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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Pharmaceutical Sciences [M. N. K., A. K. S., S. K. H., C. F. S.] and Molecular Pharmacology [P. J. H., P. J.C., L. B. R.], St. Jude Children’s Research Hospital, and Department of Pharmacology [P. J. H.] and The Center for Pediatric Pharmacokinetics and Therapeutics [P. J. H., C. F. S.], University of Tennessee, Memphis, Tennessee 38105
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9-AC has demonstrated a spectrum of activity in animal models with transplantable solid tumors including cancer of the colon, lung, breast, and malignant melanoma xenografts (3 , 4) . However, clinical trials with this compound have been quite disappointing, resulting in its discontinuation from further clinical development (5 , 6) . In contrast, two other camptothecin derivatives, topotecan and irinotecan, have shown significant clinical utility and are now approved for use by the Food and Drug Administration.
Whereas the proportion of the topotecan lactone is maintained at
40–50% in plasma, only 
10% of 9-AC exists in the lactone form in
human plasma. This contrasts with a far higher proportion of 9-AC
lactone in plasma from mice. It has been suggested that this
interspecies difference in lactone to carboxylate equilibrium accounts
for the observed responsiveness of human tumors growing in mice
compared with the nonresponsiveness of human tumors growing in patients
(7)
. However, no one has assessed the 9-AC systemic
exposure at doses associated with cytotoxicity in the xenograft model.
In previous studies, we have shown that the systemic exposure to clinically effective and tolerated doses of topotecan and irinotecan in children with solid tumors is similar to that resulting in antitumor effects observed in the xenograft models (8) . These data have been used in the design of clinical trials of these agents in children with recurrent solid tumors (9 , 10) . At initiation of this study, the activity of 9-AC in pediatric solid tumors was unknown, as was the extent of systemic exposure that would be necessary for an objective response in the xenograft model. Accordingly, the objectives of the present study were to evaluate the antitumor activity of 9-AC in a panel of pediatric solid tumor xenografts and to relate the 9-AC lactone systemic exposure, defined as the AUC, to the antitumor dose associated with tumor regression in the xenograft model. These data will be important to understanding the lack of efficacy of 9-AC in published clinical trials.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3
mm3
are implanted in the space of the dorsal
lateral flanks of the mice to initiate tumor growth. Tumor-bearing mice
are randomized into groups of seven prior to therapy.
Tumor Lines.
Tumor lines have been described previously. HC1
is a moderately differentiated adenocarcinoma of the colon derived from
an adult patient (11)
. NB-1771 and NB-SD are N-MYC
amplified stage D childhood neuroblastomas (12)
, and Rh12
is an embryonal rhabdomyosarcoma (13)
. The sensitivity of
each of these tumors to topotecan (14)
and irinotecan
(11
, 12)
has been reported previously. Once established as
xenografts, further transplantations of neuroblastoma tumors were from
mouse to mouse, rather than from cell culture to mouse. For
chemotherapy studies, all tumors were used within six passages of their
engraftment in mice. Each tumor grew routinely in >95% of recipient
mice, and all retained human origin as determined by karyotype.
Growth Inhibition Studies.
Mice bearing bilateral s.c. tumors each received the agent when tumors
were approximately 0.20–1 cm in diameter. The procedures have been
reported previously (15)
. Briefly, two perpendicular
diameters were determined at 7-day intervals using digital Vernier
calipers interfaced with a Macintosh computer. Tumor volumes were
calculated assuming tumors to be spherical using the formula
[(
/6) x d3
], where
d is the mean diameter, and mice were followed for up to 12
weeks after starting treatment.
Tumor Response.
For individual tumors, PR was defined as volume regression >50% but
with measurable tumor at all times. CR was defined as the disappearance
of measurable tumor mass at some point after initiating therapy.
Maintained complete response was CR without tumor regrowth within the
study time frame (12 weeks). The minimum effective 9-AC dose achieving
objective response (CR and PR) for individual xenograft lines (MEDOR)
was defined as the minimum dose of 9-AC producing CR or PR in all
tumors within the treatment group, respectively.
Formulation and Administration.
9-AC as a colloidal dispersion was provided by Pharmacia and
Upjohn and was reconstituted as directed. The drug was administered
i.v. on the following schedules: (dx5)1; (dx5)2; or (dx5)3. Where
indicated, cycles of therapy were repeated every 21 days over 8 weeks
for the (dx5)1 and (dx5)2 treatments or every 28 days for the (dx5)3
schedule. Either one or three cycles of treatment were administered. In
separate cohorts of mice, the drug was administered by oral gavage
using the same schedules.
