Biology Reference
In-Depth Information
selective against other serine/threonine kinases, only
showing
in toxicity. Intriguingly, it was also shown that the effects
of lower doses of gemcitabine could be enhanced by
combination with MK1775 to give greater efficacy than
could be achieved with gemcitabine alone, even at the
MTD. This offers the future possibility that combination
with MK1775 could make it possible to achieve similar
or increased antitumor activity at lower doses of chemo-
therapy. As with the
in vitro
studies previously
described, MK1776 was also demonstrated to potentiate
the effects of platinum agents and antimetabolites in
a number of xenograft models (e.g. WiDR, HeLa-luc,
MX-1, TOV21G-shp53).
In addition to the work with chemotherapeutics,
MK1775 has also been shown preclinically to enhance
the effects of radiation treatment in human lung cancer
models.
125
In these studies MK1775 was found to
increase the activity of radiation treatment both
in vitro
and
in vivo
, and the response determined to be p53-
dependent, that is, activity was enhanced in H1299
(p53 null) and CALU6 (p53 mutant) cell lines, but not
in H549 or H460 (p53 wild type) cells. In xenograft
models, treatment was associated with a rapid, transient
reduction in pCDC2 levels, increased levels of pChk,
pHH3,
g
-H2AX, and increased mitoses followed by
mitotic catastrophe. It was proposed that both cell cycle
deregulation and inhibition of DNA repair contributed
mechanistically to the observed synergy.
Development of biomarkers was focused on two
assays
80% inhibition for 8/223 kinases in the
Upstate panel at 1
m
M. Follow-up studies revealed
MK1775 was more than 10-fold selective for seven of
these eight kinases, and approximately three-fold selec-
tive against Yes (IC
50
14 nM). Importantly, MK1775 is at
least 100-fold less potent against human Myt1, which
can also suppress Cdc2 activity.
124
MK 1775 has been
shown preclinically to enhance the effects of a number
of agents including gemcitabine, cisplatin, pemetrexed,
doxorubicin, and 5-fluorouracil, both
in vitro
and
in
vivo
. The increase in activity was also shown to depend
on p53 status, being specific for cells that are deficient
in p53 function. For example, in WiDR cells (a human
colorectal cancer line with mutated p53), MK1775
inhibited phosphorylation of CDC2 (EC
50
85 nM), and
abrogated gemcitabine-induced cell cycle arrest as
determined by the induction of pHH3, a marker of
entry into mitosis (EC
50
81 nM). Similar trends were
also observed in combination with other chemothera-
peutic agents as well as in other cancer cell lines
carrying p53 mutations, e.g. H1299, TOV21G, SW948
etc. These data suggest that the approach, like that of
the checkpoint kinase inhibitors, has the potential for
very broad utility. The degree of potency, and the
degree of potentiation observed, were dependent
upon the cell line and DNA damaging agent used.
MK1775 was also shown to shift the GI
50
in combina-
tion, for example, again in WiDR cells, gemcitabine
alone had a GI
50
>
>
a p-CDC2Y15 assay to assess target modulation
and a p-HH3 assay to show checkpoint abrogation and
entry into mitosis.
In vitro
and
in vivo
studies revealed
a good correlation between reductions in p-CDC2Y15
levels (
e
100 nM, but this was enhanced to
21.5 nM and 7.1 nM in combination with 30 nM or
100 nM MK1775 respectively. It was also shown that
the combination treatment caused a dramatic increase
in sub-G1 population (55% and 59% at 30 nM and
100 nMMK1775 respectively). Treatment with gemcita-
bine alone (100 nM) induced a miminal sub-G1 popula-
tion, and caspase 3/7 activation was observed only in
combination. Again, these trends held true across
different chemotheraputic agents and p53 mutant cell
lines.
In vivo
efficacy studies in WiDR xenografts showed
that MK1775 could enhance the effectiveness of gemcita-
bine (50 mg/kg) in a dose-dependent manner when
given 24 hours post the dose of gemcitabine. This
schedule was extrapolated from earlier
in vitro
studies
that revealed this sequence to be superior to either
simultaneous or reverse sequential dosing as deter-
mined by quantification of the sub-G1 population
following treatment. Gemcitabine alone was only
moderately active in this xenograft model (T/C 35%)
but efficacy was dramatically enhanced in combination
(T/C 1% and
50%) and antitumor efficacy.
MK1775 is the subject of three clinical trials, the most
advanced being a phase II trial of a combination of
MK1775 and carboplatin in patients with p53-mutated
epithelial ovarian cancer who have previously received
first line treatment (paclitaxel
>
carboplatin combination
therapy), but showed early relapse (within 3 months).
A two-part phase I/IIa study comparing MK1775 in
combination with topotecan and cisplatin is also
ongoing. Part one of the study will define the MTD
and recommended phase II dose of the combination of
MK1775 with topotecan and cisplatin, while part two
will assess whether the triplet combination of MK1775,
cisplatin and topotecan can increase progression-free
survival (PFS) over the combination of cisplatin and top-
otecan in patients with cervical cancer. Recently, interim
data from an earlier phase I trial of MK1775 in monother-
apy and in combination with gemcitabine, cisplatin of
carboplatin in patients with advanced solid tumors
were presented.
126
The trial assessed the safety, tolera-
bility, PK and PD of MK1775 administered orally as
a monotherapy, and in combination with either gemcita-
bine, cisplatin or carboplatin. A single dose of MK1775
e
25% with 10 mg/kg and 20 mg/kg
MK1775 respectively). MK1775 dosed alone was
without significant antitumor activity (T/C 69%). The
combination was well tolerated with no overt increase
