U.S. patent application number 14/114878 was filed with the patent office on 2014-03-06 for csf-1r inhibitors for treatment of brain tumors.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Dylan Daniel, Johanna Joyce, James Sutton. Invention is credited to Dylan Daniel, Johanna Joyce, James Sutton.
Application Number | 20140065141 14/114878 |
Document ID | / |
Family ID | 46062775 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065141 |
Kind Code |
A1 |
Daniel; Dylan ; et
al. |
March 6, 2014 |
CSF-1R INHIBITORS FOR TREATMENT OF BRAIN TUMORS
Abstract
The present invention provides a compound of formula I;
##STR00001## wherein R.sup.1 is an alkyl pyrazole or an alkyl
carboxamide, and R.sup.2 is a hydroxycycloalkyl; or a
pharmaceutically acceptable salt thereof, and compositions
containing these compounds, for use to treat a brain tumor,
particularly glioblastoma. The invention provides effective
treatment of a brain tumor and can be used by oral administration
of a compound of Formula I as further described herein. The
invention also provides a method to treat a subject having a brain
tumor such as glioblastoma, wherein the method comprises
administering to the subject an effective amount of a compound of
Formula I. Gene signatures correlated with successful treatment
using these methods are also disclosed.
Inventors: |
Daniel; Dylan; (San
Francisco, CA) ; Joyce; Johanna; (New York, NY)
; Sutton; James; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Daniel; Dylan
Joyce; Johanna
Sutton; James |
San Francisco
New York
Pleasanton |
CA
NY
CA |
US
US
US |
|
|
Assignee: |
Novartis AG
Basel
CH
|
Family ID: |
46062775 |
Appl. No.: |
14/114878 |
Filed: |
May 4, 2012 |
PCT Filed: |
May 4, 2012 |
PCT NO: |
PCT/US2012/036589 |
371 Date: |
October 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61482723 |
May 5, 2011 |
|
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|
61624861 |
Apr 16, 2012 |
|
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Current U.S.
Class: |
424/133.1 ;
514/338; 514/81; 546/270.1 |
Current CPC
Class: |
C07D 417/12 20130101;
A61K 39/3955 20130101; A61P 35/00 20180101; A61K 45/06 20130101;
C07D 417/14 20130101; A61K 31/4439 20130101; A61K 31/4545 20130101;
A61P 25/00 20180101; A61K 31/175 20130101; A61P 35/04 20180101;
A61K 31/675 20130101 |
Class at
Publication: |
424/133.1 ;
546/270.1; 514/338; 514/81 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439; A61K 45/06 20060101 A61K045/06; A61K 31/175 20060101
A61K031/175; A61K 31/675 20060101 A61K031/675; A61K 39/395 20060101
A61K039/395; A61K 31/4545 20060101 A61K031/4545 |
Claims
1-24. (canceled)
25. A method to treat a brain tumor in a mammalian subject,
comprising administering to the subject an effective amount of a
compound of Formula (I): ##STR00023## wherein R.sup.1 is an alkyl
pyrazole or an alkyl carboxamide; and R.sup.2 is hydroxycyclohexyl;
or a pharmaceutically acceptable salt thereof.
26. The method of claim 25, wherein R.sup.1 is ##STR00024## wherein
R' is Me or Et.
27. The method of claim 25, wherein R.sup.2 is ##STR00025##
28. The method of claim 25, wherein the brain tumor is a
glioma.
29. The method of claim 28, wherein the glioma is glioblastoma
multiforme.
30. The method of claim 25, wherein the brain tumor is a brain
metastasis, astrocytoma, oligodendroglioma, ependymomas, or a mixed
glioma.
31. The method of claim 25, wherein the compound of formula (I) is
##STR00026## or a pharmaceutically acceptable salt thereof.
32. The method of claim 31, wherein the compound of Formula (I) is:
##STR00027## or a pharmaceutically acceptable salt thereof.
33. The method of claim 31, wherein the compound of Formula (I) is:
##STR00028## or a pharmaceutically acceptable salt thereof.
34. The method of claim 31, wherein the compound of Formula (I) is:
##STR00029## or a pharmaceutically acceptable salt thereof.
35. The method of claim 31, wherein the compound of Formula (I) is:
##STR00030## or a pharmaceutically acceptable salt thereof.
36. The method of claim 25, wherein the method further comprises
administering to the subject an effective amount of an additional
cancer therapeutic selected from bevacizumab with or without
irinotecan, a nitrosourea, a platin, an alkylating agent, a
tyrosine kinase inhibitor, Ukrain, and a cannabinoid.
37. The method of claim 25, wherein the compound of Formula (I) is
administered orally.
38. The method of claim 37, wherein the amount of the compound of
Formula (I) administered to the subject is between about 50 mg/kg
per day and about 500 mg/kg per day.
39. The method of claim 25, wherein the brain tumor is proneural
glioblastoma.
40. The method of claim 25, wherein the subject has an elevated
level of PDGF or PDGFR signaling.
41. The method of claim 25, wherein the subject is
contemporaneously treated with an inhibitor of PDGFR.
42. A compound of the formula: ##STR00031## where R' is Methyl,
Ethyl or Propyl.
43. The compound of claim 42, wherein R' is methyl.
44. A pharmaceutical composition comprising a compound of claim 42,
and at least one pharmaceutically acceptable excipient.
Description
BACKGROUND
[0001] Cancers of the brain and nervous system are among the most
difficult to treat. Prognosis for patients with these cancers
depends on the type and location of the tumor as well as its stage
of development. For many types of brain cancer, average life
expectancy after symptom onset may be months or a year or two.
Treatment consists primarily of surgical removal and radiation
therapy; chemotherapy is also used, but the range of suitable
chemotherapeutic agents is limited, perhaps because most
therapeutic agents do not penetrate the blood-brain barrier
adequately to treat brain tumors. Using known chemotherapeutics
along with surgery and radiation rarely extends survival much
beyond that produced by surgery and radiation alone. Thus improved
therapeutic options are needed for brain tumors.
[0002] Gliomas are a common type of brain tumor. They arise from
the supportive neuronal tissue comprised of glial cells (hence the
name glioma), which maintain the position and function of neurons.
Gliomas are classified according to the type of glial cells they
resemble: astrocytomas (including glioblastomas) resemble
star-shaped astrocyte glial cells, oligodendrogliomas resemble
oligodendrocyte glial cells; and ependymomas resemble ependymal
glial cells that form the lining of fluid cavities in the brain. In
some cases, a tumor may contain a mixture of these cell types, and
would be referred to as a mixed glioma.
[0003] The typical current treatment for brain cancers is surgical
removal of the majority of the tumor tissue, which may be done by
invasive surgery or using biopsy or extractive methods. Gliomas
tend to disseminate irregularly, though, and are very difficult to
remove completely. As a result, recurrence nearly always occurs
soon after tumor removal. Radiation therapy and/or chemotherapy can
be used in combination with surgical removal, but these generally
provide only modest extension of survival time. For example, recent
statistics showed that only about half of patients in the U.S. who
are diagnosed with glioblastoma are alive one year after diagnosis,
and only about 25% are still alive after two years, even when
treated with the current standard of care combination
treatments.
[0004] Glioblastoma multiforme (GBM) is the most common adult
primary brain tumor and is notorious for its lethality and lack of
responsiveness to current treatment approaches. Unfortunately,
there have been no substantial improvements in treatment options in
recent years, and minimal improvements in the survival prospects
for patients with GBM. Thus there remains an urgent need for
improved treatments for cancers of the brain such as gliomas.
[0005] Gliomas develop in a complex tissue microenvironment
comprised of many different types of cells in the brain parenchyma
in addition to the cancer cells themselves. Tumor-associated
macrophages (TAMs) are one of the prominent stromal cell types
present, and often account for a substantial portion of the cells
in the tumor tissues. Their origin is not certain: these TAMs may
originate either from microglia, the resident macrophage population
in the brain, or they may be recruited from the periphery.
[0006] TAMs can modulate tumor initiation and progression in a
tissue-specific manner: they appear to suppress cancer development
in some cases, but they enhance tumor progression in the majority
of studies to date. Indeed, in approximately 80% of the cancers in
which there is increased macrophage infiltration, the elevated TAM
levels are associated with more aggressive disease and poor patient
prognosis. Several studies have shown that human gliomas also
exhibit a significant increase in TAM numbers, which correlates
with advanced tumor grade, and TAMs are typically the predominant
immune cell type in gliomas. However, the function of TAMs in
gliomagenesis remains poorly understood, and it is currently not
known whether targeting of these cells represents a viable
therapeutic strategy. In fact, opposing effects on tumor growth
have been reported in the literature, in some cases even where a
similar experimental strategy was used to deplete macrophages in
the same orthotopic glioma implantation model. In some studies,
TNF-.alpha. or integrin .beta.3 produced by TAMs have been
implicated in the suppression of glioma growth, whereas in other
reports CCL2 and MT1-MMP have been proposed as enhancers of tumor
development and invasion.
[0007] Inhibition of CSF-1R signaling represents a novel,
translationally relevant approach that has been used in several
oncological contexts, including xenograft intratibial bone tumors.
However, it has not yet been shown to be effective in brain tumors.
Some non-brain cancers have been targeted with compounds that
affect a variety of cell types that are associated with, or
support, tumor cells rather than directly targeting the tumor cells
themselves. For example, PLX3397 is reported to co-inhibit three
targets (FMS, Kit, and Flt3-ITD) and to down-modulate various cell
types including macrophages, microglia, osteoclasts, and mast
cells. PLX3397 has been tested for treating Hodgkin's lymphoma.
However, Hodgkin's lymphoma responds well to various
chemotherapeutics, according to the PLX3397 literature, while brain
tumors are much more resistant to chemotherapeutics and have not
been successfully treated. As demonstrated herein, a CSF-1R
inhibitor had no direct effect on proliferation of glioblastoma
cells in culture, though, and it did not reduce numbers of
macrophage cells in tumors of treated animals. It is thus
surprising that, as also demonstrated herein, a CSF-1R inhibitor
can effectively inhibit growth of brain tumors in vivo, cause
reduction in tumor volume in advanced stage GBM, and even
apparently eradicating some glioblastomas.
SUMMARY OF EMBODIMENTS OF THE INVENTION
[0008] The present invention is based on demonstrations that brain
tumors, particularly glioblastoma, can be treated with an inhibitor
of CSF-1R. The effectiveness of the CSF-1R inhibitors described
herein is believed to be due to their inhibition of certain
activities of TAMs, even though it does not appear to significantly
reduce the number of TAMs present, and is likely also a function of
the demonstrated ability of these compounds to penetrate the
blood-brain barrier effectively in subjects with a brain tumor.
These methods provide much needed new therapeutic options for
patients diagnosed with brain tumors, particularly
glioblastomas.
[0009] Colony stimulating factor-1 (CSF-1), also termed macrophage
colony stimulating factor (M-CSF), signals through its receptor
CSF-1R (also known as c-FMS) to regulate the differentiation,
proliferation, recruitment and survival of macrophages. Small
molecule inhibitors of CSF-1R have been developed that block
receptor phosphorylation by competing for ATP binding in the active
site, as for other receptor tyrosine kinase inhibitors. The present
invention uses a potent, selective CSF-1R inhibitor, which
penetrates the blood-brain barrier (BBB), to block CSF-1R signaling
in glioma as illustrated in the
RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf.sup.-/- mouse model of
gliomagenesis. This genetically engineered glioma model is ideal
for preclinical testing as a model for human GBM, as it
recapitulates all features of human GBM in an immunocompetent
setting. Because it closely models human GBM, and proneural GBM in
particular, efficacy in this model is expected to translate into
clinical efficacy on human glioblastomas such as glioblastoma
multiforme and mixed gliomas.
