[CANCER RESEARCH 49, 4098-4102. August 1, 1989]

In Vitro Evaluation of the New Anticancer Agents KT6149, MX-2, SM5887,

Menogaril, and Liblomycin Using Cisplatin- or Adriamycin-resistantHuman Cancer Cell Lines1

Yuichiro Ohe,2 Kazuhiko Nakagawa, Yasuhiro Fujiwara, Yasutsuna Sasaki, Koichi Minato, Masami Bungo,

Shigeki Numi, Naoya Horichi, Masaaki Fukuda, and Nagahiro SaijoDepartment of Internal Medicine, National Cancer Center Hospital ¡Y.O., Y. F., Y. S., M. F.], and Pharmacology Division, National Cancer Center Research Institute[K. N., K. M., M. B., S. A/., N. H., N. SJ, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, and Second Department of internal Medicine, The Jikei University School ofMedicine [Y. O.], 25-8, Nishishinbashi 3-chome, Minato-ku, Tokyo 105, Japan

ABSTRACT

A new model to predict antitumor activity of new analogues wasdeveloped, and the cross-resistance against cisplatin (CDDP) and Adri-amycin (ADM) was examined. A preclinical evaluation of various newanalogues using this new model was performed. The antitumor activitiesof KT6149, MX-2 (KRN8602), SM5887, menogaril (TUT-7), and liblo-mycin (NK313) were evaluated against four non-small cell lung cancercell lines, PC-7, -9, -13, and -14; two small cell lung cancer cell lines,H69 and N231; four CDDP-resistant cell lines, PC-7/1.0, PC-9/0.5, PC-14/1.5, and H69/0.4; a human myelogenous leukemia cell line, K562; andits ADM-resistant subline, K562/ADM by clonogenic assay. The relativeantitumor activities of these new analogues were compared with those ofparental agents, mitomycin C, ADM, bleomycin, and several anticancerdrugs, CDDP, daunomycin, vindesine, and etoposide.

KT6149 was more active than mitomycin C against all lung cancer celllines and the human myelogenous leukemia cell line. Menogaril showedgreater activity than ADM, and MX-2 showed activity similar to ADM.However, the antitumor activity of SM5887 was lower than that of ADM.SM5887 and menogaril showed cross-resistance to KS62/ADM. Nevertheless, the antitumor activity against K562/ADM of MX-2 was similarto that of the parental cell lines. The activity of liblomycin was similarto that of bleomycin.

Thus, KT6149 appears to be the best analogue for use in a clinicaltrial against lung cancer. MX-2 was active even against ADM-resistantcancer cells.

The values of relative resistance to CDDP or ADM were 4.7,8.1, 7.5,20.0, and 13.6 for PC-7/1.0, PC-9/0.5, PC-14/1.5, H69/0.4, and K562/ADM, respectively. CDDP-resistant cell lines showed no cross-resistancewith other drugs in this study. K562/ADM showed cross-resistanceagainst daunomycin, etoposide, and vindesine. In contrast, mitomycin Cand bleomycin had nearly equal activity against K562 and K562/ADM.However, K562/ADM was 2.4-fold more sensitive to CDDP than itsparental cell line, K562 (I' < 0.001). These results suggested that the

mechanism of CDDP resistance is different from that of multidrugresistance.

INTRODUCTION

Although SCLC1 is responsive to initial chemotherapy, relapses are common, and only 5 to 10% of patients have long-term survival of greater than 3 yr (1). In advanced NSCLCpatients, chemotherapy has little impact on the survival rate(2). It is essential to identify new effective anticancer drugs in

Received 1/18/89; revised 4/11/89; accepted 5/3/89.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.

1This work was supported by the Comprehensive 10-Year Strategy for CancerControl and the Ministry of Health and Welfare, Japan.

2To whom requests for reprints should be addressed, at Department of InternalMedicine, National Cancer Center Hospital, 1-1, Tsukiji 5-chome, Chuo-ku,Tokyo 104, Japan.

