 at the concentration of 1

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METABOLISM OF CPT-11 IN THE MOUSE

CPT-11:

SN-38:

1= C2H5 : R2= -

Table 1 Validation of CPT-11 and SN-38 determination

Intraday (n = 3) Interday (n = 4)

% of % ofAdded (ng) Mean ±SD CV°(%) error Mean ±SD CV (%) error

CPT-111.022.045.1010.20SN-3812.625.263.0126.00.94±0.062.14

±0.085.07±0.1510.19

±0.3012.8

±0.525.1±0.362.8

±1.8126.1±0.66.43.73.02.94.21.42.80.5-7.8+4.9-0.6-0.1+

1.6-0.3-0.4+0.11.01

±0.072.09±0.085.03±0.0310.22+0.0212.7

+0.225.1±0.163.0

±0.4126.0+ 0.26.93.80.60.21.60.40.60.2-1.0+2.5-1.4+0.2+0.8-0.4-0.0+0.0

" CV, coefficient of variation.

CRT: R, = R2=H

Fig. I. Structures of CPT-11, SN-38. and CPT.

mg/ml at 37°Cfor 5 h. After incubation the serum was deproteinizedwith 4 volumes of ethanol and centrifuged. The supernatant was chro-matographed with authentic CPT-11 and SN-38 on a silica gel plate(Silica Gel 60 F254HPTLC; E. Merck AG, Darmstadt, West Germany)and developed with CHClj/methanol (2/1, v/v). The chromatogramwas observed under a 365-nm light.

HPLC was also used for the determination of CPT-11 and SN-38.Details of the HPLC conditions are given below.

Determination of CPT-11 and SN-38 in Mouse Plasma, Serum, andTissue Homogenate by HPLC. Plasma, serum, and tissue homogenatesamples containing CPT-11 and/or SN-38 were immediately diluted10-fold with 0.1 N HC1 to give final concentrations of about 10 ng/mlfor CPT-11 and of about 100 ng/ml for SN-38.

The diluted plasma or serum samples were applied to a Ci8 cassetteof an advanced automated sample processor (Analytichem International, Harbor City, CA) which was activated with 1.5 ml of methanoland water as described by Wallemacq and Lesne (19). The HPLCapparatus (Model LC-4A; Shimadzu Seisakusho) was linked to theadvanced automated sample processor, and a Ci8 reversed-phase column (LiChrosorb RP-18; 25 x 0.4 cm; Merck) with an RP-18 precol-umn was used for chromatography. The mobile phases consisted ofacetonitriles/ethanol/0.8% ammonium carbonate (2/1/1, v/v) andCH3CN/water (1/4, v/v) for CPT-11 and SN-38, respectively. The Howrate and column temperature were 1.0 ml/min and 50°Cfor CPT-11and 2.0 ml/min and 60°Cfor SN-38, respectively. A fluorospectromon-

itor (Model RF-530; Shimadzu Seisakusho) was set at an excitationwavelength of 373 nm and an emission wavelength of 428 nm for CPT-11 and at 380 nm and 540 nm for SN-38, respectively. The peak areawas integrated by a data processor (Model C-R1BS Chromatopac;Shimadzu Seisakusho). CPT-11 and SN-38 gave retention times of 6.6and 13.8 min, respectively. Calibration curves were established for eachdetermination by 0.1% or 10% mouse serum in 0.1 N HC1 containingCPT-11 or SN-38. Validations of CPT-11 and SN-38 determinationsare listed in Table 1. The limit of determination was about 1 ng forCPT-11 and about 5 ng for SN-38.

Pharmacokinetics of CPT-11 and SN-38 in Mice after i.v. Administration. CD2F, mice were given CPT-11 and SN-38 i.v. through thetail vein, and blood was collected by cardiac puncture under light etheranesthesia. The blood was immediately centrifuged, and plasma wasimmediately diluted 10-fold with 0.1 N HC1. CPT-11 and SN-38 inplasma were determined by HPLC as described above.

Plasma CPT-11 concentration-time curves were fitted to the polyex-ponential equations by MULTI weighted nonlinear regression analysis(20). A biexponential equation (Equation A) gave the best fit accordingto Akaike's information criteria.

