U.S. patent application number 13/633114 was filed with the patent office on 2013-04-11 for treating cancer with atr inhibitors.
This patent application is currently assigned to VERTEX PHARMACEUTICALS INCORPORATED. The applicant listed for this patent is John Robert Pollard, Philip Michael Reaper. Invention is credited to John Robert Pollard, Philip Michael Reaper.
Application Number | 20130089626 13/633114 |
Document ID | / |
Family ID | 47019166 |
Filed Date | 2013-04-11 |
United States Patent
Application |
20130089626 |
Kind Code |
A1 |
Pollard; John Robert ; et
al. |
April 11, 2013 |
Treating Cancer with ATR Inhibitors
Abstract
This invention relates to methods and compositions for treating
pancreatic cancer. More specifically, this invention relates to
treating pancreatic cancer with certain ATR inhibitors in
combination with gemcitabine and/or radiation therapy. This
invention also relates to methods and compositions for treating
non-small cell lung cancer. More specifically, this invention
relates to treating non-small cell lung cancer with an ATR
inhibitor in combination with cisplatin or carboplatin, etoposide,
and ionizing radiation.
Inventors: |
Pollard; John Robert;
(Abingdon, GB) ; Reaper; Philip Michael;
(Shillingford, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pollard; John Robert
Reaper; Philip Michael |
Abingdon
Shillingford |
|
GB
GB |
|
|
Assignee: |
VERTEX PHARMACEUTICALS
INCORPORATED
Cambridge
MA
|
Family ID: |
47019166 |
Appl. No.: |
13/633114 |
Filed: |
October 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61542084 |
Sep 30, 2011 |
|
|
|
Current U.S.
Class: |
424/649 ;
514/255.05; 514/255.06; 514/49 |
Current CPC
Class: |
A61K 31/555 20130101;
A61N 2005/1098 20130101; A61K 31/497 20130101; A61K 45/06 20130101;
A61P 35/00 20180101; A61K 31/4965 20130101; A61P 43/00 20180101;
A61K 31/7048 20130101; A61P 1/18 20180101; A61K 31/7068 20130101;
A61K 33/24 20130101; A61K 31/7068 20130101; A61K 2300/00 20130101;
A61K 31/7048 20130101; A61K 2300/00 20130101; A61K 31/497 20130101;
A61K 2300/00 20130101; A61K 31/4965 20130101; A61K 2300/00
20130101; A61K 31/555 20130101; A61K 2300/00 20130101; A61K 33/24
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/649 ;
514/255.05; 514/255.06; 514/49 |
International
Class: |
A61K 31/497 20060101
A61K031/497; A61K 31/7068 20060101 A61K031/7068; A61K 33/24
20060101 A61K033/24; A61K 31/4965 20060101 A61K031/4965 |
Claims
1. A method of treating pancreatic cancer in a patient by
administering to the patient an ATR inhibitor selected from
##STR00005## in combination with another cancer therapy selected
from gemcitabine, radiation therapy, or both gemcitabine and
radiation therapy together.
2. The method of claim 1, wherein the method increases the
sensitivity of pancreatic cancer cells to a cancer therapy selected
from gemcitabine or radiation therapy.
3. The method of claim 1, wherein the cancer therapy is
gemcitabine.
4. The method of claim 1, wherein the cancer therapy is radiation
therapy.
5. The method of claim 2, wherein the pancreatic cancer cells are
hypoxic pancreatic cancer cells.
6. A method of inhibiting phosphorylation of Chk1 (Ser 345) in a
pancreatic cancer cell comprising administering to a patient an ATR
inhibitor selected from ##STR00006## in combination with
gemcitabine and/or radiation.
7. The method of claim 5, wherein the cancer therapy is radiation
therapy.
8. The method of claim 5, wherein the cancer therapy is
gemcitabine.
9. The method of claim 2, wherein the cancer therapy comprises
chemoradiation.
10. The method of claim 9 wherein the chemotherapy is
gemcitabine.
11. A method of disrupting damage-induced cell cycle checkpoints by
administering to a patient an ATR inhibitor selected from
##STR00007## in combination with radiation therapy.
12. A method of inhibiting repair of DNA damage by homologous
recombination in a pancreatic cancer cell by administering an ATR
inhibitor selected from ##STR00008## in combination with radiation
treatment.
13. The method of claim 12 wherein the pancreatic cancer cells are
derived from a pancreatic cell line selected from PSN-1, MiaPaCa-2
or Panc-1.
14. The method of claim 12, wherein the pancreatic cancer cells are
in a cancer patient.
15-16. (canceled)
17. A method of treating non-small cell lung cancer in a patient
comprising administering to the patient a compound of formula 821
or 822: ##STR00009## in combination with one or more of the
following additional therapeutic agents: Cisplatin or Carboplatin,
Etoposide, and ionizing radiation.
18. The method of claim 17, comprising administering to a patient a
compound of formula 821 or 822 in combination with a cancer therapy
selected from the group consisting of Cisplatin or Carboplatin,
Etoposide, and ionizing radiation.
19. The method of claim 18, wherein the cancer therapy is Cisplatin
or Carboplatin and Etoposide.
20. The method of claim 18, wherein the cancer therapy is Cisplatin
or Carboplatin and Etoposide and ionizing radiation.
21. The method of claim 18, wherein the cancer therapy is ionizing
radiation.
22. The method according to claim 1, wherein the ATR inhibitor is
822.
23. The method according to claim 17 wherein the ATR inhibitor is
822.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application No. 61/542,084 filed
on Sep. 30, 2011, the entire contents of which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Pancreatic cancer is the tenth most common site of new
cancers and is responsible for 6% of all cancer related deaths. The
5-year survival rate is less than 5%.sub..
