Rational Design of Irinotecan Administration Based on Preclinical Models

Rational Design of Irinotecan Administration Based on Preclinical Models

ABSTRACT: Most clinical drug regimens for irinotecan (CPT-11 [Camptosar]) have been empirically based on classic in vivo pharmacokinetic and pharmacodynamic considerations. We propose an alternative approach that attempts to provide a rationally designed schedule of irinotecan administration based on preclinical data. HL60 cells grown in suspension or as subcutaneously implanted solid xenografts in nude mice served as in vitro and in vivo models to test the activity of irinotecan or its active metabolite, SN-38. For SN-38, within an effective drug concentration range, scheduling drug administration based on duration of DNA synthesis inhibition significantly potentiated cell kill in vitro, and increasing drug concentrations at suboptimal scheduling did not result in additive cell kill. These data suggested that even though high drug doses may be attainable in vivo, they may not be required to achieve maximum antitumor activity. To test this hypothesis, a sensitive in vivo model to test the toxicity and antitumor activity of CPT-11 is required, which is provided in the human myeloid HL60 xenograft model grown in nude mice. In this model, CPT-11 at a dose 50 mg/kg, daily × 5 (MTD) achieved 100% complete tumor regression. This model should be useful to test the hypothesis that for irinotecan, administration of a minimum effective dose (MED) at an optimal schedule can achieve maximum antitumor activity and should therefore prevail over the classic approach of administering the MTD. [ONCOLOGY 12(Suppl 6):22-30, 1998]


The antitumor activity of the plant alkaloid camptothecin has been
known for more than 2 decades, but initial enthusiasm for this
compound was tempered by the recognition of its severe side effects.
(Hematologic toxicities are dose-limiting.)[1-5] The discovery that
camptothecin perturbs the catalytic cycle of the topoisomerase I
enzyme offered a novel target for chemotherapy and revitalized
interest in this drug.[6-9]

Topoisomerase enzymes help regulate translation and transcription
processes by controlling the topologic structure of DNA and relaxing
the supercoiled DNA helix. The topoisomerase I catalytic cycle
involves transient covalent binding of the enzyme to DNA (cleavable
complex formation), which nicks one DNA strand. This, in turn, allows
for DNA strand passage, religation of the DNA strand break, and
subsequent release of the enzyme from the DNA. Consequently, the DNA
helix is unwound, relieving torsional stress associated with, for
instance, DNA replication. Camptothecin and most of its analogs act
by mediating stabilization of the topoisomerase I/DNA cleavable
complex,[8,9] initiating a series of events that ultimately lead to
cell death.

The renewed interest in camptothecin resulted in the development of
camptothecin derivatives, which display less severe side effects[10]
and better water solubility than the parent compound. Topotecan
(Hycamtin) and irinotecan (CPT-11 [Camptosar]) are two of the most
prominent topoisomerase I-interactive agents for which antitumor
activity has been evaluated in clinical trials.

The design of clinical drug regimens of irinotecan has been based on
classic in vivo pharmacokinetic and pharmacodynamic considerations,
which call for the administration of maximum tolerated doses (MTDs)
for any given schedule. Preclinical in vivo data of the antitumor
activity of irinotecan against human neuroblastoma xenografts,
however, have indicated that antitumor activity depends more on the
schedule of administration than on drug dose.[11]

Notwithstanding their empirical design, early phase II-III clinical
trials evaluating the topoisomerase I-interactive agents topotecan
and irinotecan have had encouraging results in patients with heavily
pretreated secondary and refractory leukemias, showing indications of
response in these otherwise poor responders.[12-16] Moreover,
currently ongoing clinical trials in colorectal cancer combining
irinotecan with fluorouracil (5-FU)/leucovorin have shown promising
response rates of up to 60%.[17-19]

Many parameters relevant for studying the action of topoisomerase
I-interactive agents have been identified. These include: catalytic
activity, protein levels, and mutations of the topoisomerase I
enzyme; topoisomerase I messenger RNA (mRNA) levels; and
DNA-topoisomerase I protein cross-links.

