U.S. patent application number 14/369348 was filed with the patent office on 2015-01-08 for methods for treatment of breast cancer nonresponsive to trastuzumab.
The applicant listed for this patent is KADMON CORPORATION, LLC. Invention is credited to Lillian Chiang, Samuel Waksal.
Application Number | 20150010545 14/369348 |
Document ID | / |
Family ID | 48698621 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010545 |
Kind Code |
A1 |
Waksal; Samuel ; et
al. |
January 8, 2015 |
METHODS FOR TREATMENT OF BREAST CANCER NONRESPONSIVE TO
TRASTUZUMAB
Abstract
The present invention provides a method of treating breast
cancer that is nonresponsive to treatment with trastuzumab,
comprising administering to a subject in need of such treatment a
therapeutically effective amount of compound
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,6aS)-2-methyloctahydro-
cyclopenta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine,
or a pharmaceutically acceptable salt thereof.
Inventors: |
Waksal; Samuel; (New York,
NY) ; Chiang; Lillian; (Princeton, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KADMON CORPORATION, LLC |
New York |
NY |
US |
|
|
Family ID: |
48698621 |
Appl. No.: |
14/369348 |
Filed: |
December 27, 2012 |
PCT Filed: |
December 27, 2012 |
PCT NO: |
PCT/US12/71883 |
371 Date: |
June 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61580543 |
Dec 27, 2011 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
514/266.2; 544/293 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 39/39558 20130101; A61K 2039/505 20130101; A61K 39/3955
20130101; A61K 2039/55511 20130101; A61K 31/517 20130101; A61K
39/39558 20130101; C07K 16/32 20130101; C07D 403/12 20130101; A61K
31/517 20130101; A61P 35/00 20180101; A61K 2300/00 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
514/266.2; 544/293 |
International
Class: |
A61K 31/517 20060101
A61K031/517; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method of treating breast cancer that is nonresponsive to
treatment with trastuzumab, comprising administering to a subject
in need of such treatment a therapeutically effective amount of a
compound of Formula 1 ##STR00003## or a pharmaceutically acceptable
salt thereof.
2. A compound of Formula 1 ##STR00004## or a pharmaceutically
acceptable salt thereof, for use in treating breast cancer that is
nonresponsive to treatment with trastuzumab.
3. Use of a compound of Formula 1 ##STR00005## or a
pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating breast cancer that is nonresponsive to
treatment with trastuzumab.
4. The method of claim 1, wherein the compound, or a
pharmaceutically acceptable salt thereof, is a stereoisomer
selected from the group consisting of
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,5r,6aS)-2-methyloctahydrocyclop-
enta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine and
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,5s,6aS)-2-methyloctahydrocyclop-
enta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine, or
a pharmaceutically acceptable salt of the stereoisomer.
5. The method as in claim 1 or 4, wherein the pharmaceutically
acceptable salt is the salt of p-toluenesulfonic acid.
6. The method as in claim 1, 4 or 5, wherein the subject is human
and the breast cancer has not been treated with trastuzumab.
7. The method as in claim 1, 4 or 5, wherein the subject is human
and the breast cancer has been treated with trastuzumab.
8. The method as in claim 1, 4 or 5, comprising co-administering
the compound of Formula 1 and trastuzumab.
9. The method as in claim 1, 4 or 5, wherein the subject is human
and the breast cancer is PTEN-negative.
10. The method as in claim 1, 4 or 5, wherein the subject is human
and the breast cancer is positive for mutations in the PIK3CA
gene.
11. The method as in claim 1, 4 or 5, wherein the subject is human
and the breast cancer expresses a truncated ErbB2 receptor that
lacks the extracellular domain to which trastuzumab binds.
12. The method as in claim 1, 4 or 5, wherein the subject is human
and the breast cancer overexpresses one or more RTKs selected from
the group consisting of EGFR family members, IGF-1R and HGFR.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application No.
61/580,543, filed Dec. 27, 2011, which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns methods for treating breast
cancer by administering the compound
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,6aS)-2-methyloctahydrocyclopent-
a[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine, or a
pharmaceutically acceptable salt thereof, to a subject in need of
such treatment. The present invention particularly concerns methods
where the breast cancer is nonresponsive to treatment with
trastuzumab.
BACKGROUND
[0003] Breast cancer is a type of cancer that forms in tissues of
the breast, usually the ducts and lobules. It occurs in both men
and women, although male breast cancer is rare. It is estimated
that in the United States approximately 230,000 new cases of breast
cancer will arise in the year 2011, and about 40,000 deaths will
occur that result from this form of cancer. See the website of the
National Cancer Institute (NCI) at www.cancer.gov.
[0004] The ErbB2 (Her2/Neu) oncogene is overexpressed in 20-30% of
human breast cancers and this overexpression is associated with
poor prognosis and poor response to chemotherapy. ErbB2 is a
185-kDa type I tyrosine kinase transmembrane receptor that is a
member of the epidermal growth factor receptor (EGFR) family. This
family includes EGFR, ErbB2, Her3 and Her4. There is no known
ligand for ErbB2, but this receptor has been shown to be the
preferential heterodimerization partner for other ErbB family
members that bind growth factors in the EGF, transforming growth
factor-.beta., and heregulin families. The ErbB2 pathway promotes
cell growth and division when it is functioning normally. While the
precise mechanism of ErbB2 pathway activation in
ErbB2-overexpressing cells is not entirely understood,
overexpression likely leads to increased cell growth. See Chan et
al., 2005, Breast Cancer Res. Treat., 91:187-201.
[0005] Trastuzumab (marketed under the name Herceptin.RTM. by
Genentech) is a recombinant humanized monoclonal antibody that
binds to the extracellular segment of the ErbB2 receptor.
Trastuzumab is used as a single agent or in combination with
chemotherapy and other targeted therapies to treat patients with
breast cancer overexpressing ErbB2. Trastuzumab shows considerable
clinical efficacy and has been shown to extend the overall survival
of certain patients with ErbB2-overexpressing breast cancer. See
Chan et al., 2005, Breast Cancer Res. Treat., 91:187-201.
[0006] Despite trastuzumab's general clinical efficacy of abut 50%
responsiveness, many patients do not respond to trastuzumab
treatment at all (de novo nonresponsiveness), or acquire
nonresponsiveness to trastuzumab treatment during the course of
treatment. Postulated mechanisms of trastuzumab nonresponsiveness
include: activation of the phosphoinositide 3-kinase (PI3K) pathway
due to, for example, mutations in the PIK3CA gene; lack or
inactivity of the tumor suppressor PTEN (phosphatase and tensin
homolog); accumulation of truncated ErbB2 receptors (p95HER2) that
cannot be inactivated by trastuzumab because they lack the
extracellular domain to which trastuzumab usually binds; and
overexpression of other RTKs that compensates for
trastuzumab-induced ErbB2 inhibition. Examples of such RTKs include
members of the epidermal growth factor receptor (EGFR) family, the
insulin-like growth factor-1 receptor (IGF-1R) and the hepatocyte
growth factor receptor (HGFR). See Zhang et al., 2011, Nat. Med.,
17(4):461-468; see also Chan et al., 2005, Breast Cancer Res.
Treat., 91:187-201.
[0007] Recently, it was demonstrated that the SRC kinase is a
common node downstream of multiple pathways that result in tumors
that are de novo nonresponsive or that have acquired
nonresponsiveness to trastuzumab. See Zhang et al., 2011, Nat.
Med., 17(4):461-468. The non-receptor tyrosine kinase SRC is a
cytoplasmic protein that consists of three domains, an N-terminal
SH3 domain, a central SH2 domain and a tyrosine kinase domain. SRC
facilitates intracellular signal transduction by interacting with
multiple RTKs through its SH2 domain and by phosphorylating and
thus activating downstream targets. Examples of pathways and
proteins activated by the SRC kinase include the AKT and the MAPK
(mitogen-activated protein kinases) pathways, FAK (focal adhesion
kinase), STAT3 (signal transducer and activator of transcription-3)
and c-MYC. These signaling pathways and proteins have diverse roles
in regulating tumor cell survival and metastasis. See Zhang et al.,
2011, Nat. Med., 17(4):461-468.
[0008] It was found that SRC is activated (i.e., phosphorylated) in
a model of acquired trastuzumab nonresponsiveness, wherein cultured
cells overexpress EGFR or IGF-1R. Moreover, SRC is activated in
PTEN-deficient cells in a model of de novo trastuzumab
nonresponsiveness and in vitro GST pull-down assays demonstrated
that SRC is a direct target of PTEN's phosphatase activity. On the
other hand, SRC is inactivated (i.e., dephosphorylated) when, for
example, the expression of EGFR is reduced, or when originally
PTEN-deficient cells are reconstituted with wildtype PTEN.
Furthermore, it was found that certain cells stably expressing a
constitutively active SRC mutant are highly resistant to
trastuzumab-mediated growth inhibition in vitro and in vivo,
suggesting that SRC activation is sufficient to confer trastuzumab
nonresponsiveness. The same study showed that SRC activity in human
cancer specimens positively correlates with a lower clinical rate
of response to trastuzumab treatment and that inhibition of SRC by
saracatinib increases responsiveness of tumors to trastuzumab. See
Zhang et al., 2011, Nat. Med., 17(4):461-468.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method of treating breast
cancer that is nonresponsive to treatment with an extracellular
HER2 antagonist, comprising administering to a subject in need of
such treatment a therapeutically effective amount of a compound of
Formula 1
##STR00001##
or a pharmaceutically acceptable salt thereof.