C = A exp(-af) + B exp(-ßt) (A)

A and B are intercepts, and a and ßare the rate constants of drugdistribution and elimination phases, respectively. The inverse square ofthe observed plasma concentration served as a weighing factor.

was calculated by Equation B.

i«,= 0.693/0 (B)

AUC was calculated by the trapezoidal rule with estimation of AUCfrom the last sampling time to infinity by Equation C.

£Cdt - C|as,/last log-linear phase slope (C)

The last log-linear phase slope in the AUC calculation of CPT-11

equals ß.CLp was calculated by Equation D.

CLp = dose/AUC (D)

yö„was calculated by Equation E of Benet and Galeazza (21).

Fdss= dose x AUMC/(AUC)2 (E)

RESULTS

Growth-inhibitory Activity of CPT-11 in Vitro. The growth-inhibitory activities of CPT-11, CPT, and SN-38 against LI 210leukemia cells and KB cells are shown in Fig. 2. CPT-11 showedvery weak growth inhibition activity in comparison with CPTand SN-38. SN-38 showed the strongest activity. ED50 valuesof CPT-11, SN-38, and CPT against KB were 1100, 0.37, and0.98 ng/ml (1600, 0.91, and 2.8 nmol), respectively. Thoseagainst L1210 were 5500, 3.6, and 3.7 ng/ml (8100, 8.7, and11 nmol), respectively.

Identification of SN-38. SN-38 was identified by a fluorescence emission spectrum, HPTLC, and HPLC. The fluorescence emission spectrum of CPT-11 in mouse serum was monitored at the beginning of incubation and after overnight incubation (Fig. 3). A peak around 520 nm appeared after overnightincubation, which was also observed in the serum containingSN-38.

CPT-11 was incubated with mouse serum overnight, and thesupernatant after ethanol precipitation was applied to HPTLCas described in "Materials and Methods." Authentic CPT-11

gave a blue spot under the 365-nm light with an Rf value of0.23 to 0.25. SN-38 gave a brownish purple spot with an Rfvalue of 0.75 to 0.8. The supernatants gave the two spots at thesame Rf values of CPT-11 and SN-38.

HPLC analysis of CPT-11 incubated in mouse serum gavethe same retention time of 13.8 min. SN-38 in mouse serumcochromatographed with the above sample gave one peak at aretention time of 13.8 min.

SN-38 Formation from CPT-11 in Mouse Serum and TissueHomogenates. CPT-11 was incubated with mouse serum at aninitial concentration of about 10 Mg/ml. The CPT-11 concen-

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METABOLISM OF CPT-ll IN THE MOUSE

A. KB

100

1010° IO1 Hf 103

Drug concentration (ng/ml)

io4

B. L 12 10

= 50S

O -

10 ' IO"1 to° 101 IO3 10105

Drug concentration (ng/mt)

Fig. 2. Growth-inhibitory activity of CPT-ll. SN-38. and CPT on KB (A)and L1210 (B) cells. KB and L1210 cells were exposed to drugs for 3 days atvarious concentrations and counted, and growth inhibition rates were calculated.Points, mean of four experiments; hars. SD. O, CPT-11; •¿�.SN-38; A, CPT.

tration decreased rapidly during the first 5 min and linearlythereafter, and SN-38 increased rapidly and then linearly withtime. The CPT-ll equivalent concentrations of CPT-ll andSN-38 were constant throughout the incubation (Fig. 4). Fig. 5shows the time course of growth-inhibitory activity of theincubated mouse serum or tissue homogenate containing CPT-11 against KB cells. The activity determined by bioassay wasmarkedly enhanced with incubation time. This activity and theactivity calculated by the ratio of SN-38 concentration, determined by HPLC, to the ED50of SN-38 showed good agreementin both serum and tissue homogenates. This result stronglysuggests that this enhanced activity was mainly due to convertedSN-38.