[0003] Current therapies involve either neoadjuvant treatment with
chemotherapy (e.g., with gemcitabine) and/or radiation therapy or
surgical removal followed by either adjuvant chemotherapy (e.g.,
with gemcitabine) or radiation therapy. Although the survival rate
with treatment of gemcitabine increases the 5-year survival from
10% to 20%, there still is a strong need for better therapies for
treating pancreatic cancer.
[0004] Several therapeutics have been tested in phase II and phase
III trials though results have not been too promising. Tipifarnib,
an oral farnesyltransferase inhibitor, did not show significant
improvement in overall survival when combined with gemcitabine.
Similarly, cetuximab, an epidermal growth factor receptor (EGRF),
also showed no clinical benefit when combined with gemcitabine.
Only a small increase in overall survival (6.24 months versus 5.91
months) was observed.
[0005] Lung cancer is the second most common form of cancer and is
the leading cause of cancer-related mortality. Non-small cell lung
cancer (NSCLC) is the most common form of lung cancer, accounting
for about 85% of all lung cancer cases. Most patients present with
advanced stage III or IV NSCLC with a 5-year survival of 24% and 4%
respectively. Because of the advanced nature of disease on
presentation, surgical resection is often not an option. For the
majority of patients therapy involves chemotherapy and/or radiation
treatment. The selection of chemotherapy is highly variable based
on disease stage, patient performance criteria and geographical
regional preference. In most cases chemotherapy is based on a
doublet that includes a platinating agent such as Cisplatin or
carboplatin and a second cytotoxic drug such as gemcitabine,
etoposide or taxotere. For a small number of patients, therapy can
include treatment with agents that target specific proteins that
are mutated or disregulated such as ALK and EGFR (eg crizotinib,
gefitinib and erlotinib). Patients are selected for these targeted
treatments based on genetic or proteomic markers. A great number of
agents have been assessed in late stage NSCLC clinical studies,
however most have shown very little benefit over chemotherapy based
treatments, with median overall survival typically less than 11
months.
[0006] Accordingly, there is a tremendous need for new strategies
to improve pancreatic and non-small cell lung cancer
treatments.
[0007] ATR ("ATM and Rad3 related") kinase is a protein kinase
involved in cellular responses to certain forms of DNA damage (eg
double strand breaks and replication stress). ATR kinase acts with
ATM ("ataxia telangiectasia mutated") kinase and many other
proteins to regulate a cell's response to double strand DNA breaks
and replication stress, commonly referred to as the DNA Damage
Response ("DDR"). The DDR stimulates DNA repair, promotes survival
and stalls cell cycle progression by activating cell cycle
checkpoints, which provide time for repair. Without the DDR, cells
are much more sensitive to DNA damage and readily die from DNA
lesions induced by endogenous cellular processes such as DNA
replication or exogenous DNA damaging agents commonly used in
cancer therapy.
[0008] Healthy cells can rely on a host of different proteins for
DNA repair including the DDR kinases ATR and ATM. In some cases
these proteins can compensate for one another by activating
functionally redundant DNA repair processes. On the contrary, many
cancer cells harbour defects in some of their DNA repair processes,
such as ATM signaling, and therefore display a greater reliance on
their remaining intact DNA repair proteins which include ATR.
[0009] In addition, many cancer cells express activated oncogenes
or lack key tumour suppressors, and this can make these cancer
cells prone to dysregulated phases of DNA replication which in turn
cause DNA damage. ATR has been implicated as a critical component
of the DDR in response to disrupted DNA replication. As a result,
these cancer cells are more dependent on ATR activity for survival
than healthy cells. Accordingly, ATR inhibitors may be useful for
cancer treatment, either used alone or in combination with DNA
damaging agents, because they shut down a DNA repair mechanism that
is more important for cellular survival in many cancer cells than
in healthy normal cells.
[0010] In fact, disruption of ATR function (e.g. by gene deletion)
has been shown to promote cancer cell death both in the absence and
presence of DNA damaging agents. This suggests that ATR inhibitors
may be effective both as single agents and as potent sensitizers to
radiotherapy or genotoxic chemotherapy.
[0011] Furthermore, solid tumors often contain regions that are
hypoxic (low oxygen levels). This is significant because hypoxic
cancer cells are known to be resistant to treatment, most notably
IR treatment, and are highly aggressive. One reason for this
observation is that components of the DDR can be activated under
hypoxic conditions and it has also been shown that hypoxic cells
are more reliant on components of the DDR for survival.
[0012] For all of these reasons, there is a need for the
development of potent and selective ATR inhibitors for the
treatment of pancreatic cancer, for the treatment of lung cancer,
and for the development of agents that are effective against both
hypoxic and normoxic cancer cells.
SUMMARY OF THE INVENTION
[0013] This invention relates to uses of ATR inhibitors for
treating pancreatic cancer and non-small cell lung cancer. With
respect to pancreatic cancer, this invention relates to methods of
treating pancreatic cancer in a patient (e.g., a human) with an ATR
inhibitor in combination with gemcitabine and/or radiation therapy.
Applicants have demonstrated synergistic efficacy of ATR inhibitors
in combination with gemcitabine and/or radiation therapy in
clonogenic and viability assays on the pancreatic cancer cell
lines, (e.g. PSN-1, MiaPaCa-2 and Panc-1) as well as in a primary
tumor line (e.g., Panc-M). Disruption of ATR activity was measured
by assessing DNA damage induced phosphorylation of Chk1 (Ser 345)
and by assessing DNA damage foci and RAD51 foci following
irradiation.