[0010] The present invention further provides a compound of Formula
1, or a pharmaceutically acceptable salt thereof, for use in
treating breast cancer that is nonresponsive to treatment with
trastuzumab.
[0011] The present invention further provides the use of a compound
of Formula 1, or a pharmaceutically acceptable salt thereof, for
the manufacture of a medicament for treating breast cancer that is
nonresponsive to treatment with trastuzumab.
[0012] The present invention also provides a method of treating
HER2-positive cancer that is nonresponsive to treatment with an
extracellular HER2 antagonist, comprising administering to a
subject in need of such treatment a therapeutically effective
amount of a compound of Formula 1, or a pharmaceutically acceptable
salt thereof.
[0013] The present invention also provides a method of treating
EGFR-dependent cancer that is nonresponsive to treatment with an
extracellular EGFR antagonist, comprising administering to a
subject in need of such treatment a therapeutically effective
amount of a compound of Formula 1, or a pharmaceutically acceptable
salt thereof.
[0014] In certain embodiments of the present invention, the
compound of Formula 1 is N-(3,4-dichloro-2-fluorophenyl)-7-({[(3
aR,5r,6aS)-2-methyloctahydrocyclopenta[c]pyrrol-5-yl]methyl}oxy)-6-(methy-
loxy)quinazolin-4-amine or
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,5s,6aS)-2-methyloctahydrocyclop-
enta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine. In
another embodiment of the present invention, the pharmaceutically
acceptable salt is the salt of p-toluenesulfonic acid.
[0015] In some embodiments of the present invention, the subject is
human and the breast cancer has not previously been treated with
trastuzumab. In other embodiments of the present invention, the
subject is human and the breast cancer has been previously treated
with trastuzumab. In other embodiments of the present invention,
the method comprises co-administering a compound of Formula 1 and
trastuzumab.
[0016] In some embodiments of the present invention, the subject is
human and the breast cancer is PTEN-negative. In other embodiments
of the present invention, the subject is human and the breast
cancer is positive for mutations in the PIK3 CA gene. In other
embodiments, the subject is human and the breast cancer expresses a
truncated ErbB2 receptor that lacks the extracellular domain to
which trastuzumab usually binds. In other embodiments of the
present invention, the subject is human and the breast cancer
overexpresses RTKs, for example members of the EGFR family, IGF-1R
and HGFR.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the effect of EXEL-7647 on SRC kinase activity
and on the phosphorylation of the SRC family protein FAK (focal
adhesion kinase).
[0018] FIG. 2 shows the effect of EXEL-7647 on ErbB2
(Her2)-phosphorylation in BT474 tumor xenografts in mice.
[0019] FIG. 3 shows the effect of EXEL-7647 on EGFR-phosphorylation
in A431 tumor xenografts in mice.
[0020] FIG. 4 shows the effect of EXEL-7647 on KDR-phosphorylation
in mouse lungs.
[0021] FIG. 5 shows the effect of EXEL-7647 on
EphB4-phosphorylation in HCT116/EphB4 xenografts in mice.
[0022] FIG. 6 shows the effect of EXEL-7647 on angiogenesis.
[0023] FIG. 7 shows the effect of EXEL-7647 on the growth of
MDA-MB-231 tumor xenografts in mice.
[0024] FIG. 8 shows inhibition of Src in traztuzumab resistant
cells by EXEL-7647 but not by other ErbB family inhibitors. A)
EXEL-7647 inhibits Src in a dose dependent manner B)
Phosphorylation of Src in JIMT-1 and HCC1954 cells following
treatment with lapatinib, erlotinib, EXEL-7647, and trastuzumab
after 18 hours of treatment. C and D) Cell proliferation assay of
JIMT-1 (C) and HCC1954 (D) cells treated with EXEL-7647. Cells were
treated with the indicated concentrations for 72 hours and cell
viability was determined.
[0025] FIG. 9 shows the effect of EXEL-7647, trastuzumab, and a
combination of EXEL-7647 and trastuzumab, on growth of trastuzumab
resistant JIMT-1 xenograft tumors.
[0026] FIG. 10 shows effects of EXEL-7647 on Her2, EGFR, and Met
activation. A) JIMT-1 and HCC1954 cells were treated for 18 hours
with the indicated compounds. Herceptin, but none of the small
molecule inhibitors, affect Her2 expression. B) The small molecule
inhibitors, but not herceptin, inhibit Her2 phosphorylation. C)
EXEL-7647 inhibits EGFR phosphorylation in HCC1954 and JIMT-1
cells. D) EXEL-7647 inhibits Met phosphorylation in HCC1954
cells.
[0027] FIG. 11 shows the effect of EXEL-7647 and AZD0530
(saracatinib) on phosphorylation of Src and its target Paxillin
phosphorylation, and on proliferation of JIMT-1 cells. A)
Phosphorylation of Src and Paxillin in JIMT-1 treated for 18 hrs.
with increasing concentrations of KD019 or AZD0530. B) Cell
proliferation assay of JIMT-1 cells treated with the indicated
concentrations of KD019 or AZD0530. Cell viability was measured by
MTS assay after 72 hrs. Error bars represent standard deviation of
the average of triplicate wells.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides a method of treating HER2
positive cancer, including breast cancer, that is nonresponsive to
treatment with an extracellular HER2 antagonist, comprising
administering to a subject in need of such treatment a
therapeutically effective amount of a compound of Formula 1
##STR00002##
or a pharmaceutically acceptable salt thereof. The chemical name of
the compound of Formula 1 is
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,6aS)-2-methyloctahydrocyclopent-
a[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine.
[0029] The compound of Formula 1, and its pharmaceutically
acceptable salts, includes stereoisomers, enantiomers,
diastereomers, racemates, and racemic or non-racemic mixtures
thereof, as well as any pharmaceutically acceptable salts of said
stereoisomers, enantiomers, diastereomers, racemates and racemic or
non-racemic mixtures.
[0030] In an embodiment of the invention, the compound of Formula 1
is N-(3,4-dichloro-2-fluorophenyl)-7-({[(3
aR,5r,6aS)-2-methyloctahydrocyclopenta[c]pyrrol-5-yl]methyl}oxy)-6-(methy-
loxy)quinazolin-4-amine or
N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,5s,6aS)-2-methyloctahydrocyclop-
enta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine, or
a pharmaceutically acceptable salt thereof. In another embodiment
of the invention, the pharmaceutically acceptable salt is the salt
ofp-toluenesulfonic acid.
[0031] As used herein, the term pharmaceutically acceptable salt(s)
includes pharmaceutically acceptable acid addition salts.
Pharmaceutically acceptable acid addition salts are salts that
retain the biological effectiveness of the free bases and that are
not biologically or otherwise undesirable, formed with inorganic
acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric acid, phosphoric acid, and the like, as well as organic
acids such as acetic acid, trifluoroacetic acid, propionic acid,
hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic
acid, pyruvic acid, lactic acid, malic acid, oxalic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid, stearic acid, and the like. A preferred
pharmaceutically acceptable acid addition salt is the salt of
p-toluenesulfonic acid.
[0032] The compound of Formula 1 and pharmaceutically acceptable
salts thereof can be manufactured using techniques commonly known
in the art. For example, said compound and pharmaceutically
acceptable salts thereof, as well as methods of manufacturing them,
are disclosed in U.S. Pat. No. 7,576,074, which is incorporated
herein by reference. U.S. Pat. No. 7,576,074 was assigned from
Exelixis, Inc. to Symphony Evolution, Inc. on Jun. 10, 2009. Kadmon
Corporation, LLC has acquired certain rights to the compound of
Formula 1 (also known as XL647, EXEL-7647 and KD-019), including
data provided in the Examples below.
[0033] Gendreau et al. describes certain research studies conducted
by Exelixis, Inc. concerning the pharmacological properties of
XL647. Specifically, this study showed that XL647 is an in vitro
inhibitor of several receptor tyrosine kinases (RTKs), including
EGFR, EphB4, KDR (VEGFR), Flt4 (VEGFR3) and ErbB2. Furthermore, in
vivo experiments showed that XL647 inhibits the activity of EGFR in
xenograft tumors derived from A431 epidermal carcinoma cells, and
that it inhibits the growth of xenograft tumors derived from
MDA-MB-231 human breast cancer cells, which overexpress VEGFR. See
Gendreau et al., 2007, Clin. Cancer Res., 13:3713-3723.
[0034] The Examples set forth below demonstrate that the compound
of Formula 1, in addition to being an inhibitor of several receptor
tyrosine kinases (RTKs), is also an inhibitor of the SRC kinase,
which is involved in multiple pathways that result in
nonresponsiveness of ErbB2-overexpressing tumors to trastuzumab.
Accordingly, the present invention now provides a method of
treating breast cancer, e.g., breast cancer, that is nonresponsive
to treatment with an extracellular HER2 antagonist, including but
not limited to trastuzumab, comprising administering to a subject
in need of such treatment a therapeutically effective amount of a
compound of Formula 1, or a pharmaceutically acceptable salt
thereof.
[0035] An extracellular HER2 antagonist is an agent that binds to
the extracellular portion of HER2 and reduces or inhibits its
function. In one embodiment, the HER2 antagonist is an antibody or
antigen binding fragment, or conjugate thereof, that binds to the
extracellular portion of HER2. In an embodiment of the invention,
the HER2 antagonist is trastuzumab. While treatment with
trastuzumab has been observed to increase HER2 phosphorylation,
trastuzumab treatment also leads to internalization and degradation
of HER2 and a reduction in HER2 signaling. Thus, trastuzumab may be
considered a HER2 antagonist according to the invention. In another
embodiment of the invention, the HER2 antagonist is trastuzumab
emtansine (trastuzumab-DM1; T-DM1). In another embodiment, the HER2
antagonist is pertuzumab.
