Intracellular Roles of SN-38, a Metabolite of the ...was applied to a phenyl-Sepharose CL-4B column (Pharmacia), and topoisomerase I was eluted stepwise between 1 and 0.5 M(NH4)2SO4.

[CANCER RESEARCH 51. 4187-4191. August 15. 1991]

Intracellular Roles of SN-38, a Metabolite of the Camptothecin Derivative CPT-11,

in the Antitumor Effect of CPT-11

Yasuyoshi Kawato,1 Masashi Animimi, Yasuhide Hirota, Hiroshi Kuga, and Keiki Sato

Exploratory Research Laboratories I, Daiichi Pharmaceutical Co.. Ltd., 16-13, Kitakasai l-Chome, Edogawa-ku, Tokyo 134, Japan

ABSTRACT

It is known that 7-ethyl-10-|4-(l-piperidino)-l-piperidino|carbonylox-ycamptothecin (CPT-11), a semisynthesized derivative of camptothecin(CPT), has a potent antitumor activity in vivo, but 7-ethyl-10-hydroxy-camptothecin (SN-38), a metabolite of CPT-11, shows much strongercytotoxicity in vitro than CPT-11. In this study, we demonstrated thatthe relaxation of SV40 DNA plasmids by type I DNA topoisomeraseprepared from P388 murine leukemia cells was inhibited by 50% by SN-38 at approximately 1 JJ.M,although CPT-11 at 1 HIMslightly inhibitedthe relaxation. SN-38 and CPT showed strong, time-dependent inhibitoryactivity against DNA synthesis of P388 cells. However, CPT-11 weaklyinhibited DNA synthesis independently of time with coincident inhibitionof the total thymidine uptake by the cells. By alkaline and neutral elutionassays, it was demonstrated that SN-38 caused much more frequent DNAsingle-strand breaks in P388 cells than did CPT-11. The same contentof SN-38 and a similar frequency of single-strand breaks were detectedin the cells treated with SN-38 at 0.1 MMor with CPT-11 at 100 JIM.Therefore, single-strand breaks by CPT-11 seem to be due to SN-38produced from CPT-11 in cells. These results indicate that CPT-11 itselfpossesses a marginal antiproliferative effect but that SN-38 plays anessential role in the mechanism of action of CPT-11.

INTRODUCTION

CPT,2 first isolated from Camptotheca acuminata (1), dem

onstrated antitumor effects on experimental tumors (2). However, it showed severe toxicity not only in animal experiments(3) but also in clinical trials (4-6), and it is not utilized clinicallyas an anticancer drug.

A semisynthesized water-soluble derivative of CPT, CPT-11,has demonstrated potent antitumor activities against severalmurine tumors after i.v., p.o., or i.p. administration (7-9). CPT-11 is converted to SN-38 in mouse serum and tissue homoge-nate (10). SN-38 possesses much stronger growth-inhibitoryactivity against tumor cells than CPT-11 in vitro and is thoughtto play an important role in the antitumor effect of CPT-11 in

v/vo (10).Recent studies revealed that CPT inhibits type I DNA topo

isomerase (topoisomerase I) through the formation of stabletopoisomerase I-DNA cleavable complexes (11-13). The anti-tumor activity of CPT analogues correlates with the drug-induced accumulation of topoisomerase I-DNA cleavable complexes (14) and with inhibitory activity against DNA relaxationby topoisomerase I (15). The mutants of yeasts lacking thisenzyme are resistant to CPT (16, 17). Furthermore, topoisomerase I prepared from CPT-resistant mammalian cells is markedly resistant to CPT or CPT-11, and/or the amount and totalactivity of this enzyme are reduced in comparison with those of

Received 1/10/91; accepted 6/4/91.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.'The abbreviations used are: CPT, camptothecin; CPT-11. 7-ethyl-10-|4-(l-

piperidino)-l-piperidino]carbonyloxy-camptothecin; SN-38, 7-ethyl-10-hydroxy-camptothecin; SSB, DNA single-strand breaks; DSB, DNA double-strand breaks;ADM, Adriamycin; IC50, IC2!, concentrations inducing 50% and 25% inhibition,respectively.

the enzyme from wild-type cells (18-22). These observationsindicate that inhibition of topoisomerase I is a principal mechanism in the cytotoxicity of CPT and its derivatives in eukar-yotes including mammalian cells.