3The abbreviations used are: SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; CDDP, cisplatin; ADM, Adriamycin; MMC, mitomycinC; LD!0, 50% lethal dose; BLM, bleomycin; FBS, fetal bovine serum, VP-16,etoposide; VDS, vindesine; DM, daunomycin; HS, horse serum; IC50, 50%inhibitory concentration; PPC, peak plasma concentration; CBDCA, carboplatin.

order to improve the survival rate in lung cancer.Although CDDP and ADM have been demonstrated to be

active drugs against SCLC, the acquired resistance to CDDPand/or ADM is a common cause of chemotherapy failure inSCLC. It is important to develop new anticancer drugs whichare active against CDDP- and/or ADM-resistant tumor cells

for the treatment of patients in whom CDDP and/or ADMtreatment was not effective.

Many new anticancer agents used in Phase II studies areineffective, and the patients who enter these studies with inactive agents sometimes have the disadvantage of a shortenedsurvival time. However, it is important not to overlook thepossible new active anticancer agents. Therefore, critical preclinical evaluation of new anticancer agents is important. Wehave developed a new model to predict the antitumor activityof new analogues using relative antitumor activity. This modelis proposed for the prediction of the cross-resistance of CDDPand ADM, using their resistant cell lines.

KT6149 is a new derivative of MMC and has been demonstrated to be more active against mouse tumors and humanxenografts than MMC.4 The LD50 value of KT6149 in mice ishigher than that of MMC. MX-2, SM5887, and menogaril arenew anthracyclines with greater antitumor activity and lowercardiac toxicity than ADM. Liblomycin is a new derivative ofBLM, and its pulmonary toxicity has been predicted to be lowerthan that of BLM (3, 4).

In order to accurately determine the preclinical evaluation ofthe new analogues, KT6149, MX-2, SM5887, menogaril, andliblomycin, the antitumor activity of these drugs against severalhuman cancer cell lines and their CDDP- or ADM-resistantcell lines was evaluated by clonogenic assay and compared tothat of their parent compounds, MMC, ADM, and BLM.

MATERIALS AND METHODS

Tumor Cell Lines and Culture. Four NSCLC-, two SCLC-, fourCDDP-resistant lung cancer cell lines, a human myelogenous leukemiacell line, and the derived ADM-resistant sublines were used in aclonogenic assay. The NSCLC cell lines, PC-7, -9, -13, and -14, werekindly donated by Professor Y. Hayata of the Tokyo Medical College.PC-7, -9, and -14 were derived from an adenocarcinoma, and PC-13was from a large cell carcinoma (5, 6). The SCLC cell lines, H69 andN231, were established at the National Cancer Institute, USA (7).CDDP-resistant cell lines, PC-7/1.0, PC-9/0.5, PC-14/1.5, and H69/0.4, were established in our laboratory (8), and they were maintainedat concentrations of 1.0,0.5, 1.5, and 0.4 ¿ig/mlof CDDP, respectively.The human myelogenous leukemia cell line, K562 (9), and its ADM-resistant subline, K562/ADM (10), were kindly donated by Dr. T.Tsuruo of the Japanese Foundation for Cancer Research. This cell linewas maintained at the concentration of 0.3 Mg/m' of ADM. Thecharacteristics of these cell lines are shown in Table 1. Prior to use inthe clonogenic assay, the cell lines were propagated by culturing in