[0036] Amplification or over-expression of HER2 has been shown to
play an important role in the pathogenesis and progression not only
of certain types of breast cancer, but other types of cancer as
well. Accordingly, the methods disclosed herein are useful to treat
HER2-positive cancers including, without limitation, breast cancer,
ovarian cancer, such as ovarian epithelial cancer, ovarian germ
cell tumor, non-small cell lung cancer, stomach cancer, esophageal
cancer, gastric cancer, uterine cancer, endometrial cancer,
prostate cancer, bladder cancer, glioblastoma, metastatic solid
tumors characterized by Her2 expression, or any other cancer that
expresses HER2. The cancers to be treated include early and late
stage cancers.
[0037] The invention also provides a method of treating cancer that
is nonresponsive to treatment with an extracellular EGFR
antagonist, comprising administering to a subject in need of such
treatment a therapeutically effective amount of a compound of
Formula 1. In an embodiment of the invention, the EGFR antagonist
is an antibody or antigen binding fragment, or conjugate thereof,
that binds to the extracellular portion of EGFR. In one embodiment,
the EGFR antagonist is cetuximab. In another embodiment, the EGFR
antagonist is mAB806, which binds to EGFR as well as the truncated
EGFRvIII mutant. In another embodiment, the EGFR antagonist is
panitumumab. In another embodiment, the EGFR antagonist is
zalutumumab. In yet another embodiment, the EGFR antagonist is
nimotuzumab. In another embodiment, the EGFR antagonist is
matuzumab. Cancers in which EGFR plays a role include, without
limitation, colorectal cancer, head and neck cancers, and non-small
cell lung cancers.
[0038] Nonresponsive, ErbB2-overexpressing cancer is either de novo
nonresponsive or has acquired nonresponsiveness to treatment with
trastuzumab. De novo nonresponsive either means that the
ErbB2-overexpressing breast cancer, in the course of treatment with
trastuzumab, does not go into partial or complete remission, or,
alternatively, that this cancer is characterized by one or more
molecular deficiencies which make it incapable of going into
partial or complete remission in response to trastuzumab treatment.
Non-limiting examples of such molecular deficiencies may include
the activation of the phosphoinositide 3-kinase (PI3K) pathway due
to, for example, mutations in the PIK3CA gene; the lack or
inactivity of the tumor suppressor PTEN; the accumulation of
truncated ErbB2 receptors; increased heregulin-mediated autocrine
signaling; and the overexpression of other RTKs, such as members of
the epidermal growth factor receptor (EGFR) family, the
insulin-like growth factor-1 receptor (IGF-1R) and the hepatocyte
growth factor receptor (HGFR). As provided further below, all of
these molecular deficiencies can be detected by standard molecular
biological techniques commonly known in the art. Acquired
nonresponsiveness, as used herein, means that the
ErbB2-overexpressing breast cancer, in the course of treatment with
trastuzumab, initially goes into remission, but then recurs. A
cancer, such as a breast cancer, that has acquired reduced
responsiveness to trastuzumab or has acquired nonresponsiveness to
trastuzumab may also be referred to as trastuzumab resistant.
[0039] EGFR-dependent cancers also acquire reduced responsiveness
or become nonresponsive to cetuximab or other therapeutic EGFR
antibodies by several mechanisms, including those set forth above
for HER2. For example, EGFR-dependent cancers may become
nonresponsive when bypassed by a HER2 signaling mechanism. De novo
or acquired nonresponsiveness may also result from mutations in
KRAS, BRAF, and NRAS.
[0040] Remission is a decrease in or disappearance of signs and
symptoms of cancer. In partial remission, some, but not all, signs
and symptoms of cancer have disappeared. In complete remission, all
signs and symptoms of cancer have disappeared, although cancer
still may be in the body. In order to determine whether breast is
in remission, the subject is generally evaluated using the same
techniques that are commonly used for initial breast cancer
detection and diagnosis, such as, for example, mammography,
ultrasound, ductography, positron emission mammography (PEM) and
magnetic resonance imaging (MRI). The thus obtained data, are then
compared with the corresponding data obtained when the breast
cancer was originally diagnosed and it is concluded, based on
standard oncological practice, whether the signs and symptoms of
breast cancer have partially or completely disappeared, i.e.,
whether the breast cancer is in partial or complete remission. The
person of skill in the art may find that the breast cancer is in
partial remission, for example, because the size of the breast
cancer is reduced by comparison to the size of the breast cancer at
the time the breast cancer was originally diagnosed. Alternatively,
the person of skill in the art may find that the breast cancer is
in partial remission because the cancer has stabilized or because
the growth of the cancer is reduced. The breast cancer may be
considered to be in remission because the signs and symptoms of the
breast cancer are reduced by, for example, 10, 20, 30, 40, 50, 60,
70, 80, 90 or 100%.
[0041] In some embodiments of the present invention, the subject is
human and the breast cancer has been previously treated with
trastuzumab. In other embodiments of the present invention, the
subject is human and the breast cancer has not previously been
treated with trastuzumab. As set forth above, whether or not breast
cancer will be nonresponsive to trastuzumab treatment can be
determined based on the presence or absence in the breast cancer of
certain molecular deficiencies.
[0042] In one embodiment of the present invention, the compound of
Formula 1, or a pharmaceutically acceptable salt thereof, is
co-administered with a HER2 antagonist. Provided its Src inhibitory
activity, the compound of Formula 1 can increase the effectiveness
of the HER2 antagonist. Alternatively or in addition,
co-administration of a compound of Formula 1 with a HER2 antagonist
can delay or prevent the onset of resistance to either agent. HER2
antagonists include, without limitation, extracellular antagonists,
such as anti-HER2 antibodies (e.g., trastuzumab, pertuzumab) and
conjugates thereof, and intracellular antagonists (e.g., lapatinib,
canertinib, neratinib, afatinib). The compound of Formula 1, or a
pharmaceutically acceptable salt thereof, and the second agent can
be administered in a single formulation or as separate
formulations. In certain embodiments, for example, the compound of
Formula 1, or a pharmaceutically acceptable salt thereof, may be
administered orally and trastuzumab intravenously. Other routes of
administration are also possible. The compound of Formula 1, or a
pharmaceutically acceptable salt thereof, can be co-administered
with trastuzumab in such a way that it is administered before or
after trastuzumab, or at the same time.
[0043] The compound of the present invention, or a pharmaceutically
acceptable salt thereof, can also be co-administered with a variety
of other drugs in the manner described above for the
co-administration with a HER2 antagonist. The term drugs as used
herein refers to any compound with therapeutically beneficial
properties. In certain embodiments of the invention, the treatment
method further comprises administering to the subject trastuzumab,
or other antibody therapeutic effective against or being developed
to treat cancer such as cetuximab or nimotuzumab (anti-EGFR
antibodies), cixutumumab (IMC-A12), ganitumab (AMG-479),
dalotuzumab (MK-0646), MEDI-573, RG-1507, and AVE-1642 (anti-IGF-1R
antibodies in clinical development). In certain embodiments of the
invention, the method further comprises administering to the
subject a small molecule tyrosine kinase inhibitor, including but
not limited to erlotinib, gifitinib (EGFR inhibitors), AP26113
(dual EGFR, ALK inhibitor), NVP-AEW541, CP-751,871, and BMS-536924
(IGF-1R inhibitors).
[0044] In certain embodiments of the invention, the treatment
method further comprises administering to the subject an antagonist
of hepatocyte growth factor (HGF) or MET tyrosine kinase disclosed
in Comoglio et al., (Nature Reviews Drug Discovery, June 2008, vol.
7, pp. 504-516, hereby incorporated by reference), including NK2 (a
fragment of HGF containing the amino terminal hairpin and the first
two Kringle domains), NK4 (a HGF fragment containing the
.alpha.-chain and not the .beta.-chain), uncleavable HGF, decoy
MET, the isolated Sema domain of MET, various fully human
monoclonal antibodies to HGF disclosed in Burgess et al. (Cancer
Res., 66:1721-1729, 2006), ficlatuzumab, TAK-701 (L2G7),
onartuzumab, ALD-805, ALD-806, rilotumumab (AMG102) (anti-HGF
monoclonal antibodies), antibodies against MET such as LY-2875358,
HuMax-cMet, LA-480, OA-5D5 and DN30, and small molecule MET
inhibitors such as K252, SU11274, PHA665752, crizotinib
(PF2341066), foretinib (XL880), ARQ197, MK2461, MP470, SGX523, and
JNJ38877605. Additional agents that may be coadministered according
to the invention include, cabozantinib (XL184), MGCD-265,
SAR-125844, E-7050, INCB-028060, EMD-94283, EMD-1214063,
EMD-1204831, LY-2801653, LY-2875358, MK8033, and AMG-208.
[0045] In certain embodiments of the invention, the treatment
method further comprises administering to the subject an agent that
modulates the PI3K/Akt or MEK pathways, including but not limited
to, dasatinib, bosutinib, saracatinib, everolimus, temsirolimus,
ridaforolimus, vemurafenib, and sorafenib.