Topoisomerase I-DNA cleavable complexes stabilized byCPT appear to be responsible for SSB in cultured cells (12, 23,24), and the production of these SSB in the S-phase may causeinterference with or the arrest of the replication fork, resultingin cell death (25, 26). These mechanisms offer plausible reasonsfor such phenomena as the inhibition by CPT, in a time-dependent and S-phase-sensitive manner, of nucleic acid synthesis (2, 27-31) and induction by CPT of the degradation ofDNA in an alkaline sucrose gradient (32-34). Therefore, inhibition of nucleic acid synthesis and induction of SSB are alsoimportant parameters for evaluating the antitumor activity ofCPT derivatives.

In this study, we have examined both the inhibitory activitiesof SN-38 and CPT-11 against topoisomerase I and againstnucleic acid synthesis and the abilities of these compounds toinduce SSB, in order to estimate which compound has a dominant role in the antitumor effect after treatment with CPT-11.

MATERIALS AND METHODS

Tumor Cells. P388 mouse leukemia cells were cultivated and maintained in RPM1 1640 (GIBCO, Grand Island, NY) supplemented withsodium bicarbonate, 10% fetal bovine serum (Biocell Lab., ÇA),20 ^M2-mercaptoethanol, and 60 ^g/ml kanamycin sulfate at 37°Cin a

humidified atmosphere of 5% CO2 in air. For the preparation oftopoisomerase I, P388 cells and Ehrlich mouse ascites tumor cells weremaintained i.p. in DBA/2 mice and in ddY mice, respectively, purchased from the Shizuoka Laboratory' Animal Center (Hamamatsu,

Japan).Chemicals and Enzymes. CPT-11, SN-38, and CPT were supplied by

Yakult Honsha Co., Ltd. (Tokyo, Japan). ADM was purchased fromKyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan). [mefA>>/-3H]Thymidine,[/m'/A>>/-14C]thymidine,[6-3H]uridine, and L-[4,5-3H(A')]leucine were

purchased from NEN Research Products (Boston, MA). SV40 DNAwas purchased from Bethesda Research Laboratories (Bethesda, MD).Proteinase K was purchased from Sigma Chemical Co., (St. Louis,MO). Topoisomerase I was prepared from P388 cells or Ehrlich ascitestumor cells, essentially as described by Ishii et al. (35). Briefly, chro-matin extracted from the cells was loaded onto a heparin-SepharoseCL-6B column (Pharmacia AB, Uppsala, Sweden), and crude topoisomerase I was eluted stepwise between 0.4 and 0.7 M NaCl in 10 mviTris(pH 7.5), 0.5 mM EDTA, and 1 mM dithiothreitol. The active fractionwas applied to a phenyl-Sepharose CL-4B column (Pharmacia), andtopoisomerase I was eluted stepwise between 1 and 0.5 M (NH4)2SO4.

Topoisomerase I Assay. One unit (the minimum amount for fullrelaxation of 0.5 ^g SV40 DNA under the conditions of this study) oftopoisomerase I, 0.5 p\ of the test compounds, and 0.5 ¿igSV40 DNAwere added sequentially to the reaction buffer, which was composed of25 mM Tris-HCl (pH 7.5), 50 m\i KC1, 5 mM MgCl2, 0.25 mM EDTAdisodium salt, 0.25 mM dithiothreitol, 15 /ag/ml bovine serum albumin,and 5% glycerol. Then, the reaction mixture (50 ¿il)was incubated for10 min at 37°C,and the reaction was terminated by treatment with 7.5

M!of a solution consisting of 1% sodium dodecyl sulfate, 20 mM EDTAdisodium salt, and 0.5 mg/ml proteinase K for an additional 30 min at37°C.The samples were mixed with 5 p\ of the loading buffer containing

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INTRACELLULAR ROLES OF SN-38 IN EFFECT OF CPT-11

