Biology Reference
In-Depth Information
to irinotecan, which primarily activates ATR-driven
checkpoints.
107,108
Early biomarker work with AZD7762 focused on
measurement of the changes in levels of both p-Chk1
(at the activation site, Ser
345
) and
g
-H2AX as a way to
determine the extent of target modulation achieved.
Significant increases in both of these markers were
observed preclinically
in vitro
using Western blotting,
and
in vivo
by immunohistochemical staining of either
xenograft or surrogate tissue biopsies (skin punch to
observe the level of staining in hair follicles). AZD7762
was studied in two phase 1 clinical trials in combination
with gemcitabine and irinotecan respectively, but devel-
opment was discontinued in April 2011 following a full
data review and assessment of risk
irinotecan (at either 50 or 100 mg/kg) resulted in
increased efficacy, with tumor growth delay and LCK
shown to be consistently doubled for the combination
groups versus irinotecan given alone. Importantly,
combination therapy was well tolerated in both studies,
again showing that Chk inhibitors can safely be adminis-
tered in combination with chemotherapeutic agents, at
least preclinically.
110
e
112
PF00477736 has also been found to enhance the effects
of docetaxel and ionizing radiation.
113
e
115
A number of
cell lines treated with docetaxel were demonstrated to
exhibit a time- and dose-dependent increase in 4N
DNA content that is consistent with G2 or mitotic arrest.
Cells treated with a combination of PF00477736 and
docetaxel were initially shown to arrest, but in contrast
to treatment with docetaxel alone, in the case of the
combination this arrest was not maintained. In addition,
and as expected from earlier results, there was an
increase in the levels of apoptosis for the combination
treatment, with apoptosis being observed earlier than
from the treatment with docetaxel alone. This potentia-
tion of activity was shown to translate into
in vivo
studies,
where in Colo-205 xenografts, PF00477736 significantly
increased the antitumor activity of docetaxel, resulting
in enhanced tumor regression and prolonged survival.
Again, there was no increase in systemic toxicity
compared with docetaxel treatment alone.
In A431 cells using an
in vitro
clonogenic survival
assay, PF00477736 induced a marked reduction in the
G2 cell cycle arrest resulting from treatment with
ionizing radiation (10 Gy), consistent with abrogation
of the G2 checkpoint. On treatment with PF00477736,
there was also a parallel increase in the amount of cells
entering mitosis following IR
.
Translating these studies
in vivo
, mice bearing subcutaneous A431 tumors were
treated with radiation alone (3 Gy daily for five days)
or in combination with PF00477736. The tumor growth
delay observed was 2.9 days for radiation alone and
6.7 days for the combination. Consistent with other
studies, the enhancement of activity seen on combina-
tion of radiation and PF00477736 was shown to be
p53-dependent.
Interestingly, PF00477736 has been shown to have
somewhat different effects on downstream protein
phosphorylation when compared with other agents in
this class. In combination studies, both
in vitro
and
in
vivo
, PF00477736 was shown to decrease levels of Chk1
phosphorylation on both Ser317 and Ser345 beginning
around 6 hours after compound administration. This is
in marked contrast to studies with other Chk inhibitors
where a robust increase in the level of p-Chk1 (Ser317)
is detected. However, in all cases the levels of
g
-H2AX
increase significantly. PF00477736 combination treat-
ment also results in a substantial increase in the level
of activated caspase 3.
benefit profile.
e
PF00477736
Early studies by Agouron (now Pfizer) on first-gener-
ation diazepinoindolone Chk1 inhibitors showed that
both potency and reasonable selectivity for Chk1 over
Chk2 could be achieved with this series. For example,
an early lead PF-394691 (EC
50
40 nM) was demonstrated
to be 300-fold selective for Chk1 over Chk2. Following
further optimization of this series, PF00477736 was
selected as a candidate for clinical trials.
109
PF00477736 (see
Figure 10.2
) is a potent ATP-compet-
itive inhibitor of Chk1 (K
i
0.49 nM), and is around
100-fold
selective
for
Chk1
over
Chk2
(K
i
47 nM), and
10,000-fold selective over Cdk1
(K
i
9.9
m
M).
In vitro
, PF00477736 releases cell cycle arrest
induced by DNA damage as shown by an increase in
the levels of p-hH3, concomitant with an increase in
sub-G1 population. This response was shown to be
general across a number of cell lines (HT-29, Colo-205,
PC-3, MDA-MB-231, and k562) and DNA-damaging
agents (e.g. gemcitabine, SN-38, carboplatin, doxoru-
bicin, and mitomycin-C). As with other inhibitors, the
most effective chemopotentiation was in combination
with gemcitabine, for example in Colo-205 or PC-3 cells
an approximately 12-fold potentiation factor was
achieved versus gemcitabine alone. PF00477736 in
combination with gemcitabine resulted in enhanced
cytotoxicity and a 78% increase in apoptosis over gem-
citabine alone, and this response was shown to be p53-
dependent, with cytotoxicity selectively enhanced in
p53-defective cancer cell
>
lines over p53-competent
normal cell lines
.
In vivo
, in studies using Colo-205 mouse xenografts,
a combination of PF00477736 (40 mg/kg) was found to
enhance the antitumor activity of gemcitabine (120 mg/
kg). There was a significant increase in LCK for the
combination over gemcitabine alone (1.2 versus 0.27,
respectively). Similarly, in HT-29 xenografts, a combina-
tion of PF00477736 (40 mg/kg) in combination with
