aiititiinior activity of a novel antitumor antibiotic ......group. sarcoma 180 (5 x 10* cells/mouse)...

[CANCER RESEARCH 47, 1516-1522, March 15, 1987]

Aiititiinior Activity of a Novel Antitumor Antibiotic, Quinocarmycin

Citrate (KW2152)Kazuhisa Fujimoto, Tetsuo Oka, and Makoto Morimoto'

Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Shimotogari, Nagaizumi-cho, Sunto, Shizuoka 411, Japan

ABSTRACT

A novel antitumor antibiotic, 2Â«,3,4,5,6,6a,7,lIA-octahydro-lI-nieth-oxy-12-methyl-3,6-imino-l//-2-oxa-llc-azanaphth(l,2,3-c</)azulene-5-carboxylic acid monocitrate (quinocarmycin citrate; KW2152) was selected for investigation in a number of experimental tumor systemsbecause of its efficacy against P388 leukemia.

In the initial studies with P388 leukemia (i.p.-i.p.), KW21S2 gave anincrease in life span of >80%. The activity was schedule dependent anddaily administration was the most effective. KW2152 caused marginalactivity against LI 210 leukemia, B16 melanoma, and M51176sarcoma,riu- effect on cultured cells suggested that KW2152 was not cross-resistant to Adriamycin (ADM) but was cross-resistant to mitomycin C(MMC); however, KW2152 caused prolongation of life span against micebearing P388/AOM or P388/MMC. In tests against human tumorsxenografted s.c. in nude mice, KVV2152significantly inhibited the growthof M VI mammary carcinoma with all tumors cured at i.v. doses of 4.4mg/kg/day and p.o. doses of 26.2 mg/kg/day given daily for 7 days.KW2152 also inhibited distinct human gastric carcinomas, St-4 and Si-15 tumors, and colon carcinoma Co-3 by daily administration for 7 days.Against Si-4. KW2I52 gave a treated versus control percentage of 27,compared to 52 for ciVdiamminedichloroplatinum. Against Co-3,KVV2152was at least as effective as MMC, ADM, cfr-diamminedichlo-roplatinum, and bleomycin, giving a treated versus control percentage of18 at a dose of 8.6 mg/kg/day given daily for 7 days. KVV2152showedgrowth inhibitory activity against cultured murine tumors and humancells. The order of in vitro efficacy of KW2I52 against murine tumors,P388 leukemia > 1,1210 leukemia, B16 melanoma, correlated with theorder of the sensitivity on the i.p.-i.p. systems of these tumors. The 50%inhibitory concentrations against P388 leukemia cells were 5.3 x 10~*and 1.1 x 10~7 M after 1 and 72 h exposure, respectively. KW2152

caused significant inhibition of RNA synthesis after a short time exposure. In P388 leukemia cells exposed for 1 h with KW2152, the 50%inhibitory concentration for RNA synthesis was 10~5M, 30-fold less than

that for DNA synthesis.White blood cell depression or platelet depression was not significant

after administration of the i.v. 10% lethal dose given daily for 7 days.Because of its good activity against human mammary tumor MX-1

and some effectiveness against other gastric and colon carcinomas andits water solubility, a novel antitumor antibiotic, KW2152, is beingdeveloped as a Phase I anticancer agent.

INTRODUCTION

Quinocarmycin citrate (KW2152), a citrate salt of quinocar-cin (DC52) (1, 2) which contains a novel skeleton with theisoquinoline structure in the molecule, was found in the culturebroth of Streptomyces melanovinaceus nov. sp. Quinocarmycincitrate was active against Bacillus subtilis, Slaphylococcus au-reus, and Klebsiella pneumoniae but not against Gram negativebacteria. The antitumor activity of quinocarcin has not beenreported.

However, since quinocarcin is not stable in aqueous solutionfor the purpose of further evaluation including clinical trials, acitrate salt of quinocarcin was synthesized with higher stability,

Received 5/27/86; revised 10/28/86; accepted 12/1/86.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.

and more than 95% activity remained after 72 h in phosphatebuffer (pH 7.2) at 37Â°C.

Saframycins (3) and naphthyridinomycin (4) were antitumorantibiotics with common pyrazinoisoquinoline ring systems intheir molecule. However, quinocarcin also has a pyrazinoisoquinoline structure completely different from both compoundsin the structure, because quinocarcin has an aromatic ring inplace of the quinone ring of saframycin and naphthyridinomycin, and interestingly it has a carboxylic acid group that isabsent in the other two compounds. Saframycin (5) was reported to be highly active against P388 leukemia and moderately active against LI210 leukemia and B16 melanoma, butantitumor activity against human tumor cells transplanted tonude mice were not reported. Naphthyridinomycin was reported to have only marginal activity (6).

This paper describes the spectrum of activity of KW2152against murine tumors and human carcinomas xenografted innude mice. We also report its growth inhibitory activity againstcultured cells and its effect on macromolecular synthesis.

