See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/6958445

Antitumor activity of TZT-1027 (Soblidotin)

Article  in  Anticancer Research · May 2006

Source: PubMed

CITATIONS READS

32 380

3 authors, including:

Watanabe Junichi
ASKA Pharmaceutical Co., Ltd.
7 PUBLICATIONS   94 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Watanabe Junichi on 19 May 2014.

The user has requested enhancement of the downloaded file.

ANTICANCER RESEARCH 26: 1973-1982 (2006)

Antitumor Activity of TZT-1027 (Soblidotin)

JUNICHI WATANABE1, MEGUMI MINAMI2 and MOTOHIRO KOBAYASHI1

1ASKA Pharmaceutical Co., Ltd., R&D Administration, 1604 Shimosakunobe,

Kawasaki-shi, Kanagawa 213-8522 Takatsu-ku;
2Daiichi Pharmaceutical Co., Ltd., R&D Division, New Product Research Laboratories III,

16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan

Abstract. Background: TZT-1027 (Soblidotin), a newly of 1.2 ÌM (4). TZT-1027 has a broad spectrum of antitumor
synthesized dolastatin 10 derivative that depolymerizes activity against various murine tumors – P388 leukemia,
microtubules, has potent antitumor activity. Materials and Colon26 and LLC carcinomas, B16 melanoma and M5076
Methods: Cell-killing kinetic analysis was performed by the sarcoma – as well as human tumor xenografts, MX-1, LX-1
colony-forming assay and the kinetics of TZT-1027 were and SBC-3 carcinomas (3, 5). Our previous results suggested
compared with those of neocarzinostatin, adriamycin and an association of the growth-inhibitory effect of TZT-1027
vincristine, known to be typical concentration-, AUC- and with the induction of apoptosis and indicated that TZT-1027
time-dependent agents, respectively. DNA fragmentation was induced apoptosis following G2/M arrest, even in the absence
detectable by electrophoresis, cytotoxicity was evaluated by of caspase-3 or Bcl-2 (6). Furthermore, TZT-1027 displayed a
MTT assay and antitumor activity was examined by measuring potent antivascular effect against advanced-stage Colon26
the tumor weight after treatment. Results: TZT-1027 exhibited adenocarcinoma (7, 8). TZT-1027 is currently undergoing
its cytocidal and apoptosis-inducing activity in a time- clinical evaluation and, in phase I clinical trials, its major dose-
dependent manner. Its growth-inhibitory effect was less limiting toxicity was neutropenia (9-14).
affected by overexpression of P-glycoprotein than that of other In this study, the cell-killing kinetics of TZT-1027 were
tubulin inhibitors and was not affected by the overexpression compared with those of positive control drugs with typical
of breast cancer resistance protein or multidrug resistance- mechanisms of action on WiDr human colon cancer cells and
associated protein. TZT-1027 exhibited potent antitumor were then analyzed to classify TZT-1027 as a concentration-,
activities in an in vivo tumor model in which vincristine and AUC-, or time-dependent drug (15). Next, the in vitro growth-
docetaxel failed to show effectiveness. Conclusion: Because its inhibitory effects of TZT-1027 against human tumor cell lines
growth-inhibitory and antitumor activities were superior to that overexpress the multidrug-efflux pumps,
those of the other drugs tested, including the tubulin inhibitors P-glycoprotein (P-gp), breast cancer resistance protein
paclitaxel, docetaxel and vincristine, TZT-1027 should be (BCRP) and multidrug resistance-associated protein (MRP)
useful in the chemotherapy of tumors that are not responsive were assessed and were compared with those of several
to other tubulin inhibitors. anticancer agents currently in clinical use. Finally, not only the
in vitro cytotoxicity, but also the in vivo antitumor activity of
TZT-1027 (Soblidotin) is a newly synthesized (1) derivative of TZT-1027 were evaluated using the murine fibrosarcoma cell
dolastatin 10, which was isolated by Pettit et al. (2) from the line Meth A.
Indian Ocean sea hare, Dolabella auricularia, in 1987. TZT-
1027 (1-10 ÌM) inhibited the polymerization of microtubule Materials and Methods
protein, with an IC50 value of 2.2 ÌM (3) and monosodium Agents. TZT-1027, its metabolites and docetaxel (DTX) were
glutamate-induced tubulin polymerization, with an IC50 value synthesized in the laboratories of ASKA Pharmaceutical Co., Ltd.
(Kawasaki, Japan) and Daiichi Pharmaceutical Co., Ltd. (Tokyo,
Japan), respectively. Adriamycin (ADM) and vinorelbine (VNB)
were purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan).
Correspondence to: Junichi Watanabe, ASKA Pharmaceutical Co., Cisplatin (CDDP) and etoposide (VP-16) were purchased from
Ltd., R&D Administration, 1604 Shimosakunobe, Takatsu-ku, Nippon Kayaku Co., Ltd. (Tokyo, Japan). Paclitaxel (PTX) was
Kawasaki-shi, Kanagawa 213-8522, Japan. Tel: +81-44-812-8632, purchased from Indena S.p.A. (Milan, Italy). Vincristine (VCR) was
Fax: +81-44-833-5310, e-mail: watanabe-j@aska-pharma.co.jp purchased from Shionogi & Co. (Osaka, Japan) and neocarzinostatin
(NCS) was purchased from Yamanouchi Pharmaceutical Co., Ltd.
Key Words: TZT-1027, Soblidotin, tubulin inhibitor, cell-killing (Tokyo, Japan). Dilutions were performed just prior to the addition
kinetics, multidrug resistance, fibrosarcoma Meth A. of the agents.