[0046] In the methods of the invention, the compound of Formula 1
can be administered by routes commonly known in the art. This
includes oral administration, or any other convenient route. The
compound of Formula 1 may also be administered together with
another biologically active agent. Administration can be systemic
or local. Various delivery systems are known, e.g., encapsulation
in liposomes, microparticles, microcapsules, capsules, and can be
used to administer the compound and pharmaceutically acceptable
salts thereof.
[0047] Methods of administration include but are not limited to
parenteral, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, oral, sublingual,
intranasal, intracerebral, intravaginal, transdermal, transmucosal,
rectally, by inhalation, or topically, particularly to the ears,
nose, eyes, or skin. The mode of administration is left to the
discretion of the practitioner. In most instances, administration
will result in the release of a compound into the bloodstream.
[0048] In specific embodiments, it may be desirable to administer a
compound locally. This may be achieved, for example, and not by way
of limitation, by local infusion, topical application, by
injection, by means of a catheter, by means of a suppository, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. In such instances, administration may
selectively target a local tissue without substantial release of a
compound into the bloodstream.
[0049] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent, or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant. In certain embodiments, a compound is formulated as a
suppository, with traditional binders and vehicles such as
triglycerides.
[0050] In another embodiment, a compound is delivered in a vesicle,
in particular a liposome (See Langer, 1990, Science 249:1527-1533;
Treat et al., in Liposomes in the Therapy of Infectious Disease and
Bacterial infection, Lopez-Berestein and Fidler (eds.), Liss, New
York, pp. 353-365 (1989); Lopez Berestein, ibid., pp. 317-327; see
generally ibid.).
[0051] In another embodiment, a compound is delivered in a
controlled release system (See, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138
(1984)). Examples of controlled-release systems are discussed in
the review by Langer, 1990, Science 249:1527-1533 may be used. In
one embodiment, a pump may be used (See Langer, supra; Sefton,
1987, CRC Crit. Ref Biomed. Eng. 14:201; Buchwald et al., 1980,
Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In
another embodiment, polymeric materials can be used (See Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev.
Macromol. Chem. 23:61; See also Levy et al., 1985, Science 228:190;
During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 71:105).
[0052] The present invention provides a method of treating breast
cancer in a subject. The term subject, as used herein, refers to
the animal being treated, wherein the animal can be a mammal such
as a human.
[0053] The therapeutically effective amount of the compound of
Formula 1 is the dose of this compound, or of a phatmaceutically
acceptable salt thereof, that provides a therapeutic benefit in the
treatment or management of cancer, delays or minimizes one or more
symptoms associated with cancer, or enhances the therapeutic
efficacy of another therapeutic agent used in the treatment or
management of cancer. The therapeutically effective amount may be
an amount that reduces or inhibits the growth of breast cancer. A
person skilled in the art would recognize that the therapeutically
effective amount may vary depending on known factors such as the
pharmacodynamic characteristics of the particular active ingredient
and its mode and route of administration; age, sex, health and
weight of the recipient; nature and extent of symptoms; kind of
concurrent treatment, frequency of treatment and the effect
desired. A person skilled in the art would also recognize that the
therapeutically effective amount, or dose, of the compound of
Formula 1 can be determined based on the disclosures in this patent
application and common knowledge in the art.
[0054] The amount of a compound, or the amount of a composition
comprising a compound, that will be effective in the treatment
and/or management of cancer can be determined by standard clinical
techniques. In vitro or in vivo assays may optionally be employed
to help identify optimal dosage ranges.
[0055] In some cases, the dosage of a compound may be determined by
extrapolating from the no-observed-adverse-effective-level (NOAEL),
as determined in animal studies. This extrapolated dosage is useful
in determining the maximum recommended starting dose for human
clinical trials. For instance, the NOAELs can be extrapolated to
determine human equivalent dosages (HED). Typically, HED is
extrapolated from a non-human animal dosage based on the doses that
are normalized to body surface area (i.e., mg/m.sup.2). In specific
embodiments, the NOAELs are determined in mice, hamsters, rats,
ferrets, guinea pigs, rabbits, dogs, primates, primates (monkeys,
marmosets, squirrel monkeys, baboons), micropigs or minipigs. For a
discussion on the use of NOAELs and their extrapolation to
determine human equivalent doses, see Guidance for Industry
Estimating the Maximum Safe Starting Dose in Initial Clinical
Trials for Therapeutics in Adult Healthy Volunteers, U.S.
Department of Health and Human Services Food and Drug
Administration Center for Drug Evaluation and Research (CDER),
Pharmacology and Toxicology, July 2005. In one embodiment, a
compound or composition thereof is administered at a dose that is
lower than the human equivalent dosage (HED) of the NOAEL over a
period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, three
months, four months, six months, nine months, 1 year, 2 years, 3
years, 4 years or more.
[0056] A dosage regime for a human subject can be extrapolated from
animal model studies using the dose at which 10% of the animals die
(LD.sub.10). In general the starting dose of a Phase I clinical
trial is based on preclinical testing. A standard measure of
toxicity of a drug in preclinical testing is the percentage of
animals that die because of treatment. It is well within the skill
of the art to correlate the LD.sub.10 in an animal study to a
maximal-tolerated dose (MTD) in humans, adjusted for body surface
area, as a basis to extrapolate a starting human dose. In some
embodiments, the interrelationship of dosages for one animal model
can be converted for use in another animal, including humans, using
conversion factors (based on milligrams per meter squared of body
surface) as described, e.g., in Freireich et al., Cancer Chemother.
Rep., 1966, 50:219-244. Body surface area may be approximately
determined from height and weight of the patient. See, e.g.,
Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537.
In certain embodiments, the adjustment for body surface area
includes host factors such as, for example, surface area, weight,
metabolism, tissue distribution, absorption rate, and excretion
rate. In addition, the route of administration, excipient usage,
and the specific disease or cancer to target are also factors to
consider. In one embodiment, the standard conservative starting
dose is about 1/10 the murine LD.sub.10, although it may be even
lower if other species (i.e., dogs) were more sensitive to the
compound. In other embodiments, the standard conservative starting
dose is about 1/100, 1/95, 1/90, 1/85, 1/80, 1/75, 1/70, 1/65,
1/60, 1/55, 1/50, 1/45, 1/40, 1/35, 1/30, 1/25, 1/20, 1/15, 2/10,
3/10, 4/10, or 5/10 of the murine LD.sub.10. In other embodiments,
an starting dose amount of a compound in a human is lower than the
dose extrapolated from animal model studies. In another embodiment,
a starting dose amount of a compound in a human is higher than the
dose extrapolated from animal model studies. It is well within the
skill of the art to start doses of the active composition at
relatively low levels, and increase or decrease the dosage as
necessary to achieve the desired effect with minimal toxicity.
[0057] In some of the embodiments of the present invention, the
compound of Formula 1, or a pharmaceutically acceptable salt
thereof, may be used at a dose of between about 0.01 mg/kg of
patient body weight per day and about 10 mg/kg of patient body
weight per day, and preferably between about 0.05 mg/kg of patient
body weight per day and about 5 mg/kg of patient body weight per
day. Accordingly, daily doses include, without limitation, 1000
mg/day, 750 mg/day, 500 mg/day, 300 mg/day, 250 mg/day, 100 mg/day,
and 50 mg/day.
[0058] The compound of the present invention, and its
pharmaceutically acceptable salts, may be formulated in a
pharmaceutical composition. In certain embodiments provided herein,
the composition may comprise said compound and a pharmaceutically
acceptable carrier, excipient, or diluent. The pharmaceutical
compositions provided herein can be in any form that allows for the
composition to be administered to a subject, including, but not
limited to a human, and formulated to be compatible with an
intended route of administration.
[0059] The ingredients of compositions provided herein may be
supplied either separately or mixed together in unit dosage form,
for example, as a dry lyophilized powder or water free concentrate
in a hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0060] Pharmaceutically acceptable carriers, excipients and
diluents include those approved by a regulatory agency of the
Federal or a state government or listed in the U.S. Pharmacopeia or
other generally recognized pharmacopeia for use in animals, and
more particularly in humans Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0061] Typical compositions and dosage forms comprise one or more
excipients. Suitable excipients are well-known to those skilled in
the art of pharmacy, and non limiting examples of suitable
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. Whether a
particular excipient is suitable for incorporation into a
pharmaceutical composition or dosage fonn depends on a variety of
factors well known in the art including, but not limited to, the
way in which the dosage form will be administered to a patient and
the specific active ingredients in the dosage form. The composition
or single unit dosage form, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering
agents.
[0062] Lactose free compositions can comprise excipients that are
well known in the art and are listed, for example, in the U.S.
Pharmacopeia (USP)SP(XXI)/NF (XVI). In general, lactose free
compositions comprise an active ingredient, a binder/filler, and a
lubricant in pharmaceutically compatible and pharmaceutically
acceptable amounts. Preferred lactose free dosage forms comprise a
compound, microcrystalline cellulose, pre gelatinized starch, and
magnesium stearate.
[0063] Further provided herein are anhydrous pharmaceutical
compositions and dosage forms comprising one or more compounds,
since water can facilitate the degradation of some compounds. For
example, the addition of water (e.g., 5%) is widely accepted in the
pharmaceutical arts as a means of simulating long term storage in
order to determine characteristics such as shelf life or the
stability of formulations over time. See, e.g., Jens T. Carstensen,
Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker,
NY, N.Y., 1995, pp. 379 80. In effect, water and heat accelerate
the decomposition of some compounds. Thus, the effect of water on a
formulation can be of great significance since moisture and/or
humidity are commonly encountered during manufacture, handling,
packaging, storage, shipment, and use of formulations.