Drug Administration and Sample Collection.
The disposition of 9-AC was evaluated after a single 9-AC dose of
1.25 mg/kg administered by direct injection (duration of infusion <1
min) into a lateral tail vein or by oral gavage. Pharmacokinetic
studies were performed in nontumor bearing-mice and mice bearing
HC1, NB-1215, and Rh12 tumors. Heparinized blood
samples (1 ml) were collected prior to the dose and at 0.25, 0.5, 1,
1.5, 2, 4, and 6 h after oral 9-AC, and prior to the dose and at
0.25, 0.5, 1, 2, 4, and 6 h after i.v. 9-AC. Blood samples were
immediately centrifuged at 5.5 x g for 2 min on a
tabletop centrifuge. The plasma was separated, and the proteins
precipitated by the addition of 600 µl of cold methanol (-30°C) to
200 µl of plasma, followed by vigorous agitation with a vortex mixer,
and centrifugation again at 5.5 x g for 2 min. The
supernatant was decanted and stored at -70°C until analysis.
9-AC HPLC Assay.
9-AC plasma concentrations were determined using a modification of the
method of Takimoto et al. (16)
. Briefly, after
the plasma sample was processed as described above, it was manually
injected onto a 5 µM Ultrasphere ODS
(Beckman, Fullerton, CA) analytical column (4.6 mm x 25 cm). The
samples were eluted with a mobile phase consisting of
acetonitrile:methanol:0.1 M ammonium acetate, pH
5.5 (25:10:65, v/v/v), at a flow rate of 1.0 ml/min. Because 9-AC has
poor fluorescence relative to the parent compound camptothecin, we used
postcolumn acidification to protonate the C-9 amino group and enhance
drug fluorescence, which is optimal at pH 1.7–2.3. Postcolumn
acidification was accomplished by in-line mixing with 0.3
M aqueous trifluoroacetic acid (flow rate, 0.3
ml/min) prior to fluorescence detection. The emission and excitation
wavelengths were 352 and 418 nm, respectively. All calibrators and
controls for the xenograft studies were prepared in murine plasma (Hill
Top Lab Animals, Inc., Scottdale, PA).
Pharmacokinetic Analysis.
Noncompartmental methods (WinNonlin Professional version 3.0;
Pharsight, Cary, NC) were used to analyze both the i.v. and oral 9-AC
plasma concentration time data from this study. The AUC was calculated
by the log-linear trapezoidal rule to the last measurable data point.
The elimination rate constant (Ke)
determined by regression analysis of the terminal phase of the plasma
concentration-time curve was used to extrapolate the AUC to infinity
(AUC0
). The
area under the first moment curve (AUMC) and mean residence
time (MRT) were also determined (17)
. For i.v.
dosing, the clearance and volume of distribution at steady-state were
determined, and for oral dosing, the apparent clearance and volume of
distribution were determined.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. When 9-AC was administered i.v. daily for 5 days for a single course,
[(dx5)1]1, no PRs were obtained at dose levels associated with
acceptable toxicity (i.e., weight loss <20%). Thus, when
9-AC was administered as a single course on a [(dx5)1]1 schedule,
the minimum effective daily dose was 
1.5 mg/kg. Similarly, no PRs
were noted in HC1, NB-1771, or NB-SD when the
5-day course was repeated every 21 days for three cycles, [(dx5)1]3.
Using the 5-day schedule on 2 consecutive weeks for one cycle,
[(dx5)2]1, resulted in objective regressions
(HC1 and Rh12) at a daily dose of 1 mg/kg. This
dose was toxic to mice bearing NB-1771 tumors. In Fig. 1
, we depict the effect of administering three cycles of therapy, where
9-AC was given i.v. for [(dx5)1]3, [(dx5)2]3 every 21 days, or
[(dx5)3]3 every 28 days for HC1 colon tumors.
Both [(dx5)2]3 and [(dx5)3]3 schedules resulted in regressions at
dose levels of 0.36 and 0.29 mg/kg, respectively. In contrast, 1.5
mg/kg [(dx5)1]3 failed to prevent tumor progression. The cumulative
doses administered for [(dx5)1]3, [(dx5)2]3, and [(dx5)3]3
schedules were 22.5, 10.8, and 13.0 mg/kg, respectively. Very similar
results were obtained with NB-1771 neuroblastoma xenografts, further
indicating marked schedule-dependent antitumor activity of 9-AC (Fig. 2)
. Responses of NB-SD xenografts to decreasing doses of 9-AC using the
[(dx5)2]3 schedule are shown in Fig. 3
. Complete regressions were obtained at 0.66 mg/kg, with some PRs at
0.44 mg/kg and only growth retardation at 0.29 mg/kg.