[0010] The invention can be practiced with any inhibitor of CSF-1R
capable of penetrating the brain. Some such compounds are the
6-O-substituted benzoxazole and benzothiazole compounds disclosed
in WO2007/121484, particularly the compounds of Formula IIa and IIb
in that reference, and the compounds disclosed herein.
[0011] In one aspect, the invention provides a method to treat a
brain tumor in a mammalian subject, comprising administering to the
subject an effective amount of a compound of Formula (I):
##STR00002##
wherein R.sup.1 is an alkyl pyrazole or an alkyl carboxamide; and
R.sup.2 is a hydroxycycloalkyl; or a pharmaceutically acceptable
salt thereof.
[0012] The method can be used to treat a patient, frequently a
human subject, who has been diagnosed with a brain tumor. Further
embodiments of the invention are described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A is a graph showing the relative amounts of Live
DAPI-positive cells in normal brain and glioblastoma tissue, as
measured by the increased proportion of cells staining positive for
CD45 (pan-leukocyte marker) and CD11b (myeloid cell marker) in the
tumor tissue. The fluorescence activated cell sorting (FACS) data
is shown, also.
[0014] FIG. 1B depicts CD68 stained brain cells from Normal Brain
tissue and from a Grade IV glioblastoma, and shows abundant
macrophage infiltration in the tumor tissue. See Example 1.
[0015] FIG. 1C depicts the increased level of mRNAs for CD68,
CSF-1R and CSF-1 relative to the housekeeping gene Ubiquitin C
(Ubc), for GBM tissue relative to normal brain tissue.
[0016] FIG. 1D shows the relative amounts of CD11b, TVA, CSF-1 and
CSF-1R in TAMs relative to tumor cells.
[0017] FIG. 2 depicts amounts of BLZ945 in Plasma, brain tissue
from the left half of a brain containing GBM, and from the right
half of the same brain with no visible GBM at several time points
after treating cohorts of mice with BLZ945.
[0018] FIG. 3A shows inhibition by BLZ945 of CSF-1R
phosphorylation, following CSF-1 stimulation, in bone-marrow
derived macrophage cells (BMDM).
[0019] FIG. 3B shows the rate of population doubling of BMDM cells
untreated, and demonstrates that treating the cells with 67 nM
BLZ945 has the same effect on this rate as absence of CSF-1
stimulation.
[0020] FIGS. 3C-3E show rate of proliferation of BMDM cells from
the Ink4a/Arf-/- mice, of CRL-2647 normal mouse brain cells, and
for two mouse GBM cell cultures.
[0021] FIG. 3F shows that the total number and size of neurospheres
was unaffected by BLZ945 at 670 nM.
[0022] FIG. 4A depicts symptom-free survival of
RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf.sup.-/- mice treated with
vehicle alone or vehicle+BLZ945. See Example 4.
[0023] FIG. 4B depicts tumor grade for treated and untreated mice
at the 26 week study endpoint. All control mice had grade Ill or IV
tumors.
[0024] FIG. 5A shows tumor size data measured by MRI for treated
and control animals during the first 6 days of treatment with
BLZ945.
[0025] FIG. 5B shows tumor volume for individual control mice
(upper graph) and treated mice (lower graph) during the first 6
days after dosing with BLZ945 started.
[0026] FIGS. 5C and 5D depict tumor volume measured by MRI in
BLZ945-treated animals beginning with large tumors (volume>40
mm.sup.3), and shows that even with large tumors, tumor volume
decreased in nearly all subjects.
[0027] FIG. 5-2 shows data on tumor volume for individual animals
in the control group for Example 5 (5-2A) and the treated group
(5-2B), and FIG. 5-2C shows the tumor size data for the large tumor
subjects treated with BLZ945 in Example 5.
[0028] FIG. 6: the first graph shows the percentage of Olig2+ cells
in the brains of animals in the vehicle, treated, and `Large tumor`
groups in Example 5. The second graph shows the fraction of tumor
cells that were actively dividing, as measured by bromodeoxyuridine
(BrdU) labeling. The third graph shows the level of apoptosis in
the tumor cells, as measured by cleaved caspase 3 (CC3) staining,
and demonstrates that BLZ945 promotes apoptosis of tumor cells.
[0029] FIG. 7A shows the steps used for FACS separation of cells
for gene expression analyses in Example 7.
[0030] FIG. 7B shows the SVM gene signature for treated and
untreated animals, from which genes upregulated and downregulated
by the treatment were identified.
[0031] FIGS. 7C-7E show selective upregulation of M2-associated
genes and EGR2 targets.
[0032] FIG. 8A graphically depicts the degree of upregulation and
statistical relevance used to classify differentially-expressed
genes in the SVM gene signature.
[0033] FIG. 8B shows the 5-gene Lasso regression signature.
[0034] FIG. 8C shows the Lasso gene signature prediction for
proneural GBM tumors in the TCGA data set.
[0035] FIG. 8D shows the Lasso gene signature prediction for
proneural GBM tumors in the combined data set.
[0036] FIG. 8E shows the SVM gene signature prediction for
proneural GBM tumors in the TCGA data set.
[0037] FIG. 8F shows the SVM gene signature prediction for
proneural GBM tumors in the combined data set.
[0038] FIG. 8G depicts the BLZ945 gene signature hazard ratios for
the TCGA and combined data sets, for proneural, classical,
mesenchymal, and neural GBM tumors, and highlights the statistical
correlation with proneural GBM across all of the data.
DETAILED DESCRIPTION
[0039] The invention provides compounds of Formula (I) for use to
treat brain tumors, and methods of using compounds of Formula (I)
for the treatment of brain tumors. The compounds of Formula (I)
have this formula:
##STR00003##
[0040] wherein R.sup.1 is an alkyl pyrazole or an alkyl
carboxamide; and
[0041] R.sup.2 is a hydroxycycloalkyl;
[0042] and include pharmaceutically acceptable salts as well as
neutral compounds of this formula.
[0043] Specific compounds within the scope of the invention are
further described below.
[0044] The treatment of a brain tumor can include inhibition of the
rate of growth of a brain tumor (slowing tumor growth), or reversal
of growth of a brain tumor (i.e., reduction in tumor volume), or
substantial elimination of the tumor, which has been demonstrated
by the treatment herein of mice having such tumors. In particular,
the treatment can slow progression or reverse progression of a
glioblastoma. It may be used in conjunction with other treatments
including removal of the bulk of a brain tumor, and may be used to
slow or reverse regrowth or to reduce the volume or mass of
residual tumorous tissue following brain tumor removal by surgical
or biopsy methods. The compounds may also be used in conjunction
with other chemotherapeutics.
[0045] The compounds of formula (I) include compounds wherein
R.sup.1 is an alkyl-substituted pyrazole or carboxamide, e.g., a
C1-C4 alkyl pyrazole or a carboxamide of the formula --C(O)NHR,
where R is a C1-C4 alkyl group. In preferred embodiments, the alkyl
group is Me or Et. Certain preferred compounds for use in the
invention are disclosed below. In some embodiments of these
methods, R.sup.1 is
##STR00004##
[0046] wherein R' is Me or Et. Preferably, the pyrazole ring is
attached at position 4, i.e.:
##STR00005##
[0047] In these compounds, R.sup.2 can be a hydroxycyclohexyl group
such as this:
##STR00006##
or a 2-hydroxycyclopent-1-yl group.
[0048] Specifically preferred compounds include any of the
following compounds, or a mixture of any two or more of these
compounds, or a pharmaceutically acceptable salt of any one of
these:
##STR00007##
[0049] Each of these compounds and their pharmaceutically
acceptable salts are preferred embodiments for purposes of the
present invention. Preferred embodiments of these compounds also
include compounds of these formulas:
##STR00008##
[0050] where R' is Me, Et or Propyl, preferably methyl. Specific
embodiments of these compounds can be of (R,R) absolute
stereochemistry or (S,S) absolute stereochemistry.
[0051] These compounds are expected to exhibit blood-brain barrier
penetration like BLZ945, based on their very similar
physicochemical properties, and are therefore suitable for use in
the present treatment methods.
[0052] Compounds of Formula (I) are known in the art, and methods
for making them are disclosed, for example, in WO2007/121484; their
usefulness to treat glioma and their penetration of the blood-brain
barrier were not previously known. Compound (1c) corresponds to
BLZ945, which was utilized for in vitro and in vivo tests described
herein.
[0053] Compounds of Formula (Ih) having the (1S,2S) stereochemistry
at the cyclohexyl ring are novel. These compounds are unexpectedly
good inhibitors of PDGFR.beta. while also inhibiting CSF-1R very
effectively (see data herein). Accordingly, the novel compounds of
this formula
##STR00009##
where R' is Me, Et or Propyl are another aspect of the present
invention that provide a dual-inhibitor effect that is expected to
increase effectiveness in the treatment methods disclosed
herein.
[0054] The compounds can be used alone or they can be formulated
into a pharmaceutical composition that also contains at least one
pharmaceutically acceptable excipient, and often contains at least
two pharmaceutically acceptable excipients. It will be understood
that pharmaceutically acceptable excipients are typically
sterilized. Some suitable excipients are disclosed herein; in some
embodiments, the compound is formulated as a composition comprising
captisol, e.g, 20% captisol.
[0055] In some embodiments, the brain tumor is selected from a
brain metastasis, an astrocytoma (including glioblastoma), an
oligodendroglioma, an ependymomas, and a mixed glioma. In preferred
embodiments, the brain tumor is a glioma, particularly glioblastoma
multiforme. In other embodiments, the brain tumor is a brain
metastasis, i.e., a metastatic tumor arising from a cancer that
originated elsewhere in the body.
[0056] In some embodiments, the patient is one having glioblastoma.
In specific embodiments, the subject is one diagnosed with
proneural glioblastoma. See Verhaak, et al., Cancer Cell
17(1):98-110 (2010). This subtype of glioblastoma tends to occur in
younger subjects and to involve mutations of TP53, IDH1 and PDGFRA.
Verhaak, et al. reported that patients with proneural glioblastoma
were less responsive than other subtypes (classical, neural,
mesenchymal) to the aggressive chemotherapies in use in 2010, and
even suggested that such treatment may be contraindicated for these
patients. The present methods are especially effective to treat
proneural glioblastoma, as demonstrated by the proneural GBM animal
model used herein. Specific genetic signatures found in TAMs in
mice treated with BLZ945 were found to match those of human
proneural glioblastoma patients who had longer than average median
survival times; this correlation did not occur when compared with
patients having other subtypes of glioblastoma. Thus the genetic
signature information can be used to select patients for treatment
with a CSF-1R inhibitor as described herein, or to assess prognosis
for a subject receiving such treatments.
[0057] In some embodiments, the method is used to treat a subject
before other treatment methods such as tumor removal. In other
embodiments, the method is used to treat a subject in conjunction
with other treatment methods such as tumor removal by either
surgical or biopsy methods, or in conjunction with radiation
therapy, or in conjunction with both tumor removal and radiation
therapy.
[0058] Optionally, other chemotherapeutic agents can be used along
with the compounds and methods disclosed above. Suitable additional
chemotherapeutic agents for use in these methods are those known in
the art as conventional ones for use in treating glioblastoma. Some
such chemotherapeutics include antiangiogenic agents, bevacizumab
with or without irinotecan, nitrosoureas such as Carmustine (BCNU),
platins such as cis-platinum (cisplatin), alkylating agents such as
temozolomide, tyrosine kinase inhibitors (gefitinib or erlotinib),
Ukrain, and cannabinoids. These additional therapeutic agents
(co-therapeutics) can be used simultaneously with the CSF-1R
inhibitor as by concurrent administration, admixing the
cotherapeutic with the CSF-1R inhibitor, or by sequential
administration. A preferred embodiment involves use of a compound
selected from those of Formula I disclosed herein, (e.g., Formula
Ia, Ib, Ic, Id, Ie, If, Ig or Ih) in combination with temozolomide
or a platin compound.