MATERIALS AND METHODS

Drugs. Quinocarmycin citrate (KW2152; M, 522.5) was isolated fromS. melanovinaceus in our laboratory. The structure is shown in Fig. I.Its isolation procedure and chemical nature have been reported previously (2). Mitomycin C and Adriamycin were obtained from KyowaHakko Kogyo Co., Ltd., Tokyo, Japan. Bleomycin, formulated forclinical use, was obtained from Nippon Kayaku Co., Ltd., Tokyo,Japan. rÃ -Diamminedichloroplatinum was synthesized in our laboratory. [6-3H]Thymidine (22 Ci/mmol), [5-3H]uridine (25 Ci/mmol), andL-[4,5-3H]leucine (54 Ci/mmol) were purchased from Amersham Inter

national, Pic (Amersham, England).Animal and Tumor Cells. Adult male BALB/c x DBA/2 F, (hereafter

called CD2F,) mice weighing 20-25 g, male C57BL/6 x DBA/2 F,(hereafter called B6D2F!> mice weighing 20-25 g, male ddY miceweighing 18-20 g were obtained from Shizuoka Agricultural Co-operative Association for Laboratory Animals, Hamamatsu, Japan. Adultmale BALB/c-nu/nw mice weighing 20-25 g were supplied by NihonClea Co., Ltd., Tokyo, Japan. P388 leukemia, LI210 leukemia, B16melanoma, M5076 sarcoma, Lewis lung carcinoma. Sarcoma 180, andhuman mammary carcinoma MX-1 cells were supplied by CancerChemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan. P388/ADM2 leukemia cells (7) and P388/MMC leukemia

cells obtained by serial passage in mice bearing P388 with MMC orADM were kindly supplied by Dr. Inaba, Cancer Chemotherapy Center,Japanese Foundation for Cancer Research. Co-3 (8), well differentiatedadenocarcinoma of colon; St-4 (8), poorly differentiated gastric ade-nocarcinoma with a few tubular structures and signet ring type cells;and Si 15 (8), mucinous adenocarcinoma of stomach, were kindlysupplied by Dr. Kubota (8), Kitazato Medical School. HeLa S, (9), KB(10), and SK-MEL-28 (11) cells were passaged in culture and periodically checked for contamination with Mycoplasma. growth properties,

2The abbreviations used are: P388/ADM. strain of P388 resistant to ADM;

P388/MMC, strain of P388 resistant to MMC; MMC, mitomycin C; ADM,Adriamycin; cw-DDP, m-diamminedichloroplatinum; BLM. bleomycin; T/C,treated/control; II),,,, dose required to kill 50% of the treated animals; LD1(hdose required to kill 10% of the treated animals; GIBCO, Grand Island BiologicalCo., Grand Island, NY; ICjo, drug concentration required for 50% growthinhibition from the cell numbers of duplicate or triplicate cultures at eachconcentration.

1516

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ANTITUMOR ACTIVITY OF QUINOCARMYCIN CITRATE

COOH

HiCO

Fig. 1. Chemical structure of KW2I52.

CHiCOOH

HOCCOOH

CHiOH

and sensitivity to several antitumor agents in our laboratory.Evaluation of Antitumor Activity. LI210 (IO5), P388 (IO6), P388/

ADM (IO6), or P388/MMC (IO6) cells were transplanted i.p. into

CD2F| mice and i.p. administration of drugs was started the day aftertumor transplantation. B16 melanoma was introduced into B6D2F|mice by i.p. administration of 0.5 ml of a 10% homogenate of tumorbivi and drug administration was started the day after tumor transplantation. M5076 (IO6)was transplanted i.p. into B6D2F, mice. P388 cells( 10*)transplanted i.v. were treated with i.v. drugs. Lewis lung carcinomawas transplanted s.c. at 8 mm3 by trocar to B6D2F, mice and drug

were administered i.v. Antitumor efficacy against the above systemswas expressed as a percentage of the mean survival time of the controlgroup. Sarcoma 180 (5 x 10* cells/mouse) was inoculated s.c. at the

axillary region in ddY mice. Drugs were administered i.v. starting theday after tumor inoculation and the tumor volume was measured onday 7. Efficacy was based on mean tumor volume as derived fromcaliper measurements of the length and width of the tumor volumecalculated using the formula for a prolate ellipsoid

Tumor volume =L (mm) x W2 (mm)

in which L is the length of the major axis and W is the length of theminor axis (12). The T/C percentage of mean tumor volume wascalculated. BALB/c-nu/nÂ« mice were given s.c. tumor fragment equivalent to 8 mm3 of MX-1, Co-3, St-4, or St-15 tumor passaged in nudemouse. When tumor volume reached to 100-300 mm3, the mice were

pair matched in groups of 6 mice each and drug or vehicle wasadministered i.v. according to the protocol. Tumor volume was calculated as shown for Sarcoma 180 cells. Drug efficacy was expressed asthe percentage of the VÂ¡Vaof the control group, where V is the meantumor volume at the day of evaluation and I â€žis the mean tumor volumeat the day of initial administration of drug.