0250-7005/2006 $2.00+.40 1973

 ANTICANCER RESEARCH 26: 1973-1982 (2006)

Table I. Human cancer cell lines used in these experiments.

Cell line Cancer type Type of drug Drug for Drug Resistance to Efflux-pump Ref.
resistance selection and concentration other drugs overexpressiona
maintenance required for
maintenance
(ng/mL)

HCT116 Human colon cancer (Parent cell line) – – – –

(carcinoma) 17
HCT116/tere-1-1 Acquired multidrug Docetaxel 10 Paclitaxel P-gp ++
resistance

PC-6 (Parent cell line) – – – MRP+/–

PC-6/Tax1-1 Acquired multidrug Paclitaxel 8 Adriamycin, MRP+/–,

resistance Vincristine P-gp +

PC-6/ADM2-1 Human lung cancer Acquired multidrug Adriamycin 10 Paclitaxel, MRP+/–,

(oat cell carcinoma) resistance Vincristine P-gp ++

PC-6/VCR29-9 Acquired multidrug Vincristine 10 Paclitaxel, MRP+/–,

resistance Adriamycin P-gp ++ 16

PC-6/VP1-1 Acquired multidrug VP-16 1000 Paclitaxel, MRP+/–,

resistance Adriamycin, P-gp +++
Vincristine, CPT-11

PC-6/SN2-5 Acquired multidrug SN-38 2 Mitoxantrone, MRP+/–,

resistance Topotecan BCRP+

PANC-1 Human pancreatic cancer Intrinsic multidrug – – Cisplatin, MRP++ 19, 20

(epithelioid carcinoma) resistance Adriamycin,
Vincristine

NCI-H460 Human lung cancer (Parent cell line) – – – –

(large cell carcinoma) 18

NCI-H460/PTX-13 Acquired multidrug Paclitaxel 20 Cisplatin, P-gp ++

resistance Adriamycin,
Vincristine

a–, negative; +/–, weakly positive; +, positive; ++ and +++, strongly positive in order of ++ < +++.

Experimental animals and cell lines. Male BALB/c mice (6 weeks PTX for PC-6/Tax1-1 and NCI-H460/PTX-13, SN-38 for
old) were purchased from Japan SLC Inc. (Shizuoka, Japan) and PC-6/SN2-5, ADM for PC-6/ADM2-1, VCR for PC-6/VCR29-9
were maintained under specific pathogen-free conditions. All the and VP-16 for PC-6/VP1-1 (16). The sublines, HCT116/tere1-1,
animal experiments were conducted in accordance with the in-house PC-6/Tax1-1, PC-6/ADM2-1, PC-6/VCR29-9, PC-6/VP1-1 and
guidelines of the Institutional Animal Care and Use Committee of NCI-H460/PTX-13 overexpressed P-gp, and PC-6/SN2-5 over-
Daiichi Pharmaceutical Co., Ltd. A human colorectal adenocarcinoma expressed BCRP (16-18). Additionally, PANC-1 overexpressed a
cell line, WiDr, a human colorectal carcinoma cell line, HCT116, a significant amount of MRP spontaneously (19, 20). All the cell
human non-small cell lung cancer cell line, NCI-H460 and a human lines, except for PANC-1 and WiDr, were cultured in RPMI 1640
pancreatic epithelioid carcinoma cell line, PANC-1, were purchased medium supplemented with 10% FBS. The PANC-1 and WiDr
from the American Type Culture Collection (Rockville, MD, USA). cell lines were cultured in Eagle’s MEM alpha medium
A human oat cell lung cancer cell line, PC-6, was purchased from supplemented with 10% FBS and MEM medium supplemented
Immuno-Biological Laboratories (Gunma, Japan) and a murine with 10% FBS, respectively. The drug-resistant sublines derived
fibrosarcoma Meth A cell line was obtained from the Institute of from HCT116, PC-6 and NCI-H460 were maintained in the
Immunological Science, Hokkaido University (Sapporo, Japan). presence of the drugs used to select them. The concentrations of
Drug-resistant sublines were established in the Daiichi the drugs used for maintenance are indicated in Table I. The
laboratories by stepwise exposure to DTX for HCT116/tere1-1, cultures were grown at 37ÆC under 5% CO2.

1974
 Watanabe et al: Antitumor Activity of TZT-1027

Figure 1. Log-log relationship between the IC90 value and exposure for
neocarzinostatin (A), adriamycin (B) and vincristine (C) against WiDr
cells. The IC90 values obtained from the concentration-survival curves for
the drugs were plotted against exposure times, on a log scale. Each value
is the mean of three determinations.