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. At the maximum tolerated dose (1.5 mg/kg/dose), no objective
regressions were obtained using a single cycle of [(dx5)1]1 therapy
in mice bearing HC1 tumors. The same schedule
[(dx5)1]3 repeated for three cycles was also ineffective. Objective
regressions were induced at acceptable levels of toxicity following a
single cycle of [(dx5)2]1 treatment in HC1,
Rh12, and NB-1771 xenografts. Three cycles, either using (dx5)2 or
(dx5)3 schedules, resulted in objective regressions of all four tumor
lines. Representative results for NB-1771 tumors showing the
dose-response relationship are presented in Fig. 4
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and Table 3
. The 9-AC clearance (CL) increased 23, 68, and 41%
in mice bearing HC1, NB-1215, and Rh12 xenografts
compared with non-tumor-bearing mice, respectively (P =
0.07; ANOVA). Moreover, 9-AC AUC0 was
decreased 81, 58, and 71% in mice bearing HC1,
NB-1215, and Rh12 xenografts compared with non-tumor-bearing mice,
respectively (P = 0.06; ANOVA). The 9-AC plasma
concentration was greater than the limit of quantitation (3 ng/ml) for
6 h after drug administration in all experiments with
tumor-bearing mice.
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. After oral administration, the median (range) percentage of AUC
extrapolated was 5.1 (range, 0.53–15.3%). 9-AC noncompartmental
pharmacokinetic parameters measured after oral administration are
presented in Table 4
. The median (range) 9-AC oral bioavailability was 0.32 (range,
0.13–0.63). The decrease in 9-AC lactone AUC after oral administration
in tumor-bearing mice, relative to non-tumor-bearing mice, was
consistent with that observed after i.v. administration
(i.e., 54, 72, and 74% in mice bearing
HC1, NB-1215, and Rh12 xenografts compared with
non-tumor-bearing mice, respectively; P = 0.07; ANOVA).
The 9-AC concentration was >3 ng/ml for 4 h after drug
administration in all tumor-bearing mice.
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) associated with the lowest dose
achieving CRs and PRs for each xenograft line was extrapolated from the
AUC measured after a dose of 1.25 mg/kg (data presented in Tables 5
and 6
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As with other camptothecin analogues, 9-AC has schedule-dependent antitumor activity. As the schedule of 9-AC administration was extended from [(dx5)1]3 to 2 weeks [(dx5)2] every 21 days or 3 weeks [(dx5)3] every 28 days, greater antitumor activity was observed. Although the daily dose for the three xenograft lines tested with the [(dx5)3] schedule was somewhat lower than for the [(dx5)2] schedule, the cumulative dose needed to attain an antitumor response was lower in the [(dx5)2] group (i.e., 13.0 versus 10.8 mg/kg, respectively).
The MEDOR of all tumors in a treatment group in our studies varied from
0.29 to 1.5 mg/kg/dose administered i.v. This range was dependent upon
the administration schedule that was used. Previous studies examining
9-AC in the xenograft model include an evaluation by Giovanella
et al. (3)
. Briefly, 9-AC at a dose of 12.5
mg/kg was administered s.c. into mice bearing HT-29 colon cancer
xenografts twice weekly for 3–6 weeks and resulted in >90% reduction
in tumor volume. Additionally, Pantazis et al.
(19)
injected 4 mg/kg 9-AC i.m. twice weekly into mice
bearing BRO melanoma xenografts and observed that the drug-treated mice
were virtually free of tumor after 40 days. However, no
pharmacokinetics results from these studies are available for
comparison with the present study.
Numerous trials of 9-AC have been conducted in adults, and the results
of these are summarized in Table 7
. Various dosing regimens, schedules, and routes of administration have
been evaluated in these clinical trials to optimize antitumor activity
and minimize toxicity. However, the antitumor responses have been very
disappointing at doses that have led to unacceptable toxicities,
primarily myelosuppression. Even with the addition of growth factor
support, which decreased the duration and severity of myelosuppression,
little increase in antitumor activity was observed at the highest 9-AC
doses.