10 HIMNa2HPO4, 31.3% sucrose, and 0.3% bromophenol blue. Relaxed(form Ir) DNA was separated from supercoiled (form I) and nicked(form II) DNA by electrophoresis on 0.8% agarose gel at 50 mA and20 V for 17 h in the presence of 2 Mg/ml chloroquine, 10 mM EDTA,30 HIMNaH2PO4, and 36 mM Tris-HCl (pH 7.8). After electrophoresis,

the gel was stained with 0.05% ethidium bromide and photographedwith UV light (302 nm). The amount of DNA was quantified using adensitometer (ACD-25D; ATTO Corp., Tokyo, Japan). The inhibition

rate of topoisomerase I activity was calculated as

1 -Fire —F\,

x 100 (%)

where F\n and F,rc are the ratios of relaxed DNA to total DNA treatedwith topoisomerase I in the presence and absence of a test compound,respectively. F¡,is the proportion of relaxed DNA in untreated DNA.The ICso of each test compound was estimated from the dose-response

curve.Incorporations of Thymidine, Uridine, and Leucine. P388 cells at 2 x

10" cells/ml were cultured for 2 days and further incubated with thetest compounds for a given period at 37°C.The cells were treated with

['Hjthymidine (0.45 MCi/ml) for the last 15 min in the incubationperiod or with [3H]uridine (0.9 /iCi/ml) or [3H]leucine (1.8 MCi/ml) for

an additional 60 min. These cells were washed twice with ice-coldphosphate-buffered saline and were solubilized by treatment with 0.5 NNaOH for 10 min on ice. After addition of the same volume of 20%trichloroacetic acid to the solubilized cells on an ice bath, the samplewas filtered through a membrane filter (OA5-^m pore size; Millipore

Corp., Bedford, MA). The residue on the filter was washed twice withice-cold 5% trichloroacetic acid. The radioactivities remaining on thefilter (acid-insoluble fraction) and in the filtrate (acid-soluble fraction)

were measured with a liquid scintillation counter (Liquid ScintillationSystem, LSC-700; Aloka Co., Ltd., Tokyo, Japan). The radioactivitiesof the acid-insoluble fraction and of both fractions were considered tobe proportional to the syntheses of macromolecules and to the cellularuptakes of their precursors, respectively. The ICso of each test compound for macromolecule synthesis was evaluated from the dose-

response curve.DNA Strand Breaks. SSB and DSB were measured by alkaline elution

assay (36) and neutral elution assay (37), respectively. In both assays,the elution was carried out at a rate of 0.03 to 0.04 ml/min, and thefractions were collected at intervals of 90 min. The DNA strand breakfrequency was calculated as described by Zwelling et al. (38).

Intracellular Content of SN-38. An equal number of P388 cells (1 xIO7cells) incubated with 100 MMCPT-11 or 0.1 MMSN-38 for l h at37°Cwere washed once with ice-cold phosphate-buffered saline. Half

of the cells were disrupted by alternately freezing in a dry ice-methanol

bath and thawing in a water bath twice (within 3 min) and then werecentrifuged (7000 x g; 5 min). To stop the formation of excessive SN-

38, 75 ¡Aof the supernatant were mixed with an equal volume of 90%methanol, 0.2% phenylmethylsulfonyl fluoride, and 0.1 N HC1. Thesame procedure was performed on the other half without the freezingand thawing. SN-38 in the mixture was isolated by high-performance

liquid chromatography using a TSK gel ODS 80TM column (TosohCorp., Tokyo, Japan) and an eluting solution composed of 0.001 NHC1 and CH3CN:H2O (2:1, v/v) and was then quantified by fluorospec-

trometry at an excitation wavelength of 380 nm and an emissionwavelength of 556 nm (Fluorescence Spectrophotometer F1000; Hitachi, Ltd., Tokyo, Japan). The intracellular content of SN-38 was

determined as

[SN-38]rr - [SN-38]c

where [SN-38JFT and [SN-38]C are the amounts of SN-38 in cells

processed with and without freezing and thawing, respectively.

RESULTS

Effects of CPT-11 and SN-38 on Activity of Topoisomerase I.SN-38 caused the strongest inhibition of the relaxation of SV40DNA by topoisomerase I prepared from P388 cells, followedby CPT and then CPT-11 (Fig. 1). In this experiment, therespective IC50of SN-38, CPT, and CPT-11 were 0.74, 2.3, and>1000 /UM,as listed in Table 1. Each compound showed asimilar relative activity against the enzyme prepared from Ehrlich tumor cells.