4 Personal communication.

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PRECLINICAL EVALUATION OF NEW AGENTS ON RESISTANT CELLS

Table 1 Characteristics and plating cell numbers of human cancer cell lines

CelllinePC-7PC-7/1.0PC-9PC-9/0.5PC-

13PC-14PC-14/1.5H69H69/0.4N231K562K562/ADMHistologyAdeno"AdenoAdenoAdenoLargeAdenoAdenoSmall,

classicSmall,classicSmall,classicCMLCMLPrior

chemotherapy>rAro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacendione hydrochloride (SM5887), 1-con-O-methylnogarol (menogaril, TUT-7), and 3-|(Ar-methyl-A'-j3/-[bis-(3",4"-diben7.yloxybenzyl)aniino]propyl|amino|| propylaminobleomy-

cin hydrochloride (liblomycin, NK313) are shown in Fig. 1.The drugs were obtained from the following sources: KT6149, MMC,

and ADM from Kyowa Hakko Co., Ltd., Tokyo; MX-2 (KRN8602)from Kirin Co., Ltd., Tokyo; SM5887 from Sumitomo Co., Ltd.,Osaka; menogaril (TUT-7) from Taihou Co., Ltd., Tokyo; liblomycinand BLM from Nippon Kayaku Co., Ltd., Tokyo; CDDP and VP-16from Bristol Myers, Co., Ltd., New York, NY; YDS from ShionogiCo., Ltd., Osaka; and DM from Meiji Co., Ltd., Tokyo, Japan.

All drugs except CDDP were dissolved in distilled water and storedat -20°C.The drugs were diluted with RPMI 1640 plus 10% FBS just

prior to each experiment.Clonogenic Assay. The clonogenic assay used in this study was a

modification of the double agar method developed by Hamburger andSalmon (11-13). Tumor cells were harvested from the cell culture and

counted. Viable cells were determined by the trypan blue dye exclusionmethod. The number of cells plated was determined according to thegrowth rate and the plating efficiency of each cell line (Table 1). Allassays were performed using 6-well plate dishes (Linbro, VA; No. 76-058-05). Tumor cells plated in the top layer consisted of RPMI 1640plus 10% FBS, 0.3% agar, and the appropriate concentration of eachdrug. All experiments were performed with continuous drug exposure.One ml per well was layered onto the previously prepared underlayer.The underlayer contained 0.5% agar in enriched McCoy's Medium 5A(Gibco). Enriched McCoy's Medium 5A consisted of 50 ml of heat-inactivated FBS, 25 ml of heat-inactivated HS, 4 ml of 2.2% sodiumpyruvate, 4 ml of 200 HIMglutamine, 0.8 ml of 2.1% serine, 4 ml of100 units/ml of penicillin, and 100 ¿ig/mlof streptomycin, mixed with400 ml of McCoy's Medium 5A. All assays were performed in triplicate,

and the value obtained from one experiment represents the mean of thetriplicate assay. Plates were incubated in a humidified atmosphere of5% CO2 at 37'C. After 7 to 21 days, the number of colonies was

counted once very 2 to 3 days with a CP-2000 automatic colony counter(Shiraimatsu Instrument, Osaka, Japan). Colonies larger than 50 urnin diameter were regarded as positive. The percentage of colony inhibition was determined by the following formula.

% of colony inhibition

= 100- no. of maximal colonies/test dishno. of maximal colonies/control dish

x 100

Mitomycin C

SMS887MX-2

MenogarilAdricmycin

Liblomycin

Bl«omycm

Fig. 1. Chemical structure of KT6149, MMC, MX-2, SM5887, menogaril,ADM, liblomycin, and BLM.

Analysis. The IC5ovalue was determined graphically from the dose-response curve at three or four drug concentration points. The experiments were repeated 3 to 6 times for each drug and each cell line. Therelative antitumor activity, calculated by the following formula (14),was used to compare the antitumor effect of each drug.

Relative antitumor activitypeak plasma concentration Qjg/ml)

1C..,,values Gig/ml)

The PPC of MMC, CDDP, VP-16, YDS, ADM, DM, menogaril,

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and BLM has been reported in previous Phase I studies (Table 2) (15).However, accurate pharmacokinetic data of KT6149, MX-2, SM5887,and liblomycin have not been reported in humans. Therefore, the PPCof KT6149, MX-2, SM5887, and liblomycin was obtained by thefollowing formula (16).