[0059] In addition, macrophages have been implicated in reduced
therapeutic responses in breast cancer and increased
revascularization in glioblastoma xenografts following radiation
therapy. Since these macrophage effects reduce the efficacy of
other therapies, compounds of the invention, which inhibit
macrophage activities in glioblastoma in vivo, may be expected to
provide a synergistic effect when used in combination with other
therapeutic agents or radiation therapy.
[0060] In some embodiments, the methods described herein are
practiced with a compound of Formula (Ic). In other embodiments,
the methods may be practiced with a compound of Formula (I) that is
not the compound of Formula (Ic), such as the other species
disclosed herein.
[0061] In some embodiments, the compound of Formula (I) also
inhibits at least one other target to provide enhanced antitumor
effects. For example, compounds of these formulas:
##STR00010##
also inhibit PDGFR at concentrations achieved in typical
therapeutic dosages such as those described herein. Accordingly,
these compounds can be used where a dual mechanism of action is
desired, and can be used in any of the methods described above.
[0062] Exemplary compounds of Formula Ig and Ih are included in the
following table to illustrate the relative activities on CSF-1R and
PDGFR. Many such compounds are known in the art, see WO2007/066898,
and methods to make these compounds are also well known. The
compounds of Formula I are quite active on CSF-1R regardless of the
stereochemistry at the cyclohexyl ring as shown in the table below.
Among the various isomers, the S,S isomers are also highly active
on PDGFR-.beta. as well as on CSF-1R, and thus may act on gliomas
by two mechanisms to provide enhanced efficacy.
TABLE-US-00001 (Ig) ##STR00011## (Ih) ##STR00012## CSF-1R Compound
R' Stereochem. IC-50 (.mu.M) PDGFR-.beta. IC-50 (.mu.M) Ig-A Me
(1R,2R) 0.001 5.9 Ig-B Et (1R,2R) 0.006 .mu.M 13.9 Ig-C Pr (1R,2R)
0.008 7.7 Ig-D Me (1S,2S) 0.0008 0.048 Ig-E Me (1R,2S) 0.006 6.6
Ig-F Me (1S,2R) 0.001 0.78 Ih-A Me (1R,2R) 0.0009 0.74 Ih-B Et
(1R,2R) 0.003 1.7 Ih-C Pr (1R,2R) 0.007 1.5 Ih-D Me (1S,2S) 0.001
0.02 Ih-E Me (1S,2R) 0.002 0.63
[0063] The following enumerated embodiments are representative of
the invention:
[0064] 1. A method to treat a brain tumor in a mammalian subject,
comprising administering to the subject an effective amount of a
compound of Formula (I):
##STR00013##
wherein R.sup.1 is an alkyl pyrazole or an alkyl carboxamide; and
R.sup.2 is a hydroxycycloalkyl; or a pharmaceutically acceptable
salt thereof.
[0065] 2. The method of embodiment 1, wherein R.sup.1 is
##STR00014##
[0066] wherein R' is Me or Et.
[0067] 3. The method of embodiment 1 or 2, wherein R.sup.2 is
##STR00015##
[0068] 4. The method of any of the preceding embodiments, wherein
the brain tumor is a glioma, preferably proneural glioblastoma.
[0069] 5. The method of embodiment 4, wherein the glioma is
glioblastoma multiforme.
[0070] 6. The method of any of embodiments 1-3, wherein the brain
tumor is a brain metastasis, astrocytoma (including glioblastoma),
oligodendroglioma, ependymomas, or a mixed glioma.
[0071] 7. The method of any of the preceding embodiments, wherein
the compound of formula (I) is
##STR00016##
or a pharmaceutically acceptable salt thereof; or an isolated
stereoisomer of one of these.
[0072] 8. The method of embodiment 7, wherein the compound of
Formula (I) is:
##STR00017##
[0073] 9. The method of embodiment 7, wherein the compound of
Formula (I) is:
##STR00018##
[0074] 10. The method of embodiment 7, wherein the compound of
Formula (I) is:
##STR00019##
[0075] 11. The method of embodiment 7, wherein the compound of
Formula (I) is:
##STR00020##
[0076] 12. The method of any of the preceding embodiments, wherein
the method further comprises administering to the subject an
effective amount of an additional cancer therapeutic an
antiangiogenic agents, bevacizumab with or without irinotecan,
nitrosoureas such as Carmustine (BCNU), platins such as
cis-platinum (cisplatin), alkylating agents such as temozolomide,
tyrosine kinase inhibitors (gefitinib or erlotinib), Ukrain, and
cannabinoids.
[0077] 13. The method of any of the preceding embodiments, wherein
the compound of Formula (I) is administered orally.
[0078] 14. The method of any of the preceding embodiments, wherein
the amount of the compound of Formula (I) administered to the
subject is between about 50 mg/kg per day and about 500 mg/kg per
day, or between 5-500 mg/kg, or between 100 and 300 mg/kg per
day.
[0079] 15. The method of any of the preceding embodiments, wherein
the subject has proneural glioblastoma.
[0080] 16. The method of any of the preceding embodiments, wherein
the subject is one selected because the subject has an elevated
level of PDGF or PDGFR signaling.
[0081] 17. The method of any of the preceding embodiments, wherein
the subject is contemporaneously treated with an inhibitor of
PDGFR, or is treated with a CSF-1R inhibitor having sub-nanomolar
activity as an inhibitor of PDGFR, e.g., compound (Id) or (If).
[0082] 18. The method of any of the preceding embodiments, wherein
the subject is a human.
[0083] 19. A compound of embodiment 1 for use to treat a brain
tumor.
[0084] 20. The compound of embodiment 19, wherein the brain tumor
is glioblastoma.
[0085] 21. The compound of embodiment 20, wherein the glioblastoma
is proneural glioblastoma.
[0086] 22. The compound of embodiment 20, which is formulated for
use with a cotherapeutic agent.
[0087] 23. A compound of the formula:
##STR00021##
where R' is Me, Et or Propyl.
[0088] 24. The compound of embodiment 23, wherein R' is Me.
[0089] 25. A pharmaceutical composition comprising the compound of
embodiment 23 or 24, and at least one pharmaceutically acceptable
excipient.
[0090] As used herein, the terms "salt" or "salts" refers to an
acid addition or base addition salt of a compound of the invention.
"Salts" include in particular "pharmaceutically acceptable salts".
The term "pharmaceutically acceptable salts" refers to salts that
retain the biological effectiveness and properties of the compounds
of this invention and, which typically are not biologically or
otherwise undesirable.
[0091] Pharmaceutically acceptable acid addition salts can be
formed with inorganic acids and organic acids, e.g., acetate,
aspartate, benzoate, besylate, bromide/hydrobromide,
bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate,
chloride/hydrochloride, chlortheophyllonate, citrate,
ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate,
hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate,
laurylsulfate, malate, maleate, malonate, mandelate, mesylate,
methylsulphate, naphthoate, napsylate, nicotinate, nitrate,
octadecanoate, oleate, oxalate, palmitate, pamoate,
phosphate/hydrogen phosphate/dihydrogen phosphate,
polygalacturonate, propionate, stearate, succinate,
sulfosalicylate, tartrate, tosylate and trifluoroacetate salts.
[0092] Inorganic acids from which salts can be derived include, for
example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like.
[0093] Organic acids from which salts can be derived include, for
example, acetic acid, propionic acid, glycolic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic
acid, and the like. Pharmaceutically acceptable base addition salts
can be formed with inorganic and organic bases.
[0094] Inorganic bases from which salts can be derived include, for
example, ammonium salts and metals from columns I to XII of the
periodic table. In certain embodiments, the salts are derived from
sodium, potassium, ammonium, calcium, magnesium, iron, silver,
zinc, and copper; particularly suitable salts include ammonium,
potassium, sodium, calcium and magnesium salts.
[0095] Organic bases from which salts can be derived include, for
example, primary, secondary, and tertiary amines, substituted
amines including naturally occurring substituted amines, cyclic
amines, basic ion exchange resins, and the like. Certain organic
amines include isopropylamine, benzathine, cholinate,
diethanolamine, diethylamine, lysine, meglumine, piperazine and
tromethamine.
[0096] The pharmaceutically acceptable salts of the present
invention can be prepared by conventional chemical methods.
Generally, such salts can be prepared by reacting free acid forms
of these compounds with a stoichiometric amount of the appropriate
base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or
the like), or by reacting free base forms of these compounds with a
stoichiometric amount of the appropriate acid. Such reactions are
typically carried out in water or in an organic solvent, or in a
mixture of the two. Generally, use of non-aqueous media like ether,
ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable,
where practicable. Lists of additional suitable salts can be found,
e.g., in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack
Publishing Company, Easton, Pa., (1985); and in HANDBOOK OF
PHARMACEUTICAL SALTS: PROPERTIES, SELECTION, AND USE by Stahl and
Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
[0097] Any formula given herein is intended to represent unlabeled
forms as well as isotopically labeled forms of the compounds.
Isotopically labeled compounds have structures depicted by the
formulas given herein except that one or more atoms are replaced by
an atom having a selected atomic mass or mass number. Examples of
isotopes that can be incorporated into compounds of the invention
include isotopes of hydrogen, carbon, nitrogen, oxygen,
phosphorous, fluorine, and chlorine, such as .sup.2H, .sup.3H,
.sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.18F, .sup.31P,
.sup.32P, .sup.35S, .sup.36Cl, .sup.125I respectively. In preferred
embodiments, the compounds of the invention are unlabeled, i.e.,
they comprise approximately natural isotope abundances for all
atoms. In other embodiments, the compounds of the invention are
labeled by selective incorporation of an enriched non-natural
isotope for one atom in the compound of Formula (I). The invention
includes various isotopically labeled compounds as defined herein,
for example those into which radioactive isotopes, such as .sup.3H
and .sup.14C, or those into which non-radioactive isotopes, such as
.sup.2H and .sup.13C are present. Such isotopically labelled
compounds are useful in metabolic studies (with .sup.14C), reaction
kinetic studies (with, for example .sup.2H or .sup.3H), detection
or imaging techniques, such as positron emission tomography (PET)
or single-photon emission computed tomography (SPECT) including
drug or substrate tissue distribution assays, or in radioactive
treatment of patients. In particular, an .sup.18F or labeled
compound may be particularly desirable for PET or SPECT studies.
Isotopically-labeled compounds of formula (I) can generally be
prepared by conventional techniques known to those skilled in the
art in view of the description of synthesis of the compounds of
Formula I in, for example, U.S. patent publication no.
US200810045528 (WO2007/121484). BLZ945 is described in that
reference as well as several of its isomers. Examples 173 and 174
in that reference describe synthesis of pyrazole compound (Ie)
using 1R,2R-aminocyclohexanol, and can be adapted for synthesis of
other pyrazole compounds of Formula I, both labeled and unlabeled.
The same publication at page 163 describes synthesis of both 1R,2R-
and 1S,2S-aminocyclohexanol, which can readily be substituted into
the method of Example 173 to produce (If) and other compounds of
Formula I, both labeled and unlabeled.
[0098] Further, substitution with heavier isotopes, particularly
deuterium (i.e., .sup.2H or D) may afford certain therapeutic
advantages resulting from greater metabolic stability, for example
increased in vivo half-life or reduced dosage requirements or an
improvement in therapeutic index. It is understood that deuterium
in this context is regarded as a substituent of a compound of the
formula (I). The concentration of such a heavier isotope,
specifically deuterium, may be defined by the isotopic enrichment
factor. The term "isotopic enrichment factor" as used herein means
the ratio between the isotopic abundance and the natural abundance
of a specified isotope. If a substituent in a compound of this
invention is denoted deuterium, such compound has an isotopic
enrichment factor for each designated deuterium atom of at least
3500 (52.5% deuterium incorporation at each designated deuterium
atom), at least 4000 (60% deuterium incorporation), at least 4500
(67.5% deuterium incorporation), at least 5000 (75% deuterium
incorporation), at least 5500 (82.5% deuterium incorporation), at
least 6000 (90% deuterium incorporation), at least 6333.3 (95%
deuterium incorporation), at least 6466.7 (97% deuterium
incorporation), at least 6600 (99% deuterium incorporation), or at
least 6633.3 (99.5% deuterium incorporation).