Antitumor Activity in Vivo. Fig. 6 shows the dose-responsecurves of the antitumor activity of SN-38 and CPT in comparison with that of CPT-ll. CPT-ll gave a higher T/C valuethan SN-38 or CPT at the doses examined when administeredi.p. on Days 1, 5, and 9. The maximum T/C values of CPT,SN-38, and CPT-11 were 288, 286, and 500% at the total dosesof 100, 400, and 200 and 400 mg/kg, respectively. Forty-daysurvivors were observed in the CPT-ll- and SN-38-treatedgroups. In the 200- and 400-mg/kg CPT-ll groups, all theanimals survived to the end of the experiment (40 days). CPT-

n3in

300 400 500 600

Emission wavelength (nm)Fig. 3. Fluorescence emission spectra of SN-38 in mouse serum and of CPT-

11 in mouse serum at the beginning of and after overnight incubation. Thefluorescence spectrum was determined at an excitation wavelength of 300 nm.Curve A, CPT-11 in mouse serum just after incubation started; Curve B, SN-38in lOîcmouse serum; Curve C. CPT-11 in mouse serum after overnight incubationat 37°C.A peak appeared at about 520 nm.

12

10( )

g3-

I 5cCDÃœCoo

10 20 30 45

Incubation time (min)

60

Fig. 4. Formation of SN-38 from CPT-ll in mouse serum. CPT-ll wasincubated in mouse serum at 37°C.CPT-11 and SN-38 concentrations weredetermined by HPLC. O. CPT-11; ».SN-38; A. CPT-11 equivalent concentrationcalculated from the concentrations of CPT-11 and SN-38. Only trace amounts ofSN-38 were degraded from CPT-11 in water, saline solution, phosphate-bufferedsaline, and heat-inactivated 50% mouse serum at 37'C for 6 h. These degradations

were much lower than that shown in Fig. 4.

11 was found to be superior to SN-38 and CPT in bothantitumor activity and toxicity.

Plasma Pharmacokinetics of CPT-ll and SN-38 after i.v.Administration. Plasma concentrations of CPT-11 and its metabolite SN-38 after i.v. administration at the doses of 10, 20,and 40 mg of CPT-11 per kg are shown in Fig. 7. The phar-

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METABOLISM OF CPT. 11 IN THE MOUSE

S¿ 4

6 12 18 24Incubation time (hr)

500

g 300

O

200

100

6 12 18Incubation time (hr)

24

Total dose (mg/kg)

Fig. 6. Antitumor activity of CPT-11, SN-38, and CPT. On Day 0, 1 X 10sL1210 cells were inoculated i.p. into mice. SN-38 or CPT was given i.p. on Days1, 5. and 9. Median survival of drug-treated animals relative to that of controlanimals (T/C) was plotted against the total dose of the drugs. Numbers inparentheses, number of 40-day survivors per 6 animals. Experiments were performed as three different experiments for each drug. O, CPT-11; •¿�,SN-38: A.CPT.

50

6 12 18Incubation time (hr)

24

Fig. 5. Enhancement of growth-inhibitory activity in mouse serum and intissue homogenates. CPT-11 was incubated in serum (A), liver homogenate (B),or intestine homogenate (C) at the concentration of 100 (jg/ml up to 24 h. O,activity determined by bioassay; •¿�,predicted activity based on the SN-38 concentration determined by HPLC; , activity expected from CPT-11 at a concentration of 100 jig/ml; . activity expected from SN-38 with an equimolarconcentration of CPT-11.

10O)CPlasma

concentratiopbi

-1V*»V1*b

T4-i1-

\*1T,1it0.1 -

fiti0.05 •¿�

macokinetic parameters calculated are listed in Table 2.CPT-11 decayed biphasically at the doses examined with a

f./,,,of 0.8 to 1.1 h. AUCs were 3 to 23.5 ^g-h/ml and increased8 times, while the dose increased 4 times from 10 to 40 mg/kg.In contrast, CLp decreased from 3.4 to 1.8 liters/kg-h. Dosedependency of CPT-11 was detected by the normalization ofAUC and CLp to the dose. Fdssvalues were 3.3 to 2.6 liters/kgand were relatively constant, though a tendency to decreasewith an increase in dose was observed.