Known resistance mechanisms against topoisomerase I-interactive
agents include decreased topoisomerase I protein levels[20] due to
posttranslational or posttranscriptional changes or gene
rearrangements[21] and topoisomerase I mutations[22,23] affecting
cleavable complex formation (decreased catalytic activity) or binding
of topoisomerase I-interactive agent.[23,24] (Catalytic activity is
preserved, but the topoisomerase I agent-induced stabilization of
cleavable complex formation is reduced.) One mechanism specifically
related to irinotecan resistance is a change in the activity of
carboxylesterase,[25] the enzyme required for the metabolic
conversion of irinotecan to its active metabolite, SN-38. Although
many of the above resistance mechanisms have been described in
different cell line models, their prognostic value for in vivo
sensitivity to topoisomerase I-interactive agents has yet to be established.

The demonstration that (temporary) inhibition of DNA/RNA synthesis
could protect from topoisomerase I agent-induced toxicity but not
from drug-induced formation of protein/DNA cross-links has led to the
"DNA-replication fork collision" model. This model assumes
that the protein-DNA cross-links are prelethal lesions, which become
lethal only when they encounter a moving DNA replication fork. This
encounter results in irreparable double-strand DNA breaks[26-28] and
chromosomal aberrations.[29-33]

Supporting evidence for this model comes from drug interaction
studies of topoisomerase I- and topoisomerase II-interactive
agents.[34,35] These studies have shown that simultaneous exposure of
cells to camptothecin and etoposide results in antagonistic effects,
which can be changed to additive effects if the drug exposures are
separated by an interval that exceeds the time necessary to restore
drug-induced inhibition of DNA and RNA synthesis.[35]

These data suggest that, to a certain extent, inhibition of DNA/RNA
synthesis may provide a cell with an opportunity to restore the
DNA/protein cross-link lesions before a fatal collision with DNA
replication forks occurs. The fact that topoisomerase I- and
topoisomerase II-interactive agents are effective as single agents
despite concomitant drug-induced inhibition of DNA/RNA synthesis
appears to be contradictory. We propose, therefore, that the extent
and duration of DNA/RNA-synthesis must be taken into consideration.

Evidence that these parameters may play a role in ultimate
drug-induced cytotoxicity may come from another study exploring the
interaction between topoisomerase I- and topoisomerase II-interactive
agents, which showed that simultaneous drug exposure results in drug
synergism[36] rather than antagonism. In this study, very low
concentrations of the drugs were used (< IC10, or the
concentration inhibiting 10% of cell growth). One theory that may
explain the two seemingly contradictory findings suggests that, at
the low drug concentrations used in the latter study, the inhibition
of DNA/RNA synthesis is insufficient to interfere with the moving
replication forks and to affect drug efficacy.

This article describes a series of experiments designed to determine
how exposure to topoisomerase I inhibitors (specifically,
camptothecin and irinotecan) affect DNA synthesis, and how the extent
and duration of this inhibition may be used to optimize scheduling of
these drugs. In addition, a preclinical in vivo model is presented in
which a 100% complete response (CR) rate can be achieved with
irinotecan in a single-drug regimen. This model should prove to be
useful to test the hypotheses developed from in vitro data in an in
vivo setting.

Materials and Methods

Cell Lines and Xenografts

The human myeloid leukemia cell line HL60 was propagated in RPMI1640
media supplemented with 10% heat-inactivated fetal bovine serum. Cell
cultures were maintained at exponential growth by maintaining cell
densities below 1 × 106 cells/mL.

Xenografts were initially established by subcutaneous (SC) injection
of 200 mL of a cell suspension (1× 107 cells/mL) in
8- to 12-week-old, female athymic nude mice (Sprague Dawley Inc.,
Indianapolis, Indiana). Subsequently, xenografts were maintained for
several generations by SC transplantation of 50-mg nonnecrotic solid
tumor tissue.


Camptothecin and SN-38 for the in vitro studies were donated by
BioNumerik Pharmaceuticals, Inc. (San Antonio, Texas). Stock
solutions of each drug (5 mmol) were made in 100% dimethyl sulfoxide
(DMSO) and stored at -20 °C until use. Drug dilutions were made
from these frozen stocks with media to achieve the desired final
concentrations. Irinotecan for the in vivo studies was donated by the
Pharmacia & Upjohn Company (Kalamazoo, Michigan) as a sterile
solution of 20 mg/mL in 0.9% saline.


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