[0099] As used herein, the term "pharmaceutically acceptable
excipients" includes any and all solvents, dispersion media,
coatings, surfactants, antioxidants, preservatives (e.g.,
antibacterial agents, antifungal agents), isotonic agents,
absorption delaying agents, salts, preservatives, drug stabilizers,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, and the like and combinations
thereof, as would be known to those skilled in the art (see, for
example, Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, pp. 1289-1329). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated.
[0100] The term "a therapeutically effective amount" of a compound
of the present invention refers to an amount of the compound of the
present invention that will elicit the biological or medical
response of a subject, for example, reduction or inhibition of an
enzyme or a protein activity, or ameliorate symptoms, alleviate
conditions, slow or delay disease progression, or prevent a
disease, etc. In one non-limiting embodiment, the term "a
therapeutically effective amount" refers to the amount of the
compound of the present invention that, when administered to a
subject, is effective to (1) at least partially alleviating,
inhibiting, preventing and/or ameliorating a condition, or a
disorder or a disease (i) mediated by CSF-1R, or (ii) associated
with CSF-1R activity, or (iii) characterized by activity (normal or
abnormal) of CSF-1R; or (2) reducing or inhibiting the activity of
CSF-1R; or (3) reducing or inhibiting the expression of CSF-1R. In
another non-limiting embodiment, the term "a therapeutically
effective amount" refers to the amount of the compound of the
present invention that, when administered to a cell, or a tissue,
or a non-cellular biological material, or a medium, is effective to
at least partially reducing or inhibiting the activity of CSF-1R;
or at least partially reducing or inhibiting the expression of
CSF-1R. The meaning of the term "a therapeutically effective
amount" as illustrated in the above embodiment for CSF-1R also
applies by the same means to any other relevant
proteins/peptides/enzymes, such as PDGFR and the like.
[0101] As used herein, the term "subject" refers to an animal.
Typically the animal is a mammal. A subject also refers to for
example, primates (e.g., humans, male or female), cows, sheep,
goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the
like. In certain embodiments, the subject is a primate. In
preferred embodiments, the subject is a human.
[0102] As used herein, the term "inhibit", "inhibition" or
"inhibiting" refers to the reduction or suppression of a given
condition, symptom, or disorder, or disease, or a significant
decrease in the baseline activity of a biological activity or
process.
[0103] As used herein, the term "treat", "treating" or "treatment"
of any disease or disorder refers in one embodiment, to
ameliorating the disease or disorder (i.e., slowing or arresting or
reducing the development of the disease or at least one of the
clinical symptoms thereof). In another embodiment "treat",
"treating" or "treatment" refers to alleviating or ameliorating at
least one physical parameter including those which may not be
discernible by the patient. In yet another embodiment, "treat",
"treating" or "treatment" refers to modulating the disease or
disorder, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter), or both. In yet another embodiment, "treat", "treating"
or "treatment" refers to preventing or delaying the onset or
development or progression of the disease or disorder. In reference
to a brain tumor, `treating` typically includes either slowing rate
of growth of a tumor or of regrowth of a tumor after the bulk of
the tumor has been removed, or reducing the size of the tumor or of
remnants of the tumor after the bulk of the tumor has been
removed.
[0104] As used herein, a subject is "in need of" a treatment if
such subject would benefit biologically, medically or in quality of
life from such treatment. Typically the subject has been diagnosed
with a brain tumor, frequently a form of glioblastoma, and
preferably with glioblastoma multiforme.
[0105] As used herein, the term "a," "an," "the" and similar terms
used in the context of the present invention (especially in the
context of the claims) are to be construed to cover both the
singular and plural unless otherwise indicated herein or clearly
contradicted by the context.
[0106] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed.
[0107] In some embodiments, the present invention utilizes a
pharmaceutical composition comprising a compound of the present
invention and a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition can be formulated for particular
routes of administration such as oral administration, parenteral
administration, and rectal administration, etc. In addition, the
pharmaceutical compositions of the present invention can be made up
in a solid form (including without limitation capsules, tablets,
pills, granules, powders or suppositories), or in a liquid form
(including without limitation solutions, suspensions or emulsions).
The pharmaceutical compositions can be subjected to conventional
pharmaceutical operations such as sterilization and/or can contain
conventional inert diluents, lubricating agents, or buffering
agents, as well as adjuvants, such as preservatives, stabilizers,
wetting agents, emulsifiers and buffers, etc.
[0108] In some embodiments, the pharmaceutical composition
comprises at least one additional chemotherapeutic agent such as
temozolomide, in an effective amount.
[0109] Typically, the pharmaceutical compositions are tablets or
gelatin capsules comprising the active ingredient together with at
least one excipient, such as captisol (used in the Examples herein)
one of the following: [0110] a) diluents, e.g., lactose, dextrose,
sucrose, mannitol, sorbitol, cellulose and/or glycine; [0111] b)
lubricants, e.g., silica, talcum, stearic acid, its magnesium or
calcium salt and/or polyethyleneglycol; for tablets also [0112] c)
binders, e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone; if desired; [0113] d) carriers such as an
aqueous vehicle containing a co-solvating material such as
captisol, PEG, glycerin, cyclodextrin, or the like; [0114] e)
disintegrants, e.g., starches, agar, alginic acid or its sodium
salt, or effervescent mixtures; and/or [0115] f) absorbents,
colorants, flavors and sweeteners.
[0116] Tablets may be either film coated or enteric coated
according to methods known in the art.
[0117] Preferably, the compound or composition is prepared for oral
administration, as a tablet or capsule, for example, or as a
solution or suspension of the compound of Formula (I), optionally
packaged in a single-dose container such as a capsule.
[0118] Suitable compositions for oral administration include an
effective amount of a compound of the invention in the form of
tablets, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsion, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use are prepared according to any
method known in the art for the manufacture of pharmaceutical
compositions and such compositions can contain one or more agents
selected from the group consisting of sweetening agents, flavoring
agents, coloring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets may
contain the active ingredient in admixture with nontoxic
pharmaceutically acceptable excipients which are suitable for the
manufacture of tablets. These excipients are, for example, inert
diluents, such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate; granulating and
disintegrating agents, for example, corn starch, or alginic acid;
binding agents, for example, starch, gelatin or acacia; and
lubricating agents, for example magnesium stearate, stearic acid or
talc. The tablets are uncoated or coated by known techniques to
delay disintegration and absorption in the gastrointestinal tract
and thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate can be employed. Formulations for oral use can
be presented as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent, for example, calcium
carbonate, calcium phosphate or kaolin, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil medium,
for example, peanut oil, liquid paraffin or olive oil.
[0119] In some embodiments, the compound or composition is prepared
to be administered by injection. Certain injectable compositions
are aqueous isotonic solutions or suspensions, and suppositories
are advantageously prepared from fatty emulsions or suspensions.
Said compositions may be sterilized and/or contain adjuvants, such
as preserving, stabilizing, wetting or emulsifying agents, solution
promoters, salts for regulating the osmotic pressure and/or
buffers. In addition, they may also contain other therapeutically
valuable substances. Said compositions are prepared according to
conventional mixing, granulating or coating methods, respectively,
and contain about 0.1-75%, or contain about 1-50%, of the active
ingredient.
[0120] In some embodiments, the compound or composition is prepared
to be administered topically. Suitable compositions for transdermal
application include an effective amount of a compound of the
invention with a suitable carrier. Carriers suitable for
transdermal delivery include absorbable pharmacologically
acceptable solvents to assist passage through the skin of the host.
For example, transdermal devices are in the form of a bandage
comprising a backing member, a reservoir containing the compound
optionally with carriers, optionally a rate controlling barrier to
deliver the compound of the skin of the host at a controlled and
predetermined rate over a prolonged period of time, and means to
secure the device to the skin.
[0121] Suitable compositions for topical application, e.g., to the
skin and eyes, include aqueous solutions, suspensions, ointments,
creams, gels or sprayable formulations, e.g., for delivery by
aerosol or the like. Such topical delivery systems will in
particular be appropriate for dermal application, e.g., for the
treatment of skin cancer, e.g., for prophylactic use in sun creams,
lotions, sprays and the like. They are thus particularly suited for
use in topical, including cosmetic, formulations well-known in the
art. Such may contain solubilizers, stabilizers, tonicity enhancing
agents, buffers and preservatives.
[0122] As used herein a topical application may also pertain to an
inhalation or to an intranasal application. They may be
conveniently delivered in the form of a dry powder (either alone,
as a mixture, for example a dry blend with lactose, or a mixed
component particle, for example with phospholipids) from a dry
powder inhaler or an aerosol spray presentation from a pressurized
container, pump, spray, atomizer or nebulizer, with or without the
use of a suitable propellant.
[0123] In some embodiments, the effective amount of the compound of
Formula (I) is between about 10 mg/kg per day, and about 500 mg/kg
per day. In particular embodiments, the effective amount is between
about 25 mg/kg per day and about 300 mg/kg per day, such as about
100 to about 250 mg/kg per day. The dosage may be administered in
1-4 doses per day, or it may be administered on alternating days.
In a preferred embodiment, the dosage is about 200 mg/kg per day,
and is administered in one or two oral doses per day.
[0124] The present invention further provides anhydrous
pharmaceutical compositions and dosage forms comprising the
compounds of the present invention as active ingredients, since
water may facilitate the degradation of certain compounds.
[0125] Anhydrous pharmaceutical compositions and dosage forms of
the invention can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
An anhydrous pharmaceutical composition may be prepared and stored
such that its anhydrous nature is maintained. Accordingly,
anhydrous compositions are 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.
[0126] The invention further provides pharmaceutical compositions
and dosage forms that comprise one or more agents that reduce the
rate by which the compound of the present invention as an active
ingredient 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,
etc.
[0127] The compounds and methods described herein are useful to
treat a variety of brain tumors, based on their demonstrated
ability to penetrate the blood-brain barrier and to inhibit
accumulation of TAMs in and/or around a tumor in the brain. In some
embodiments, the brain tumor is a metastasis of a cancer that
originated elsewhere in the body. In other embodiments, the brain
tumor is a glioma such as glioblastoma multiforme.
[0128] The compounds of formula I in free form or in salt form,
exhibit valuable pharmacological properties, e.g. CSF-1R and
optionally PDGFR modulating properties, e.g. as indicated in in
vitro and in vivo tests as provided in the next sections and are
therefore indicated for therapy.
[0129] Thus, as a further embodiment, the present invention
provides the use of a compound of formula (I) or in therapy. In a
further embodiment, the therapy is selected from a disease which
may be treated by inhibition of CSF-1R. In another embodiment, the
disease is selected from the afore-mentioned list, suitably any
brain tumor, more suitably a glioblastoma such as glioblastoma
multiforme.
[0130] In another embodiment, the invention provides a method of
treating a disease which is treated by inhibition of CSF-1R,
comprising administration of a therapeutically acceptable amount of
a compound of formula (I) or any of the embodiments of these
compounds disclosed herein. In a further embodiment, the disease is
selected from the afore-mentioned list, suitably a brain tumor,
such as one of the gliomas, specifically including glioblastoma
multiforme.
[0131] The pharmaceutical composition or combination of the present
invention can be in unit dosage of about 1-1000 mg of active
ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or
about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50
mg of active ingredients. The therapeutically effective dosage of a
compound, the pharmaceutical composition, or the combinations
thereof, is dependent on the species of the subject, the body
weight, age and individual condition, the disorder or disease or
the severity thereof being treated. A physician, clinician or
veterinarian of ordinary skill can readily determine the effective
amount of each of the active ingredients necessary to treat or
inhibit the progress of the disorder or disease based on the
present disclosure.