[0014] With respect to non-small cell lung cancer, his invention
relates to methods of treating non-small cell lung cancer with an
ATR inhibitor in combination with cisplatin or carboplatin,
etoposide, and ionizing radiation. Applicants have demonstrated
synergy of ATR inhibitors in combination with cisplatin, etoposide,
gemcitabine, oxaplatin and irinotecan in viability assays against a
panel of 35 human lung cancer cell lines as well as demonstrated in
vivo efficacy in a lung cancer mouse model in combination with
cisplatin.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1. VE-821 radiosensitises pancreatic tumour cells.
[0016] A) Western blot analysis of Chk1 inhibition.
[0017] Cells were treated with 100 nM gemcitabine for 1 h, 1 .mu.M
VE-821 was added 1 h later and cells were irradiated (6 Gy) 1 h
after that. Drugs were left for the duration of the experiment and
cells were lysed at 2 h post-irradiation and subjected to Western
blot analysis.
[0018] B) VE-821 radiosensitizes pancreatic tumour cells but not
normal fibroblasts.
[0019] PSN-1, Panc-1, MiaPaCa-2 pancreatic cancer cell lines and
MRC5 fibroblasts were treated with increasing concentrations of
VE-821 for 96 h combined with or without 4 Gy radiation at 1 h
after VE-821 addition. Cell viability was measured after 8 days and
shown as normalized to DMSO-treated cells.
[0020] C) Scheduling of VE-821 affects radiosensitivity.
[0021] PSN-1 cells were plated as single cells, treated with 1
.mu.M VE-821 at different time points in relation to 4 Gy
irradiation and assessed for colony formation after 10 days. The
survival fraction at 4 Gy for each of the treatment schedules was
determined by taking into account the relevant plating efficiency
of unirradiated cells.
[0022] D) Clonogenic survival of cells pancreatic cancer cells in
response to ATR inhibition.
[0023] Cells were treated with 1 .mu.M VE-821 4 h after plating and
1 h prior to irradiation. Drug was removed after 72 h and
colony-forming ability was assessed after 10 to 21 days. (n=3). *,
P<0.05; **, P<0.01 over DMSO-treated control.
[0024] FIG. 2. VE-821 radiosensitises pancreatic tumour cells under
hypoxic conditions.
[0025] A) clonogenic survival curves of cells treated with 1 .mu.M
VE-821 and irradiation under hypoxic conditions. Plated cells were
transferred to hypoxia (0.5% O.sub.2) and acclimatised for 6 h.
VE-821 (1 .mu.M) was then added at 1 h prior to irradiation and
left for 72 h upon which the medium was replaced. Cells were
transferred to normoxia at 1 h post-irradiation.
[0026] B) clonogenic survival of cells after irradiation with 6 Gy
and treatment with 1 .mu.M VE-821 in oxic and hypoxic (0.5%
O.sub.2) conditions, as described above and in FIG. 1 (n=3). *,
P<0.05; **, P<0.01; ***, P<0.001 over DMSO-treated
control.
[0027] FIG. 3. VE-821 sensitises pancreatic cancer cells to
gemcitabine treatment.
[0028] A) clonogenic survival of cells treated with gemcitabine and
1 .mu.M VE-821. Cells were treated with increasing concentrations
of gemcitabine for 24 h followed by 72 h treatment of 1 .mu.M
VE-821. Colony forming ability was assessed after 10 to 21
days.
[0029] B) clonogenic survival of cells treated with gemcitabine in
hypoxia. Plated cells were transferred to hypoxia (0.5% O.sub.2)
and acclimatised for 6 h. Cells were then treated with increasing
concentrations of gemcitabine for 24 h followed by 72 h treatment
of 1 .mu.M VE-821. Hypoxic cells were transferred to normoxia 1 h
after VE-821 addition.
[0030] C) clonogenic survival after treatment with 20 nM
gemcitabine and VE-821 in oxic and hypoxic (0.5% O.sub.2)
conditions, as described above.
[0031] D) clonogenic survival of cells treated with gemcitabine and
irradiation. PSN-1 and MiaPaCa-2 cells were treated with 5 nM or 10
nM gemcitabine, respectively, for 24 h, medium was then replaced
and 1 .mu.M VE-821 was added from 1 h prior to 72 h post 4 Gy
irradiation. Colony forming ability was assessed after 10 to 21
days (n=3). *, P<0.05; **, P<0.01; ***, P<0.001 over
DMSO-treated control.
[0032] FIG. 4. VE-821 perturbs the irradiation-induced cell cycle
checkpoint in pancreatic cancer cells.
[0033] VE-821 (1 .mu.M) was added 1 h prior to 6 Gy irradiation and
left for the duration of the experiment. Cells were lifted and
fixed at 12 h or 24 h after irradiation, stained with propidium
iodide and analysed for cell cycle distribution by flow cytometry
(n=3)
[0034] FIG. 5. VE-821 increases 53BP1 and .gamma.H2AX foci number
and reduces RAD51 foci formation.
[0035] Cells were treated with 1 .mu.M VE-821 at various time
points in relation to 6 Gy irradiation, as indicated, and fixed at
24 h post-irradiation. Subsequently, cells were stained for (A)
.gamma.H2AX and (B) 53BP1 foci and the percentage of cells with
more than 7 and 5 foci per cell was quantitated, respectively. C,
for analysing Rad51 foci formation, cells were fixed at 6 h
post-irradiation and the percentage of cells with more than 9 foci
per cell was quantitated. Representative images are shown on the
right (n=3). *, P<0.05
Supplementary Figures
[0036] Suppl FIG. 1. Effect of VE-821 incubation time on plating
efficiency.