Cell Culture. P388, P388/MMC, P388/ADM, L1210, and Sarcoma180 cells were transplanted to the culture medium with Roswell ParkMemorial Institute Medium 1640 (GIBCO) supplemented with 10%fetal bovine serum (GIBCO), 100 units/ml of penicillin, and 100 jig/ml of streptomycin. B16, HeLa S3 (9), KB (10), and SK-MEL-28 (11)cells were cultured in Eagle's minimum essential medium (Nissui

Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetalbovine serum (GIBCO) and 60 ng/ml of kanamycin.

The cultures were incubated at 37Â°Cin a humidified atmosphere of

5% CO2. For the drug treatment experiments, P388, P388/ADM,P388/MMC, LI210, and Sarcoma 180 cells (2 x IO4 cells) were

cultured in Falcon 2058 culture tubes (Falcon Plastics, Oxnard, CA)containing 1 ml of growth medium. B16, HeLa S., KB, and SK-MEL-28 cells were cultured in Nunclon 24-well multidishes (Nunc, Roskilde,Denmark) containing 1 ml of growth medium. For the short exposuretime experiments (l h exposure) cells were transferred to the freshgrowth medium and incubated for another 71 h. The monolayer cellswere washed with phosphate buffered salts solution (Flow Laboratories,United Kingdom) and incubated with 0.05% trypsin (Difco Laboratories, Detroit, MI)-0.02% EDTA (Wako Pure Chemical Industries Co.,Ltd., Osaka, Japan). Monolayer cells treated as described above orsuspension cells were counted with a Toa Micro-Cell Counter (ToaMedical Electronics Co., Ltd., Kobe, Japan).

The growth inhibitory activity was expressed at the ICso.Kffect on the Incorporation of labeled Precursors into Nucleic Acids

and Proteins. For the time response studies, P388 cells (10s/ml) inexponential phase were cultured simultaneously with KW2152 ( 10~5or10~4M) and [6-3H]thymidine (1 nCi/m\) Or [5-3H]uridine (l ^Ci/ml) orL-[4,5-3H]leucine (1 ^Ci/ml) for various time periods. The radioactivity

incorporated into the trichloroacetic acid insoluble fraction was counted

in a liquid scintillation counter and the average radioactivities of 3tubes were calculated. For the dose-response studies, P388 cells (IO5/

ml) were cultured in growth medium simultaneously with variousconcentrations of KW2152 and |6-'H]thymidine (l ÃŸC\/m\)or [5-3H]-uridine (1 jiCi/ml) or t.-[4,5-3H]leucine (1 nCi/ml) for 1 h. The radio

activity incorporated into the trichloroacetic acid insoluble fraction wascounted. The average radioactivities of 3 tubes at each point werecalculated and a percentage of the radioactivity of the control groupwas determined.

Toxicity Testing. The i.v. LDW and LD10 values of KW2152 aftersingle and daily administration were determined in ddY and CD2F,mice after 30 days of observation and were calculated by probit analysis.

Hematological measurements were determined 3, 5, 7, 10, 14, 17,and 23 days after initial drug administration. The retroorbital sinusblood samples of CD2Fi mice were counted for WBC and RBC with aToa Micro-Cell Counter and for platelets with a Toa Platelet Counter(Toa Medical Electronics Co., Ltd.). KW2152 was injected at the i.v.1.1)ii, or 1/1.57 I 1) i,dose daily for 7 days and mitomycin C was givenin a single i.v. LD,0 dose.

RESULTS

Schedule Dependency of KW2152 against P388 Leukemia.KW2152 showed good activity against i.p. P388 leukemia inmice, consistently giving increased survival times of >80% (T/C percentage > 180) at doses of 2.1-6.1 mg/kg/day given i.p.daily for 7 days. Representative results from several experiments are shown in Table 1. The activity of KW2152 wasschedule dependent with optimal results obtained with dailyadministration for 7 days. The highest T/C percentage (215)was achieved with a dose of 6.1 mg/kg/day administered i.p.daily for 7 days. Intermittent scheduling resulted in decreasedefficacy with the highest T/C percentage (170), but singleinjection gave only marginal activity with the highest T/Cpercentage (130) found with 16.4 mg/kg. Daily injection couldgive more than a 2-fold higher total dose than single administration (Table 1).

Effect of KVV2152 on Murine Tumors. From the scheduledependency test on P388, antitumor test against murine tumorswas evaluated by daily injection. KW2152 showed marginalactivity against i.p. 11210 leukemia in mice and i.p. B16melanoma in mice with T/C percentages of 139 (7.3 mg/kg/day) and 140 (3.7 mg/kg/day), respectively.

Against the M5076 sarcoma, KW2152 showed marginalactivity (T/C percentage, 125 at 3.7 mg/kg/day for 10 days).

KW2152 showed activity against i.v. P388 leukemia in mice.Daily i.v. administration for 7 days (5.7 mg/kg/day) gave a T/C percentage of 157 in one test.

