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Experimental Therapeutics |
Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Saitama 335-8505 [S. O., T. Y., S. Y., S. K., N. T., T. K., K. T.], and Discovery Research Laboratory, Osaka 532-8505, [M. H., Y. S., J. I.], Japan
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ABSTRACT |
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INTRODUCTION |
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To alter the pharmacokinetic behavior of a parent anticancer drug, thereby decreasing the toxicity and/or increasing the therapeutic efficacy of the agent, many macromolecules have been used as drug carriers (6 , 7) . This concept of a macromolecular prodrug is attractive and could well form the basis of a new generation of antitumor agents. Styrene maleic anhydride neocarzinostatin is the first example of a drug-polymer conjugate approved in Japan as a drug for primary liver cancer. PK1 is another example, which comprises doxorubicin covalently bound to N-(2-hydroxypropyl)methacrylamide copolymer by a peptidyl linker. The Phase I study demonstrated that PK1 decreases important dose-limiting toxicities while maintaining antitumor activity (8) . Tumor targeting of these conjugates is assisted by the enhanced permeability and retention effect; tumors usually have a hyperpermeable vasculature, which allows longer circulating macromolecules to pass out through the leaky vessels into the tumor tissue, from where there is no readily available return lymphatic route (9) .
In an attempt to overcome the side effects and improve the pharmacological profile of the CPT analogue, T-0128 is designed as a macromolecular prodrug, which consists of CM dextran and T-2513, linked through the triglycine spacer. T-2513 was chosen as a parent CPT analogue for T-0128 from our chemical libraries for the following reasons: (a) an analogue with great potency is favorable; (b) an amino group is needed for the binding to a peptidyl linker, without the opening of the lactone ring, known to be crucial for the activity (10) ; and (c) a good hydrophobic/hydrophilic balance is required for the synthesis of a conjugate with high water solubility and high drug content. On the other hand, a longer circulation is realized by dextran with appropriate anionic charges. The reason is that macromolecules greater than roughly Mr 70,000 with weak anionic charges are known to circulate in the plasma for a long time because of small hepatic uptake and urinary excretion clearances (11 , 12) . Therefore, CM groups were introduced to the commercially available dextran with a molecular size of about Mr 110,000. Dextrans have high water solubility and low in vivo toxicity (13) . Furthermore, CM groups are available for drug fixation through a peptidyl linker. These favorable properties supported the choice of CM dextran as a drug carrier for T-0128.
The purpose of the present study is to test the concept that the rational design of a CPT analogue-polymer conjugate would achieve the tumor targeting of the active drug, resulting in improved therapeutic efficacy. To this end, we examined the preclinical profile of the antitumor activity of T-0128 against Walker-256 carcinoma in Wistar rats and a panel of human tumor xenografts in nude mice. The efficacy of T-0128 was compared with those of T-2513, CPT-11, and topotecan. In addition, we investigated the plasma and tissue (tumor and nontumor) pharmacokinetics of both released and polymer-bound T-2513s after the administration of T-0128 to rats bearing Walker-256 carcinoma. These data are also compared with those obtained after dosing of T-2513 itself to tumor-bearing rats using the same dosage. Results support potential advantages of T-0128 attributable to the passive tumor targeting.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of T-0128.
Molecular weight and size distributions of T-0128 were determined
by the use of gel permeation chromatography, coupled with the
multiangle laser light scattering detector at 40°C (15)
.
The chromatographic system consisted of a gel permeation chromatography
system 21 (Shodex, Tokyo, Japan), G3000PWXL and G4000PWXL columns
300 x 7.8 mm (Tosoh, Osaka, Japan), a DAWN DSP-F laser
photometer (Wyatt Technology, Santa Barbara, CA), and a Wyatt Optilab
DSP interferometric refractometer. Data were collected and handled by
ASTRA software (Wyatt Technology). A 0.2 M phosphate buffer
(pH 6.9) at a flow rate of 0.8 ml/min was used as a mobile phase.
Normalization of the instrument was carried out by Shodex pullulan P-82
standard series. The weight-average molecular weight of T-0128 was
Mr 
130,000 with narrow dispersity
(Mw/Mn < 1.3). The content of T-2513 was
4.5–5.5% w/w, determined by measuring absorbance at 360 nm in water.