Log(PPC) = 0.755 x log(mice LD,0) -0.788

Statistical Analysis. AH data were analyzed for significance by thetwo-tailed Student t test. P values were calculated by comparison ofexperimental groups.

RESULTS

Comparison of the IC5()Values of Several Anticancer DrugsUsed Clinically. The IC5»values for MMC, CDDP, VP-16,YDS, BLM, DM, and ADM against six human lung cancercell lines and four cell lines selected for CDDP resistance andagainst the human myelogenous leukemia cell line and itsADM-resistant subline were determined by clonogenic assay(Tables 3 to 6).

PC-7/1.0, PC-9/0.5, PC-14/1.5, and H69/0.4 were moreresistant to CDDP than the parental cell lines, with the relativeresistance (IC5o of resistant cell line/IC50 of parental cell line)of 4.7, 8.1, 7.5, and 20.0, respectively. As shown in Table 7, nocross-resistance was observed between the parental and theCDDP-resistant cell line to other drugs in this study. K562/ADM was 13.6 times more resistant to ADM than its parentalcell line, and 40.8-, 11.1-, and 771.4-fold resistant to DM, VP-

Table 2 Peak plasma concentration and predicted peak plasma concentration ofseveral anticancer agents

LD!0 values of new agents were obtained from the respective pharmaceuticalcompany.

Table 4 Comparison o//C50 values and relative antitumor activity (peak plasmaconcentration/ICyJ between MMC and KT6I49 of human cancer cell lines and

the CDDP- or ADM-resistant sublines

AgentsMMCKT6149ADM

DMMX-2(KRN8602)

SM5887Menogaril(TUT-7)BLMLiblomycin

(NK313)CDDPVP-16VDSLD50

inmice(mg/kg)8.422.523

8.64.93

42(152)"303

11326.81056.3Peak

plasma concentration(jig/ml)0.52

(10 mg/m2 i.v.)1.71(predicted)0.60

(60 mg/m2 i.v.)0.41 (100 mg/m2 i.v.)

0.54 (predicted)2.73 (predicted)0.85 (200 mg/m2i.v.)3.00(15

units/m2 i.v.)

5.78(predicted)2.49

(100 mg/m2i.v.)30.0

(130 mg/m2i.v.)0.883

(2.0 mg/m2 i.v.)

°Total dose of 5 days' administration.

Table 3 /C50 values of human cancer cell lines and its CDDP- or ADM-resistantsublines to several anticancer agents

Values were obtained from more than 3 experiments.

IC50values Oig/ml)

CDDP DM VDS VP-16

PC-7PC-7/1.0PC-9PC-9/0.5PC-

13PC-14PC-14/1.5H69H69/0.4N231K562K562/ADM0.472.20.191.60.80.64.90.071.40.61.70.09"1.10.100.40.20.21.40.040.90.30.20.7

0.10.024

+0.0010.024±0.0120.13

±0.070.16±0.060.37

±0.620.190.150.210.110.410.050.120.070.130.110.230.042.2

0.20.00170.00250.0140.0370.00370.0070.0110.0050.00320.0070.00231.8±++±¿+±-t-0.00050.00030.0070.0160.00190.0020.0060.002±0.0016+++0.0020.00010.80.26

±0.110.273.82.10.472.31.70.430.50.201.10.20.070.30.10.060.10.40

±0.100.5±0.16±2

IC50values(jig/ml)PC-7PC-7/1.0PC-9PC-9/0.5PC-

13PC-14PC-14/1.5H69H69/0.4N231K562K562/ADMMMC0.0240.0400.0400.0390.0440.250.210.0320.060.0490.180.005"0.0180.0120.0100.0150.000.070.0180.040.0230.090.28

0.09KT61490.049

±0.0190.046±0.0190.06

±0.010.07±0.010.07

±0.030.17±0.050.13

±0.070.010±0.0040.06

±0.040.041±0.0250.08±0.010.13

±0.04Relative

anlitu-moractivityMMC21.413.013.013.512.02.12.516.58.010.62.91.9KT614935.137.529.325.324.89.913.5171.028.142.222.313.5

' Mean ±SD.