For comparison of the inhibitory activities of SN-38 andCPT-11, the IC25 value for topoisomerase I of P388 was calculated similarly to that of the IC50. This value of CPT-11 was0.72 HIM,whereas that of SN-38 was 0.20 MM(about 3600-foldstronger than CPT-11).

CPT-11 dose dependently shifted the position of relaxedDNA in the direction of nicked DNA, as shown in Fig. 1. SN-38 and CPT showed no effect on the position of relaxed DNAin the experiments described above.

Effects of CPT-11 and SN-38 on DNA, RNA, and ProteinSyntheses and on Cellular Uptakes of Their Precursors. Thedose-dependent effects of 2- or 3-h treatment of CPT-11, SN-38, or CPT on DNA, RNA, and protein syntheses are shownin Fig. 2. CPT-11, SN-38, and CPT dose-dependently inhibitedDNA synthesis. The synthesis was decreased to 20% of the

12345Form n *•

Form Ir

Form I *•

Form n >•

Form Ir

Form I »•

Form n >•

Form Ir

6 7 8 9 10

11 12 13 14 15

Form I *-

Fig. 1. Inhibition of topoisomerase I-induced relaxation of supercoiled DNAby CPT-11, SN-38, and CPT. SV40 DNA was treated with no agent and noenzyme (Lane 1); or with no agent (Lane 2); with 0.1, 0.3, or 1.0 mM CPT-11(Lanes 3-5, respectively); with 0.1, 0.3, 1.0, 3.0, or 10 JÕMSN-38 (Lanes 6-10,respectively); and with 0.3,1.0, 3.0,10, or 30 MMCPT (Lanes 11-15, respectively)in the presence of 1 unit of topoisomerase I.

Table 1 Inhibitory activity of SN-38, CPT, and CPT- II against relaxation ofsupercoiled DNA by topoisomerase 1

Topoisomerase 1 prepared from P388 cells or from Ehrlich ascites tumor cellswas treated with each agent for 10 min at 37°C,using SV40 DNA as the substrate.The inhibition rate of the enzyme activity was evaluated as described in "Materialsand Methods."

AgentSN-38

CPTCPT-1 11CP3880.74(1.0)

2.3(3.1)>1000(NC)*so"

(MM)Ehrlich1.9(1.0)

7.5 (3.9)>1000(NC)

°Numbers in parentheses, ratios of IC50of respective agents to that of SN-38.* NC, not calculable.

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INTRACELLULAR ROLES OF SN-38 IN EFFECT OF CPT-ll

oCoo"o

ss

I 10 100 0.01 O.I I 0.01 0.1

Concentration (/¿M)

Fig. 2. Dependence of inhibition of DNA, RNA, and protein syntheses onconcentrations of CPT-ll (A), SN-38 (fi), and CPT (C). Respective syntheseswere determined by Ihe degrees of incorporation of [5H]thymidine (O). |'H|-uridine (•),and (3H]leucine (•)into acid-insoluble fractions of P388 cells treatedwith each agent at 37°Cfor 2 (DNA synthesis) or 3 (RNA and protein syntheses)

h.

Table 2 Inhibitor)' effects of SN-38, CPT, and CPT-ll on DNA and RNAsynthesis in P388 cells

DNA and RNA syntheses were measured at 37'C from the respective incorporations of [5H]thymidine and [3H]uridine into the acid-insoluble fractions, asdescribed in "Materials and Methods."

AgentSN-38

CPTCPT-ll1CDNA0.077(1.0)

0.18(2.3)19 (-250)so"

(MM)RNA1.3(1.0)2.4(1.9)

61 (-50)

" Numbers in parentheses, ratios of IC50of respective agents to that of SN-38.

control at 100 MMof CPT-11. At a concentration of 1 MM,SN-38 and CPT reduced the synthesis to 12 and 21 % of the control,respectively. The inhibitory effect of each compound on RNAsynthesis was less than that on DNA synthesis. No inhibitionwas observed in protein synthesis after the treatment with anyof the compounds. Respective IC50 values of CPT-11, SN-38,

and CPT in DNA synthesis were 19, 0.077, and 0.18 MM,aslisted in Table 2.