1.0x10–6 to 9.3x10–16 g/mL; ADM, 1.0x10–5 to 1.5x10–10 g/mL; NCS,

1.0x10–6 to 2.0x10–9 g/mL; and VCR, 1.0x10–5 to 2.4x10–12 g/mL).
Subsequently, the medium was removed by aspiration, the dish was
washed twice with 2 mL of PBS, 2 mL of fresh MEM was added and
the cells were incubated continuously for colony formation for up to
10 days. On the tenth day, the medium was removed by aspiration
and the dish was washed once with 2 mL of PBS, 1 mL of fixing stain
(10% formalin solution and 0.05% crystal violet solution were mixed
at a ratio of 2:1) was added and the dish was placed at room
temperature for 15 minutes. After the fixing stain had been removed
by aspiration, the dish was washed with water and air-dried. The
number of colonies was counted using a Colony Counter (Sekisui
Chemical Co., Ltd., Tokyo, Japan). Surviving fractions (rates of
colony formation, %) were expressed as percentages of the colonies
of cells treated with drug to those of untreated cells. Cell-killing
kinetics were obtained using a log-log plot of drug concentration
versus exposure time to derive the drug concentration (IC90) at which
the number of colonies decreased to one-tenth the number of
colonies present in the absence of drug. As a result, when the IC90
value was constant, the drug was classified as a concentration-
dependent drug; when the gradient was –1, the drug was classified as
Colony-forming assay. The experiment was performed according to an AUC-dependent drug; and when the gradient was steeper than –1,
the method of Inaba et al. (21). That is, WiDr cells (200 cells/1.8 mL) the drug was classified as a time-dependent drug. Based on these
were seeded in a 35-mm dish containing MEM. On the following day, results, the cell-killing kinetics of TZT-1027 were compared to those
0.2 mL of the drug at each concentration was added to the medium of the positive control drugs and the drugs which had cell-killing
and incubated at 37ÆC for 1 to 96 hours under 5% CO2 (TZT-1027, kinetics similar to those of TZT-1027 were identified.

1975
 ANTICANCER RESEARCH 26: 1973-1982 (2006)

Figure 3. Agarose gel electrophoresis of DNA extracted from P388

leukemia cells treated with TZT-1027. The P388 leukemia cell line was
treated for the indicated times in the presence or absence of TZT-1027
(1x10–9 g/mL). M: molecular size marker, C: untreated control.

on a 2% agarose gel using a mini-gel electrophoresis system and the

gels were stained with ethidium bromide (0.5 Ìg/mL). The DNA
bands were visualized under UV-light and photographed with
Polaroid type 667 film. A Hind III digest of lambda DNA served as a
wt. standard.

MTT assay. On day 0, the cells (except for Meth A cells) were plated
into 96-well plates at a density of approximately 500 cells/well
(NCI-H460 and NCI-H460/PTX-13), 1000 cells/well (HCT116 and its
sublines), 2000 cells/well (PANC-1), or 5000 cells/well (PC-6 and its
Figure 2. Concentration-survival curves for various lengths of exposure time sublines) in RPMI 1640 supplemented with 10% FBS. The Meth A
to TZT-1027 (A). WiDr cells were exposed to various concentrations of TZT- cells were plated on day 1 at a density of 1000 cells/well. On day 1,
1027 for various time-periods and the surviving fractions were evaluated by a each drug-diluted solution was added to individual wells. After 3 days
colony assay. Log-log relationship between the IC90 value and exposure time of culture, viable cells were quantified by means of the MTT assay
for TZT-1027 against WiDr cells (B). The IC90 values obtained from the (22). T/C (%) was calculated by the following equation from the OD
concentration-survival curves for TZT-1027 were plotted against exposure value of each well:
times on a log scale. Each value is the mean of three determinations. T/C (%) = [(SV on day 4 – SV on day 1)/(CV on day 4 – CV on day
1)]x100
SV = OD value of treated well, CV = OD value of untreated control
well.
DNA fragmentation. Untreated and drug-treated cells were harvested, The concentrations of a compound and T/C (%) values were
the cell pellets were washed with ice-cold PBS three times and cellular plotted on a graph and the concentration that produced 50%
DNA was extracted and purified using Sepa Gene (Sanko Junyaku growth inhibition (GI50 value) was calculated automatically from
Co., Ltd., Tokyo, Japan). The samples were next dissolved in TE the graph. The resistance factor (R f), which represents the
buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) and incubated degree of resistance to drug treatment for each resistant subline,
with RNase A (20 Ìg/mL) at 37ÆC for 30 minutes. The DNA was was determined by the following formula: R f = GI 50 value for
purified once again and the purity was determined spectro- each drug for the resistant cell line / GI50 value for the parental
photometrically. DNA (2 Ìg) was electrophoresed at 50 V for 30 min cell line.

1976
 Watanabe et al: Antitumor Activity of TZT-1027

Table II. In vitro growth inhibition caused by various anticancer agents against multidrug-resistant cell lines that overexpress P-gp.