[0037] PSN-1 cells were plated as single cells, treated with 1 uM
VE-821 for various time periods and assessed for colony formation
after 10 days.
[0038] Suppl FIG. 2.
[0039] VE-821 perturbs the irradiation-induced G2/M checkpoint in
pancreatic cancer cells in hypoxic conditions.
[0040] Cells were pre-incubated under hypoxic (0.5% O.sub.2)
conditions for 6 h and 1 .mu.M VE-821 was added 1 h prior to
irradiation (6 Gy). Cells were transferred to normoxia 1 h after
irradiation and were lifted and fixed at 12 h or 24 h after
irradiation, stained with propidium iodide and analysed for cell
cycle distribution by flow cytometry (n=3).
[0041] FIG. 1X. Dose response relationship for radiosensitivity
induced by Compounds 821, 822, 823, and 824.
[0042] Small scale clonogenic survival assays were performed on
HeLa cells treated with the different ATR inhibitors at increasing
concentrations followed by irradiation at 6Gy. Data is plotted as
decrease in clonogenic survival in relation to the DMSO-treated
cells for both irradiated (SF 6Gy, pink line) and unirradiated
cells (plating efficiency, PE; blue line). A high degree of
increased radiosensitivity can be seen as a large decrease in
survival after irradiation accompanied by a small decrease in
unirradiated survival at a specific drug concentration.
[0043] FIG. 2X. Assessment of radiosensitivity in tumour cells and
normal cells. [0044] A) Clonogenic survival after drug treatment in
the absence of irradiation. PSN1 and MiaPaca cells were plated at
low densities, treated with the drugs indicated and assessed for
clonogenic survival. [0045] B) Clonogenic survival of PSN1,
MiaPaca, and MRC5 cells pretreated with Compounds 821, 822, 823 and
824 drugs followed by irradiation. Cells were plated at low
densities, treated with drugs indicated 1 h prior to irradiation
and assessed for clonogenic survival.
[0046] FIG. 3X. Assessment of dependency of drug addition and
removal timing on radiosensitivity.
[0047] MiaPaca cells were plated at low densities and drug was
added at various time points in relation to the 4Gy radiation
treatment: 1 h prior to IR, 5 min after IR, 2 h or 4 h after IR;
and removed at various time points: 5 min after, 1 h after, or 19 h
after IR. Clonogenic survival was assessed after 14 days. Results
are shown as the surviving fraction at 4Gy (top panel) or the
percentage radiosensitisation (middle panel) compared to the
DMSO-treated cells. The different treatment schedules did not cause
differences in plating efficiency (bottom panel).
[0048] FIG. 4X. DNA damage foci analysis after Compound 822
treatment and irradiation. [0049] A) Assessment of gH2AX, 53BP1
foci at 24 h after IR at 6Gy and of RAD51 foci at 6 h after IR.
MiaPaca cells were treated with 80 nM Compound 822 1 h prior or 1 h
post irradiation and drug was washed away at 5 min after or 1 h
after IR. Cells were fixed after 6 h (for RAD51 foci) or 24 h (for
gH2AX and 53BP1 foci). The percentage of cells containing more than
a certain number of foci was quantitated. [0050] B) Time course of
DNA damage foci. Cells were treated as in A and fixed at the time
points shown followed by staining for gH2AX, 53BP1 and RAD51 foci.
Data is shown as the mean number of foci at a particular time point
(upper panels) or the percentage of cells containing more than a
certain number of foci (lower panels).
[0051] FIG. 5X. Cell cycle analysis of Compound 822-treated cells
after 6Gy irradiation.
[0052] PSN1 cells were treated with 40 nM Compound 822 1 h prior to
6Gy irradiation in triplicate wells. Cells were lifted and fixed at
several time points after IR, stained with propidium iodide and
analysed by flow cytometry. [0053] A) Cell cycle histogram plots.
Fitted peaks are coloured red for G1 phase, shaded for S-phase, and
green for G2/M phase. One out of three wells is shown for each time
point and treatment. [0054] B) Average cell cycle percentages over
time. Cell cycle percentage values were obtained from fitted
histogram plots (n=3) and plotted for control-treated and Compound
822-treated cells.
[0055] FIG. 6X. MiaPaCa Tumor Volume over Time for Compound
822.
[0056] FIGS. 7X and 8X. PSN-1 Tumor Volume over Time for Compound
822.
[0057] FIG. 1Y. Lung Cancer Cell Screen: VE-822 Synergizes with
Chemotoxics Across a Panel of Lung Cancer Cell Lines in Lung Cell
Viability Assay
[0058] FIG. 2Y. Lung Cancer Cell Screen: VE-822 Exhibits Greater
than 3-fold Synergy with Chemotoxics in Lung Cancer Cell Lines in a
Cell Viability Assay
[0059] FIG. 3Y. Pancreatic Cancer Cell Screen: VE-822 Synergizes
with Cisplatin and Gemcitabine in Pancreatic Cancer Cell Lines in a
Cell Viability Assay
[0060] FIG. 4Y. Pancreatic Cancer Cell Screen: VE-822 Exhibits
Greater than 3-fold Synergy with Chemotoxics in Pancreatic Cancer
Cell Lines a Cell Viability Assay
[0061] FIG. 5Y. Effect of VE-822 and cisplatin on tumor volume and
body weight in a primary adenocarcinoma NSCLC xenograft in SCID
mice.
[0062] FIG. 6Y: Effect of VE-822 administered PO q2d at 10, 30 or
60 mg/kg in combination with gemcitabine (15 mg/kg IP q3d) on the
tumor volume of mice bearing PSN1 pancreatic cancer xenografts.