T-0128 contained <0.05% of the free drug in relation to its total
T-2513 content. The degree of substitution of the carboxymethylated
groups was determined by measuring the sodium content using an atomic
absorption spectrophotometer, when CM dextran was isolated as a sodium
salt after the reaction of dextran with chloroacetic acid in alkaline
solution. The degree was 0.40–0.45 per glucose moiety. The chemical
structure and characteristics of T-0128 are presented in Fig. 1
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In Vitro Cytotoxicity.
WiDr, HT-29, SK-BR-3, MKN-1, SK-LU-1, LX-1, KB, and HeLaS3 cells were
maintained in the recommended media at 37°C under atmosphere of 5%
CO2. Exponentially growing cells were typically
seeded in 24-well culture plates (20000 cells/well) and cultured in
Eagle’s MEM supplemented with nonessential amino acids (Life
Technologies, Inc., Tokyo, Japan) and 10% heat-inactivated FCS
(CosmoBio, Tokyo, Japan) for 48 h. Cells were then treated with
drugs in the serum-free medium. After 24 h of exposure to drugs,
cells were cultured in the fresh medium with FCS for additional 24 h and then trypsinized and counted by Coulter counter (Coulter
Electronics Coulter Z1, Krefeld, Germany). Cell survival was plotted
relative to the controls incubated in the medium in the absence of the
drug. The mean drug concentration required to inhibit cell growth by
50% (GI50) was determined from the dose-response
curves.
Topoisomerase I Inhibitory Activity.
The ability of CPT analogues to inhibit topoisomerase I was quantified
in the cleavable complex assays as described elsewhere
(16)
.
Cell Cycle Analysis.
The effect of T-2513 on cell cycle progression was assayed by flow
cytometry using a Cycle Test Plus DNA Reagent kit (Becton Dickinson,
Mansfield, MS).
Toxicity Dose-Finding Studies.
Healthy male SD rats (Charles River Japan, Yokohama, Japan) and healthy
male BALB/c mice (Nippon SLC, Hamamatsu, Japan) were used for toxicity
dose-finding studies. CPT analogues were dissolved in 0.9% NaCl
solution and administered i.v. Rats were observed for at least 28 days.
The MTD was defined as the maximum dose that caused no drug-related
lethality and that produced animal body weight loss of <20% of
original weight. The body weight of mice was monitored two or three
times weekly for at least 28 days. The loss of >30% of body weight
was considered to be lethal. The MTD was defined as the maximum dose
that caused sufficient morbidity as determined by body weight loss.
Evaluation of Antitumor Activity against Walker-256 Carcinoma.
Walker-256 carcinoma cells (Sasaki Institute, Tokyo, Japan), grown for
7 days in the peritoneal cavity of female Wistar rats (Nippon SLC),
were injected s.c. to the inguinal region of 6-week-old female Wistar
rats at a dose of 1 x 107
cells/rat. After 3 days (day 0), CPT analogues were dissolved in 0.9%
NaCl solution and administered once i.v. with five rats/group. Control
rats were injected with 0.9% NaCl solution. The rats were killed on
day 4, and the tumors were removed and weighed. The efficacy of each
drug was assessed as T/C, where T and
C were the tumor weight of the treated and control groups,
respectively, with a ED50 being calculated from
T/C by using the probit method.
Evaluation of Antitumor Activity in Human Tumor Xenograft Models.
Tumor fragments (3 x 3 x 3 mm) of MX-1,
LX-1, HT-29, WiDr, and St-4, obtained from each tumor line were
implanted s.c. to the right flank of male 8-week-old BALB/c
nu/nu mice (Nippon SLC), and the tumor growth was followed
by measurements of tumor diameters with a sliding caliper two or three
times a week. The tumor volume (TV) was calculated according
to the formula: TV = L x W2
/2, where L and
W are the major and minor dimensions, respectively. Drug
treatment with five or six mice/group started on days 8 to 15 after the
transplantation, when the tumor volume was 100–300
mm3. Each drug was injected i.v. once, three
times with a 4-day interval, or once a week for 3 or 4 weeks. Control
mice were treated with 0.9% NaCl solution. The efficacy of the drug
was assessed as relative tumor volume to the original. The tumor growth
inhibition rate (IR) was calculated from the equation:
IR (%) = (1 - T/C) x 100, where T
and C were the tumor volume of the treated and control
groups, respectively. Complete tumor regression refers to a tumor that
is not palpable for a period of time; and cure is defined as an
established tumor that is not palpable for >30 days after the final
measurement day. These days for MX-1, LX-1, HT-29, WiDr, and St-4 were
30, 21, 32, 37, and 42 days, respectively, after the first drug dosing.
