Biology Reference
In-Depth Information
In an
in vivo
pharmacodynamic model, hollow fibers
containing HT29 cells are treated with topotecan
in vitro
to induce a robust G2/M arrest. The hollow fibers are
then implanted under the skin of a nude mouse prior
to treatment with AZD7762. Thirty hours later the fibers
are retrieved and the cells analyzed by FACS to deter-
mine the extent to which arrest has been released. This
model was extremely useful since it allowed for potency
ranking of multiple compounds
in vivo
in a highly effi-
cient manner. In this hollow fiber assay, AZD7762 was
shown to overcome topotecan-induced cell cycle arrest
in a dose-dependent manner.
101
Additionally, studies in xenograft models have
confirmed the ability of AZD7762 to increase the efficacy
of DNA-damaging agents preclinically. AZD7762 has
been shown to potentiate the effects of gemcitabine, iri-
notecan, and radiation in a number of xenograft models,
including some using patient-derived xenografts in an
attempt to more closely mimic the clinical situation.
Early xenograft studies used an H460 dominant nega-
tive p53 cell line and demonstrated that, in the nude
mouse, neither gemcitabine (60 mg/kg) nor AZD7762
(25 mg/kg) had a significant effect on tumor growth
when dosed on an intermittent schedule for five treat-
ment cycles (each cycle giving two doses of AZD7762
at 4 and 16 hours post gemcitabine). In contrast, the
combination treatment significantly reduced tumor
growth and gave an increased log cell kill (LCK) (0.9
for the combination versus 0.1 for gemcitabine alone).
Similarly, combination of AZD7762 (25 mg/kg) with iri-
notecan (25 and 50 mg/kg) over four treatment cycles
(each cycle giving two doses of AZD7762 at 2 and 14
hours post irinotecan) resulted in a marked increase in
efficacy for the combination therapy versus irinotecan
given alone. In this case, while both doses of irinotecan
given as a single agent caused a significant, dose depen-
dent reduction in tumor growth, this was greatly
increased such that in combination tumor-free survival
was achieved in 5/9 animals (25 mg/kg irinotecan
group) and 8/9 animals (25 mg/kg irinotecan group).
Neither dose of irinotecan given as monotherapy
resulted in tumor-free survival (0/9 animals in both
groups
).
Given the different levels of activity for gemcitabine
and irinotecan as single agents, coupled with the
inability to easily use gemcitabine at MTD in the nude
mouse (because of very rapid clearance) there was
concern that the high degree of potentiation for the
combination with gemcitabine could merely be a reflec-
tion of the use of a suboptimal dose of chemotherapeutic
agent. To address this, a xenograft study was under-
taken in the nude rat, where the pharmacokinetics of
gemcitabine are very different and thus allow it to be
used at the MTD (10 mg/kg). Again, in this setting,
gemcitabine alone showed very little effect, suggesting
that the H460 DN p53 cell line is fairly resistant to gem-
citabine as a single-agent therapy. As before, there was
increased efficacy for the combination of gemcitabine
with AZD7762 (growth delay 8.9 days versus 1.7 days
for gemcitabine alone). It should be noted that the use
of gemcitabine at the MTD limited the study to three
cycles as control-treated animals were not able to
tolerate additional treatment cycles owing to a marked
weight loss.
More recently, AZD7762 has also been demonstrated
to enhance both radiation, and gemcitabine-radiation
therapy both
in vitro
and in HT29 and MiaPaca-2, as
well as patient-derived xenografts. Excitingly, this
work extended to triplet combinations of AZD7762,
radiation, and gemcitabine, and provides support for
the hypothesis that Chk1 inhibitors may be able
to be developed using more sophisticated dosing
schedules.
103,104
Activation of Chk1 by radiation was found to be rapid
and to persist for several hours following radiation treat-
ment showing pathway activation and supporting the
notion that AZD7763 would sensitize tumor cells to
radiation in a similar manner to that observed for cyto-
toxic chemotherapies. Again, similarly to the studies
with gemcitabine and iriniotecan, pilot studies in combi-
nation with radiation showed that AZD7762 treatment
following radiation was more effective than pretreat-
ment. The concentration of AZD7762 found to give the
best enhancement of the effects of radiation, while
having minimal cytotoxicity alone was 100 nM, thus
studies were carried out by treating cells with
AZD7762 for one hour prior to radiation,
then for
24 hours after
followed by clonogenic
survival
assessment.
The combination of AZD7762 with radiation was
shown to be more effective than radiation treatment
alone in p53-mutated tumor cell lines from a variety
of tumor types as shown by dose modification factors
(DMFs) of 1.6
1.7. The DMF is determined by calcu-
lating the ratio of radiation doses at the 10% survival
(control radiation dose divided by the radiation plus
AZD7762 dose), thus DMFs
e
1 indicate an increase
in radiosensitivity. This response was found to be
p53-dependent as p53 wild-type tumor lines had
much lower DMFs of 1.1
>
1.2. Finally, in a matched
pair of cell lines (H460 versus H460 d/n p53) DMFs
of 1.11 and 1.58 respectively were obtained. In addition
it was demonstrated that AZD7762 did not enhance the
radiosensitivity of normal human fibroblasts (DMF
1.05), and that AZD7762 treatment alone exhibited
little cytotoxicity in any of the lines studied.
AZD7762 treatment was shown to abrogate radiation
induced G2 delay, as shown in flow cytometry studies,
inhibit radiation damage repair (assessed by
g
-H2AX),
and suppress radiation-induced cyclin B expression,
e