The time-dependent effects of each test compound on DNAsynthesis and cellular thymidine uptake are illustrated in Fig.3. The inhibitory effect of CPT-11 against DNA synthesis wastime independent, although those of SN-38 and of CPT weretime dependent. Moreover, the 20-min treatment with CPT-11reduced the cellular thymidine uptake to 67 and 25% at 10 and100 MM,respectively. This reduction also occurred in a time-

independent manner. The cellular uptake was not inhibited bythe 60-min treatment with 1 MMof SN-38 or CPT, whereasDNA synthesis was strongly inhibited. The cellular uridineuptake was also inhibited by CPT-ll but was not suppressedby the other compounds. The cellular leucine uptake was notaffected by any of the compounds (data not shown).

DNA Strand Breaks by CPT-ll and SN-38. After a 1-htreatment, CPT-11 caused SSB much less frequently than SN-38 and ADM, as demonstrated in Fig. 4. The ability of CPT-11 above 400 MMto produce SSB was saturated at about 400rad-equivalents (Fig. 4A). SSB frequencies induced by 0.1 UMSN-38 and by 100 MMCPT-11 were 74 ±2 and 115 ±22 rad-equivalents, respectively (Table 3). A similar frequency (118rad-equivalents) was induced by l UMADM. ADM also inducedDSB at 2 MM,but no obvious DSB were detected after treatments with either SN-38 at 1 MMor CPT-11 at 1 ITIM(data notshown). The relationship between SSB frequency and DSBfrequency is illustrated in Fig. 5, which reveals that few or no

SSB arising from DSB are included in the apparent SSB induced by SN-38 or CPT-11 (38).

Relationship of SSB Frequency and Intracellular Content ofSN-38. Table 3 compares the effects of the 1-h treatment with100 MMCPT-11 and that with 0.1 MMSN-38 on the frequencyof SSB and on the intracellular content of SN-38. No significantdifferences in either parameter were observed between thesetwo treatments. These results indicate that SN-38 has a potencyto induce SSB that is 1000 times stronger than that of CPT-11and that most SSB induced by CPT-11 are due to SN-38.

DISCUSSION

CPT-11 shows potent antitumor activities in antitumor testsin vivo (7-9). SN-38, a metabolite of this compound, possessesa much higher cytotoxicity against tumor cells in vitro (10),although it seems to be less effective in vivo (7), in comparisonwith CPT-11. CPT, the mother compound of CPT-11, is knownas a specific inhibitor of topoisomerase I (23). The inhibitionof this enzyme is thought to be responsible for the cytotoxicity

oo*-oè?

120

Time (min)

ou"o

i?

100 120

Time (min)

oo

100

so

20 40 60 80 100 120

Time (min)Fig. 3. Time courses of inhibition of DNA synthesis and cellular thymidine

uptake by CPT-ll, SN-38, and CPT. P388 cells were incubated for variousperiods with A, CPT-11 at 10 (•)and 100 (O) „M:B. SN-38 at 0.1 (•).1.0 (O),and 10 (A) J<M;or C CPT at 0.1 (•)and 1.0 (O) MM.DNA synthesis ( ) andcellular thymidine uptake (—) were determined by [3H]thymidine incorporationsinto the acid-insoluble fraction and into both acid-soluble and acid-insolublefractions, respectively.

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INTRACELLULAR ROLES OF SN-38 IN EFFECT OF CPT-II

200 -

600 800 1000 2000

1000

800

600

400

200

0.5 1.5 2.0

Concentration (/¿M)Fig. 4. Dependence of DNA SSB frequency on concentrations of CPT-11 (A)

and SN-38 and ADM (fi). P388 cells were treated with CPT-11 (O), SN-38 (D),or ADM (A) for l h at 37°C.The SSB were measured by alkaline elution assays.SSB frequencies (rad-equivalents) were determined as described by Zwelling et al.(38). Bars, SD.

Table 3 DNA SSB frequencies and contents of SN-38 in P388 cells treated with100 »MCPT-II and O.I IM SN-38

P388 cells were treated with each agent for l h at 37"C. Values represent themeans ±SD (n = 3) and were statistically analyzed by Student's t test.