Pharmacokinetic Studies.
The tumor-bearing rats received an i.v. bolus injection of either
T-2513 or T-0128. Both drugs were administered at doses equivalent to 1
mg of T-2513/kg. A dose of T-0128 is hereafter always expressed as
T-2513 equivalent mg/kg of body weight per injection. For T-2513
treatment, 27 rats were divided into nine groups
(n = 3), corresponding to 0.017, 0.083, 0.25,
0.5, 1, 2, 4, 6, and 24 h, for terminal blood and tissue sample
collection, whereas for T-0128 treatment, 24 rats were divided into
eight groups (n = 3), corresponding to 0.083,
1, 3, 6, 12, 24, 48, and 72 h. Tumor, liver, spleen, kidney,
heart, lung, duodenum, lymph nodes, and femoral bone marrow were
removed and weighed. Immediately before sampling, the rats were
anesthetized with ether, and blood was removed from abdominal aorta
using a heparinized syringe. Plasma was harvested by centrifugation.
Samples were stored at -80°C until analysis.
Determination of Drug Concentration in Plasma and Tissue.
Tissue samples were homogenized on ice using a Polytron mixer
(Kinematica, Lucerne, Switzerland) and suspended in PBS (pH 7.0) at a
concentration of 25% w/w. By using aliquots of the homogenates and
plasma (100 µl), the concentrations of free T-2513 (that released
in vivo from T-0128) and polymer-bound T-2513 (that
remaining bound to CM dextran) were determined by HPLC analysis. For
free T-2513, proteins were precipitated with acetonitrile (300 µl),
before adding 180 ng/ml 10-hydroxyethyloxy-CPT/35
mM formic ammonium buffer (pH 3.0; 100 µl) as
an IS. After the addition of 0.1 M HCl (100
µl), the sample was vortexed for 10 s and centrifuged for 3 min
at 5000 x g. Prior to analysis, the clear
supernatant (100 µl) was diluted with the formic ammonium buffer.
To permit accurate quantitation of polymer-bound T-2513, T-2513 must
first be released from the conjugate, because T-0128 is weakly
fluorescent. Preliminary experiments revealed that exposure of samples
(100 µl) containing T-0128 to 6 M HCl (200 µl; final
concentration, 4 M) at 100°C for 4 h is sufficient
to release 100% of T-2513, and T-2513 is stable during this
hydrolysis. Therefore, a hydrolysis time of 4 h was routinely used
to permit the quantitation of polymer-bound T-2513. After the
hydrolysis, 6 M NaOH (200 µl) was added to neutralize the
acid, followed by a dilution with the formic ammonium buffer. After the
addition of 18 ng/ml IS/acetonitrile (100 µl), the hydrolysate was
filtered through a Millipore KC 0.45 µm filter (Bedford, MA) and
analyzed.
HPLC Chromatography.
Reversed-phase HPLC was performed at 40°C on an Inertsil ODS-2 column
150 x 4.6 mm (GL Sciences, Tokyo, Japan) preceded by a
Merck LiChrospher 100RP-18 guard cartridge (Darmstadt, Germany). T-2513
and the hydrolysate of polymer-bound T-2513 were eluted with 35
mM formic ammonium buffer (pH 3.0):acetonitrile (80:20,
v/v) and the buffer:acetonitrile (85:15, v/v), respectively, using a
Hitachi L-6200 pump (Tokyo, Japan) at a flow rate of 1.0
ml/min. Detection was carried out using a Shimadzu RF-550 fluorescence
detector (Kyoto, Japan) with excitation and emission wavelengths of 360
and 420 nm, respectively. Samples (20 µl, prepared as described
above) were injected into the HPLC system with a Tosoh AS-8000
automatic sampler. Peak data were recorded by a Shimadzu CR-6A
integrator. For every sample, the content of T-2513 was calculated by
measuring the relevant peak area and calibrating against the
corresponding IS peak area. Polymer-bound T-2513 was determined by
subtraction of free T-2513 from total T-2513 of the hydrolysate.
Data Analysis.