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400 or 708 ng/ml·h. These
investigators were able to achieve a higher 9-AC systemic exposure than
other investigators who administered lower doses over longer times. The
fact that higher systemic exposures could be tolerated in these
patients is consistent with our data that protracted dosing may provide
an overall greater exposure to the drug. Although this study used a
schedule more consistent with the antitumor properties of 9-AC, the
mean daily 9-AC lactone systemic exposure was probably below that
necessary for antitumor responses, based upon the results of our study
(Table 5)
. The antitumor responses observed after oral 9-AC were similar to those observed after i.v. administration. Although the dose associated with the oral MEDOR was higher than that required for i.v. administration, tumor regression was observed in a schedule-dependent manner, and these responses were induced at acceptable levels of toxicity to the mouse. In a Phase I trial, Mani et al. (21) administered oral 9-AC (formulated in PEG-1000) to 16 adult patients with a variety of solid tumors. The dosages ranged from 0.06 to 0.2 mg/kg daily for 5 days every 2 weeks. No objective responses or cumulative toxicities were observed; however, the investigators concluded that the formulation was not suitable for further development because of poor bioavailability and variable elimination. These clinical characteristics of oral 9-AC administration, taken together with the results from the xenograft model, would predict that it would not be possible to attain the 9-AC systemic exposure necessary for antitumor responses.
In the only published trial in children, Langevin et al. (22) administered 9-AC to children with recurrent solid tumors by a 72-h infusion every 21 days. The 9-AC disposition was analyzed at doses of 36, 43, and 62 µg/m2/h (22) , and the 9-AC lactone AUC values for the course reported were 233, 169, and 493 ng/ml·h, respectively. The authors reported one PR, and as in the adult Phase I trials, neutropenia and thrombocytopenia were dose limiting. As a consequence, the authors recommended a Phase II dose of 52 µg/m2/h on this schedule.
The schedule of 9-AC administration associated with the most antitumor
efficacy in these xenograft models was dailyx5 for 2 weeks repeated
every 21 days [(dx5)2]3. On the basis of the data presented in Table 5
, the dose required for antitumor activity on this schedule ranged
from 0.36 to 0.66 mg/kg/day. The 9-AC lactone daily systemic exposure
corresponding to these doses ranged from 69 to 158 ng/ml·h. Thus, the
cumulative 9-AC lactone systemic exposure associated with antitumor
efficacy in the xenograft model ranged from 690 to 1580 ng/ml·h for
the course of therapy. This is compared with the maximum tolerated 9-AC
systemic exposure from either adult or pediatric clinical trials of
continuous infusion 9-AC of 126 or 493 ng/ml·h. Thus, it is not
surprising that few antitumor responses have been observed with 9-AC
because hematopoietic toxicity limits the ability of patients to
tolerate the extent of 9-AC systemic exposure that is associated with
antitumor response in the xenograft model.
In summary, we have shown that 9-AC has antitumor activity in a panel of pediatric solid tumors when administered in a schedule-dependent manner. Antitumor responses were noted in the xenograft model at 9-AC doses that were associated with acceptable toxicity. However, 9-AC lactone systemic exposure required to induce tumor responses in the xenograft were greater than the plasma systemic exposures reported at maximally tolerated 9-AC doses in patients. These data support the concept of determining systemic exposures associated with MEDOR in mice bearing human tumor xenografts. Although clinical trials using 9-AC have been disappointing, our results demonstrate the potential usefulness of determining systemic exposures necessary to achieve an antitumor effect. By deriving preclinical data relating systemic exposure to antitumor activity in xenograft models, informed decisions might be made regarding the clinical development of new agents for the treatment of childhood and adult cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a grant from Pharmacia and
Upjohn Pharmaceutical Co., USPHS Award CA 23099, Cancer Center Support
Grant CA 21765, and American Lebanese Syrian Associated Charities. 
2 To whom requests for reprints should be
addressed, at Department of Pharmaceutical Sciences, St. Jude
Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.
Phone: (901) 495-3665; Fax: (901) 525-6869; E-mail: clinton.stewart{at}stjude.org
3 The abbreviations used are: 9-AC,
9-amino-20(S)-camptothecin; AUC, area under the plasma
concentration-time curve; PR, partial response; CR, complete response;
MEDOR, minimum effective dose of 9-AC achieving objective response;
(dx5)1, daily x 5 for 1 week; (dx5)2, daily x 5 for 2
consecutive weeks; (dx5)3, daily x 5 for 3 consecutive weeks.
Received 5/ 8/00; revised 9/27/00; accepted 10/ 5/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and NB-1215 (
) tumor-bearing animals.