DETAILED DESCRIPTION OF THE INVENTION
[0063] One aspect of this invention provides methods for treating
pancreatic cancer in a patient by administering to the patient an
ATR inhibitor in combination with another known pancreatic cancer
treatment. One aspect of the invention includes administering the
ATR inhibitor in combination with gemcitabine. In some embodiments,
the pancreatic cancer comprises one of the following cell lines:
PSN-1, MiaPaCa-2 or Panc-1. According to another aspect, the cancer
comprises the primary tumor line Panc-M.
[0064] Another aspect of the invention provides methods for
treating cancer (e.g., pancreatic or non-small cell lung) in a
patient by administering to the patient an ATR inhibitor in
combination with radiation therapy.
[0065] Another aspect of the invention provides methods for
treating non-small cell lung cancer in a patient by administering
to the patient an ATR inhibitor in combination with cisplatin or
carboplatin, etoposide, and/or ionizing radiation. Applicants have
demonstrated synergy of ATR inhibitors in combination with
cisplatin, etoposide, gemcitabine, oxaliplatin and irinotecan in
viability assays against a panel of 35 human lung cancer cell lines
as well as demonstrated in vivo efficacy in a lung cancer mouse
model in combination with cisplatin. This invention also relates to
the use of ATR inhibitors in combination with cisplatin or
carboplatin, etoposide, and/or ionizing radiation for treating
non-small cell lung cancer.
[0066] Examples of ATR inhibitors are shown in Table 1 below:
TABLE-US-00001 TABLE 1 ##STR00001## 821 ##STR00002## 822
[0067] The terms referring to compounds 821 and 822 are
interchangeable with VE-821 and VE-822, respectively.
[0068] Another aspect provides a method of treating pancreatic
cancer by administering to pancreatic cancer cells an ATR inhibitor
selected from a compound in Table 1 in combination with one or more
cancer therapies. In some embodiments, the ATR inhibitor is
combined with chemoradiation, chemotherapy, and/or radiation
therapy. As would be understood by one of skill in the art,
chemoradiation refers to a treatment regime that includes both
chemotherapy (such as gemcitabine) and radiation. In some
embodiments, the chemotherapy is gemcitabine.
[0069] Yet another aspect provides a method of increasing the
sensitivity of pancreatic cancer cells to a cancer therapy selected
from gemcitabine or radiation therapy by administering an ATR
inhibitor selected from a compound in Table 1 in combination with
the cancer therapy.
[0070] In some embodiments, the cancer therapy is gemcitabine. In
other embodiments, the cancer therapy is radiation therapy. In yet
another embodiment the cancer therapy is chemoradiation.
[0071] Another aspect provides a method of inhibiting
phosphorylation of Chk1 (Ser 345) in a pancreatic cancer cell
comprising administering an ATR inhibitor selected from a compound
in Table 1 after treatment with gemcitabine (e.g., 100 nM) and/or
radiation (e.g., 6 Gy) to a pancreatic cancer cell.
[0072] Another aspect provides method of radiosensitizing hypoxic
PSN-1, MiaPaCa-2 or PancM tumor cells by administering an ATR
inhibitor selected from a compound in Table 1 to the tumor cell in
combination with radiation therapy.
[0073] Yet another aspect provides a method of sensitizing hypoxic
PSN-1, MiaPaCa-2 or PancM tumor cells by administering an ATR
inhibitor selected from a compound in Table 1 to the tumor cell in
combination with gemcitabine.
[0074] Another aspect provides a method of sensitizing PSN-1 and
MiaPaCa-2 tumor cells to chemoradiation by administering an ATR
inhibitor selected from a compound in Table 1 to the tumor cells in
combination with chemoradiation.
[0075] Another aspect provides a method of disrupting
damage-induced cell cycle checkpoints by administering an ATR
inhibitor selected from a compound in Table 1 in combination with
radiation therapy to a pancreatic cancer cell.
[0076] Another aspect provides a method of inhibiting repair of DNA
damage by homologous recombination in a pancreatic cancer cell by
administering an ATR inhibitor selected from a compound in Table 1
in combination with one or more of the following treatments:
chemoradiation, chemotherapy, and radiation therapy.
[0077] In some embodiments, the chemotherapy is gemcitabine.
[0078] Another aspect provides a method of inhibiting repair of DNA
damage by homologous recombination in a pancreatic cancer cell by
administering an ATR inhibitor selected from a compound in Table 1
in combination with gemcitabine and radiation therapy.
[0079] In some embodiments, the pancreatic cancer cells are derived
from a pancreatic cell line selected from PSN-1, MiaPaCa-2 or
Panc-1.
[0080] In other embodiments, the pancreatic cancer cells are in a
cancer patient. In other embodiments, the cancer cells are part of
a tumor.
[0081] Another embodiment provides methods for treating non-small
cell lung cancer in a patient by administering to the patient an
ATR inhibitor in combination with other known non-small cell lung
cancer treatments. One aspect of the invention includes
administering to a patient an ATR inhibitor in combination with
cisplatin or carboplatin, etoposide, and/or ionizing radiation.
[0082] Another aspect provides a method of treating non-small cell
lung cancer by administering to a patient an ATR inhibitor selected
from a compound in Table 1 in combination with one or more cancer
therapies. In some embodiments, the ATR inhibitor is combined with
chemoradiation, chemotherapy, and/or radiation therapy. As would be
understood by one of skill in the art, chemoradiation refers to a
treatment regime that includes both chemotherapy (such as
cisplatin, carboplatin, or etoposide) and radiation. In some
embodiments, the chemotherapy comprises Cisplatin or carboplatin,
and etoposide.