[0064] Anhydrous compositions and dosage forms provided herein can
be prepared using anhydrous or low moisture containing ingredients
and low moisture or low humidity conditions. Compositions and
dosage forms that comprise lactose and at least one compound that
comprises a primary or secondary amine are preferably anhydrous if
substantial contact with moisture and/or humidity during
manufacturing, packaging, and/or storage is expected.
[0065] An anhydrous composition should be prepared and stored such
that its anhydrous nature is maintained. Accordingly, anhydrous
compositions are preferably packaged using materials known to
prevent exposure to water such that they can be included in
suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers (e.g., vials), blister packs, and strip packs.
[0066] Further provided herein are compositions and dosage forms
that comprise one or more agents that reduce the rate by which a
compound will decompose. Such agents, which are referred to herein
as "stabilizers," include, but are not limited to, antioxidants
such as ascorbic acid, pH buffers, or salt buffers.
[0067] The compositions and single unit dosage fauns can take the
faun of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Such compositions
and dosage forms will contain a therapeutically effective amount of
a compound preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper
administration to the patient.
[0068] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms, in
which case solid excipients are employed. If desired, tablets can
be coated by standard aqueous or nonaqueous techniques. Such dosage
forms can be prepared by any of the methods of pharmacy. In
general, pharmaceutical compositions and dosage forms are prepared
by uniformly and intimately admixing the active ingredients with
liquid carriers, finely divided solid carriers, or both, and then
shaping the product into the desired presentation if necessary. For
example, a tablet can be prepared by compression or molding.
Compressed tablets can be prepared by compressing in a suitable
machine the active ingredients in a free flowing form such as
powder or granules, optionally mixed with an excipient. Molded
tablets can be made by molding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent.
[0069] Examples of excipients that can be used in oral dosage forms
provided herein include, but are not limited to, binders, fillers,
disintegrants, and lubricants. Binders suitable for use in
pharmaceutical compositions and dosage forms include, but are not
limited to, corn starch, potato starch, or other starches, gelatin,
natural and synthetic gums such as acacia, sodium alginate, alginic
acid, other alginates, powdered tragacanth, guar gum, cellulose and
its derivatives (e.g., ethyl cellulose, cellulose acetate,
carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),
polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch,
hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),
microcrystalline cellulose, and mixtures thereof.
[0070] Examples of fillers suitable for use in the pharmaceutical
compositions and dosage forms provided herein include, but are not
limited to, talc, calcium carbonate (e.g., granules or powder),
microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre gelatinized starch,
and mixtures thereof. The binder or filler in pharmaceutical
compositions provided herein is typically present in from about 50
to about 99 weight percent of the pharmaceutical composition or
dosage form.
[0071] Suitable foams of microcrystalline cellulose include, but
are not limited to, the materials sold as AVICEL PH 101, AVICEL PH
103 AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation,
American Viscose Division, Avicel Sales, Marcus Hook, PA), and
mixtures thereof. A specific binder is a mixture of
microcrystalline cellulose and sodium carboxymethyl cellulose sold
as AVICEL RC 581. Suitable anhydrous or low moisture excipients or
additives include AVICEL PH 103.TM. and Starch 1500 LM.
[0072] Disintegrants are used in the compositions provided herein
to provide tablets that disintegrate when exposed to an aqueous
environment. Tablets that contain too much disintegrant may
disintegrate in storage, while those that contain too little may
not disintegrate at a desired rate or under the desired conditions.
Thus, a sufficient amount of disintegrant that is neither too much
nor too little to detrimentally alter the release of the active
ingredients should be used to form solid oral dosage forms provided
herein. The amount of disintegrant used varies based upon the type
of formulation, and is readily discernible to those of ordinary
skill in the art. Typical pharmaceutical compositions comprise from
about 0.5 to about 15 weight percent of disintegrant, specifically
from about 1 to about 5 weight percent of disintegrant.
[0073] Disintegrants that can be used in pharmaceutical
compositions and dosage forms provided herein include, but are not
limited to, agar, alginic acid, calcium carbonate, microcrystalline
cellulose, croscarmellose sodium, crospovidone, polacrilin
potassium, sodium starch glycolate, potato or tapioca starch, pre
gelatinized starch, other starches, clays, other algins, other
celluloses, gums, and mixtures thereof.
[0074] Lubricants that can be used in pharmaceutical compositions
and dosage forms proVided herein include, but are not limited to,
calcium stearate, magnesium stearate, mineral oil, light mineral
oil, glycerin, sorbitol, mannitol, polyethylene glycol, other
glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated
vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil,
sesame oil, olive oil, corn oil, and soybean oil), zinc stearate,
ethyl oleate, ethyl laureate, agar, and mixtures thereof.
Additional lubricants include, for example, a syloid silica gel
(AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Md.), a
coagulated aerosol of synthetic silica (marketed by Degussa Co. of
Plano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold
by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at
all, lubricants are typically used in an amount of less than about
1 weight percent of the pharmaceutical compositions or dosage forms
into which they are incorporated.
[0075] A compound can be administered by controlled release means
or by delivery devices that are well known to those of ordinary
skill in the art. Examples include, but are not limited to, those
described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;
3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767,
5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of
which is incorporated herein by reference. Such dosage forms can be
used to provide slow or controlled release of one or more active
ingredients using, for example, hydropropylmethyl cellulose, other
polymer matrices, gels, permeable membranes, osmotic systems,
multilayer coatings, microparticles, liposomes, microspheres, or a
combination thereof to provide the desired release profile in
varying proportions. Suitable controlled release formulations known
to those of ordinary skill in the art, including those described
herein, can be readily selected for use with the active ingredients
of the invention. The invention thus encompasses single unit dosage
forms suitable for oral administration such as, but not limited to,
tablets, capsules, gelcaps, and caplets that are adapted for
controlled release.
[0076] All controlled release pharmaceutical products have a common
goal of improving drug therapy over that achieved by their non
controlled counterparts. Ideally, the use of an optimally designed
controlled release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition in a minimum amount of time. Advantages of
controlled release formulations include extended activity of the
drug, reduced dosage frequency, and increased patient compliance.
In addition, controlled release formulations can be used to affect
the time of onset of action or other characteristics, such as blood
levels of the drug, and can thus affect the occurrence of side
(e.g., adverse) effects.
[0077] Most controlled release formulations are designed to
initially release an amount of drug (active ingredient) that
promptly produces the desired therapeutic effect, and gradually and
continually release of other amounts of drug to maintain this level
of therapeutic effect over an extended period of time. In order to
maintain this constant level of drug in the body, the drug must be
released from the dosage form at a rate that will replace the
amount of drug being metabolized and excreted from the body.
Controlled release of an active ingredient can be stimulated by
various conditions including, but not limited to, pH, temperature,
enzymes, water, or other physiological conditions or agents.
[0078] In some embodiments of the present invention, the breast
cancer is PTEN-negative. The term PTEN-negative refers to breast
cancer in which at least some cancer cells lack any detectable
amount of human PTEN protein or contain a significantly reduced
amount, or in which at least some cancer cells lack a human PTEN
gene, carry a null-mutation in the human PTEN gene or carry a
mutation that significantly reduces the expression and/or function
of the human PTEN protein. The PTEN tumor suppressor gene has been
extensively researched. See. e.g., Li et al., 1997, Science
275:1943-1947; Steck et al., 1997, Nat. Genet. 15: 356-362.
Molecular biological, immunohistochemical and other methods for
detecting human PTEN protein, or the absence thereof, in tumor
tissue and for detecting mutations in the human PTEN gene are
commonly known in the art and disclosed, for example, in U.S. Pat.
No. 7,981,616, the disclosure of which is incorporated herein by
reference. For instance, a breast cancer biopsy sample can be
obtained and analyzed immunohistochemically for human PTEN
expression by using a PTEN-specific antibody and appropriate
secondary detection reagents. PTEN-specific antibodies are
available commercially from many sources, including, Abcam and Cell
Signaling Technology. Human PTEN protein expression thus determined
can be classified as absent or reduced by comparison to, for
example, the expression of internal molecular markers, e.g., actin
and other ubiquitously expressed proteins, by comparison to PTEN
expression in normal tissue surrounding the tumor, or as set forth
in U.S. Pat. No. 7,981,61. Mutations of the human PTEN gene can be
detected, for example, as described below for the analysis of the
human PIK3CA gene.
[0079] In other embodiments of the present invention, the breast
cancer is positive for mutations in the human PIK3CA gene. A breast
cancer that is positive for mutations in the PIK3CA gene includes
at least some cancer cells that carry a mutation in the human
PIK3CA gene or carry more than two alleles of this gene. The human
PIK3CA gene encodes the p110.alpha. protein, which is a catalytic
subunit of class I phosphatidylinositol 3-kinases (PI3-kinases).
See, e.g., Baselga, 2011, The Oncologist, 16(Suppl. 1):12-19, and
references therein.
[0080] Molecular biological and other methods for detecting such
mutations and genetic amplifications are commonly known in the art
and disclosed, for example, in U.S. Pat. No. 8,026,053, the
disclosure of which is incorporated herein by reference. For
example, a breast cancer biopsy sample can be obtained and genomic
and/or RNA can be extracted therefrom. The genomic DNA can then be
analyzed by PCR, DNA sequencing and Southern blotting, for example,
to detect point mutations, larger rearrangements or gene
amplifications in/of the human PIK3CA gene. Alternatively, the RNA
can be reverse transcribed and the resulting cDNA analyzed for such
mutations. See, e.g., Sambrook et al., Molecular cloning: a
laboratory manual, Cold Spring Harbor Press, 2001. Body fluid
biomarkers can also be tested, including circulating tumor cells,
nucleic acids (DNA and RNA) originating from tumor cells and
circulating in serum or plasma, urine, and saliva.