°Mean ±SD.

16, and VDS, respectively. MMC and BLM had almost thesame activity against K562 and K562/ADM. However, K562/ADM was 2.4-fold more sensitive to CDDP than its parentalcell line, K562 (P < 0.001) (Fig. 2).

Comparison of the Antitumor Activity of KT6149 and MMC.As shown in Table 4, KT6149 and MMC had nearly the sameIC5()values. In a previous Phase I study, PPC of MMC wasreported to be 0.52 Mg/ml (15). However, PPC of KT6149 inhumans has not been reported yet, and therefore the predictedPPC was calculated from the LD50 value of mice using theabove-mentioned formula (16). The predicted PPC of KT6149was 1.71 ng/m\ (Table 2). As shown in Table 4, KT6149 showeda higher relative antitumor activity than MMC in all cell lines.

Comparison of the Antitumor Activity of New Anthracyclinesand ADM. MX-2, SM5887, menogaril, and ADM were aseffective against CDDP-resistant sublines as the parental celllines. MX-2 and menogaril had similar IC50 values to ADM inhuman lung cancer cell lines and its CDDP-resistant sublines.SM5887 showed higher IC5values than that of ADM in alllung cancer cell lines in this study (Table 5). In a previousPhase I study, the PPC values of menogaril and ADM werereported to be 0.85 and 0.60 tig/m\, respectively (15). Thepredicted PPC values MX-2 and SM5887, calculated from theLD50 values of mice (16), were 0.54 and 2.73 (ig/m\, respectively. Menogaril had a higher relative antitumor activity thanADM, while MX-2 had activity similar to ADM. However, theantitumor activity of SM 5887 was lower than that of ADM inlung cancer cell lines (Table 5). ADM, SM5887, and menogarilwere less effective against K562/ADM than its parental cellline, K562. Nevertheless, MX-2 had the same antitumor activityagainst K562/ADM to its parental cell line (Tables 4 and 7).

Comparison of the Antitumor Activity of Liblomycin andBLM. IC50values of liblomycin against human lung cancer celllines and human myelogenous leukemia cell lines were slightlyhigher than those of BLM (Table 6). The reported PPC ofBLM is 3.00 Mg/ml (15), and the predicted PPC of liblomycin,from LD5o values of mice, is 5.78 ng/ml. Liblomycin had arelative antitumor activity similar to BLM.

DISCUSSION

MMC is considered to be one of the active anticancer agentsagainst NSCLC. KT6149 is a derivative of MMC with lesstoxicity than MMC in mice. KT6149 showed no cross-resistance and no collateral sensitivity against CDDP- or ADM-resistant sublines. KT6149 was more active than MMC againstall lung cancer cell lines, and thus it can be concluded by this

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Table 5 Comparison of 1C¡avalues and relative antitumor activity between ADM and new anlhracyclines of human cancer cell linesand the CDDP- or ADM-resistant sublines

IC50values (tig/ml) Relative antitumor activity

ADM MX-2 SM5887 Menogaril ADM MX-2 SM5887 Menogaril

PC-7PC-7/1.0PC-9PC-9/0.5PC-

13PC-14PC-14/1.5H69H69/0.4N231K562K562/ADM0.12

±0.01°0.09

±0.060.6±0.40.28±0.060.24±0.020.8±0.20.48±0.320.18

±0.050.14+0.050.19

±0.120.14+0.081

.9 ±0.90.120.110.210.310.210.340.310.370.120.230.180.220.070.030.030.090.020.140.100.150.090.010.080.020.8