Pharmacokinetic parameters were determined from the average
concentration of three rats at each time point using the software
WinNonlin (Scientific Consulting, Alexandria, VA). The plasma
concentration-time profiles of polymer-bound T-2513, and of T-2513
after dosing of T-2513 itself, were well described by a biexponential
equation. The parameters obtained include the area under the
concentration-time curve extrapolated to infinity using a fitted model
(AUC0–
), the mean residence time
(MRT), total plasma clearance
(CLtotal), the volume of central
compartment (Vc), the volume of distribution at steady state
(Vss), the half-life of initial phase
[T1/2(
)], and the half life of
terminal phase [T1/2 (ß)]. The
AUC0-t values of polymer-bound T-2513 and
T-2513 in organs were calculated using the linear trapezoidal rule. The
area under the first moment curve (AUMC0-t)
was also calculated similarly. The MRT was determined by dividing
AUMC0-t by
AUC0-t.
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RESULTS |
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summarizes the results, indicating that T-2513 has a potent and broad
cytotoxicity, although it is less potent than SN-38. In contrast,
T-0128 is 1000-fold less potent than T-2513. The
IC50s (equivalent to T-2513) of T-0128 against
WiDr, SK-BR-3, and HeLaS3 were 17, 39, and 53 µg/ml, respectively.
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Toxicity Dose-Finding Studies.
To compare the antitumor effects of T-2513 with CPT-11 and topotecan at
their optimal doses and to evaluate the improvement in efficacy
attributable to the conjugation (T-0128), preliminary studies in
healthy rats and BALB/c mice were set up to assess their MTDs. In the
case of rats, the MTDs after a single i.v. injection of T-0128, T-2513,
and CPT-11 were found to be comparable, i.e., 100 (based on
the amount of T-2513 bound to CM dextran), 60, and 80 mg/kg,
respectively. In contrast, topotecan at 30 mg/kg caused 25% body
weight loss with diarrhea. A similar trend toward the order of the MTDs
was observed in the case of mice. The MTDs after a single i.v.
injection of T-0128, T-2513, and CPT-11 were apparently comparable,
i.e., 100, 80, and 100 mg/kg, respectively. In comparison,
the MTD of topotecan was <20 mg/kg. With four times weekly schedule,
the MTD of T-0128 was 100 mg/kg. In both animals, the main adverse
effects of T-0128 and T-2513 were myelosuppression and gastrointestinal
toxicity. Also, our pilot study showed that the MTD of T-0128 in an
St-4 tumor xenograft model was 75 mg/kg weekly for 4 weeks or 100 mg/kg
weekly for 3 weeks.
Antitumor Activity against Walker-256 carcinoma.
The ability of T-0128 and T-2513 to affect the growth of rat Walker-256
carcinoma was evaluated. As shown in Fig. 2
, T-0128 was able to shrink the tumor to 50% of the control at 2.3
mg/kg (ED50). In contrast, the
ED50 of T-2513 was 23 mg/kg; T-0128 was 10-fold
as active as T-2513. CPT-11 at 60 mg/kg produced about a 50% reduction
in the tumor growth, whereas topotecan at 12 mg/kg produced an 80%
reduction. Table 2
compares the therapeutic indexes (MTD/ED50) of
the four drugs, clearly indicating the superiority of T-0128 over
T-2513, CPT-11, and topotecan. These results mean that the use of
macromolecular carrier increases the potency and the range of
therapeutic doses of T-2513.
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shows the tumor growth and body weight change curves of nude mice given
a single i.v. administration of T-0128, as compared with controls or
animals given CPT-11 weekly at 60 mg/kg for 4 weeks. A single injection
of T-0128 at 4 mg/kg produced a transient inhibition of tumor growth.
T-0128 at 6 mg/kg (one-fiftieth of its MTD with three times weekly
schedule) achieved complete tumor inhibition in five of the six mice,
whereas no obvious decrease in the body weight was detected. CPT-11 at
60 mg/kg with four times weekly schedule produced not only a comparable
tumor reduction but also >11% body weight loss. T-2513 and topotecan
were less active than CPT-11 (data not shown).
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, T-0128 at 10 mg/kg once a week for 3 weeks (one-tenth of its MTD)
regressed the tumor completely and inhibited the body weight loss
attributable to cachexia. In contrast, CPT-11 at a dose of 100 mg/kg
with the same schedule was only able to slow tumor growth, with
virtually no regression of the tumor. T-2513 and topotecan were less
potent than CPT-11.