Table 1 Effect ofKW2152 on survival of mice with P3SX leukemiaThe effects of dose and schedule on the efficacy of KW2I52 against P388

leukemia were determined. P388 cells (1 x I0*/mouse) were implanted intoCD2Fi mice (6 mice/group) on day 0 and i.p. treatment was initiated on day Iaccording to the schedules shown below.

Dose (mg/kg/day)Control24.616.412.38.26.14.12.11.00.510.26Day

1Mean

survivaldays Â±SD10.0

Â±0.84.5Â±3.813.0

Â±3.5NT11.8Â±

1.1NT11.7

Â±0.811.7Â±0.5NTNTNTT/C(%)45130118117117Days

1,5,Mean


Â±0.8NT"NT6.2

Â±0.417.0Â±015.3

Â±1.815.3Â±1.213.8Â±1.712.5Â±1.012.0Â±1.6NT9T/C

(%)62170153153138125120Days

1-7Mean


Â±0.8NTNTNT19.8

+8.921.5Â±2.519.2

Â±1.318.0Â±2.315.3

Â±1.413.8Â±2.313.0Â±1.2T/C(%)198215192180153138130

1NT, not tested.

1517on July 7, 2020. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from



KW2152 was not effective against s.c. Lewis lung carcinomain mice on the prolongation of life span. KW2152 also did notshow activity against s.c. Sarcoma 180 by daily i.v. administration (Table 2).

KW2152 was active against Â¡.p.P388/MMC in mice with aT/C percentage of 168 at a dose of 5.4 mg/kg/day. It was alsoeffective against i.p. P388/ADM in mice with a T/C percentageof 168 at a dose of 3.6 mg/kg/day by daily administration for7 days (Table 3).

The growth inhibitory effects of KW2152 on P388 leukemiaand the above resistant cell lines were examined in culture.KW2152 gave IC50 value similar to those P388 leukemia cellsand P388/ADM cells after 1 and 72 h exposure; however,KW2152 was 6- and 45-fold less effective on P388/MMC cellsthan P388 leukemia cells, after 72 and l h exposure, respectively (Table 4).

Although KW2152 showed similar T/C percentages againstP388/ADM and P388/MMC, efficacy against cells in culturesuggested that KW2152 might not show cross-resistance toADM but might show cross-resistance to MMC.

Table 2 Effect ofKW2152 on murine tumorsLI210cells (10*/mouse) were implanted i.p. into CD2Fi mice (6 mice/group).

BI6 cells (0.5 ml of 10% homogenate) were implanted i.p. into B6D2F, mice(6 mice/group) on day 0. M5076 cells (10'/mouse) were implanted Â¡.p.intoB6D2Fi mice (6 mice/group). Against these models drug was administered i.p.on day I or daily for 7 or 10 days from day 1. P388 (10*/mouse) were implantedi.v. into < I >-1 i mice and drug was administered i.v. Lewis lung carcinoma cells(8-mm3 block) and Sarcoma 180 cells (5 x lO'/mouse) were implanted intoB6D2F, mice (6 mice/group) and ddY mice (6 mice/group), respectively, anddrug was administered i.v.

Tumor Drug Schedule of Dose (mg/ Mean survival T/CTumor site route treatment days Kg/day) days Â±SD (%)

L1210

BI6

M5076P388Lewis

lungcarci

nomaS180i.p.

i.|i.p. i.|i.p.i.]i.p.

i.]i.p. i.i.p.i.|i.p.

i.]i.p.i.|i.p.i.i.v.

i.i.v.i.i.v.i.s.c.

i.s.c.i.s.c.i.S.C.

i.s.c.

i.s.c.i.).

).1>.1-7

>.>.1).

1-10

J.).1).1-1011-711-101i.

1-514.77.314.7

3.77.43.711.75.911.45.711.75.97.2

Â±0.78.2Â±1.210.0Â±2.415.1

Â±2.917.5Â±4.4

21.0Â±1.223.0

Â±1.325.3Â±3.229.8

Â±3.811.0

Â±0.812.3+0.517.3

Â±0.825.5

Â±3.428.3Â±1.328.0Â±1.5100

113139100

116140100

10612510011215710011111010071Â°59

" Ratio of tumor volume of treated group to that of control group.

Table 3 Effect ofKH'2152 against CD2F, mice bearing P388/MMC ana P388/

ADMP388. P388/MMC, and P388/ADM cells (1 x lO'/mouse) were implanted

into CD2F, mice on day 0 (6 mice/group). Treatment was initiated on Day 1.