[0132] The above-cited dosage properties are demonstrable in vitro
and in vivo tests using advantageously mammals, e.g., mice, rats,
dogs, monkeys or isolated organs, tissues and preparations thereof.
The compounds of the present invention can be applied in vitro in
the form of solutions, e.g., aqueous solutions, and in vivo either
enterally, parenterally, advantageously intravenously, e.g., as a
suspension or in aqueous solution. The dosage in vitro may range
between about 10.sup.-3 molar and 10.sup.-9 molar concentrations. A
therapeutically effective amount in vivo may range depending on the
route of administration, between about 0.1-500 mg/kg, typically
10-400 mg/kg, or between about 100-300 mg/kg, or between 1-100
mg/kg. In some embodiments, a dose of about 200 mg/kg is suitable
for treatment of glioblastoma, and can be administered orally.
[0133] The activity of a compound according to the present
invention can be assessed by the following in vitro & in vivo
methods.
[0134] Using the test assay methods described in US20080045528, the
compounds of the invention can be shown to inhibit CSF-1R. As
described herein these compounds readily traverse the blood-brain
barrier, and also inhibit or reverse growth of a tumor in the
brain. Preferably the tumor is detectable by known methods, and
progress of treatment can be monitored by known methods. In some
embodiments, the progress of the treatment is monitored by using
MRI (magnetic resonance imaging) to determine the size of the tumor
and any metastases.
[0135] The compound of the present invention may be administered
either simultaneously with, or before or after, one or more other
therapeutic agents such as the cotherapeutic agents described
herein. The compound of the present invention may be administered
separately, by the same or different route of administration, or
together in the same pharmaceutical composition as the other
agents.
[0136] In one embodiment, the invention provides a product
comprising a compound of formula (I) and at least one other
therapeutic agent as a combined preparation for simultaneous,
separate or sequential use in therapy. In one embodiment, the
therapy is the treatment of a disease or condition mediated by
inhibition of CSF-1R. Products provided as a combined preparation
include a composition comprising the compound of formula (I) and
the other therapeutic agent(s) together in the same pharmaceutical
composition, or the compound of formula (I) and the other
therapeutic agent(s) in separate form, e.g. in the form of a
kit.
[0137] In one embodiment, the invention provides a pharmaceutical
composition comprising a compound of formula (I) and another
therapeutic agent(s). Optionally, the pharmaceutical composition
may comprise a pharmaceutically acceptable excipient, as described
above, or more than one such cotherapeutic agent.
[0138] In one embodiment, the invention provides a kit comprising
two or more separate pharmaceutical compositions, at least one of
which contains a compound of formula (I). In one embodiment, the
kit comprises means for separately retaining said compositions,
such as a container, divided bottle, or divided foil packet. An
example of such a kit is a blister pack, as typically used for the
packaging of tablets, capsules and the like.
[0139] The kit of the invention may be used for administering
different dosage forms, for example, oral and parenteral, for
administering the separate compositions at different dosage
intervals, or for titrating the separate compositions against one
another. To assist compliance, the kit of the invention typically
comprises directions for administration.
[0140] In the combination therapies of the invention, the compound
of the invention and the other therapeutic agent may be
manufactured and/or formulated by the same or different
manufacturers. Moreover, the compound of the invention and the
other therapeutic may be brought together into a combination
therapy: (i) prior to release of the combination product to
physicians (e.g. in the case of a kit comprising the compound of
the invention and the other therapeutic agent); (ii) by the
physician themselves (or under the guidance of the physician)
shortly before administration; (iii) in the patient themselves,
e.g. during sequential administration of the compound of the
invention and the other therapeutic agent.
[0141] Accordingly, the invention provides the use of a compound of
formula (I) for treating a disease or condition mediated by CSF-1R,
wherein the medicament is prepared for administration with another
therapeutic agent, including one of the additional chemotherapeutic
agents disclosed herein as suitable for use in combination with
compounds of Formula I. The invention also provides the use of
another therapeutic agent for treating a disease or condition
mediated by CSF-1R wherein the medicament is administered with a
compound of formula (I).
[0142] The invention also provides a compound of formula (I) for
use in a method of treating a disease or condition mediated by
CSF-1R], wherein the compound of formula (I) is prepared for
administration with another therapeutic agent. The invention also
provides another therapeutic agent for use in a method of treating
a disease or condition mediated by CSF-1R, wherein the other
therapeutic agent is prepared for administration with a compound of
formula (I). The invention also provides a compound of formula (I)
for use in a method of treating a disease or condition mediated by
CSF-1R wherein the compound of formula (I) is administered with
another therapeutic agent. The invention also provides another
therapeutic agent for use in a method of treating a disease or
condition mediated by CSF-1R, wherein the other therapeutic agent
is administered with a compound of formula (I).
[0143] The invention also provides the use of a compound of formula
(I) for treating a disease or condition mediated by CSF-1R wherein
the patient has previously (e.g. within 24 hours) been treated with
another therapeutic agent. The invention also provides the use of
another therapeutic agent for treating a disease or condition
mediated by CSF-1R wherein the patient has previously (e.g. within
24 hours) been treated with a compound of formula (I).
[0144] In one embodiment, the other therapeutic agent is selected
from an antiangiogenic agents, bevacizumab with or without
irinotecan, nitrosoureas such as Carmustine (BCNU), platins such as
cis-platinum (cisplatin), alkylating agents such as temozolomide,
tyrosine kinase inhibitors (gefitinib or erlotinib), Ukrain, and
cannabinoids. In some embodiments, the other agent is a
cotherapeutic agent selected from: an antiangiogenic compound, a
cannabinoid, and temozolomide.
[0145] Specific individual combinations which may provide
particular treatment benefits include compound Ia, Ib, Ic, Id, Ie,
If, Ig, or Ih, in combination with temozolomide. This combination
may be administered orally as described herein to treat various
brain tumors, such as glioblastoma multiforme.
[0146] In addition to the treatment methods, compounds and
pharmaceutical composition, certain gene signature changes
associated with efficacy of the CSF-1R compounds for treatment of
GBM have also been identified. The Examples below provide
information about these changes and identify gene signatures or
biomarkers that can be used in conjunction with the treatment
methods disclosed herein. As will be evident to the skilled reader,
the Lasso signature and SVM signature data provided herein can be
used in the determination of a prognosis for a patient treated with
these methods by obtaining a sample from the patient and comparing
gene expression data for the sample against the gene expression
changes and signatures disclosed herein as correlating with
positive prognosis and/or prolonged survival.
EXAMPLES
[0147] Compounds of the invention were prepared according to
methods known in the art, particularly those described in
WO2007/121484.
[0148] The compounds and/or intermediates were characterized by
high performance liquid chromatography (HPLC) using a Waters
Millenium chromatography system with a 2695 Separation Module
(Milford, Mass.). The analytical columns were reversed phase
Phenomenex Luna C18-5, 4.6.times.50 mm, from Alltech (Deerfield,
Ill.). A gradient elution was used (flow 2.5 mL/min), typically
starting with 5% acetonitrile/95% water and progressing to 100%
acetonitrile over a period of 10 minutes. All solvents contained
0.1% trifluoroacetic acid (TFA). Compounds were detected by
ultraviolet light (UV) absorption at either 220 or 254 nm. HPLC
solvents were from Burdick and Jackson (Muskegan, Mich.), or Fisher
Scientific (Pittsburgh, Pa.).
[0149] Mass spectrometric analysis was performed on one of two LCMS
instruments: a Waters System (Alliance HT HPLC and a Micromass ZQ
mass spectrometer; Column: Eclipse XDB-C18, 2.1.times.50 mm;
gradient: 5-95% (or 35-95%, or 65-95% or 95-95%) acetonitrile in
water with 0.05% TFA over a 4 min period; flow rate 0.8 mL/min;
molecular weight range 200-1500; cone Voltage 20 V; column
temperature 40.degree. C.) or a Hewlett Packard System (Series 1100
HPLC; Column: Eclipse XDB-C18, 2.1.times.50 mm; gradient: 5-95%
acetonitrile in water with 0.05% TFA over a 4 min period; flow rate
0.8 mL/min; molecular weight range 150-850; cone Voltage 50 V;
column temperature 30.degree. C.). All masses were reported as
those of the protonated parent ions.
[0150] Analytical Data for Compound (If): HPLC retention time 1.93
min. Molecular Ion (MH+): m/z=422.1 (LC/MS RT=0.50 min).
Example 1
Macrophage Numbers are Increased in a Mouse Model of Gliomagenesis
Compared to Normal Brain
[0151] This example demonstrated the contribution of
tumor-associated macrophages (TAMs) to gliomagenesis in the
RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf.sup.-/- mouse model. In these
mice, when tumor development is induced in adults, the vast
majority of lesions that develop are high-grade glioblastoma
multiforme (GBM), which histologically models human GBM. FIG. 1.
(A) Cerebrum/forebrain from uninjected Nestin-Tv-a;Ink4a/Arf-/-
mice (normal brain) or grade IV tumors (GBM) from symptomatic
RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf-/- (PDG) mice were processed
to a single cell suspension with papain for flow cytometry (n=5
each). There was a significant increase in CD45+ leukocytes from
3.6.+-.0.6% to 13.1.+-.2.0%. CD11b+ myeloid cells/macrophages
accounted for the overwhelming majority of leukocytes (89.9-98.5%
of CD45+ cells), with a 3.8-fold increase in CD45+CD11b+ cells in
the tumors (12.7.+-.2.0%) compared to normal brain (3.3.+-.0.5%),
and no differences in the populations of CD45+CD11b-cells. (B)
Normal brain or GBM tissue sections from symptomatic PDG mice were
immunofluorescently co-stained for CSF-1R, CD68 (macrophages), and
DAPI. (C) Normal brain and GBM tumors (n=3 each) were used for RNA
isolation, cDNA synthesis, and qPCR. Assays were run in triplicate
and expression normalized to ubiquitin C (Ubc) for each sample.
Expression is depicted relative to normal brain. (D) Normal brain
or GBM tissue sections from symptomatic PDG mice were stained for
CSF-1R in combination with the macrophage markers F4/80 and CD11b
as well as F4/80, CD11b, and CD68 in combination with Iba-1
(macrophages/microglia). DAPI was used for the nuclear
counterstain. Scale bar, 50 .mu.m. Data are presented as mean+SEM.
P values were obtained using unpaired two-tailed Student's t-test;
*P<0.05; **P<0.01.
[0152] Numbers of macrophage cells were substantially higher in GBM
tissue relative to normal brain, as shown by staining with the
macrophage-specific antibody CD68 (FIG. 1B). This was confirmed by
flow cytometry analysis, in which tumor-associated leukocytes
(CD45.sup.+) constitute 13.1% of the tumor mass, and the vast
majority are macrophages (CD11b.sup.+) (FIG. 1A). Expression
analysis of normal brain compared to GBM revealed that the mRNA
level of CSF-1 and CSF-1R, as well as CD68, increases in tumors
(FIG. 1C).
[0153] The different cell type-specific populations were also from
GBMs to determine the source of CSF-1 and its receptor. The purity
of the distinct populations was confirmed by expression of the TVA
receptor only in the tumor cell fraction and CD11b solely in the
TAMs. While CSF-1 was expressed by both tumor cells and TAMs,
CSF-1R was only expressed by TAMs (FIG. 1D). The first column in
each group of three in FIG. 1D is Mixed cells, the second is
FACS-purified tumor cells, and the third is FACS-purified TAMs;
Mixed cells are set to 1 to normalize the data. The graphs show no
CD11b expression in tumor cells and no CSF-1R expression in tumor
cells, while TVA stains tumor cells only, not TAMs, and CSF-1 is
present in approximately equal amounts in both tumor and TAM cells.