[0081] In other embodiments of the present invention, at least some
cancer cells of the breast cancer express a truncated ErbB2
receptor that lacks the extracellular domain to which trastuzumab
usually binds. The ErbB2 receptor and its gene have been
extensively researched. Molecular biological, immunological and
other methods for detecting truncated ErbB2 receptor protein or
mutations in the gene that result in a truncated ErbB2 receptor are
commonly known in the art.
[0082] In other embodiments of the present invention, the breast
cancer overexpresses RTKs, for example members of the EGFR family,
IGF-1R and HGFR. The EGFR family, IGF-1R and HGFR have been
extensively researched. Molecular biological, immunological and
other methods for detecting overexpression or amplification of any
of these receptors in breast cancer are commonly known in the
art.
[0083] Throughout this application, various publications are
referenced. These publications are hereby incorporated into this
application by reference in their entireties to more fully describe
the state of the art to which this invention pertains. The
following examples further illustrate the invention, but should not
be construed to limit the scope of the invention in any way.
EXAMPLES
Example 1
EXEL-7647 Inhibits SRC Kinase Activity
[0084] Inhibition of SRC kinase activity by EXEL-7647 was measured
using in vitro kinase assays. These experiments showed that
EXEL-7647 inhibited SCR kinase activity with an IC.sub.50 of 10.3
nM.+-.2.0 (data not shown). In addition, the effect of EXEL-7647 on
the phosphorylation of the SRC-family protein FAK (focal adhesion
kinase) was measured in cell culture (FIG. 1). Specifically,
DLD1-PTK2 cells were treated with EXEL-7647 at concentrations of
1.6, 4.7, 14, 41, 123, 370, 1111, 3333 or 10,000 nM for one hour in
serum-free DMEM and harvested. The phosphorylation status of the
tyrosine at amino acid position 861 of the FAK protein
("FAK-PY861") in the treated cells was then determined by standard
Western blotting using phosphorylation-specific antibodies, as
indicated in FIG. 1. The Western blot was quantified using a
Typhoon scanned image and ImageQuant software and the IC.sub.50
value for EXEL-7647-mediated inhibition of FAK-phosphorylation
calculated. EXEL-7647 inhibited phosphorylation of FAK at the
tyrosine at amino acid position 861 with an IC.sub.50 of about 1
.mu.M. The kinase inhibitor staurosporine (EXEL 00878128) was used
as a positive control. The inhibition of FAK-autophosphorylation of
the tyrosine at ammino acid position 397 ("FAK-PY397") was also
assayed, as indicated in FIG. 1, but not further quantified. The
identifier EXEL-02377647:9 in FIG. 1 refers to compound
EXEL-7647.
Example 2
EXEL-7647 Inhibits Growth of Breast Cancer Xenografts
[0085] The human breast cancer cell line BT474 expresses high
levels of ErbB2 (Her2), a significant fraction of which is
constitutively phosphorylated even in the absence of an exogenous
ligand. The phosphorylation of ErbB2 is a measure of its activity.
Athymic nude mice bearing tumor xenografts derived from the human
breast cancer cell line BT474 were treated orally on three
consecutive days with 3, 10, 30 or 100 mg/kg of EXEL-7647. The
tumors were harvested 1 hr after the final dose was administered
and their weights were measured. The calculated ED.sub.50 of
EXEL-7647-mediated inhibition of tumor growth was 30 mg EXEL-7647
per kg of body weight (data not shown).
Example 3
EXEL-7647 Inhibits ErbB2 (Her2) Phosphorylation
[0086] Tumors generated as described in Example 2 above were
assayed individually or in treatment groups for total and
phospho-ErbB2 levels by standard Western blotting (FIG. 2, top left
panel). Tumors from mice treated with vehicle alone served as the
negative control. The detection of actin served as a control for
protein integrity and concentration. It is readily apparent that
XL647 induced a dose-dependent decrease in total and phospho-ErbB2
levels. Decreases were calculated by reference to the vehicle
control. For example, a dose of 100 mg/kg of EXEL-7647,
administered on three consecutive days, resulted in a reduction of
phospho-ErbB2 of approximately 70%.
[0087] The plasma concentrations of EXEL-7647 in the mice carrying
the analyzed tumors (see above) were determined as well and
correlated with the corresponding phospho-ErbB2 levels (FIG. 2, top
right panel). These measurements revealed that EXEL-7647 inhibited
the accumulation of phospho-ErbB2 in the tumors with an IC.sub.50
of 3.58 .mu.M, and that a plasma concentration of EXEL-7647 of
about 5.37 .mu.M resulted in an approximately 69% reduction of
phospho-ErbB2.
[0088] The kinetics of the decrease of phospho-ErbB2 in tumors in
response to single doses of EXEL-7647 was also determined (FIG. 2,
bottom left panel). Tumors were generated as described above. Tumor
bearing mice were subjected to a single dose of EXEL-7647 (30 or
100 mg/kg), and tumors were dissected and lysates prepared 4, 24 or
48 hours after dosing. Total and phospho-ErbB2 content in the
lysates was determined and decreases in ErbB2 levels calculated as
described above. Overall, total and phospho-ErbB2 amounts were not
significantly reduced in response to a dose of 30 mg/kg of
EXEL-7647. In response to a dose of 100 mg/kg, however, there was a
significant reduction, especially after 24 and 48 hours. The
histogram in FIG. 2, bottom right panel, shows the kinetics of
phospho-ErbB2 decrease in response to a dose of 100 mg/kg of
EXEL-7647. As can be seen, the decrease of phospho-ErbB2 is
statistically significant (p<0.05).
Example 4
EXEL-7647 Inhibits EGFR Phosphorylation
[0089] The ability of EXEL-7647 to inhibit the EGF-induced
phosphorylation of EGFR was validated as follows. Athymic nude mice
bearing tumor xenografts derived from the human epithelial
carcinoma cell line A431 were treated orally with 3, 10, 30 or 100
mg/kg EXEL-7647. Three and half hours after EXEL-7647 treatment,
EGF was administered intravenously to induce EGF-phosphorylation.
The tumors were then harvested and their phospho-EGFR (pEGFR)
content was measured. Tumors from mice treated with vehicle alone
served as the negative control and tumors from mice treated with
vehicle and EGFR served as the positive control. The histogram in
FIG. 3, left panel, shows the decrease of EGF-induced
EGFR-phosphorylation in the tumors in response to the different
doses of EXEL-7647 administered. For example, a dose of 100 mg/kg
EXEL-7647 resulted in a reduction of EGF-induced
EGFR-phosphorylation of about 90% by comparison to the positive
control. As can be seen, the reduction of EGF-induced
EGFR-phosphorylation in response to the different EXEL-7647 doses
administered is statistically significant (p<0.05).
[0090] The plasma concentrations of EXEL-7647 in the mice carrying
the analyzed tumors (see above) were determined as well and
correlated with the corresponding reduction of phospho-EGFR levels
(FIG. 3, right panel). These measurements revealed that EXEL-7647
inhibited the phosphorylation of EGFR in the tumors with an
IC.sub.50 plasma concentration of 0.72 .mu.M.
Example 5
EXEL-7647 Inhibits KDR Phosphorylation
[0091] Highly vascularized tissue such as the lung of mice contains
significant levels of KDR, but only a small fraction of it is in
the phosphorylated form. Intravenous administration to mice of 10
.mu.g of VEGF, the ligand of KDR, increased the amount of
phospho-KDR (pKDR) in their lungs about 1.5 fold after 30 minutes.
The ability of EXEL-7647 to inhibit the VEGF-induced activation of
the KDR receptor (determined by measuring the receptor's level of
phosphorylation) was validated as follows. Mice were treated orally
with a single dose of 10, 30 or 100 mg/kg of EXEL-7647. Three and
half hours after EXEL-7647 treatment, 10 .mu.g of VEGF were
administered intravenously. The lungs were then harvested and
lysates from each dosage group (n=5) pooled. Lysates were assayed
for total KDR and pKDR levels by standard Western blotting (FIG. 4,
left panel). Lungs from mice treated with vehicle alone served as
the negative control and lungs from mice treated with vehicle and
VEGF served as the positive control. It is readily apparent that
administration of EXEL-7647 resulted in a dose-dependent decrease
of VEGF-induced KDR-phosphorylation. Decreases were calculated by
reference to the vehicle control. For example, a single dose of 100
mg/kg EXEL-7647 completely suppressed VEGF-induced KDR receptor
phosphorylation to baseline levels. Quantification of p-KDR in
lysates from individual mice confirmed both the statistical
significance of VEGF-induced KDR-phosphorylation (p<0.0005) and
the statistical significance of the complete inhibition of this
induction by the administration of 100 mg/kg of EXEL-7647
(p<0.001) (data not shown).
[0092] The plasma concentrations of EXEL-7647 in the treated mice
(see above) were determined as well and correlated with the
corresponding decrease of VEGF-induced KDR-phosphorylation measured
in lysate pools (FIG. 4, right panel). These measurements revealed
that EXEL-7647 decreased the VEGF-induced KDR receptor
phosphorylation with an IC.sub.50 of about 1.23 .mu.M, and that a
plasma concentration of about 3.36 .mu.M of EXEL-7647 resulted in
the complete inhibition of KDR-phosphorylation in response to VEGF
treatment.