Drug HCT116 HCT116/tere1-1 PC-6 PC-6/ADM2-1 PC-6/TAX1-1 PC-6/VCR29-9 PC-6/VP1-1

GI50 GI50 Rf GI50 GI50 Rf GI50 Rf GI50 Rf GI50 Rf

TZT-1027 0.026 1.08 42 0.023 0.917 39 0.327 14 0.686 29 9.89 417

Paclitaxel 0.073 493 6772 0.923 529 573 263 285 541 586 >10000 >10834
Docetaxel 0.160 68.5 428 0.419 97.9 234 42.7 102 93.7 224 1150 2745
Adriamycin 19.6 270 13.8 6.21 238 38 52.5 8.5 143 23 2510 404
Vincristine 1.71 198 116 0.423 224 530 56.7 134 122 288 877 2073
Etoposide 463 1990 4.3 162 868 5.4 286 1.8 531 3.3 3080 19
Cisplatin 776 373 0.48 182 494 2.7 119 0.65 99.3 0.55 267 1.5

GI50: ng/mL.
The resistance factor (Rf) was determined from the following formula:
Rf = GI50 value for each drug for the resistant cell line / the GI50 value for the parental cell line.

Antitumor activity against murine fibrosarcoma Meth A. The Meth A Table III. In vitro growth inhibition caused by various anticancer agents
cells maintained in vivo were harvested and washed with Hank’s against a human lung cancer cell line, PC-6, its camptothecin-resistant
Balanced Saline Solution (HBSS). The washed cells were re- subline that overexpresses BCRP and a human pancreatic cancer cell line,
suspended in HBSS and were inoculated subcutaneously (s.c.) into PANC-1, that spontaneously overexpresses MRP.
BALB/c mice (1x106 cells/0.1 mL/mouse) on day 0. When the mean
tumor volume had reached approximately 120 mm3 on day 7 after Drug PC-6 PC-6/SN2-5a PANC-1b
GI50 GI50 Rf GI50
tumor inoculation, the drugs were administered. The volume
administered to each mouse was calculated from the body weight
TZT-1027 0.010 0.006 0.63 0.029
measured prior to each administration (10 mL/kg). TZT-1027, DTX,
Paclitaxel 0.307 0.321 1.05 1.38
or VCR and ADM were administered on a q7dx2 schedule (once Docetaxel 0.331 0.280 0.85 1.90
every 7 days for a total of two administrations), on a q2dx3 schedule Adriamycin 5.22 6.35 1.22 109
(once every 2 days, for a total of three administrations), or on a qdx1 Vincristine 0.257 0.193 0.75 6.23
schedule (single administration), respectively. On the twenty-first day Etoposide 88.6 82.6 0.93 5630
after the tumor inoculation, the animals were sacrificed by cervical Cisplatin 177 87.8 0.50 846
vertebrae dislocation and the tumors were excised and weighed. The
antitumor activity of each treatment was evaluated using tumor aMRP+/–, BCRP+, bMRP+++.
weight. The tumor growth inhibition ratio (IR) was calculated by GI50: ng/mL.
means of the following formula: IR (%) = [1 – (TWt/TWc)]x100 The resistance factor (Rf) was determined from the following formula:
(TWt: the mean tumor weight of a treated group, TWc: the mean Rf = GI50 value for each drug for the resistant cell line / the GI50 value
tumor weight of the control group). When the IR was 58% or higher, for the parental cell line.
the therapy was considered effective (23). The rate of body weight
loss (BWL) was calculated by the following formula: BWL (%) = (1
– BWn/BWs)x100 (BWn, the mean body weight of mice on day n;
BWs, the mean body weight of mice on the day administration
started). BWLmax = the maximum value of BWL caused by length of exposure (data not shown) and the cell-killing
administration of the test compound. A BWLmax <0 indicated no kinetics also showed a constant IC90 value for exposure times
body weight loss. Groups with BWLmax values of greater than 20% of 1 to 96 hours (Figure 1A). These results confirmed that
or with death caused by toxicity were defined as "toxic groups". The this drug is a concentration-dependent drug. In the case of
statistical analysis of tumor weight was conducted with the EXSAS
ADM, an antitumor antibiotic, a line with the gradient of –1
version 6.10 (Arm Corp.; Osaka, Japan), based on SAS release 8.2
(SAS Institute Japan, Tokyo, Japan). Dunnett's test was used to
(Y=–1.138X–5.614, r=0.990) was obtained from the results
assess differences in mean tumor weight, and p values less than 5% of the cell-killing kinetics (Figure 1B), showing the typical
were considered statistically significant with regard to tumor weight. pattern of concentration and time dependence, that is, an
AUC-dependent drug. The results of the cell-killing kinetics
Results for VCR, an antimicrotubule agent like TZT-1027, indicated
that the gradient of the line (Y=–2.429X–5.091, r=0.998) for
Cell-killing kinetics. The cell-killing kinetics are shown in the exposure times of 3 to 24 hours was steeper than –1,
Figures 1 and 2B. The concentration-survival curve of TZT- indicating the typical pattern of a time-dependent drug
1027 is shown in Figure 2A. NCS, an antitumor antibiotic, (Figure 1C). Based on the mechanisms classified by Ozawa
showed no difference in effective doses, regardless of the et al. (15), the cell-killing kinetics of TZT-1027 were studied.

1977
 ANTICANCER RESEARCH 26: 1973-1982 (2006)

Table IV. In vitro cytotoxicity of TZT-1027 and its metabolites against human cancer cell lines.