The AUC of SN-38 was 0.41, 0.71, and 1.08 ng-h/ml afterthe administration of 10, 20, and 40 mg of CPT-11 per kg,respectively. The half-times of the terminal log-linear phase ofSN-38 concentration were 2.2 to 3.4 h.

Plasma SN-38 Concentration Profile after i.v. Administrationof SN-38. Fig. 8 shows the plasma concentration profile afteri.v. administration of SN-38 (10 mg/kg), which was quitedifferent from that of SN-38 after the administration of CPT-

0.02

Time after administration (hr)

Fig. 7. Plasma concentration profiles of CPT-11 and the metabolite SN-38 inmice. CPT-11(10, 20. and 40 mg/kg) was administered to female CD2F, micei.v. Animals were sacrificed to collect blood samples at various time intervals bycardiac puncture. Plasma CPT-11 and SN-38 concentrations were determined byHPLC. Points, mean of all the animals in three experiments (n = 7 to 12); bars,SD. Open and closed symbols are concentrations of CPT-11 and SN-38, respectively, after i.v. administration. D. 10 mg/kg; A, 20 mg/kg; O, 40 mg/kg.

11. Plasma SN-38 decayed triexponentially with tv,„,#, and y of

1.6 min, 7 min, and 1.7 h, respectively. The plasma concentration decreased 1/1000 within 1 h. The AUC was 1.35 ¿ig-h/mland was mainly attributed to the high concentration just afteradministration.

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METABOLISM OF CPT-II IN THE MOUSE

DISCUSSION

A new water-soluble CPT derivative, CPT-11, showed verystrong antitumor activity against mouse tumors when administered by various routes (Fig. 6) (16,18). In spite of the activityin vivo, its in vitro activity was very poor (Fig. 2), if based onthe assumption that the antitumor activity of the drug in vivoshould directly reflect in vitro activity. We found that SN-38was formed from CPT-11 in mouse serum and tissue homoge-nate and that the growth-inhibitory activity determined bybioassay and that calculated from the SN-38 concentrationshowed good agreement (Fig. 5). Therefore SN-38 was thoughtto play an important role in the in vitro antitumor activity ofCPT-11 when it was incubated in serum or liver and intestinehomogenates. It was expected that SN-38 would result in higherantitumor activity in vivo than CPT-11, but this was not thecase. SN-38 had lower am humor activity than CPT-11 (Fig. 6).The effective concentration was calculated from the plasma

Table 2 Pharmacokinetic parameters of CPT-11 and SN-38 after i.v.administration of CPT-11 to mice

ParameterCPT-11i«,(h)AUC(^g.h/ml)*CLp

(liter/kg-h)F„„(liter/kg)SN-38r«

(h)AUC^g.h/ml)100.95

±0.22°2.96

±0.123.38±0.153.34

±0.242.

16±0.660.41±0.06Dose

(mg/kg)200.83

±0.047.65±1.092.65±0.372.84±0.173.01

±1.550.71±0.24401.07

+0.1323.45±7.751.82

±0.552.59±0.943.40

±0.561.08±0.11

" Mean ±SD of three experiments.* AUC from Time 0 to infinity.

30

10

D)3-

C<DO

oo

CO«

z(O

«tEM

Sío.

0.5

0.1

0.05

0.01

0.004

1

Time after administration (hr)Fig. 8. Plasma concentration profile of SN-38 after i.v. administration of SN-

38. SN-38 (10 mg/kg) was injected i.v. into female CD2F, mice. The SN-38concentration was determined by the same method as described in the legend forFig. 7. Points, mean of all the animals in three different experiments (n = 12 to15); ftars, SD.