Example 6
EXEL-7647 Inhibits EphB4 Phosphorylation
[0093] The ability of EXEL-7647 to inhibit the activity of the
EphB4 receptor (determined by measuring the receptor's level of
phosphorylation) was validated as follows. A cell line expressing
high levels of EphB4 was derived by transfecting the human colon
carcinoma line HCT116 with a drug selectable marker and an
expression vector encoding EphB4. The resulting EphB4 expressing
cells (HCT116/EphB4) grow as xenografts in immunocompromised mice.
Analysis of lysates from these xenografts showed that a significant
amount of EphB4 in the cells is constitutively phosphorylated at a
tyrosine residue. Attempts to further stimulate
EphB4-phosphorylation by intravenous or intratumoral injection of
Eph A2 were not successful (data not shown).
[0094] Mice bearing HCT116/EphB4 xenografts were dosed orally on
three consecutive days with 3, 10, 30 or 100 mg/kg EXEL-7647.
Tumors were harvested 1 hr after the final dose and assayed
individually or in treatment groups for total and phospho-EphB4
levels by standard Western blotting (FIG. 5, left panel). Tumors
from mice treated with vehicle alone served as the negative
control. The detection of actin served as a control for protein
integrity and concentration. EXEL-7647 induced a dose-dependent
decrease in phospho-EphB4 levels. Specifically, a dose of 100 mg/kg
of EXEL-7647, administered on three consecutive days, resulted in a
reduction of phospho-EphB4 of approximately 70%.
[0095] The plasma concentrations of EXEL-7647 in the mice carrying
the analyzed tumors (see above) were determined as well and
correlated with the corresponding reductions of phospho-EPHB4
levels (FIG. 5, right panel). These measurements revealed that a
EXEL-7647 plasma concentration of about 3 .mu.M reduced the
phosphorylation of EphB4 in the tumors by approximately 70%. A 50%
inhibition of EphB4-phosphorylation was predicted to occur at a
EXEL-7647 plasma concentration of about 2.4 .mu.M.
Example 7
EXEL-7647 Inhibits Angiogenesis
[0096] The ability of EXEL-7647 to inhibit angiogenesis was
validated by in vitro and in vivo experiments, as shown below (FIG.
6; Table 2). Endothelial tube formation and cell migration assays
were performed to test the effect of EXEL-7647 on in vitro models
that reflect aspects of endothelial cell function thought to
contribute to angiogenesis in vivo. When plated on a confluent
layer of normal human diploid fibroblasts, human microvascular
endothelial cells (HMVECs) form extensive networks of tubules in
response to VEGF over a 7-10 day period. Tubules were stained and
quantified using an antibody that recognizes the endothelial cell
marker CD31, as illustrated in FIG. 6, left panel. EXEL-7647
inhibited VEGF-induced tubule formation with an IC.sub.50 of
approximately 0.22 .mu.M, which was similar to the IC.sub.50 values
obtained using the receptor phosphorylation assays discussed above
in Examples 3-6. The IC.sub.50 for cytotoxic effects of EXEL-7647
on HMVECs, as determined by Alamar blue staining, was about 1.3
.mu.M, approximately 5-fold higher than the IC.sub.50 with which
EXEL-7647 inhibits VEGF-induced tubule formation (data not
shown).
[0097] A second assay, the so-called "scratch assay," was employed
to examine the effects of EXEL-7647 on murine endothelial cells
(FIG. 6, right panel). In this assay, a cell-free zone was
scratched into a monolayer of cells and the ability of EXEL-7647 to
block VEGF-stimulated migration of murine MS1 endothelial cells
into the cell-free zone was measured. In the absence of VEGF,
migration of cells bordering the scratch into the cell-free space
was minimal during the 24 hrs time-course of the experiment. VEGF
greatly stimulated migration, resulting in a nearly complete
closure of the scratch within that time frame. EXEL-7647 inhibited
cellular migration into the scratch with an IC.sub.50 of about 0.12
.mu.M, as determined from a six-point dose response. (FIG. 6, right
panel). This is consistent with EXEL-7647 inhibiting the murine KDR
receptor and the human KDR receptor to a similar extent. No
evidence for cytotoxicity of EXEL-7647 was found in this assay at
concentrations below 1.1 .mu.M.
[0098] Anti-angiogenic effects of EXEL-7647 were also studied in
vivo. Tumor xenografts derived from the human breast cancer cell
line MDA-MB-231 were established in athymic female mice and allowed
to reach a total weight of 100 mg. The mice were then treated
orally on fourteen consecutive days with doses of 10, 30 or 100
mg/kg of EXEL-7647. The tumors were harvested and their weights
were measured after the last dosage had been administered. Tumors
derived from mice treated with vehicle alone served as negative
controls. Tumor growth was inhibited significantly by all three
dosage regimens (data not shown). Specifically, the 100 mg/kg
dosage resulted in a complete cessation of tumor growth (tumor
weight at start of study=100.6.+-.8.7 mg, tumor weight at end of
study 112.+-.16.2 mg). The 30 and 100 mg/kg regimens also
significantly increased the percentage of total tumor necrosis when
compared to the necrosis in control tumors treated with vehicle
alone (Table 2 below). A statistically significant increase in
tumor necrosis was not observed at the lower dose of 10 mg/kg in
this model. Furthermore, the amount of CD31-positive blood vessels
was significantly decreased in the viable tissue of those tumors
that were derived from mice subjected to any of the three dosage
regimens tested (Table 2 below). Finally, the percentage of cells
expressing Ki67, a marker for cell proliferation, was significantly
reduced in the tumors that were derived from mice subjected to any
of the three dosage regimens tested (Table 2). This indicated that
these tumors contained fewer proliferating cells.
TABLE-US-00001 TABLE 2 Anti-Angiogenesis and Other Effects of XL647
in vivo CD31 Ki67 Necrosis CD31 Analysis Ki67 Expression KD-019
Necrosis fold Analysis % Expression % Dose % increase increase MVC*
Reduction % of Cells Reduction Vehicle 15.7 (7.3) N.A. 18.5 (6.4)
N.A. 49.4 (10.2) N.A. 10 mg/kg 25.0 (14.2) 1.6 12.93 (2.3) 30.23
29.8 (13.3) 39.6 30 mg/kg 30.6 (11.3) 2.0 8.18 (1.4) 55.83 28.3
(10.4) 42.6 100 mg/kg 71.1 (8.1) 4.5 1.625 (1) 91.23 24.87 (4.9)
49.6 *MVC stands for mean vessel count. **Values are means with the
standard deviations being in parentheses.
Example 8
EXEL-7647 Inhibits Growth of Breast Cancer Xenografts
[0099] The ability of EXEL-7647 to inhibit the growth of tumors
derived from the human breast cancer cell line MDA-MB-231 was
validated as follows. Tumor xenografts derived from MDA-MB-231
cells were established in athymic female mice and allowed to reach
a total weight of 100 mg. The mice were then treated orally on up
to twenty-eight consecutive days with doses of 10, 30 or 100 mg/kg
of EXEL-7647. The tumors were harvested on specific days after
treatment with XL647 had begun and their weights were measured, as
indicated in FIG. 7. Tumors derived from mice treated with vehicle
alone served as negative controls. Tumor growth was inhibited
significantly by the administration of the 30 and the 100 mg/kg of
EXEL-7647 dosages (FIG. 7). Specifically, the 100 mg/kg dosage
resulted in a complete arrest of tumor growth (starting tumor
weight 94.+-.9 mg, final tumor weight 117.+-.33 mg). The calculated
ED.sub.50 of EXEL-7647-mediated inhibition of tumor growth was 22.9
mg of XL647 per kg body weight.
Example 9
EXEL-7647 Suppresses Trastuzumab Resistant Cell Proliferation
[0100] A significant proportion of ERBB2 positive breast cancer
patients does not respond or becomes resistant to trastuzumab
treatment. Resistance arises largely via genetic alteration in RTKs
and other signaling molecules downstream of the receptors or via
upregulation of the activity of other RTKs as a compensatory
mechanism. EXEL-7647 is potently active in models of trastuzumab
resistance, as demonstrated by growth inhibition of JIMT-1 and
HCC1954 trastuzumab resistant cancer cell lines.
[0101] JIMT-1 and HCC1954 cells were seeded into 96-well plates
(Costar), in Dulbecco's Modification of Eagle's Medium (DMEM,
Invitrogen) containing 10% Fetal Bovine Serum (heat inactivated
FBS, Hyclone), 1% Penicillin-streptomycin (Hyclone). 18 hours after
seeding, cells were treated with the compounds for 72 hours.
Triplicate wells were used for each compound concentration. The
control wells were treated with 0.2% DMSO media. The cultures were
incubated at 37.degree. C., 5% CO2 and the quantity of
proliferating cells was determined using the "CellTiter 96 AQueous
Non-Radioactive Cell Proliferation Assay kit" (Promega). Following
incubation with the substrate solution, the plate was read using
Infinite M1000 plate reader (Tecan). IC.sub.50 values were
calculated based on the GraphPad Prism software analysis.
Percentage inhibition of cell proliferation was calculated as
[1-(treated cells/control cells).times.100].
[0102] Treatment of either cell line with increasing concentrations
of trastuzumab had no impact of the rate of cellular proliferation,
confirming that JIMT-1 and HCC1954 cell lines are not responsive to
trastuzumab treatment. However, EXEL-7647 strongly inhibited
proliferation of these cells (Table 3 and 4).