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summarizes the antitumor activities of T-0128, compared with CPT-11 or
T-2513. T-0128 demonstrated a strong antitumor activity in all tumor
models evaluated. It is important to note that T-0128 consistently
produced regression of tumor xenografts, which are highly refractory to
CPT analogues such as CPT-11 and T-2513, with IR values of >96%. This
significant effectiveness of T-0128 is exemplified by the results of
St-4 gastric tumor and HT-29 colorectal tumor. Against St-4, the MTDs
of CPT analogues only resulted in a delay in tumor growth. However,
T-0128 given at four-fifths of its MTD (at 60 mg/kg weekly for 4 weeks)
cured the mice. As shown in Fig. 5
, a single injection of T-0128 at 60 mg/kg obtained an IR of >90%
against HT-29 tumor. Three injections of T-0128 at the same total dose
(at 20 mg/kg weekly for 3 weeks) regressed the tumor more effectively.
The optimal schedule for administering T-0128 was not determined here.
Therefore, the dose required to achieve cures may be further reduced
with schedule optimization.
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, the plasma concentration-time profile of polymer-bound T-2513 is
compared with that of T-2513 after dosing of T-2513. It was clearly
shown that CM dextran endows T-2513 with an extremely long-term
circulation. When T-0128 was administered, polymer-bound T-2513
continued to circulate at a high concentration for an extended period
in the bloodstream, whereas there was virtually no free drug in the
plasma during the experiment. In contrast, T-2513 was cleared rapidly
from the blood. The mean plasma concentration-time profiles of both
drugs showed a biphasic decline, and pharmacokinetic parameters are
summarized in Table 4
.
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shows the tumor concentration-time profiles of polymer-bound T-2513 and
released T-2513 after dosing of T-0128. These profiles in several
tissues are summarized in Fig. 8
. As was expected, there was evidence that the liberation of T-2513 from
T-0128 occurred in various tissues. High levels of released T-2513 were
particularly observed in the tumor, spleen, liver, and lymph nodes
(data not shown for lymph nodes). The profiles of free T-2513 in all
tissues measured reached their peaks 24 h. On the other hand, the
polymer-bound T-2513 profiles were dependent on organs. The
concentrations in the tumor, liver, spleen, and lymph nodes peaked
12 h and subsequently declined very slowly, suggesting the cellular
uptake and retention of T-0128. In contrast, the profiles in the
kidney, heart, lung, and duodenum (data not shown for duodenum), not
corrected by remaining blood in the tissue, showed a decline similar to
that of the plasma. For comparison, the profiles of T-2513 after the
administration of T-2513 itself are also shown in Figs. 7
8
. T-2513
was cleared rapidly from the body. At 24 h after dosing, no
retention of T-2513 in the tumor was observed, as in all of the tissues
measured. To compare the tissue accumulations of released T-2513 with
those of T-2513 after dosing T-2513 itself, the AUC values in each
tissue are summarized in Table 5
. T-0128 increased the relative presence of T-2513 particularly in the
tumor, liver, spleen, and lymph nodes. The accumulations were attained
by factors ranging from 52 to 86, as determined by AUC evaluation,
whereas the AUC values in the kidney and the heart were unaffected.
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DISCUSSION |
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T-2513 had a broad cytotoxicity against a range of human tumor cell lines, whereas T-0128 was inactive in vitro. However, when used in vivo, the efficacy of T-0128 was 10-fold superior to that of T-2513 against Walker-256 carcinoma in Wistar rats. Although the MTDs of T-0128 and T-2513 were comparable, the conjugate gained a significant advantage in the tumor-bearing rats in terms of therapeutic index (MTD/ED50). Furthermore, T-0128 demonstrated a strong antitumor activity against a panel of human tumor xenografts that were mostly refractory to CPT-11, T-2513, and topotecan. Overall, T-0128 was more active and had a much broader range of therapeutic doses than T-2513 in vivo.
The improvements in potency of a drug through the drug-polymer conjugation, especially in the case of doxorubicin (17) , have been reported elsewhere. An example is PK1, a doxorubicin-N-(2hydroxypropyl)methacrylamide copolymer conjugate. Another example, BR96-doxorubicin immunoconjugate, was shown to exhibit a strong antitumor activity in a panel of human tumor xenografts (18) . It is true that the use of drugs with an established clinical profile, such as doxorubicin, may offer a safety advantage over more potent but less defined agents. However, we believe that the rational design of a drug-polymer conjugate, including the optimization of a drug, will achieve the maximal therapeutic efficacy. This is because the chemical structures of conventional chemotherapeutics are mostly restricted for the linkage to a polymeric carrier. On the basis of this belief, we synthesized and evaluated T-0128. The results described here proved our concept and encouraged us to develop this conjugate.