[0083] Yet another aspect provides a method of increasing the
sensitivity of non-small cell lung cancer cells to a cancer therapy
selected from cisplatin or carboplatin, etoposide, and ionizing
radiation by administering to a patient an ATR inhibitor selected
from a compound in Table 1 in combination with one or more cancer
therapy.
[0084] In some embodiments, the cancer therapy is cisplatin or
carboplatin. In other embodiments, the cancer therapy is radiation
therapy. In yet another embodiment the cancer therapy is
etoposide.
[0085] In some embodiments, the cancer therapy is a combination of
cisplatin or carboplatin, etoposide, and ionizing radiation. In
some embodiments the cancer therapy is cisplatin or carboplatin and
etoposide. In other embodiments the cancer therapy is cisplatin or
carboplatin and etoposide and ionizing radiation. In yet other
embodiments the cancer therapy is cisplatin or carboplatin and
ionizing radiation.
[0086] Another aspect provides a method of inhibiting
phosphorylation of Chk1 (Ser 345) in a non-small cell lung cancer
cell comprising administering to a patient an ATR inhibitor
selected from a compound in Table 1. In some embodiments, the ATR
inhibitor is administered in combination with gemcitabine (e.g.,
100 nM), cisplatin or carboplatin, etoposide, ionizing radiation or
radiation (e.g., 6 Gy) to a non-small cell lung cancer cell.
[0087] In other embodiments, the non-small cell lung cancer cells
are in a cancer patient.
[0088] In some embodiments, the ATR inhibitor is
##STR00003##
[0089] In other embodiments, the ATR inhibitor is
##STR00004##
Uses
[0090] Another aspect provides use of an ATR inhibitor selected
from a compound in Table 1 in combination with gemcitabine and
radiation therapy for treating pancreatic cancer.
[0091] Another aspect provides use of an ATR inhibitor selected
from a compound in Table 1 in combination with cisplatin or
carboplatin, etoposide, and ionizing radiation for treating
non-small cell lung cancer.
[0092] In some embodiments, the ATR inhibitor is Compound VI-821.
In other embodiments, the ATR inhibitor is Compound VI-822.
Manufacture of Medicaments
[0093] Another aspect provides use of an ATR inhibitor selected
from a compound in Table 1 in combination with gemcitabine and
radiation therapy for the manufacture of a medicament for treating
pancreatic cancer.
[0094] Another aspect provides use of an ATR inhibitor selected
from a compound in Table 1 in combination with cisplatin or
carboplatin, etoposide, and ionizing radiation for the manufacture
of a medicament for treating non-small cell lung cancer.
[0095] In some embodiments, the ATR inhibitor is Compound VI-821.
In other embodiments, the ATR inhibitor is Compound VI-822.
EXAMPLES
[0096] The examples are for the purpose of illustration only and
are not to be construed as limiting the scope of the invention in
any way.
Cell Viability Assays
[0097] MiaPaCa-2, PSN-1, Panc1 and MRC5 cells (5.times.104) were
plated in 96-well plates and after 4 h treated with increasing
concentrations of VE-821 at 1 h before irradiation with a single
dose of 6 Gy. Medium was replaced 96 h post-irradiation at which
point viability was measured using the using the Alamar Blue assay
(Resazurin substrate, SIGMA). Cells were allowed to proliferate and
cell viability was again analyzed at day 8 for the different
treatment conditions. Cell viability and surviving fraction were
normalized to the untreated (control) group.
Clonogenic Survival Assay
[0098] Logarithmically growing cells were plated in triplicate in
6-well tissue culture dishes under oxic (21% O.sub.2) or hypoxic
conditions (0.5% O.sub.2) using an InVivo2 300 chamber (Ruskinn
Technology, UK). Cells were incubated for 6 hours before
irradiation under oxia or hypoxia using tightly sealed chambers.
The target O.sub.2 level was achieved within 6 h of gassing and
maintained during irradiation, as confirmed by an OxyLite oxygen
probe (Oxford Optronix). Cells irradiated under hypoxia were
exposed to normoxia at 1 h post-irradiation. As standard, VE-821 (1
.mu.M) was added 1 h prior to irradiation (6 Gy) and was washed
away 72 h after irradiation. For the chemotherapy experiments,
cells were initially exposed to increasing concentrations of
gemcitabine (5, 10 and 20 nM) for 24 h before addition of the
VE-821 (1 .mu.M) for another 72 h. The effect of triple combination
of irradiation with VE-821 and gemcitabine was examined as well.
Cells were incubated for 10-21 days until colonies were stained
with 0.5% crystal violet and counted in a CellCount automated
colony counter (Oxford Optronix). Clonogenic survival was
calculated and data were fitted in the GraphPad Prism 4.0 (GraphPad
Software, CA).
Western Blot
[0099] MiaPaCa-2 and PSN-1 cells were exposed to gemcitabine and/or
1 .mu.M VE-821 drug 1 h prior to irradiation with a single dose of
6 Gy. Cells were lysed in RIPA buffer 2 h post-irradiation and
subjected to SDS-PAGE electrophoresis and immunoblotting.
Chemoluminescence (SuperSignal, Millipore) and film exposure was
used to detect antibody binding. Exposed film was digitized and
figures were assembled using Microsoft PowerPoint.
Nuclear Foci Analysis
[0100] Cells growing in 96-well plates were treated with 1 .mu.M
VE-821 drug 1 h prior to 6 Gy irradiation and fixed in 3%
formaldehyde at multiple time points. Cells were subsequently
pearmeabalised and blocked in PBS with 0.1% Triton 1% BSA (w/v).