Drug HCT116 [Colon] NCI-H460 [Lung] NCI-H460/PTX-13 [Lung]

P-gp (–) P-gp (–) P-gp (+)

GI50 Ratio GI50 Ratio GI50 Ratio

TZT-1027 0.172±0.045 1.0 0.227±0.069 1.0 1.69±0.41 1.0

Ma 0.181±0.029 1.1 0.271±0.038 1.2 9.04±0.79 5.4
Ma(R) 0.194±0.010 1.1 0.285±0.002 1.3 12.9±0.58 7.6
Ma(S) 0.162±0.013 0.9 0.230±0.016 1.0 11.8±1.50 7.0
Mb 0.262±0.029 1.5 0.512±0.085 2.3 24.0±1.73 14
Mc 0.488±0.030 2.8 0.928±0.061 4.1 25.5±0.88 15
Md 0.446±0.042 2.6 1.04±0.153 4.6 36.4±3.51 22
Mg 0.172±0.045 1.0 0.982±0.075 4.3 48.4±5.62 29

GI50: ng/mL
Ratio = GI50 for each metabolite/that for TZT-1027.

TZT-1027 showed low cell-killing effects to cancer cells at Table V. In vitro cytotoxic activity of tubulin inhibitors against the murine
exposure times of 1 to 12 hours. However, this drug showed fibrosarcoma cell line Meth A and the human lung cancer cell line PC-6.
dramatically enhanced cell-killing effects at exposure times of
Drug Meth A PC-6
12 hours or longer, demonstrating high time-dependence
(Figure 2A). TZT-1027 exerted its cell-killing effect, GI50 Ratio GI50 Ratio
regardless of time, at exposure times of 1 to 12 hours,
indicating a fast-acting pattern (concentration-dependent TZT-1027 0.012 1 0.024 1
pattern) at first glance. However, the gradient of the line
Vincristine 2.72 221 0.423 18
(Y=–7.273X–0.347, r=0.919) at exposure times of 12 to 24 Vinorelbine 2.15 175 0.055 2
hours was far steeper than –1 (Figure 2B), and was greater
than that of VCR. Therefore, it was concluded that TZT- Docetaxel 1.1 89 0.419 18
1027 was a time-dependent antitumor agent, with a higher Paclitaxel 6.59 536 0.923 39
time dependence than VCR.
GI50: ng/mL.
Ratio = (GI50 of each agent for Meth A or PC-6)/(GI50 of TZT-1027
Analysis of DNA fragmentation. DNA fragmentation of the
for Meth A or PC-6).
P388 leukemia cell line was induced by TZT-1027 at a
concentration of 1x10–9 g/mL within 6 hours and this DNA
fragmentation, the hallmark of apoptosis, increased time- inhibitors, VCR (116- to 2073-fold), DTX (102- to 2745-fold)
dependently (Figure 3). After 24 hours of treatment with and PTX (285- to >10000-fold). PC-6/SN2-5 overexpressed
TZT-1027, DNA fragmentation was maximally increased. BCRP, which conferred the subline with resistance to
camptothecin derivatives and mitoxantrone (Table I).
In vitro growth inhibition against various multidrug-resistant However, it showed no cross resistance to TZT-1027 or other
human cancer cell lines that overexpress P-gp, BCRP, or MRP. anticancer agents, including other tubulin inhibitors (Table
The in vitro growth-inhibitory effects of the drugs were III). PANC-1, the human pancreatic epithelioid carcinoma cell
examined using five P-gp-overexpressing cell lines that were line that spontaneously overexpresses MRP, was used to
derived from two parental cell lines: the human colon cell line, examine the in vitro growth-inhibitory effects more fully.
HCT116, and the human lung cancer cell line, PC-6 (Table I). Compared with the parental HCT116 and PC-6 cell lines, this
These variants showed cross-resistance to TZT-1027, as well as cell line showed a similar high susceptibility to TZT-1027, but
to VCR, ADM, PTX and DTX, which are known substrates of was less sensitive to ADM, VCR and VP-16 (Table III).
P-gp. For these sublines, the degrees of resistance to these
drugs correlated well with the level of P-gp which each subline In vitro cytotoxicity of TZT-1027 and its metabolites against
expressed (Table II). The Rf values for TZT-1027 ranged from human cancer cell lines. The in vitro cytotoxicities of TZT-1027
14- to 417-fold; this range is higher than the range for VP-16 and its metabolites were examined against the human colon
(1.8- to 19-fold), equivalent to the range for ADM (8.5- to 404- cancer cell line, HCT116, the human lung cancer cell line, NCI-
fold), but significantly lower than the ranges for the tubulin H460 and its P-gp-overexpressing subline, NCI-H460/PTX-13

1978
 Watanabe et al: Antitumor Activity of TZT-1027

Table VI. In vivo antitumor effect TZT-1027 against murine fibrosarcoma Meth A solid tumors subcutaneously implanted into male BALB/c mice.