These findings were confirmed by immunostaining, and all
CSF-1R.sup.+ cells were also positive for CD68 (not shown). This
demonstrates that any effects on tumorigenesis following CSF-1R
inhibition in this model are macrophage dependent.
Example 2
Analysis of the CSF-1R Inhibitor BLZ945: Pharmacokinetics and
Cell-Based Assays
[0154] BLZ945 (Compound Ic) has been disclosed as a selective c-fms
(CSF-1R) kinase inhibitor for the suppression of tumor-induced
osteolytic lesions in bone. BLZ945 is an ATP competitive inhibitor
that inhibits CSF-1R in biochemical assays at 1 nM, and inhibits
CSF-dependent cell proliferation at an IC-50 of about 67 nM. By
comparison, the IC50 values for most of >200 miscellaneous
kinases tested are >10 .mu.M (10,000 nM), and for cKIT and
PDGFR.beta. the IC-50's are 3.5 .mu.M (3500 nM) and 5.9 .mu.M (5900
nM) respectively. When screened against several hundred kinases in
the Ambit.RTM. kinase array, the compound showed activity lower
than 50% of control only against CSF-1R, PDGFR.alpha. and
PDGFR.beta., and the activity on the two PDGFRs was far lower than
its activity on CSF-1R in direct inhibition assays. As discussed
herein, compounds like BLZ945 but having the (S,S) stereochemistry
exhibit activity against PDGFR.beta. at levels similar to their
high level of activity on CSF-1R.
[0155] Mice having GBM detectable in only the right half of their
brains were treated with BLZ945, and the concentration of compound
in plasma, and in the right and left halves of the brain were then
measured at various time points (15 mins, 2 hr, 8 hr, 24 hr). As
FIG. 2 shows, the plasma concentration rises rapidly to a little
over 100 uM and remains above 50 uM at 8 hr, then declines to a low
level by 24 hr. The concentration in brain tissue follows a similar
pattern: it remains a little lower than the plasma level, but rises
well above 50 uM at the 15 min and 2 hr time points. This shows
that BLZ945 crosses the blood-brain barrier (BBB), and that
concentrations sufficient to inhibit macrophage growth and/or
survival can be achieved in the brain. It also shows that the
compound penetrates at similar levels into tumor-containing and
tumor-free halves of the brain, suggesting that penetration may not
depend on a lesion in the BBB caused by the presence of the tumor.
This demonstrates sufficiently rapid penetration of the blood-brain
barrier to provide therapeutically effective drug levels in the
brain, well above the levels needed to effectively inhibit
macrophages in culture.
Example 3
Inhibitory Activity of BLZ945 Against Different Cell Types In
Vitro
[0156] Bone marrow-derived macrophages (BMDMs) were isolated and
differentiated as previously described in the literature, and were
then treated with 67 nM BLZ945. BLZ945 caused a clear inhibition of
CSF-1R phosphorylation following CSF-1 stimulation (FIG. 3A) at
each time point (1.5 min, 3 min, 5 min).
[0157] The effects of BLZ945 on macrophages were also examined: a
range of doses, from 67 nM to 6700 nM dramatically blocked
macrophage survival, comparable to the effects of CSF-1 withdrawal
(FIG. 3B).
[0158] BMDMs from Ink4a/Arf null mice (the genetic background of
the GBM model), were also tested in the presence and absence of
BLZ945. FIG. 3C shows that these BMDMs, like those from the
wild-type mice, were substantially inhibited by concentrations of
BLZ945 of 67 nM and above (FIG. 3D). Thus, BLZ945 is an effective
inhibitor of CSF-1R signaling, which leads to a complete block in
macrophage viability. FIGS. 3C-3E demonstrate that proliferation of
BMDM cells from the Ink4a/Arf-/- mice as strongly inhibited at
concentrations of BLZ945 of 67 nM and above, as were CRL-2467 cells
(normal mouse brain), while even at 6700 nM it has little or no
effect on proliferation of four mouse and one human glioblastoma
cell cultures
[0159] To determine the lack of a direct effect of BLZ945 on tumor
cells, a human glioma cell line and a series of primary tumor cells
and neurospheres were treated with BLZ945 at similar concentrations
to those found effective against macrophage growth. U87-MG cells,
derived from a human GBM, which have been shown to be dependent on
PDGFR signaling in culture and in vivo, were not affected by BLZ945
treatment at the same doses as above (FIG. 3E). Similarly, the
formation of secondary neurospheres from primary neurospheres
(derived from mouse RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf.sup.-/-
GBMs) was not altered by BLZ945 treatment (FIG. 3F). Neither the
number nor the size of neurospheres were significantly affected by
BLZ945. Finally, the effects of BLZ945 on multiple tumor cell lines
that were established from secondary mouse GBM neurospheres were
examined, and again, there were no differences (FIG. 3F).
Collectively, these experiments demonstrate that the effects of
CSF-1R inhibition by BLZ945 are specific to macrophages, with no
discernible direct consequences on tumor cells.
Example 4
Treatment with the CSF-1R Inhibitor BLZ945 Blocks Glioma
Progression
[0160] Given the potent inhibitory effects of BLZ945 in macrophage
cell-based assays, and its demonstrated ability to cross the
blood-brain barrier, it appeared desirable to test this inhibitor
in preclinical trials in the
RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf.sup.-/- model. These
genetically engineered mice were injected at 5-6 weeks of age with
RCAS-PDGF-B-HA virus-infected DF-1 cells to initiate glioma
formation as described (Hambardzumyan, et al., Transl. Oncol., vol.
2, 89-95 (2009). At 2.5 weeks following tumor initiation, cohorts
of mice were dosed via oral gavage daily with either 200 mg/kg
BLZ945 in 20% captisol, or the vehicle (20% captisol) as a control.
The mice were subsequently evaluated for symptom-free survival. The
median survival in the vehicle treated cohort was 5.71 weeks (40
days), whereas 64.4% of the BLZ945 treated cohort were still alive
at the trial endpoint of 26 weeks post-injection (31-32 weeks of
age) (FIG. 4A, P<0.0001). This endpoint was chosen because mice
in the Ink4a/Arf.sup.-/- background start developing spontaneous
tumors, mostly lymphomas and sarcomas, around 30 weeks of age,
which would complicate interpretation of the glioma phenotype in
longer studies. The data in FIG. 4A shows that none of the control
mice (vehicle only) were symptom free by 8 weeks after virus
injection, while over half of the treated mice were symptom free at
the endpoint of 26 weeks. Note: 4 treated mice were sacrificed at
12 weeks for histology studies. Of these, 3 were tumor free, and
one had a grade II glioma.
[0161] Tumor Grades were determined for the mice in both cohorts of
mice (see FIG. 4B). All vehicle-treated mice at end stage had
high-grade tumors, with Grade IV GBM lesions in 13 of 14 mice. In
contrast, the BLZ945 treated animals had significantly less
malignant tumors: 80% were either Grade II or tumor free; the
remaining 20% had Grade III tumor. In 56% of the mice alive at the
26-week trial endpoint, there were no detectable lesions (FIG. 4B).
Five of the BLZ945 treated mice were sacrificed as symptomatic
during the trial (n=5), and compared to the group that were still
asymptomatic when sacrificed at the end of the trial (n=9). In both
groups, there was still a significant decrease in tumor grade
compared to the vehicle-treated animals. This shows a dramatic
increase in survival and reduction in tumor malignancy in this
long-term trial with BLZ945 treatment.
Example 5
MRI Imaging to Monitor Effects of BLZ945 on Tumor Growth
[0162] A short-term, 7 day trial of BLZ945 in tumor-induced mice
was monitored by regular MRI scans to measure tumor size changes
during a short treatment period when tumor growth is normally
rapid. Tumor volume in the
RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf.sup.-/- mice was determined by
MRI, and mice were added to the trial when this was at least 4.5
mm.sup.3 or greater. Mice were treated with BLZ945 or the vehicle
control for 7 days, as described above. MRI scans were performed on
the day before treatment was initiated, at the mid-point of the
treatment, and at the day before the end of the trial period.
Vehicle-treated mice showed a progressive, dramatic increase in
tumor volume over this short trial, as shown in FIG. 5A, with the
average tumor volume increasing about 5-fold. BLZ945 treatment
blocked tumor progression as determined by MRI (FIG. 5A), with no
increase in tumor size over the same short period. Treated
subjects, (lower line) showed little or no tumor enlargement, while
tumor volume increased sharply in the vehicle-treated controls.
FIG. 5B shows tumor volume for individual control mice (upper
graph) and treated mice (lower graph) during the first 6 days after
dosing with BLZ945 started. Nearly all of the BLZ945 treated
animals show little or no increase in tumor size, while all of the
control animals show large increases in tumor volume.
[0163] As shown in FIG. 5B, untreated tumors increased by about
150-850% in volume during this time, while tumor size was reduced
in 7 of 11 treated animals and only two of the treated animals had
tumor volume increases over 50%. FIGS. 5-2A and 5-2B depict the
tumor volume data for all 11 test and control animals, and show
that treatment largely stopped tumor size increases, while
untreated tumors grew substantially in the 6-day treatment. These
results indicate that CSF-1R signaling, and the presumed
contribution of CSF-1R-dependent macrophages, is critical for
glioma progression in this mouse model, and that BLZ945 can prevent
growth of a brain tumor in a highly relevant mammalian model for
human glioblastoma.
[0164] In a second in vivo test on larger tumors in the same GBM
model ("large tumor" cohort), mice with tumor volumes of 48.7 to
132 mm.sup.3 were treated with BLZ945, and changes in tumor volume
were monitored by MRI over a span of 6 days. Tumor volume actually
decreased in nearly all test animals, and 6 of 18 treated mice had
a reduction of at least 30% in tumor size (FIGS. 5D and 5-2C).
Control animals were not included in this test, because they would
not have been expected to survive to the endpoint.
Example 6
Analysis of Hallmark Capabilities of Cancer in BLZ945 Treated
Tumors
[0165] The identification of a striking effect of CSF-1R inhibition
on gliomagenesis led us to investigate the underlying mechanisms
for this response and determine how BLZ945 treatment affected
several of the hallmark capabilities of cancer. The analyses were
performed on tissues from the short-term trial (see Example 5), so
that tumors from the different treatment groups could be compared
at the same defined endpoint. Tumor cell density was examined using
the oligodendrocyte marker Olig2, which has previously been used to
identify glioma cells. Olig2 was significantly reduced in the
BLZ945 treated group compared to the vehicle controls, showing that
BLZ945 significantly reduced numbers of tumor cells. (FIG. 6A).
[0166] Analysis of the proportion of Olig2+ cells that were
proliferating, as determined by bromodeoxyuridine (BrdU)
incorporation, revealed a significant reduction in the BLZ945 group
(FIG. 6B). Again, BLZ945 significantly reduced proliferation of
tumor cells.
[0167] The level of apoptosis in these cells was assessed, also.
Apoptotic cells were counted as those that had cytoplasmic cleaved
caspase-3 (CC3)+ staining and condensed nuclei. As shown in FIG.
6C, the CSF-1R inhibitor treatment caused an increase in apoptosis
at the earlier time point in particular, although little staining
was observed in the Day 7 large tumor cohort.
[0168] The following table summarizes the histological analyses
performed on the samples from Example 5:
TABLE-US-00002 TABLE 1 Histologic analyses. 8LZ945 BLZ945 BLZ945,
BLZ945, Large, Large, Parameter Vehicle Day 3 Day 7 Day 3 Day 7
Tumor Volume +498% -- +0.68% -- -24.3% (Day -1 vs Day 6) Total
DAPI* Cells -- -72% -80% -40% -65% Tumor Cells -- -27% -77% -14%
-73% (% Olig2*) Proliferation -- -91% -67% -98% -94% (%
BrdU*Olig2*) Apoptosis (% CC3*) -- +17-fold .sup. +6-fold .sup.