Tumor Drug Schedule of Dose(mg/ T/CTumor site Drug route treatment days kg/day) (%)

P388i.p.P388/MMC

i.p.P388/ADM

i.p.KW2152MMCADMKW2152MMCKW2152ADMi.p.i.p.i.p.i.p.i.p.Â¡.p.i.p.i.p.i.p.i.p.7.1-7

5.46.012.07.1-7

5.42.014.3-7

3.68.0132198164215111168104124168108

Effect of KW2152 on Human Tumors. KW2152 was evaluatedagainst MX-1 breast, Co-3 colon, St-4 gastric, and St-15 gastriccarcinomas under the growth of tumor cells transplanted tonude mice. Against the MX-1 human mammary carcinoma,KW2152 demonstrated significantly good activity after i.v.administration. Daily administration gave significant reductionof tumor volume and all tumors were not palpable on day 13at 5.9 and 4.4 mg/kg/day, respectively. The regrowth of tumorwas not observed until day 25 after starting drug administration.Single injection of KW2152 also gave temporary tumor regression, but tumor growth started with almost the same growthrate in the untreated control. MMC used as a positive controlproduced significant regression of tumor with all tumors curedat a dose of 6.0 mg/kg i.v., but the regression of tumor wasmore rapid with KW2152 than with MMC (Fig. 2A). KW2152was also significantly effective against MX-1 p.o. Because adose of 26.2 mg/kg/day cured all mice, an approximately 6-fold higher dose was required p.o. than i.v. to obtain the sameextent of effect. MMC was also effective p.o. with an approximately 5-fold dose i.v. needed (Fig. 2B).

Against St-4 human gastric carcinoma, KW2152 demonstrated moderate activity. A dose of 8.6 mg/kg/day with dailyi.v. administration produced the decrease in tumor size and T/C percentage value was 27 on day 10, but daily administrationof 5.7 mg/kg/day or a single administration (17.1 mg/kg) wasnot effective. MMC showed a growth inhibiting effect with aT/C percentage value of 32 after a single i.v. administration.KW2152 did not show a significant duration of activity, givingmaximum effect on day 10 and rapid growth of tumor thereafterwith daily administration for 7 days starting from day 0 (Fig.3).

Against St-15 human gastric carcinoma, KW2152 showedmoderate activity. Tumor growth was not observed until day 8by daily administration and T/C percentages were 29 and 35on day 13 at doses of 8.6 and 5.7 mg/kg/day, respectively.However, the regrowth of tumor was shown thereafter. Singlei.v. injection of MMC (6.0 mg/kg) was more effective with notumor growth until day 26 (Fig. 4).

Against Co-3, human colon carcinoma, KW2152 showedmoderate activity at doses of 5.7-8.6 mg/kg/day and was moreactive than MMC. T/C percentages for KW2152 were 18 (8.6mg/kg/day) and 30 (5.7 mg/kg/day) on day 11, and those forMMC were 41 and 39 on days 11 and 21, respectively (Fig. 5).

The antitumor activity of KW2152 against 4 human tumorxenografts was compared with that of established antitumoragents (Table 5). MMC, ADM, m-DDP, and BLM were administered at their optimal doses and schedules as determinedfrom reported values and preliminary lethality tests in nudemice conducted in our laboratory.3 As reported previously (14),MX-1 was relatively sensitive to various anticancer agents, butnot as many compounds cured all mice. MMC and m-DDPcured all mice, but ADM did not give significant growth inhibition against MX-1. The activity of KW2152 against MX-1was comparable to those of MMC and m-DDP. Against Co-3,only KW2152 showed tumor reduction with a T/C percentageof 18 and was more effective than MMC, ADM, m-DDP, andBLM which gave T/C percentages of 30-47.

Against St-4, MMC produced an effective response, but cis-DDP showed a marginal response with a T/C percentage of 52;however, KW2152 caused significant growth inhibition of St-4. MMC and m-DDP caused a significant tumor growth inhibition (T/C percentage = 7 for MMC and 9 for m-DDP)

3 Unpublished data.

1518




Table 4 Growth inhibitory activity ofKW 2152 in P38S, P388/MMC, and FÃŒSSIADM leukemia cells in cultureTumor cells (1-2 x 104/ml)wereseededin plastic tubescontaining 1 ml of growth medium,and gradedconcentrationof drug wereadded 24 h after the cellswere

seeded.After l h of pulseor 72 h of continuousdrug exposurethe tumor cellswerecounted, and 1CÂ»wasdeterminedas describedin "Materials and Methods."

KW2152Cell

lineP388

P388/MMCP388/ADM1

h (XKTÂ»)1.1

Â±0.6*49.4Â±13.7Â»'e

4.9 Â±l.7e72

h (xIO"7)1.6

Â±0.410.1 Â±5.1'

1.9 Â±0.5MMC1

h (xIO"Â«)3.7

Â±1.0599 Â±180e

5.8 Â±2.372

h (x10-")5.0

Â±1.5337 Â±150e9&Â±l.tfADM1

h (XIO"7)4.3

Â±0.510.3 Â±2.4e280 Â±75'72

h (xIO'Â«)2.9

Â±0.85.2 Â±1.0e175

Â±Kr"

' Mean Â±SD of four to six experiments.* P < 0.05 versus P388 leukemia cells by Student's t test.