Cells were incubated with primary antibody overnight at 4.degree.
C. and after a PBS wash incubated with fluorescently labeled
secondary antibody followed gy a PBS wash and nuclear staining with
DAPI. Images were acquired and foci quantitated using an IN Cell
Analyzer 1000 automated epifluorescence microscope and analysis
software (GE Healthcare, Cahlfont St. Giles, UK)
Cell Cycle Analysis
[0101] Cells growing in 6-well dishes were treated with 1 .mu.M
VE-821 drug 1 h prior to 6 Gy irradiation. Cells were incubated for
6 h before irradiation under oxia (21% O.sub.2) or hypoxia (0.5%
O2) using tightly sealed chambers. At multiple time points, cells
were lifted in trypsin and fixed in 70% ethanol and stored at
4.degree. C. Cells were incubated with propidium iodide (50
.mu.g/ml in PBS containing 200 .mu.g/ml RNAse) for 1 h at room
temperature and analysed by flow cytometry (FACSort, Becton
Dickinson). Cell cycle phase was quantitated using ModFit Cell
Cycle Analysis software.
Cell Seeding and Compound Addition for Lung Cancer Cell Screen
[0102] All cell lines were seeded in 30 .mu.l of tissue culture
medium containing 10% FBS into 384-well opaque-bottom assay plates.
The seeding density was based on the logarithmic growth rate of
each cell line. After 24 hours, compound stock solutions were added
to each well to afford a matrix consisting of 5 concentrations for
VE-822 and 6 concentrations for chemotoxics. Each well contains
either, agent alone or a combination of both agents. The final
concentration range for VE-822 was 25 nM-2 .mu.M. The concentration
ranges for the chemotoxics were as follows: Etoposide, 10 nM-10
.mu.M; Gemcitabine, 0.16 nM-160 nM; Cisplatin, 20 nM-20 .mu.M;
Oxaliplatin, 40 nM-40 .mu.M; Irinotecan (SN-38), 0.12 nM-120 nM.
The cells were then incubated for 96 hours at 37.degree. C. in an
atmosphere of 5% CO.sub.2 and 95% humidity.
Cell Seeding and Compound Addition for the Pancreatic Cancer Cell
Screen
[0103] All cell lines were seeded in 30 .mu.l of tissue culture
medium containing 10% FBS into 384-well opaque-bottom plates. The
seeding density was based on the logarithmic growth rate of each
cell line. After 24 hours, compound stock solutions were added to
each well to afford a matrix consisting of 9 concentrations for
VE-822 and 7 concentrations for Gemcitabine and Cisplatin. Each
well contains either, agent alone or a combination of both agents.
The final concentration ranges were as follows: VE-822, 0.3 nM-2
.mu.M; Gemcitabine, 0.3 nM-0.22 .mu.M; Cisplatin, 30 nM-20 .mu.M.
The cells were then incubated for 96 hours at 37.degree. C. in an
atmosphere of 5% CO.sub.2 and 95% humidity.
Cell Viability Assay
[0104] This assay measures the number of viable cells in a culture
based on the quantitation of ATP, which is present in metabolically
active cells.
[0105] CellTiter-Glo Reagent (Promega, Madison, Wis., USA) was
prepared according to the manufacturer's instructions and added 96
hours after compound addition (25 .mu.l/well) to measure cell
viability. Luminescence signal was measured with the PHERAStarFS
(BMG Labtech, Cary, N.C., USA) automated plate reader. All cell
lines were screened in duplicate.
[0106] Raw luminescence CellTiter-Glo (CTG) values were normalized
to the mean CTG value for the negative control DMSO-treated samples
on each assay plate. IC.sub.50 values for chemotoxic alone were
calculated using DMSO-normalized cell survival values for the
samples treated with chemotoxic compound alone. To determine
fraction of cell survival in the presence of VE-822, raw CTG values
were normalized to the mean CTG value for the samples exposed to
the same concentration of VE-822 in the absence of the chemotoxic
compound. VE-822-treated chemotoxic IC.sub.50 values were
calculated using VE-822-normalized cell survival values for all
samples treated with the chemotoxic at a given concentration of
VE-822. A 3.times. or greater reduction in IC.sub.50 was used to
identify strongly synergistic effects between VE-822 and
chemotoxics.
Primary Adenocarcinoma NSCLC Xenograft Model
[0107] Tumor tissue was excised from a patient with a poorly
differentiated adenocarcinoma. This tumor tissue was implanted
subcutaneously in the flank of a SCID mouse and passaged twice
before compound testing. For compound testing passage-two tumor
tissue was implanted subcutaneously in the flank of SCID mice and
tumors grown to a volume of about 200 mm.sup.3 Cisplatin was dosed
alone at either 1 or 3 mg/kg ip, once per week (ip, q7d, on day 2
of each week) for two weeks. VE-822 was dosed as a solution alone
at 60 mg/kg po on 4 consecutive days per weekly cycle (qd4, dosed
on days 1, 2, 3 and 4 each week). Two combination groups received
cisplatin at 1 or 3 mg/kg plus VE-822 at 60 mg/kg po on the same
schedule as the single agent group. A control group received
vehicle alone (10% Vitamin E TPGS in water, po qd4). All drug
treatment was stopped on Day 28. Vehicle, cisplatin (1 mg/kg) and
VE-822 (60 mg/kg) groups were sacrificed and the remainder
monitored for a further 40 days to assess tumor re-growth.