+9-fold .sup. +2-fold Vasculature -- -- -17% -- -67% (CD31 MVD)
Macrophages -- .sup. +3-fold .sup. +2-fold .sup. +2-fold .sup.
+4-fold (% CD68*) Phagocytic -- +2.6-fold +3.0-fold +2.2-fold
+4.1-fold index Phagocytic -- +11.5-fold +5.0-fold +7.1-fold
+6.0-fold Capacity
Tumor volume change is volume at endpoint (day 6) relative to day
one, and the reported changes are relative to the control (vehicle)
group.
[0169] Together, these analyses demonstrate that inhibition of
CSF-1R signaling effectively blocks the growth and malignancy of
gliomas through a combined effect on reducing tumor cell
proliferation and increasing cell death.
[0170] In summary, these data demonstrate that the CSF-1R inhibitor
BLZ945 is a potent new therapy that blocks tumor progression in a
very aggressive glioma model in mice. The compound dramatically
enhanced survival in a preclinical mouse model of gliomagenesis,
and sharply reduced tumor growth rates and also reduced tumor size
over a short and longer test period. In the long term test, BLZ945
appears to eliminate visible tumors in significant numbers of mice,
and sharply reduces the tumor grade in most of the treated
mice.
[0171] Since increased macrophage infiltration has been shown to
correlate with malignancy in human gliomas, the potency of BLZ945
in this mouse model, apparently due to therapeutic targeting of
TAMs in subjects with GBM, is expected to translate into efficacy
against glioblastoma in other mammals, including humans. Since
myeloid cells, including macrophages, have been implicated in
blunting chemotherapeutic response in breast cancer models and in
enhancing the adaptive response following irradiation in GBM
xenograft models, this and similar CSF-1R inhibitors may be
effective in combination with therapies directed against the cancer
cells in gliomas, a possibility that merits further investigation.
In particular, compounds such as
##STR00022##
offer the ability to target CSF-1R and PDGFR at similar
concentrations, and thus may be even more effective than BLZ945.
Indeed, compound (Id) inhibits PDGFR with an IC50 only about 4-fold
higher than its IC50 for CSF-1R. Thus a therapeutically effective
concentration of either of these compounds is expected to affect
both target sites, and to exhibit synergistic activity on
gliomas.
Example 7
[0172] To investigate the molecular mechanisms whereby
BLZ945-treated TAMs can elicit such a striking anti-tumor response
in vivo, despite a lack of evident depletion of TAMs or any direct
antiproliferative effect on human GBM cells, CD11b.sup.+Gr-1.sup.-
TAMs were isolated from mice treated with vehicle or BLZ945, and
microarray expression profiling was performed (see FIG. 7).
Microarray analysis identified 257 genes as significantly
differentially expressed between the groups: 52 genes were
upregulated and 205 downregulated (FIGS. 7B; also 8A). Among these,
gene set enrichment analysis (GSEA) revealed that targets of Egr2,
a transcription factor downstream of CSF-1R signaling, were
downregulated in BLZ945 treated TAMs (FIG. 7C). Disproportionately,
genes associated with M2 phase were upregulated (FIGS. 7D and
7E).
Example 8
Gene Expression Changes Induced by the CFR-1R Inhibitor
[0173] Lasso regression modeling was employed to determine the
minimal number of genes that best discriminated the two treatment
groups. This identified a 5-gene signature for BLZ945 treatment
comprised of adrenomedullin (Adm), arginase 1 (Arg1), the clotting
factor F13a1, mannose receptor C type 1 (Mrc1/CD206), and the
protease inhibitor serpinB2 (FIG. 8B). Interestingly, each of these
genes has been associated with alternatively activated/M2
macrophage polarization, and 4 of 5 genes are downregulated
following BLZ945 treatment. SerpinB2 (also known as PAI2), the only
upregulated gene in the 5-gene signature, generally correlates
positively with increased survival, particularly in breast cancer
patients.
[0174] In many tissue contexts TAMs have been found to be more M2
polarized, which has been linked to their immunosuppressive and
pro-tumorigenic functions. Further, macrophages in human gliomas
exhibit an M2-like phenotype, determined by increased levels of the
scavenger receptors CD163 and CD204, which are associated with
higher tumor grade. Given the striking enrichment for M2 genes in
the restricted 5-gene signature, the 257-gene list was examined to
determine if there were additional M2-associated markers altered
following BLZ945 treatment. This revealed 10 further genes [Alox15
(arachidonate 15-lipoxygenase); Cdh1 (cadherin); Cd163 (CD163
antigen); Fpr2 (formyl peptide receptor 2); Hmox1 (heme oxygenase
(decycling) 1); il1b (interleukin 1 beta); and Stab1 (stabilin 1)],
the majority of which were downregulated (FIG. 7D, table 2).
Classically activated/M1 polarization genes were not
correspondingly upregulated, with the exception of
interleukin-1-beta receptor (FIG. 7E). These data suggest that in
response to CSF-1R inhibition by BLZ945, TAMs lose their M2
polarization and may gain anti-tumorigenic functions.
[0175] This also suggests that monitoring these gene expression
changes as biomarkers may provide valuable prognosis information
for treatment of glioma patients with CSF-1R inhibitors. Treated
subjects whose gene expression profiles change in the same or a
similar pattern as these observed changes may be expected to
respond positively to treatment with the CSF-1R inhibitor, and
those who do not exhibit such gene expression changes may need to
receive an alternative or additional treatment due to a negative
prognosis on the CSF-1R inhibitor alone.
TABLE-US-00003 TABLE 2 Differential gene expression as a result of
CSF-1R inhibitor treatment. Fold Change Nominal Symbol Description
BLZ945-Vehicle P value Akap12 A kinase (PRKA) anchor protein
(gravin) 12 -2.85 1.31E-04 Abhd15 abhydrolase domain containing 15
-2.48 1.36E-05 Acp5 acid phosphatase 5, tartrate resistant -2.36
2.08E-03 Aoah acyloxyacyl hydrolase -2.43 3.83E-06 Ada adenosine
deaminase -3.00 2.28E-07 Arxes1 adipocyte-related X-chromosome
expressed sequence 1 -2.23 1.68E-03 Arxes2 adipocyte-related
X-chromosome expressed sequence 2 -2.96 3.37E-04 Adm * #
adrenomedullin -10.85 2.60E-09 Aldh1a2 aldehyde dehydrogenase
family 1, subfamily A2 -2.18 8.36E-04 Apbb2 amyloid beta (A4)
precursor protein-binding, family 2.27 2.97E-06 B, member 2 Anln
anillin actin binding protein -2.99 1.38E-04 Asb10 ankyrin repeat
and SOCS box-containing 10 2.10 1.14E-03 Asb11 ankyrin repeat and
SOCS box-containing 11 2.19 3.00E-04 Mki67 antigen identified by
monoclonal antibody Ki 67 -7.18 2.78E-05 Apob apolipoprotein B
-2.92 3.42E-05 Apoc1 apolipoprotein C-I 3.21 1.56E-06 Apoc4
apoplipoprotein C-IV 3.14 1.91E-04 Alox15 # arachidonate
15-lipoxygenase 4.24 8.85E-03 Arg1 * # arginase, liver -8.48
5.07E-03 Aspm asp (abnormal spindle-like microcephaly -2.22
1.02E-03 associated (Drosophila) Aurka aurora kinase A -2.23
1.30E-03 Aurkb aurora kinase B -2.71 4.19E-06 Birc5 baculoviral IAP
repeat-containing 5 -6.13 3.00E-06 Bambi BMP and activin
membrane-bound inhibitor, homolog (Xenopus laevis) 2.64 6.53E-05
Bub1 budding uninhibited by benzimidazoles 1 homolog (S.
cerevisiae) -2.72 4.19E-06 Cdh1 # cadherin 1 -6.43 1.70E-04 Cdh2
cadherin 2 -2.23 6.25E-04 Camkk1 calcium/calmodulin-dependent
protein kinase -2.13 2.69E-08 kinase 1, alpha Calml4
calmodulin-like 4 -2.06 2.12E-05 Chst2 carbohydrate
sulfotransferase 2 2.44 5.14E-04 Cbr2 carbonyl reductase 2 -4.15
2.93E-07 Cpa3 carboxypeptidase A3, mast cell 2.17 6.30E-04 Ctnnd2
caterin (cadherin associated protein), delta 2 -2.94 8.46E-07 Ctsf
cathepsin F 2.10 1.53E-04 Cd163 # CD163 antigen -2.65 3.87E-07 Cd22
CD22 antigen 2.35 1.09E-05 Cd244 CD244 natural killer cell receptor
2B4 -2.71 1.11E-07 Cd38 CD38 antigen -3.72 4.44E-05 Cd5 CD5 antigen
3.62 2.96E-05 Cd83 CD83 antigen 2.28 2.53E-05 Cd93 CD93 antigen
-2.42 2.30E-07 Cks1b CDC28 protein kinase 1b -2.54 1.71E-06 Cdc20
cell division 20 homolog (S. cerevisiae) -2.75 1.16E-04 Cdc45 cell
division cycle 45 homolog (S. cerevisiae) -2.03 9.78E-08 Cdc6 cell
division cylce 6 homolog (S. cerevisae) -3.67 8.12E-08 Cdca5 cell
division cycle associated 5 -2.24 6.77E-06 Cenpe centromere protein
E -4.18 1.96E-05 Cenpk centromere protein K -2.45 1.46E-05 Cep55
centrosomal protein 55 -2.40 8.23E-05 Ccr1 chemokine (C-C motif)
receptor 1 -4.56 6.86E-05 Cxcr7 chemokine (C-X-C motif) receptor 7
-2.26 8.65E-03 Cspg5 chondroitin sulfate proteoglycan 5 -2.61
1.09E-05 Clu clusterin -2.34 3.55E-04 F3 coagulation factor III
-2.11 4.58E-03 F9 coagulation factor IX 2.12 5.92E-04 F13a1 * #
coagulation factor XIII, A1 subunit -10.66 1.39E-09 Col11a1
collagen, type XI, alpha 1 -3.49 3.09E-04 Col14a1 collagen, type
XIV alpha 1 -2.65 1.37E-06 Cfp complement factor properdin -2.64
2.60E-04 Cntn1 contactin 1 -4.93 2.80E-08 Cpne2 copine II -2.20
1.13E-05 Crybb1 crystallin, beta B1 -2.83 2.44E-05 Clec4n C-type
lectin domain family 5, member n -6.53 4.34E-10 Ccna2 cyclin A2
-3.90 1.19E-05 Ccnb1 cyclin B1 -3.55 2.25E-05 Ccnb2 cyclin B2 -4.53
1.16E-05 Ccnd1 cyclin D1 -3.01 1.06E-08 Ccnd2 cyclin D2 -3.34
1.36E-05 Ccne2 cyclin E2 -5.28 3.67E-08 Ccnf cyclin F -2.30
1.48E-04 Cdk1 cyclin-dependent kinase 1 -2.18 2.75E-05 Cst7
cystatin F (leukocystatin) 2.62 2.29E-07 Cyp4v3 cytochrome P450,
family 4, subfaimly v, 2.14 1.15E-05 polypeptide 3 Cpeb1
cytoplasmic polyadenylation element binding 2.86 1.97E-05 protein 1
Ckap2 cytoskeleton associated protein 2 -2.17 1.80E-04 Ddhd1 DDHD
domain containing 1 2.06 4.86E-03 Dner delta/notch-like EGF-related
receptor -2.68 2.65E-04 Dck deoxycytidine kinase -2.07 2.50E-04
Depdc1a DEP domian containing 1a -2.81 9.05E-05 Dhfr dihydrofolate
reductase -2.40 5.79E-06 Prim1 DNA primase, p49 subunit -2.76
2.87E-07 D17H6S56E-5 DNA segment, Chr 17, human D6S56E 5 -2.01
1.66E-03 Ddit4 DNA damage-inducible transcript 4 -2.43 5.07E-06
Dusp1 dual specificity phosphatase 1 2.33 3.55E-04 E2f8 E2F
transcription factor 8 -2.71 1.20E-05 Ect2 ect2 oncogene -3.19
1.65E-04 Emb embigin -2.59 9.66E-05 Eepd1
endonuclease/exonuclease/phosphatase family 2.70 1.62E-06 domain
containing 1 Ezh2 enhancer of zeste homolog 2 (Drosophila) -2.54
1.36E-05 Etl4 enhancer trap locus 4 2.41 1.24E-05 Eps8 epidermal
growth factor receptor pathway substrate 8 -2.51 4.00E-06 Emp1
epithelial membrance protein 1 -3.19 6.42E-04 Ephx1 epoxide
hydrolase 1, microsomal 2.76 1.75E-04 Ero1l ERO1-like (S.