Drug Route Treatment Total dose Tumor weight BWLmaxb Toxic

schedule (mg/kg) (%)[day] deathc
Mean±SE (g) Sig. IR(%)a

Experiment 1
Control - - - 3.02±0.30 0 <0 0/6
TZT-1027 i.v. q7d x 2 4 0.08±0.01 *** 98 11.7[9] 0/6
2 0.69±0.17 *** 77 4.5[9] 0/6
Experiment 2
Control - - - 4.34±0.38 0 <0 0/6
Docetaxel i.v. q2d x 3 80 1.19±0.30 *** 73 34.4[21] 0/6
65 1.55±0.22 *** 64 24.7[18] 0/6
50 2.11±0.30 *** 51 16.1[18] 0/6
31 2.94±0.40 * 32 3.2[16] 0/6
Vincristine i.v. qd x 1 2 4.10±0.27 ns 6 16.7[11] 0/6
Adriamycin i.v. qd x 1 12 1.17±0.33 *** 73 4.8[16] 0/6

aIR%: Tumor growth inhibition rate = [1–(mean tumor weight of treated group)/ (that of the untreated control group)] x 100. When the IR value
was 58% or higher, the drug was considered effective.
bBWLmax: Maximum value for rate of body weight loss (%); numbers in parentheses denote the day. <0: no loss.
cNumber of mice that died/number of mice used.
Asterisks (***) and (***, *) indicate statistically significant differences from the untreated control indicated by Dunnett's test, p < 0.001 and by
Student’s t-test, p<0.001 and p<0.05, respectively; ns, no significance.

Figure 4. Meth A tumors excised on day 21 (median sagittal plane views). TZT-1027 dose-dependently inhibited the growth of Meth A tumors
subcutaneously implanted into BALB/c mice. The antitumor activity was characteristically accompanied by ischemic necrosis of the tumors.

(Table IV). The seven metabolites of TZT-1027, Ma, Ma(R), In vitro cytotoxic activity of TZT-1027, VCR, VNB, DTX and
Ma(S), Mb, Mc, Md and Mg, showed equivalent or somewhat PTX against the Meth A and PC-6 cell lines. The GI50 values
weaker cytotoxicity against HCT116 and NCI-H460 compared of all five drugs against the murine fibrosarcoma cell line
to that shown by TZT-1027 (ratio: 0.9 to 4.6). They also Meth A and the human lung cancer cell line PC-6 are shown
showed weaker cytotoxicity against NCI-H460/PTX-13 in Table V. TZT-1027 exhibited the strongest cytotoxic
compared with that of TZT-1027 (ratio: 5.4 to 29). activity among the drugs tested: the GI50 values for TZT-
Furthermore, there was no difference in the cytotoxicities of 1027 were 0.012 against Meth A cells and 0.024 ng/mL
Ma, Ma(R) and Ma(S). against PC-6 cells. Other tubulin-polymerization inhibitors,