PSN1 Pancreatic Cancer Xenograft Model
[0108] PSN1 cells (1.times.10.sup.6 cells per mouse) were implanted
as a mixture in Matrigel (100 .mu.l per mouse) into the flank of
female nude MF1 mice and grown to a volume of about 200 mm.sup.3
prior to compound administration. Gemcitabine was dosed alone at 15
mg/kg ip, once every three days (ip, q3d) in 0.5% methylcellulose
in water for a maximum of 10 cycles. VE-822 was dosed, as a
suspension in 0.5% methylcellulose in water, alone at either 10, 30
or 60 mg/kg po every other day for 28 days (po q2d). Three
combination groups received gemcitabine at 15 mg/kg plus VE-822
either at 10, 30 or at 60 mg/kg po on the same schedule as the
single agent groups. A control group received vehicle alone (0.5%
methylcellulose ip q3d). All drug treatment was stopped on Day 30.
Vehicle and VE-822 groups were sacrificed on day 13 due to
excessive tumor volumes.
Results
Compounds VE-821 and VE-822 Sensitize Pancreatic Cancer Cells to
Radiation Therapy
[0109] Compound VI-821 inhibits phosphorylation of Chk1 (Ser 345)
after treatment with gemcitabine (100 nM), radiation (6 Gy) or both
(see FIG. 1A). Compound VI-821 radiosensitises pancreatic tumour
cells but not normal cells. When cells were irradiated in the
presence of Compound VI-821, a decrease in surviving fraction was
observed and this radiosensitising effect increased as the drug
incubation time after irradiation was extended (see FIG. 1C).
[0110] Compound VI-821 radiosensitises tumour PSN-1, MiaPaCa-2 and
PancM cells under hypoxic conditions (see FIG. 2A-B). Compound
VI-821 also sensitises normoxic and hypoxic cancer cells to
gemcitabine (see FIG. 3B-C). Compound VI-821 potentiates the effect
of chemoradiation in both PSN-1 and MiaPaCa-2 cancer cells (see
FIG. 3D). Compound VI-821 disrupts damage-induced cell cycle
checkpoints (see supplementary FIG. 2). Compound VI-821 inhibits
repair of DNA damage by homologous recombination (see FIGS. 5A, 5B,
and 5C).
[0111] Results for Compounds 821 and 822 are shown in FIGS. 1X to
8X and 1Y to 6Y. VE-821 and VE-822 sensitize cancer cells to
radiation therapy (see FIGS. 1X-5X).
VE-822 Enhances the Antitumor Effects of Cancer Therapies in
Xenograft Models
[0112] VE-822 enhances the antitumor effects of ionizing radiation
in a MiaPaCa pancreatic cancer xenograft model (see FIG. 6X) and in
a PSN-1 pancreatic cancer xenograft model (see FIGS. 7X and
8X).
[0113] VE-822 enhances the antitumor effects of cisplatin in a
primary adenocarcinoma NSCLC xenograft model. FIG. 5Y shows the
effect of VE-822 and cisplatin on tumor volume and body weight in a
primary adenocarcinoma NSCLC xenograft in SCID mice. Data are
mean.+-.sem, n=9-10. Black filled circles are vehicle treatment;
Red filled diamonds are Cisplatin treatment (1 mg/kg q7d); Blue
filled diamonds are Cisplatin treatment (3 mg/kg q7d); Green filled
squares are VE-822 treatment (60 mg/kg qd4); Green empty triangles
are Cisplatin (1 mg/kg) and VE-822 (60 mg/kg qd4); Blue empty
triangles are Cisplatin (3 mg/kg) and VE-822 (60 mg/kg qd4) (see
FIG. 5Y).
[0114] VE-822 also enhances the antitumor effects of gemcitamine in
a PSN1 pancreatic cancer xenograft model. FIG. 6Y shows the effect
of VE-822 administered PO q2d at 10, 30 or 60 mg/kg in combination
with gemcitabine (15 mg/kg IP q3d) on the tumor volume of mice
bearing PSN1 pancreatic cancer xenografts. Data shown are mean
tumor volume.+-.SEM (n=8 per group). Red filled circles are VE-822
treatment; Black filled squares are vehicle treatment; Green filled
circles are gemcitabine treatment; Blue filled circles are
gemcitabine and VE-822 (10 mg/kg) treatment; Red filled circles are
gemcitabine and VE-822 (30 mg/kg) treatment; Pink filled circles
are gemcitabine and VE-822 (60 mg/kg) treatment;
VE-822 Synergizes with Chemotoxics Across a Panel of Lung Cancer
Cell Lines
[0115] The heat map represents the maximum shift in IC.sub.50 of
each chemotoxic achieved when combined with VE-822 for 96 hours.
Colors represent an IC.sub.50 shift range from -10 (antagonism,
blue) to 10 (synergy, red) (see FIG. 1Y). VE-822 exhibits greater
than 3-fold synergy with cisplatin, etoposide, gemcitabine,
oxaplatin and irinotecan in lung cancer cell lines (see FIG.
2Y).
VE-822 Synergizes with Cisplatin and Gemcitabine in Pancreatic
Cancer Cell Lines.
[0116] The heat map represents the maximum shift in IC.sub.50 of
each chemotoxic achieved when combined with VE-822 for 96 hours.
Colors represent an IC.sub.50 shift range from -10 (antagonism,
blue) to 10 (synergy, red) (see FIG. 3Y).
[0117] While we have described a number of embodiments of this
invention, it is apparent that our basic examples may be altered to
provide other embodiments that utilize the compounds, methods, and
processes of this invention. Therefore, it will be appreciated that
the scope of this invention is to be defined by the appended claims
rather than by the specific embodiments that have been represented
by way of example herein.
* * * * *