20 30

Time after initial treatment ( days }Fig. 2. Effect of KW2152 on human mammary carcinoma MX-1. MX-1 cells

(8-mm1 fragment) were implanted s.c. into male BALB/c-nu/nu mice (6 mice/group). In i. when tumor volume reached to 217 Â±69 (SD) mm1, i.v. treatment

was initiated. Untreated control (â€¢);KW2152.17.6 mg/kg on day 0 (â€¢):KW2152,5.9 mg/kg/day daily administration for 7 days from day O(O); KW2152, 4.4 mg/kg/day daily i.v. administration for 7 days from day 0 (A); MMC, 6.0 mg/kg onday 0 (A). In />'.p.o. treatment was initiated when tumor volume reached to 184Â±51 mm1. Untreated control (â€¢);KW2152. 65.5 mg/kg on day 0 (â€¢);KW2152,

26.2 mg/kg/day daily administration for 7 days from day O (O); KW2152, 19.7mg/kg/day daily administration for 7 days from day 0 (A); MMC, 30 mg/kg onday 0 (A).

10

1.0

0 10 20

Timeafterinitialtreatment

30

days )

Fig. 3. Effect of KW2I52 on the growth of s.c. implanted human gastriccarcinoma St-4 (8-mm1 fragment/mouse). Drug administration was started onday 0 when tumor volume reached to 195 Â±49 mm1. Untreated control (â€¢);KW2152. 17.1 mg/kg on day 0 (â€¢);KVV2152, 8.6 mg/kg/day daily for 7 days(O); KW2152. 5.7 mg/kg/day daily for 7 days (A); MMC, 6.0 mg/kg on day 0(A).

30

Time after initial treatment ( days )

Fig. 4. Effect of KW2152 on the growth of s.c. implanted human gastriccarcinoma St-15 (8-mm1 fragment/mouse). Drug administration (i.v.) was startedon day 0 when tumor volume reached 189 Â±53 mm3. Untreated control (â€¢);KW2152, 20 mg/kg on day 0 (â€¢);KW2152, 8.6 mg/kg/day daily for 7 days (O);KW2152, 5.7 mg/kg/day daily for 7 days (A); MMC, 6.0 mg/kg on day 0 (A).

0 10

Timeafterinitialtreatment( days)Fig. 5. Effect of KW2I52 on the growth of s.c. implanted human colon

carcinoma Co-3 (8-mm1 fragment/mouse). Drug administration (i.v.) was startedon day 0 when tumor volume reached to 162 Â±41 mm3. Untreated control (â€¢);KW2152, 20 mg/kg on day 0 (â€¢);KW2152, 8.6 mg/kg/day daily for 7 days (O);KW2152. 5.7 mg/kg/day daily for 7 days (A); MMC, 6.0 mg/kg on day 0 (A).

against St-15. KW2152 was efficacious with T/C percentagevalue of 29, but less effective than MMC and cis-DDP.

Growth Inhibitory Effect of KVV2152 on Murine and HumanTumor Cell Lines in Culture. The growth inhibitory effect ofKW2152 against 4 murine tumor cell lines and 3 human tumorcell lines was tested with 1 and 72 h exposures. KW2152 showedcomparable potency against the murine tumors P388 leukemia,L1210 leukemia, B16 melanoma, and Sarcoma 180 with 72 hexposure; IC50ranged from 0.11 to 3.4 Â¿JM.After l h exposure,the ICso was 5.3 x IO"6 M for P388 leukemia but was 30- and

40-fold greater for LI210 and B16, respectively. The ratio of




Table 5 Response of human tumor xenografls to established anticancer agentsGroups of five or six BALB/c-nu/nu mice received s.c. tumor implants (8-mm3 block/mouse). Treatment with several doses and schedules of each drug was initiated

when tumor volume reached to 100-300 mm3 (day 0). KW2152 was administered i.v. daily for 7 days from day 0. MMC and ADM were administered i.v. on day 0.m-DDP and BLM were administered i.v. on days 0, 4, and 8. Therapeutic effects were calculated as indicated in "Materials and Methods." Data obtained with the

most effective dose are shown.T/C %' (dose,schedule)DrugKW2I52

MMCADMrÃ¡-DDPBLMMX-I0

(4.4 mg/kg, days 0-6)0 (6.0 mg/kg, day 0)

77 (10.0 mg/kg, day 0)0(7.1 mg/kg, days 0, 4, 8)

NTCo-318

(8.6 mg/kg, days 0-6)38 (6.0 mg/kg, day 0)47 (15.0 mg/kg, day 0)30(7.1 mg/kg, days 0, 4, 8)34 (80.0 mg/kg, days 0, 4. 8)St-427

(8.6 mg/kg, days 0-6)32 (6.0 mg/kg, day 0)

NT*

52(7.1 mg/kg, days 0, 4, 8)NTSt-1529

(8.6 mg/kg, days0-6)7(6.0 mg/kg, day 0)

NT9(7.1 mg/kg, days 0, 4, 8)

NT* T/C %, percentage of treated versus control tumor volume.* NT. not tested.