1979
 ANTICANCER RESEARCH 26: 1973-1982 (2006)

VCR and VNB, and the tubulin-depolymerization amount of P-gp expressed by the tumor cells. The degree of
inhibitors, DTX and PTX, also showed similar potent resistance to TZT-1027 of the five P-gp-overexpressing
activity against PC-6 cells, with GI50 values below 1 ng/mL, sublines ranged from 14- to 417-fold of the parental cell
but all failed to do so against Meth A. VCR and VNB were lines. However, these values were lower than those of VCR
175-fold or less effective, DTX was about 90-fold less (from 116- to 2073-fold), DTX (from 102- to 2475-fold), or
effective and PTX was about 500-fold less effective than PTX (from 285- to 10000-fold or more). These results
TZT-1027 against the Meth A cells. indicated that TZT-1027 is 6-20 times less affected by P-gp
than other tubulin inhibitors. TZT-1027, as well as three
In vivo antitumor activity of TZT-1027 against Meth A solid other tubulin inhibitors, showed potent in vitro growth-
tumors subcutaneously implanted into BALB/c mice. The in inhibitory effects on BCRP-overexpressing cells that are
vivo antitumor activity of TZT-1027 against Meth A solid resistant to camptothecin derivatives and mitoxantrone.
tumors subcutaneously implanted into mice is shown in TZT-1027, PTX and DTX also potently inhibited the in vitro
Table VI. TZT-1027 induced significant antitumor activity growth of a tumor cell line that spontaneously overexpresses
at doses of 2 mg/kg/day (maximum tolerated dose, [MTD]) MRP and shows low sensitivity to several antitumor agents,
and 1 mg/kg/day (1/2 MTD), with IR values of 98% and including VCR. These results suggest that the growth-
77%, respectively. There were no toxic deaths or severe inhibitory activity of TZT-1027 was less affected by the
body weight loss (<20%) associated with treatment with overexpression of P-gp in tumor cells than the growth-
TZT-1027. The antitumor activity of TZT-1027 was inhibitory activity of other tubulin inhibitors currently
accompanied by ischemic necrosis of the tumors (Figure 4), available clinically, and that the activity of TZT-1027 was
probably related to the induction of local circulatory not affected by the overexpression of BCRP or MRP.
disturbances, as reported elsewhere (7). Single intravenous The cytotoxicity of TZT-1027 metabolites was examined
administration of VCR resulted in no antitumor activity, against a human colon cancer cell line (HCT116), a human
with an IR of 6%, even though considerable body weight lung cancer cell line (NCI-H460) and its P-gp-overexpressing
loss occurred (BWLmax: 16.7%). DTX was administered subline, NCI-H460/PTX-13. Five metabolites (Ma, Mb, Mc,
intravenously on a schedule of q2d x 3 (every other day for Md and Mg) and the enantiomers of Ma, a main metabolite,
a total of three times). DTX showed antitumor activities at Ma(R) and Ma(S) showed equivalent or somewhat weaker
total doses of 65 and 80 mg/kg, with IRs of 64% to 73%, cytotoxicity compared to that of TZT-1027 against HCT116
but because the BWLmax values exceeded 20%, these were and NCI-H460 and were more affected by the overexpression
defined as toxic doses. At total doses of 50 mg/kg (defined of P-gp than was TZT-1027. Furthermore, there were no
as the MTD) and below, DTX failed to show significant significant differences in the GI50 values of Ma, Ma(R) and
antitumor activity. ADM, a positive control, exhibited Ma(S). Therefore, it may be assumed that the antitumor
significant antitumor activity, with an IR of 73% at a non- efficacy in clinical trials was exerted by the parent compound
toxic dose. and not by its metabolites.
The in vitro cytotoxicity and in vivo antitumor activity of
Discussion TZT-1027 were evaluated using the murine fibrosarcoma
cell line Meth A. The in vitro cytotoxicity of TZT-1027 was
The cell-killing kinetics of TZT-1027 were first compared with compared with that of the vinca alkaloids (VCR and VNB)
those of positive control drugs that have typical mechanisms and the taxanes (PTX and DTX). When the GI50 values
of action on human colon cancer (WiDr) cells and analyzed were compared, TZT-1027 showed much greater
to determine which type, in the classification by Ozawa et al. cytotoxicity than these other tubulin inhibitors. TZT-1027
(15), is consistent with the cell-killing kinetics of TZT-1027. was approximately 200-fold more cytotoxic than the vinca
These results revealed that TZT-1027 was a highly time- alkaloids and 90- to 500-fold more potent than the taxanes.
dependent antitumor agent. Furthermore, it was found that Therefore, the in vitro cytotoxic effect of TZT-1027 was
DNA fragmentation, induced by TZT-1027, was increased superior to that of clinically available tubulin-polymerization
time-dependently. Previous studies have shown that TZT-1027 or -depolymerization inhibitors. The in vivo antitumor
induced growth arrest at the G2/M-phase of the cell cycle (6). activity of TZT-1027 was investigated in BALB/c mice
Therefore, we confirmed that TZT-1027 exerted its effects in implanted with a Meth A solid tumor. TZT-1027 at the
a time-dependent manner and, rather than a single, high-dose MTD and at 1/2 MTD exhibited strong antitumor activities
administration, divided administration of small amounts or without death caused by toxicity or significant body weight
intermittent administration was considered to be the optimum loss, even though VCR and DTX failed to show
administration schedule. effectiveness in the same tumor model. The antitumor
The in vitro growth-inhibitory effects of TZT-1027, as well activities were accompanied by ischemic necrosis of the
as of other tubulin inhibitors, declined depending on the tumors, probably related to the induction of local circulatory