The ultimate effectiveness of T-0128 in the tumors is likely a result of various factors. On the basis of the pharmacokinetic data in this study and information available on the pharmacological and physico-chemical properties of CPT analogues, several hypotheses might apply.
(a) Tumor targeting of T-0128 attributable to the enhanced
permeability and retention effect should account for the efficacy. It
is interesting to compare the amounts of free T-2513 found in
Walker-256 carcinoma after the administration of T-0128 and T-2513.
Comparing the two profiles of free T-2513 levels in tumors, it is clear
that the AUC value for T-0128 is 50 times greater than that for
T-2513 (Table 5)
. This remarkable elevation in AUC is sufficient to
explain the increased efficacy of T-0128. Our preliminary study also
confirms the tumor accumulation of T-0128 and the release of T-2513 in
a panel of human tumor xenografts.
(b) A slow prolonged release of T-2513 is likely to be
responsible for the improvement of therapeutic efficacy
(19)
. Successful CPT-based chemotherapy, which is only
effective during the comparatively short S-phase of the cell cycle,
will require continuous presence of the drug (20)
. In
fact, the time period of exposure to T-2513 is a great determinant of
cytotoxicity. T-2513 is more active with >24-h incubation in
vitro (data not shown). Therefore, the potential therapeutic
benefit of T-0128 lies in a very long tumor exposure time of T-2513,
reflected by the significant extension in MRT (Table 5)
.
(c) Part of the increased efficacy of T-0128 might be explained based on the pH-mediated structural change of the parent drug. Under acidic conditions, CPT analogues are known to exist predominantly in the ring-closed lactone form, whereas at neutral or alkaline pH, they are converted into the ring-open carboxylate form, which is a much less potent inhibitor of topoisomerase I (21) . Therefore, the cellular uptake of T-0128, probably followed by exposure to acidic medium in lysosomes, might make the lactone form of released T-2513 predominant, close to and inside the target cells, promoting antitumor activity (22) .
Having considered the mechanisms for the efficacy of T-0128, we should
now focus on the toxicity of T-0128. The principal dose-limiting
toxicities of both T-0128 and T-2513 are myelosuppression and diarrhea.
The reasons for their comparable MTDs and toxicities (Table 2)
are not
yet clear. However, pharmacokinetic studies in tumor-bearing rats may
give a clue. One possible explanation is hepatic and splenic
accumulations after dosing of T-0128 (Fig. 8
and Table 5
). These
accumulations may sacrifice the advantage of the conjugate of reducing
peak drug concentrations, resulting in the close MTD. In particular,
hepatic accumulation of released T-2513 may produce a continuous drug
exposure of the intestine attributable to the pharmacokinetic property
of CPT analogues (23)
. Therefore, the use of a carrier
with less hepatic clearance might increase the range of therapeutic
doses of T-0128.
In summary, T-0128 consistently produced regression of tumor xenografts that are highly refractory to CPT analogues. Altered pharmacokinetic properties of T-0128 in terms of plasma half-life, tumor targeting, and kinetics of T-2513 release may account for the effectiveness. Results demonstrate the broad range of therapeutic doses that were achieved with T-0128, suggesting that T-0128 is a promising candidate in clinical trials for the treatment of solid cancer. However, the precise mechanisms for the action of T-0128 are still unclear. Thus, to elucidate them, we have been looking into the cellular uptake of T-0128 and the release of T-2513, in addition to further toxicity studies. The paper is now in preparation.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd.,
Kashima 3-16-89, Yodogawa-ku, Osaka 532-8505, Japan. Phone:
81-6-6300-2777; Fax: 81-6-6300-2582; E-mail: m-harada{at}tanabe.co.jp 
2 The abbreviations used are: CPT, camptothecin;
AUC, area under the concentration-time curve; CM, carboxymethyl; HPLC,
high-performance liquid chromatography; MRT, mean residence time; MTD,
maximal tolerated dose; SN-38, 7-ethyl-10-hydroxy-camptothecin; IS,
internal standard.
Received 11/12/99. Accepted 4/ 4/00.
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), CPT-11 (
), and topotecan (
) against Walker-256
carcinoma in rats. Four days after the drug injection, the excised
tumor weight of each treated group (T) was compared with
that of the control group (C). Data
points, mean of five rats; bars, SE.