-3^

Time after administration (hr)

Fig. 9. Effective concentration of the drug in plasma of drug-treated mice.The CPT-11 and SN-38 concentrations after 10 mg/kg of CPT-11 administrationin Fig. 7 and the SN-38 concentration after 10-mg/kg administration in Fig. 8were divided by the ED50 values of CPT-11 or SN-38 against LI210. The ED50value is 1 for the effective concentration. A. SN-38 administration: O. CPT-11after CPT-11 administration; •¿�.SN-38 after CPT-11 administration.

concentrations of CPT-11 and SN-38 after the i.v. administration of CPT-11 and from that of SN-38 after the i.v. administration of SN-38. Fig. 9 shows the effective concentrations ofCPT-11 and SN-38. When SN-38 was administered, an effective concentration higher than the ED5(,was maintained for 2.5h. That of SN-38 was higher than the ED95 for at least 5 h afterinjection of CPT-11. The effective concentration of CPT-11was below the ED50just after administration. The contributionof CPT-11 to the in vivo antitumor activity was very small,

though it was not negligible.Concerning the mechanism of the antitumor activity of CPT

and its derivatives, it has been reported that the antitumoractivity of CPT is time dependent rather than dose dependent,except at extremely high concentrations of the drug (22). Thiswas also observed with SN-38.1 Maintenance of the effectiveconcentration of SN-38 by i.v. administration of CPT-11 is apreferable explanation for the mechanism of action.

The plasma SN-38 concentration profile after the i.v. administration of CPT-11 was quite different from that after i.v.administration of SN-38 (Figs. 7 and 8). The rapid decrease inSN-38 concentration after CPT-11 administration was due tothe formation of SN-38 in the blood (serum) within a fewminutes as shown in Fig. 4. Then at higher doses of CPT-11(20 and 40 mg/kg), the SN-38 level was maintained for thenext 1 to 4 h. Maintenance of this concentration is presumedto be caused by two mechanisms. One is that the concentrationis the sum of the linear formation of SN-38 from CPT-11 inthe blood (Fig. 4) and in the liver (Fig. 5). The other alternativeis that SN-38 elimination from the plasma is saturated orinhibited by coexisting CPT-11. The details of the mechanismthat maintains plasma SN-38 concentration for several hoursand of the contribution of the liver in the production of SN-38are not yet clear and require further investigation.

3 Unpublished data.

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METABOLISM OF CPT-11 IN THE MOUSE

Plasma CPT-11 concentration-time curves after i.v. administration are quite different from those of CPT (23), ECPT(11), and SN-38 (Fig. 8). No significant decrease in concentration was observed just after the injection of CPT-11. Thedecrease in concentration after 0.5 to l h is a general characteristic of sodium salts of CPT and its derivatives, because thehalf-times of SN-38 and ECPT are very similar (11). The dosedependency of the AUC of CPT-11 exhibited a concave curve,and that of SN-38 after the i.v. administration of CPT-11showed a convex curve (Table 2). This suggests that there is asaturable process during the formation of SN-38. An increasein the dose of CPT-11 from 20 to 40 mg/kg did not raise theSN-38 concentration level, while the SN-38 concentration wasmaintained, but lengthened the period of maintenance from 1to 3 h. This is presumed to be favorable from the standpoint ofthe mechanism of action for SN-38 (Fig. 7).

The phenolic compounds phenol and naphthol have beenreported to decay very fast and to be metabolized into conjugates in the lung (24, 25). SN-38 has a phenolic hydroxyl group(Fig. 1), and it has been shown in rats to be metabolized intoglucuronide, which has only 1/100 of the antitumor activity invitro* On the assumption that SN-38 metabolism in mice is

the same as in rats, the following explanation for the superiorantitumor activity in vivo and weaker toxicity of CPT-11 thanSN-38 is possible. Some CPT-11 is metabolized to SN-38 afterits administration, and the concentration is maintained forhours at the level necessary to exhibit antitumor effects; maintenance of this level is not realized after the administration ofSN-38 because of its rapid disappearance from the plasma. SN-38, which is active against tumor cells but toxic to normal cells,is detoxified by conjugation and then excreted. The ratio ofconjugated SN-38 to free SN-38 after CPT-11 administrationis higher than that of SN-38.' Weaker CPT-11 toxicity, compared with SN-38, is presumed to be due to the levels of SN-38 glucuronide and free SN-38. CPT-11, which has high anti-tumor activity and low toxicity, is thought to be suitable forclinical use.

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1990;50:1715-1720. Cancer Res   Norimasa Kaneda, Hiroshi Nagata, Tomio Furuta, et al.   Analogue CPT-11 in the MouseMetabolism and Pharmacokinetics of the Camptothecin

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