TABLE-US-00002 TABLE 3 Inhibition of JIMT-1 cell proliferation (%
inhibition) Trastuzumab EXEL- 1.0 .mu.g/ 5.0 .mu.g/ 7647 DMSO 0.2
.mu.g/ml 0.5 .mu.g/ml ml 2.0 .mu.g/ml ml DMSO 0 1.9 3.4 4.4 -1.8
-6.0 0.2 .mu.M 15.7 7.9 7.6 11.0 11.1 0.5 .mu.M 20.3 4.6 4.7 12.0
14.0 1.0 .mu.M 22.7 11.8 14.3 17.4 18.9 2.0 .mu.M 28.8 28.1 27.1
27.5 27.6 5.0 .mu.M 81.7
TABLE-US-00003 TABLE 4 Inhibition of HCC1954 cell proliferation (%
inhibition) Trastuzumab EXEL- 1.0 .mu.g/ 5.0 .mu.g/ 7647 DMSO 0.2
.mu.g/ml 0.5 .mu.g/ml ml 2.0 .mu.g/ml ml DMSO 0 -6.9 -6.4 -6.5
-15.5 -13.9 0.2 .mu.M 22.5 10.9 12.9 17.8 23.5 0.5 .mu.M 32.6 14.3
14.4 20.3 30.4 1.0 .mu.M 38.1 25.0 27.1 29.1 39.1 2.0 .mu.M 43.6
42.6 44.3 44.9 46.7 5.0 .mu.M 80.4
[0103] To compare the two cell lines directly, total protein levels
of SRC family kinases as well as the levels of activating
phosphorylation of these proteins were analyzed in both cells
lines. Cells were washed twice with ice cold PBS and lysed in RIPA
buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5%
sodium deoxycholate, 0.1% SDS, containing protease and phosphatase
inhibitor cocktail). Cell lysate was collected after centrifugation
(12,000 rpm, 15 minutes) and protein concentrations were measured
using BCA reagent (Thermo Fisher Scientific). Equal amounts of
proteins were separated on SDS-PAGE and transferred onto a PVDF
membrane (Millipore). Membranes were blocked with 5% milk in PBS
containing 0.1% Tween 20 (PBST) for 1 hour, and then probed with
primary antibodies overnight at 4.degree. C. Membranes were washed
with PBST and incubated with secondary antibodies for 1 hour at
room temperature. After 3.times. washes in PBST, blots were
visualized with enhanced chemiluminescence reagent following the
manufacturer's instructions (Thermo Fisher Scientific).
[0104] Phosphorylation at tyrosine 416 in the activation loop of
the kinase domain of Src correlates with greater activity
demonstrated by higher levels of phosphorylation of target proteins
including Paxillin. JIMT-1 cells appeared to have significantly
greater amounts of SRC proteins than HCC1954 cells (FIG. 8).
Consistent with this, treatment with EXEL-7647 had a significantly
greater effect on the pSRC levels in HCC 1954 cells, where a
considerable reduction of phosphor-Src (Tyr416) was achieved by
treatment with as low as 0.1 .mu.M EXEL-7647 (FIG. 8A).
Importantly, while 4 hr treatment with 1.0 .mu.M EXEL-7647
significantly reduced Src activation, treatment of both cells lines
with 5 or 10 .mu.M EXEL-7647 completely abolished phosphorylation
of Tyr416, suggesting complete Src inhibition (FIGS. 8A and B).
Inhibition of Src activity was further confirmed by the decrease in
phosphorylation of Paxillin (Tyr118) (FIG. 8A). To confirm that Src
inhibition was direct, SRC phosphorylation in EXEL-7647 treated
cells was compared to cells treated with other ERBB family
inhibitors (Fib. 8B). Only EXEL-7647, and not lapatinib or
erlotinib, was able to inhibit SRC activity in both cell lines. In
addition, EXEL-7647 had far greater effects on the proliferation of
both cell lines in comparison to the other small molecule ERBB
inhibitors (FIGS. 8C and D), indicating its ability to treat cells
which do not respond to RTK blockade.
Example 10
EXEL-7647 Inhibits Trastuzumab Resistant JIMT-1 Tumor
Xenografts
[0105] Female severe combined immunodeficient mice (Fox Chase
SCID.RTM., C.B-17/Icr-Prkdc.sup.scid, Charles River Laboratories)
were ten weeks old, with a body weight range of 17.6 to 20.8 grams
on Day 1 of the study. Treatment began on Day 1 in four groups of
mice (n=12) with upstaged subcutaneous JIMT-1 tumors (196-405
mm.sup.3). Mice were scheduled to receive EXEL-7647 (80 mg/kg p.o.
qd.times.35) with and without trastuzumab (20 mg/kg i.p.
biwk.times.5). The experiment included a vehicle-treated control
group and a trastuzumab monotherapy group. During the study, the
EXEL-7647 dosing schedule was modified to once daily on Days 1-19,
28, 29, 32-36, and 39-42, due to toxicity. Tumors were measured
twice per week until the study was ended on Day 42. Treatment
outcome was determined from percent tumor growth inhibition (%
TGI), which evaluated the percent differences in median tumor
volumes (MTVs) between treated and control mice at the end of daily
dosing (Day 18) and at the end of the study (Day 42), with
differences between groups deemed statistically significant at
P<0.05 using the Mann-Whitney U-test. A regimen that produced
TGI of 60% or more was considered to have potential therapeutic
activity. Mice were also monitored for complete regression (CR) and
partial regression (PR) responses, and for the frequency of 30%
tumor volume (TV) regression from Day 1. The 30% TV regressions
were deemed statistically significant at P<0.05 using the
chi-square test. Treatment tolerability was assessed by body weight
measurements and frequent observation for clinical signs of
treatment related side effects.
[0106] Once established, JIMT-1 tumors are completely resistant to
trastuzumab treatment and proliferated at a rate similar to the
vehicle control animals. In contrast, 80 mg/kg dose of EXEL-7647
administered orally on a once-daily schedule either alone or in
combination with trastuzumab (20 mg/kg i.p. bwk.times.5) prevented
tumor growth (FIG. 9).
Example 11
EXEL-7647 Targets Her2, EGFR and Indirectly Inhibits Met
Activation
[0107] Recent studies indicate that trastuzumab activates
phosphorylation of Her2, while simultaneously increasing its
internalization and degradation. Consistently, treatment with
trastuzumab lead to significant downregulation of total ERBB2
levels in JIMT-1 cells (FIG. 10A, right panel). Like other ERBB
family small molecule inhibitors, EXEL-7647 had no effect on the
total levels of ERBB2 in these cells, suggesting that receptor
turnover does not account for the anti-proliferative qualities of
the drug. In HCC1954, no effect on the levels of ERBB2 was observed
with any of the treatments, likely due to extremely high expression
levels of the receptor in these cells. ERBB2 phosphorylation was
elevated in both cell lines following trastuzumab treatment and
inhibited by small molecule inhibitiors, including EXEL-7647 (FIG.
10B).
[0108] Similar results were obtained with the activation of EGFR.
While nether trastuzumab nor small molecules changed the overall
levels of the receptor in either of the cell lines tested, marked
effects were observed on activating EGFR phosphorylation. Here too,
HCC1954 expressed the receptor at far greater levels than JIMT-1
cells, and while treatment with trastuzumab had little effect on
EGFR phosphorylation, lapatinib, erlotinib and EXEL-7647 inhibited
EGFR activity (monitored via phosphorylation of Tyrl 068) to a
similar extent. Based on the above-mentioned results, we concluded
that neither the inhibition of ERBB2 or EGFR activity nor the
effects on receptor turnover fully account for the
anti-proliferative activity of EXEL-7647.
[0109] A high level of active Met phosphorylated at tyrosine sites
1234 and 1235 was also observed in HCC1954 (FIG. 10D) but not in
JIMT-1 cells (data not shown). Aberrant activation of Met receptor
tyrosine kinase has been linked with trastuzumab resistance. While
Met is not a direct target of the molecule, EXEL-7647 effectively
inhibited Met activation in HCC 1954 cells (FIG. 10D) Inhibition of
Met in these cells may be due to receptor hetero-oligomerization,
as a similar level of inhibition was observed with lapatinib, which
does not have direct Met inhibitory activity.
Example 12
EXEL-7647 Effectively Targets Multiple Kinases
[0110] The activity of EXEL-7647 was compared to a selective SRC
inhibitor (AZD0530, saracatinib). When compared for their ability
to inhibit SRC, AZD0530 exhibited a stronger dose dependent
response. In JIMT-1 cells 1.0 .mu.M AZD0530 treatment suppressed
Src activity, and although some inhibition was observed at 1.0
.mu.M of EXEL-7647, higher concentrations were required for
equivalent inhibition (FIG. 11A). However, in JIMT-1 cells the more
potent inhibition of SRC did not directly translate into inhibition
of cell growth. In cell proliferation assays, AZD0530 was less
effective against JIMT-1 cells than EXEL-7647 (FIG. 11B). The
effectiveness of EXEL-7647 is likely a result of its ability to
inhibit multiple kinases.
[0111] JIMT-1 and HCC1954 cells are two examples of trastuzumab
resistance, and represent escape mechanisms for trastuzumab
treatment. The experiments presented demonstrate that SRC
activation plays an important role development of resistance.
EXEL-7647 effectively inhibits proliferation of ERBB2 positive,
trastuzumab refractory breast cancer cells.
* * * * *
References