ICjo values of l h exposure to that of 72 h was about 50 forP388 but was in the range of 200-600 for other 3 murine tumorcells. Against human tumor cell lines, KW2152 also showedsignificant potency comparable to that against murine tumorcell lines by 72 h exposure; IC50 values were 0.10-0.16 nM.After l h exposure, IC50for HeLa S3 and KB cells were 10-foldsmaller than that for SK-MEL-28 cells (Table 6).

DNA, RNA, and Protein Synthesis. The effect on the synthesisof DNA, RNA, and protein of P388 leukemia cells was measured by the incorporation of [6-'H]thymidine, [5-'H]uridine,and L-[4,5-3H]leucine, respectively. Change was observed insynthesis of macromolecules exposed continuously to 10~5 Mor 10~4M KW2152 and radioactive precursors for various times.

Rapid inhibition of RNA synthesis was observed after slightincrease of RNA synthesis at 15 min. At 30 min RNA synthesiswas inhibited to 50% of control with 10~5M KW2152. On the

other hand, DNA and protein synthesis were not inhibited at60 min with 10-4-10-5 M KW2152 (Fig. 6A).

A dose-response relationship was measured after l h exposure to various concentrations of KW2152 and with radioactiveprecursors. Significant RNA selective inhibition was observedin which inhibition of RNA synthesis gradually increased asthe concentration of KW2152 increased from 3 x 10~6to 10"*M at an IC50 of 1.2 x 10~5 M which was comparable to the

concentration that inhibited 50% of in vitro cell growth forKW2152 with l h exposure. DNA synthesis was not significantly inhibited, as compared with RNA synthesis. The IC50ofKW2152 for DNA synthesis was 3.4 X IO'4 M after l h

exposure. Protein synthesis was not significantly inhibited withless than 20% inhibition after exposure of the cells to a KW2152concentration of 3 x 10~4 M, the concentration that inhibits

50% of DNA synthesis (Fig. 65).General Toxicity of KVV2152in Mice. LDÂ«,was 13.9 and 6.5

mg/kg/day by an i.v. single administration and an i.v. dailyadministration for 7 days, respectively. Successive administration could attain higher total dose than a single administration.

Table 6 Evaluation of the cellular cylotoxicity ofKW2152 in vitroTumor cells (2 to 5 x IO4)were seeded in plastic tubes or dishes containing I

ml of growth medium. Graded concentrations of drug were added 24 h after thecells were seeded. After l h pulse or 72 h drug exposure, the tumor cells werecounted and the K '<â€žwas determined as described in "Materials and Methods."

(A) Time response (B) Dose response

CelllineP388

L1210B16Sarcoma180HcLaS,

KBSK-MEL-28Ih5.3

x IO"*1.5 x 10-"2.1 x IO-41.9 xIO'37.6

x IO"56.0 x 10-*7.5 x IO"41CÂ»

(M)72

h1.1x10-'7.2x IO"7

7.3 x IO'73.4 x10-Â«1.6X

IO-7l.Ox IO"71.0 x IO'7

s -X

Ã‹

15 30 60

Time ( min )

100

50

0 10~s 10"* IO'3

Concentration ( M )Fig. 6. Effect of KW2152 on the incorporation of labeled precursors into

nucleic acids and proteins. In .1. KW2I52 and [6-3H]thymidine (top) or |5-3H|-uridine (middle) or L-|4,5-3H]leucine (bottom) were added to P388 leukemia cells(1 x 10') simultaneously and the radioactivity was counted at indicated time.Control (â€¢);KW2152, IO"1 M (â€¢):KW2152, 10~' M (A). In B. various concentrations of KW2152 and (6-3H]thymidine (â€¢)or |5-3Hluridine (A) or L-|4,5-3H1-leucine (â€¢)were added to P388 leukemia cells (1 x 10s) simultaneously and the

radioactivity was counted after pulse labeling for 1 h.

120

100

80

60

40

20

1000

800

600

400

200

7 10 14 17 23

Time after initial treatment ( days )

Fig. 7. Effect of KW2152 on WBC and platelets in CD2F, mice. CD2F, micewere given injections of KW2152 or MMC from day 0. and WBC (A) and platelet(B) counts were performed at indicated days. Untreated control (â€¢);KW2152,6.5 mg/kg/day daily for 7 days (O): KW2152, 5.8 mg/kg/day daily for 7 days(A); MMC, 5.6 mg/kg on day 0 (A).

WBC and platelets were measured periodically after i.v.administration of LDio and 1/1.57 x LD50 doses in CD2Fjmice. WBC nadir occurred on day 3 with 66% of mean control




WBC by successive administration of LD,0 dose (6.5 mg/kg/day). The WBC of mice treated with a single administration ofMMC (5.6 mg/kg) reached nadir with 57% of control on day 3and recovered on day 14.

Platelets decreased to 541 x 10' cells/mm3 on day 3 after an

LDin dose of daily administration for 7 days. On the otherhand, MMC gave a significant and prolonged suppression ofplatelets with 159 x IO3cells/mm3 on day 10 and the suppres

sion did not recover until day 23 (Fig. 7). The leukopenia andthrombocytopenia caused by KW2152 were not significant ascompared with those caused by MMC. KW2152 caused noabnormal values in erythrocyte counts after daily administrationfor 7 days (data not shown).