1980
 Watanabe et al: Antitumor Activity of TZT-1027

disturbances. In summary, these results showed that 12 Yamamoto N, Andoh M, Kawahara M, Fukuoka M and Niitani
TZT-1027 inhibited the growth of murine fibrosarcoma in H: Phase I study of TZT-1027, an inhibitor of tubulin
vivo as well as in vitro. polymerization, given weekly x 3 as a 1-hour intravenous infusion
in patients (pts) with solid tumors [abstract 420]. Proc Am Soc
In conclusion, the presented results show that TZT-1027
Clin Oncol, 2002.
had strong cytotoxicity, not only against parental cancer 13 Scfoeffski P, Thate B, Beutel G, Bolte O, Otto D, Hofmann M,
cells, but also against various multidrug-resistant cancer Ganser A, Jenner A, Cheverton P, Wanders J, Oguma T, Atsumi
cells and that its cytotoxic activity was superior to that of R and Satomi M: Phase I and pharmacokinetic study of
other anticancer agents tested, including the tubulin TZT-1027, a novel synthetic dolastatin 10 derivative, administered
inhibitors PTX, DTX and VCR against murine as a 1-hour intravenous infusion every 3 weeks in patients with
fibrosarcoma in vitro and in vivo. Therefore, we anticipate advanced refractory cancer. Ann Oncol 15: 671-679, 2004.
14 De Jonge MJA, Van der Gaast A, Planting AST, Van Doorn L,
that TZT-1027 should be useful for the chemotherapy of
Lems A, Boot I, Wanders J, Satomi M and Verweij J: Phase I and
cancers that are not responsive to other tubulin inhibitors. pharmacokinetic study of the dolastatin 10 analogue TZT-1027,
given on days 1 and 8 of a 3-week cycle in patients with advanced
References solid tumors. Clin Cancer Res 11: 3806-3813, 2005.
15 Ozawa S, Sugiyama Y, Mitsuhashi J and Inaba M: Kinetic analysis
1 Miyazaki K, Kobayashi M, Natsume T, Gondo M, Mikami T, of cell killing effect induced by cytosine arabinoside and cisplatin
Sakakibara K and Tsukagoshi S: Synthesis and antitumor activity in relation to cell cycle phase specificity in human colon cancer
of novel dolastatin 10 analogs. Chem Pharm Bull 43: 1706-1718, and Chinese hamster cells. Cancer Res 49: 3823-3828, 1989.
1995. 16 Ishii M, Iwahana M, Mitsui I, Minami M, Imagawa S, Tohgo A
2 Pettit GR, Kamano Y, Herald CL et al: The isolation and and Ejima A: Growth inhibitory effect of a new camptothecin
structure of a remarkable marine animal antineoplastic analog, DX-8951f, on various drug-resistant sublines including
constituent: dolastatin 10. J Am Chem Soc 109: 6883-6885, 1987. BCRP-mediated camptothecin derivative-resistant variants
3 Kobayashi M, Natsume T, Tamaoki S, Watanabe J, Asano H, derived from the human lung cancer cell line PC-6. Anticancer
Mikami T, Miyasaka K, Miyazaki K, Gondo M, Sakakibara K and Drugs 11: 353-362, 2000.
Tsukagoshi S: Antitumor activity of TZT-1027, a novel dolastatin 17 Shionoya M, Jimbo T, Kitagawa M, Soga T and Tohgo A: DJ-927,
10 derivative. Jpn J Cancer Res 88: 316-327, 1997. a novel oral taxane, overcomes P-glycoprotein-mediated multidrug
4 Natsume T, Watanabe J, Tamaoki S, Fujio N, Miyasaka K and resistance in vitro and in vivo. Cancer Sci 94: 459-466, 2003.
Kobayashi M: Characterization of the interaction of TZT-1027, a 18 Ochi Y, Minami M and Tohgo A: The orally effective taxane
potent antitumor agent, with tubulin. Jpn J Cancer Res 91: 737- DJ-927 has little ability to induce drug resistance in human non-
747, 2000. small cell lung cancer cells. Eur J Cancer Suppl 2: 161, 2004.
5 Natsume T, Koh Y, Kobayashi M, Fukumoto H, Takahashi F, 19 Miller DW, Fontain M, Kolar C and Lawson T: The expression
Nakamura T, Ohe Y, Saijo N and Nishio K: Enhanced antitumor of multidrug resistance-associated protein (MRP) in pancreatic
activities of TZT-1027 against TNF-· or IL-6 secreting Lewis lung adenocarcinoma cell lines. Cancer Lett 107: 301-306, 1996.
carcinoma in vivo. Cancer Chemother Pharmacol 49: 35-47, 2002. 20 Anderson KM, Alrefai WA, Anderson CA, Ho Y, Jadko S, Ou
6 Watanabe J, Natsume T, Fujio N, Miyasaka K and Kobayashi M: D, Wu YB and Harris JE: A response of Panc-1 cells to
Induction of apoptosis in human cancer cells by TZT-1027, an cisplatinum, assessed with a cDNA array. Anticancer Res 22: 75-
antimicrotubule agent. Apoptosis 5: 345-353, 2000. 81, 2002.
7 Otani M, Natsume T, Watanabe J, Kobayashi M, Murakoshi M, 21 Inaba M, Mitsuhashi J and Ozawa S: Kinetic analysis of
Mikami T and Nakayama T: TZT-1027, an antimicrotubule agent, 5-fluorouracil action against various cancer cells. Jpn J Cancer
attacks tumor vasculature and induces tumor cell death. Jpn J Res 85: 187-193, 1994.
Cancer Res 91: 837-844, 2000. 22 Mosmann T: Rapid colorimetric assay for cellular growth and
8 Kobayashi M, Natsume T, Watanabe J, Fujio N, Mikami T, survival: application to proliferation and cytotoxicity assays. J
Miyazaki K and Tsukagoshi S: Activity of a novel antitumor agent, Immunol Methods 65: 55-63, 1983.
TZT-1027. Nippon Yakurigaku Zasshi (Japanese) 114 Suppl: 230- 23 Fujita F, Fujita M, Taguchi T, Shimozuma K, Sakamoto Y,
235, 1999. Kimoto Y et al: Multifactorial analysis of parameters influencing
9 Niitani H, Hasegawa K, Furuse K, Fukuoka M, Horikoshi N and chemosensitivity of human cancer xenografts in nude mice. Int J
Kudoh S: Phase I studies of TZT-1027, a novel inhibitor of Cancer 43: 637-644, 1989.
tubulin polymerization [abstract 360]. Proc 10th NCI-EORTC
Symposium on New Drug in Cancer Therapy, 1998.
10 Horti J, Juhasz E and Bodrogi I: Preliminary results of a phase I
trial of TZT-1027, an inhibitor of tubulin polymerization, in
patients with advanced non-small cell lung cancer [abstract 2744].
Proc Am Assoc Cancer Res, 2002.
11 Horti J, Juhasz E, Bodrogi I and Ikeda S: A phase I trial of
TZT-1027, an inhibitor of tubulin polymerization, in patients with
advanced non-small cell lung cancer (NSCLC) [abstract 256]. Proc
AACR-NCI-EORTC International Conference, Molecular Received January 30, 2006
Targets and Cancer Therapeutics, 2003. Accepted March 20, 2006

1981

View publication stats