±0.20.7±0.21.6

±0.52.2+0.131.9+0.42.5+0.12.1

±0.32.1±0.21.2

+0.41.5+0.80.8

±0.12.3+ 0.150.090.110.260.50.130.250.190.240.150.060.091.20.030.050.040.30.080.010.060.030.060.010.010.74.96.41.02.12.60.81.33.44.43.24.30.34.45.12.51.72.61.61.71.54.62.43.02.53.44.01.71.21.41.11.31.32.31.83.61.29.17.93.31.66.83.54.53.55.S13.39.80.7

" Mean + SD.

Table 6 Comparison o//C!0 values and relative anlitumor activity between BLMand liblomycin of human cancer cell lines and the CDDP- or

ADM-resistantsublinesIC50

values(fig/ml)PC-7

PC-7/1.0PC-9PC-9/0.5PC- 13PC- 14PC-14/1.5H69H69/0.4N231K562K562/ADMBLM0.26

±0.05"

0.43 ±0.231.2 ±0.71.9 ±1.0

0.18 ±0.060.7 ±0.4

0.27 ±0.090.21 +0.110.13 + 0.04

0.7 ±0.20.7 ±0.80.7 ±0.37Liblomycin0.6

±0.20.36 ±0.15

1.0 ±0.51.8 ±0.7

0.36 ±0.300.9 ±0.60.6 ±0.1

0.43 ±0.220.22 ±0.17

1.2 ±0.81.3 ±1.31.1 +0.4Relative

antitumoractivityBLM11.5

6.92.51.6

16.44.2

11.014.323.74.34.14.3Liblomycin10.1

16.15.83.2

16.16.59.9

13.626.3

4.94.65.3

" Mean ±SD.

Table 7 Relative resistance C/C50values of resistant sublines/ICX values ofparental cell lines) of several anticancer agents to CDDP- or ADM-resistant

human cancer cell lines

CDDPMMCKT6149ADMDMMX-2SM5887MenogarilBLMLiblomycinVDSVP-

16*/>1log. A more accurate means of PPC prediction must be devised.However, it may be possible to compare the clinical benefits ofsimilar compounds, such as CDDP versus CBDCA, MMCversus KT6149, ADM versus other anthracyclines, and BLMversus liblomycin.

We used CDDP-resistant cell lines and attempted to detectnoncross-resistant analogue agents against CDDP. The CDDP-resistant human lung cancer sublines, PC-7/1.0, PC-9/0.5, PC-14/1.5, and H69/0.4, were more resistant to CDDP than theparental cell lines. Cross-resistance to other agents was nottypically observed in parental or CDDP-resistant cell lines inthis experimental system.

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ADM-resistant cancer cells have shown cross-resistance tonot only other anthracyclines and Vinca alkaloids but also otheranticancer agents. It is also important to identify effectiveagents against ADM-resistant sublines in the screening of newanticancer anthracyclines. Therefore, we used the ADM-resistant human myelogenous leukemia subline, K562/ADM, in thisstudy. K562/ADM was 13.6-fold more resistant to ADM thanits parental cell line and showed cross-resistance with DM, VP-16, and YDS. In contrast, MMC and BLM had almost thesame efficacy against K562 as K562/ADM. Moreover, K562/ADM was 2.4-fold more sensitive to CDDP than its parentalcell line, K562. These results suggest that the mechanism ofCDDP resistance is different from that of multidrug resistance.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the review and criticism of Dr.M. Terada, Dr. B. E. Johnson, and Dr. H. Tapiero.

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1989;49:4098-4102. Cancer Res Yuichiro Ohe, Kazuhiko Nakagawa, Yasuhiro Fujiwara, et al. Adriamycin-resistant Human Cancer Cell LinesSM5887, Menogaril, and Liblomycin Using Cisplatin- or

Evaluation of the New Anticancer Agents KT6149, MX-2,In Vitro

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