Agent

SSB frequency"(rad-equivalents)

SN-38 content*(pmol/107 cells)

CPT-11SN-38

115±2274 ±2

Not significant 0.16 ±0.0110.17 ±0.005

Not significant

" Calculated according to the method of Zwelling et al. (38).* Measured by high-performance liquid chromatography and fluorospectro-

metry as described in "Materials and Methods."

of CPT (14-23, 25, 26). Therefore, it is important to evaluatethe effects of CPT-11 and SN-38 on this enzyme. Andoh et al.(18) reported that the activity of topoisomerase I of a humanlymphoblastic leukemia cell line was inhibited by SN-38 butnot by CPT-11. In the present study, we have demonstratedthat SN-38 has a stronger inhibitory effect on topoisomerase Iof P388 cells than CPT, whereas CPT-11 only slightly inhibitsthe relaxation of DNA (Fig. 1). Using IC25s, the inhibitoryactivity of CPT-11 was about 3600 times less than that of SN-38. CPT-11 produced a 28% inhibition even at 1 mM, and theconcentration of SN-38 producing a 28% inhibition was estimated at 0.23 /iM from the dose-response curve. When 1 mMof CPT-11 was incubated under the same conditions as wereused for the topoisomerase I assay, the concentration of SN-38was 0.16 ±0.01 ßM(triplicate tubes), which is close to 0.23 UM.This result indicates that the inhibition of topoisomerase I byCPT-11 is mainly attributable to the SN-38 derived from CPT-11. Therefore, it is believed that SN-38 plays a major role in

the cytotoxicity of CPT-11 on the molecular level.The results listed in Table 3 support this idea. CPT-11 at

100 /UMis contaminated with only 5 nM of SN-38, and theculture medium increased the concentration of SN-38 to lessthan 10 nM under the conditions under which SSB were tested.However, the treatment with CPT-11 at 100 /UMinduced asimilar frequency of SSB and gave an intracellular content ofSN-38 similar to those after treatment with SN-38 at 0.1 /¿M.These observations indicate that SN-38 was produced fromCPT-11 in cells and that the SSB induced by CPT-11 wereprincipally due to this SN-38.

The inhibitory effects of CPT-11 on DNA synthesis and onRNA synthesis were approximately 250 and 50 times less thanthose of SN-38, respectively (Table 2). It is impossible toexplain these results using the intracellular content of SN-38,because a concentration of CPT-11 1000 times higher than thatof SN-38 was required to obtain a similar intracellular contentof SN-38 (Table 3). Therefore, CPT-11 seems to have its owninhibitory effects on DNA and RNA syntheses at high concentrations (>10 UM). However, these effects have different characteristics from those of SN-38 and CPT. The inhibitory effectsof CPT-11 were time independent, whereas those of the othercompounds were time dependent. Moreover, CPT-11 inducedcoincident time-independent inhibitions of the cellular uptakesof thymidine and uridine, which were not induced by either SN-38 or CPT. When the cells were treated with SN-38 or CPT,the radioactivity of the acid-soluble fraction increased andcompensated for the decrease in that of the acid-insolublefraction, but it was unchanged after treatment with CPT-11.These results suggest that CPT-11 suppresses independently oftime the regulatory mechanism(s) of the transport of nucleicacid precursors and that this suppression relates to the apparentinhibition of nucleic acid synthesis.

Even if CPT-11 possesses unexpected action(s) on nucleicacid synthesis, this compound itself appears to have a marginalantiproliferative effect, because the concentrations required toshow such actions are much higher (>250 times) than those atwhich SN-38 affects topoisomerase I and DNA synthesis and

m(AQ

inJÉ•«J

' C; v

co: >I

•?:*

o•o

600

DNA single-strand breaks [SSBJ(rad-equivalents)

Fig. 5. Relationship of DNA SSB and DSB in P388 cells induced by CPT-11(•),SN-38 (D), and ADM (A). If the solid line of an agent crosses the areabetween the broken lines, all apparent SSB induced by the agent are consideredto arise from DSB (38). Bars, SD.

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INTRACELLULAR ROLES OF SN-38 IN EFFECT OF CPT-11

induces DNA strand breaks. Furthermore, as stated above, it isbelieved that topoisomerase I inhibition and SSB after treatment with CPT-11 depend principally on SN-38. Therefore, atthe intracellular level, it appears that SN-38 plays a dominantrole in the antitumor effect caused by CPT-11.

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1991;51:4187-4191. Cancer Res   Yasuyoshi Kawato, Masashi Aonuma, Yasuhide Hirota, et al.   Derivative CPT-11, in the Antitumor Effect of CPT-11Intracellular Roles of SN-38, a Metabolite of the Camptothecin

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