The main toxic signs observed in a single LD,0 dose werefailure to thrive, diarrhea, and decrease of thymus weight. Thesemain toxic signs of KW2152 were not clearly observed by adaily administration for 7 days except the decrease in thymusweight (data not shown).

DISCUSSION

KW2152 showed marked activity against P388 leukemia butmarginal activity against LI210 leukemia, B16 melanoma, andM5076 sarcoma in the i.p.-i.p. system. These results suggestedthat antitumor spectrum of KW2152 against murine tumorsystem tested was not remarkable among established antitumoragents. The growth inhibiting activity against cultured cellsshowed that P388 leukemia cells were sensitive to KW2152with an ICso of 5.3 x IO"6 M by l h exposure, but LI210

leukemia and B16 melanoma cells required 30- to 40-fold highconcentration to obtain 50% inhibition of cell growth. Growthinhibitory activity of KW2152 against cultured cells correlatedto the in vivo antitumor activity in the i.p.-i.p. system with thesame murine tumor cells.

In spite of its narrow spectrum against murine tumor models,KW2152 showed marked activity against s.c. human tumorstransplanted to nude mice. At optimal doses, KW2152 suppressed the growth of MX-1 human mammary carcinoma withall mice cured by daily administration for 7 days. Both i.v. andp.o. administration were markedly effective against MX-1; however, a 5- to 6-fold dose was required by p.o. administration toobtain the same extent of tumor growth inhibition produced byi.v. administration. It was active against Co-3 human coloncarcinoma and St-4 gastric carcinoma which had been reportedto belong to the insensitive human tumors against establishedanticancer drugs on the schedule that s.c. transplanted tumorswere treated when tumor volume was 100-300 mm3 (15-17).From our studies. MMC, ADM, c/s-DDP, and BLM gave amoderate effect with T/C percentage of 30-47 against Co-3.Against St-4, KW2152 was active, with a T/C percentage of 27comparable to that of MMC, and more effective than cw-DDP.KW2152 produced moderate effectiveness against St-15 humanstomach carcinoma which was significantly sensitive to MMCand a's-DDP. The predictive value of s.c. human tumor xeno-

grafts for clinical efficacy is not well established, but the characteristics of the antitumor spectrum of K.W2152 which israther effective to human tumor xenografts as compared withmurine tumors, are interesting in the further evaluation of thiscompound. Because KW2152 showed a significant effect onP388/ADM leukemia in vitro and in vivo, the pleiotropicallyresistant line of P388 leukemia (18) was suggested not to becross-resistant to KW2152. KW2152 was 45 times less effectiveto P388/MMC than to P388; however, it gave a significantprolongation of life span of mice bearing P388/MMC compa

rable to that of mice bearing P388. The reason of the discrepancy between in vivo and in vitro was not certain, but thedistribution of i.p. P388/MMC cells in mice might be differentfrom that of P388 cells.

The schedule dependency of KW2152 suggested that it mightact like an antimetabolite; however, the competition of variousnucleosides to the growth inhibitory activity of KW2152 onHeLa S3 cells were not observed in our preliminary study.3

From these results, antimetabolite activity of KW2152 couldnot be proved.

Synthesis of RNA is sensitive to KW2152 in P388 leukemiacells. DNA synthesis was shown to be almost 30-fold lesssensitive to K.W2152 than RNA synthesis from dose-responseexperiments. In B. subtilis, DNA synthesis was significantlyinhibited at concentrations which did not inhibit RNA or protein synthesis, suggesting primary inhibition of DNA synthesisby quinocarcin in bacteria (19). Although at present there is noway to explain this different effect, some drugs such as ADMaffect DNA and RNA synthesis to a different degree dependentupon the species and system tested (20-22).

Saframycin was reported to inhibit RNA synthesis moresignificantly than DNA synthesis in LI210, and vice versa inB. subtilis (23). These results might suggest that the isoquino-line moiety of KW2152 and saframycin was important forinhibition of RNA synthesis. From the preliminary flow cytom-etry analysis of DNA distribution of HeLa Sj cells treated withKW2152, delay of S phase progression was observed. Preliminary toxicology studies indicate that bone marrow toxicity isnot significant for this compounds. Gastrointestinal toxicityand bone marrow toxicity are more significant after a singleinjection than after daily administration for 7 days. We arecurrently investigating the biochemical mechanism of the anti-tumor effect of KW2152 as well as its tissue distribution patternand its toxicology profile. Such information will be importantin designing clinical protocols with this compound, which isbeing developed as a Phase I agent.

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1987;47:1516-1522. Cancer Res Kazuhisa Fujimoto, Tetsuo Oka and Makoto Morimoto Quinocarmycin Citrate (KW2152)Antitumor Activity of a Novel Antitumor Antibiotic,

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