cerevisiae) -2.64 1.07E-05 Fam20c family with sequence similarity
20, member C 2.79 3.62E-06 Fabp3 fatty acid binding protein 3,
muscle and heart 2.93 4.99E-06 Fabp7 fatty acide binding protein 7,
brain -6.77 9.66E-06 Fbxo32 F-box protein 32 2.54 1.79E-05 Fbn2
fibrillin 2 -2.13 3.89E-03 Fap fibroblast activation protein -2.25
1.46E-03 Fpr2 # formyl peptide receptor 2 -2.83 6.68E-05 Fhl1 four
and 3 half LIM domains 1 -2.02 5.40E-03 Gja1 gap junction protein,
alpha 1 -2.78 1.97E-03 Gpnmb glycoprotein (transmembrane) nmb 3.22
3.98E-05 Ggta1 glycoprotein galactosyltransferase alpha 1, 3 -2.40
2.12E-06 Gpm6a glycoprotein m6a -5.35 3.23E-06 Gzma granzyme A 3.55
4.11E-03 Gadd45a growth arrest and DNA-damage-inducible 45 alpha
2.40 2.77E-04 Gap43 growth associated protein 43 -2.56 7.42E-05
Gdf3 growth differentiation factor 3 -3.33 1.40E-07 Gem GTP binding
protein (gene overexpressed in skeletal muscle) 2.17 7.03E-04
Hspa1a heat shock protein A -4.38 1.45E-05 Hspa1b heat shock
protein 1B -8.71 1.88E-08 Hsp90aa1 heat shock protein 90, alpha
(cytosolic), class A member 1 -2.23 1.01E-03 Hells helicase,
lymphoid specific -3.59 9.76E-06 Hmox1 # heme oxygenase (decycling)
1 -2.90 7.05E-05 Hmgb3 high mobility group box 3 -2.42 2.32E-06
Hmgn5 hign-mobility group nucleosome binding domain 5 -2.58
1.79E-05 Igj immunoglobulin joining chain 3.36 4.53E-03 Ikbke
inhibitor of kappaB kinase epsilon 2.38 1.50E-04 Igf1 insulin-like
growth factor 1 2.13 8.56E-05 Igfbp2 insulin-like growth factor
binding protein 2 -3.54 1.24E-06 Igfbp3 insulin-like growth factor
binding protein 3 -6.53 1.05E-05 Itgam integrin alpha M -2.25
2.27E-04 Itgax integrin alpha X 2.08 1.36E-03 Ifitm1 interferon
induced transmembrane protein 1 -5.18 1.21E-04 Ifitm2 interferon
induced transmembrane protein 2 -2.82 1.54E-03 Ifitm3 interferon
induced transmembrane protein 3 -2.06 4.73E-03 Ifitm6 interferon
induced transmembrane protein 6 -4.14 6.14E-04 Il1b # interleukin 1
beta 2.06 4.50E-04 Il18bp interleukin 18 binding protein 3.66
4.53E-04 Il7r interleukin 7 receptor -2.01 4.96E-03 Kpna2
karyopherin (importin) alpha 2 -2.36 1.98E-05 Khdrbs3 KH domain
containing, RNA binding, signal -2.10 3.94E-04 transduction
associated 3 Kirb1a killer cell lectin-like receptor subfamily B
member 4.30 9.00E-05 1A Kif11 kinesin family member 11 -2.57
9.00E-05 Pbk PDZ binding kinase -5.63 9.20E-07 Pttg1 pituitary
tumor-transforming gene 1 -2.83 2.73E-06 Plac8 placenta-specific 8
-2.79 6.64E-03 Pdgfra platelet derived growth factor receptor,
alpha polypeptide -3.16 4.84E-06 Pf4 platelet factor 4 -2.96
1.21E-05 Pdgfc platelet-derived growth factor, C polypeptide -2.25
2.98E-03 Ptn pleiotrophin -3.21 4.42E-04 Pdpn podoplanin -2.01
3.51E-04 Plk1 polo-like kinase 1 (Drosophila) -2.68 5.72E-05 Pola1
polymerase (DNA directed), alpha 1 -2.37 3.52E-06 Pold2 polymerase
(DNA directed), delta 2, regulatory -2.02 5.72E-06 subunit Pole
polymerase (DNA directed), epsilon -2.27 5.96E-05 Kcnk2 potassium
channel, subfamily K, member 2 -2.13 8.52E-05 Prickle1 prickle
homolog 1 (Drosophila) 2.32 1.75E-04 P4ha2 procollagen-proline,
2-oxoglutarate 4-dioxygenase -3.54 1.25E-06 (proline
4-hydroxylase), alpha II polypeptide Ptger4 prostaglandin E
receptor 4 (subtype EP4) 2.43 5.12E-05 Pmepa1 prostate
transmembrace protein, androgen induced 1 -2.35 5.85E-04 Psmb7
proteasome (prosome, macropain) subunit, beta -2.17 4.27E-03 type 7
Prc1 protein regulator of cytokinesis 1 -3.06 1.26E-04 Ptprz1
protein tyrosin phosphatase, receptor type Z, -3.66 4.43E-05
polypeptide 1 P2ry12 purinergic receptor P2Y, G-protein coupled 12
-2.55 1.86E-04 Rab34 RAB34, member of RAS oncogene family 2.08
3.95E-05 Racgap1 Rac GTPase-activating protein 1 -2.56 2.92E-05
Rad51ap1 RAD51 associated protein 1 -2.61 1.26E-07 Rad51 RAD51
homolog (S. cerevisiae) -2.90 3.33E-06 Ranbp1 RAN binding protein 1
-2.01 6.62E-07 Rfc4 replication factor C (activator 1) 4 -2.17
4.24E-05 Rbp1 retinol binding protein 1, celluluar -4.22 1.88E-05
Rrm1 ribonucleotide reductase M1 -2.14 1.79E-05 Rrm2 ribonucleotide
reductase M2 -8.23 1.04E-07 2310016C08Rik RIKEN cDNA 2310016C08
gene -2.14 1.12E-04 2810417H13Rik RIKEN cDNA 2810417H13 gene -3.96
2.33E-07 4930583H14Rik RIKEN cDNA 4930583H14 gene -2.37 1.25E-05
Rbm3 RNA binding motif protein 3 -2.20 2.23E-09 Slfn4 schlafen 4
-3.35 3.42E-03 Stil Scl/Tal1 interrupting locus -3.15 2.15E-06
Serpinb2 * # serine (cysteine) peptidase inhibitor, clade B, member
2 6.20 1.12E-02 member 2 Serpinb6b serine (or cysteine) peptidase
inhibitor, clade B, 2.03 1.22E-03 member 6b Smyd2 SET and MYND
domain containing 2 -2.25 6.90E-04 Sh3bgr SH3-binding domain
glutamic acid-rich protein 3.33 6.84E-07 Sh3bgrl SH3-binding domain
glutamic acid-rich protein like -2.02 4.68E-04 Shcbp1 Shc
SH2-domain binding protein 1 -4.72 1.74E-06 Slamf8 SLAM family
member 8 2.81 5.20E-03 Snrpa1 small nuclear ribonucleoprotein
polypeptide A' -2.04 1.48E-06 Slc2a5 solute carrier family 2
(facilitated glucose -3.46 8.15E-07 transporter), member 5 Slc39a4
solute carrier family 39 (zinc transporter), member 4 2.44 7.83E-05
Slc6a1 solute carrier family 6 (neurotransmitter transporter, -2.09
5.66E-04 GABA), member 1 Sparcl1 SPARC-like 1 -3.23 1.06E-03 Spon1
spondin 1, (f-spondin) extracellular matrix protein -2.50 9.32E-05
Sox2 SRY-box containing gene 2 -2.50 5.13E-03 Stab1 # stabilin 1
-2.64 3.92E-06 Stmn1 stathmin 1 -2.52 5.52E-05 Smc2 structural
maintenance of chromosomes 2 -2.87 7.80E-05 Smc4 structural
maintenance of chromosomes 4 -3.20 2.26E-04 St14 suppression of
tumorigenicity 14 (colon carcinoma) 2.47 4.21E-06 Tiparp
TCDD-inducible poly(ADP-ribose) polymerase -2.06 1.25E-03 Tnc
tenascin C -2.56 2.48E-03 Tk1 thymidine kinase 1 -3.39 1.24E-07
Tipin timeless interacting protein -2.50 6.24E-06 Tfpi2 tissue
factor pathway inhibitor 2 -2.60 3.99E-03 Timp1 tissue inhibitor of
metalloproteinase 1 -2.07 1.64E-04 Top2a topoisomerase (DNA) II
alpha -2.11 2.13E-05 Topbp1 topoisomerase (DNA) II binding protein
1 -2.37 9.96E-06 Tpx2 TPX2, microtubule-associated protein homolog
(Xenopus laevis) -2.52 1.16E-05 Tcf19 transcription factor 19 -2.32
1.67E-06 Tgfbi transforming growth factor, beta induced -3.23
1.68E-06 Tgm2 transglutaminase 2, C polypeptide -2.94 2.86E-03
Tmem119 transmembrane protein 119 -3.12 1.71E-06 Tmem163
transmembrane protein 163 2.20 5.42E-03 Trps1 trichorhinophalangeal
syndrome I (human) -2.97 1.68E-07 Trim59 tripartite
motif-containing 59 -2.78 3.02E-04 Ttk Ttk protein kinase -2.63
3.35E-05 Tubb2c tubulin, beta 2C -2.13 3.84E-06 Ube2c
ubiquitin-conjugating enzyme E2C -3.98 1.12E-04 Uhrf1
ubiquitin-like, containing PHD and RING finger domains, 1 -2.96
3.73E-07 Ung uracil DNA glycosylase -2.50 5.81E-07 Wdhd1 WD repeat
and HMG-box DNA binding protein 1 -2.01 2.52E-04 Zwilch Zwilch,
kinetochore associated, homolog -4.32 3.61E-08 (Drosophila) *
Component of Lasso regression signature of response to BLZ945.
# Relevant M2 macrophage-associated genes.
[0176] In the Table, downregulated genes are given a negative `fold
change` number, while upregulated genes have positive values.
Nominal p values are from Student's two-tailed t-test.
[0177] In addition, gene signatures generated from BLZ945-treated
TAMs in mice appear to be associated with differential survival in
GBM patients. A support vector machine (SVM) and the Lasso
signature were used to analyze GBM data from The Cancer Gene Atlas
(TCGA) and a second combined series of GBM datasets and segregate
patients into either `BLZ945` or `Vehicle` classifiers. These
analyses revealed an increase in median survival ranging from 10
months in TCGA proneural patients using the Lasso signature (FIGS.
8C and 8D) to 31.5 months in the combined datasets with the SVM
signature (FIGS. 8E and 8F). Interestingly, this increase in
survival was not evident in other subtypes of GBM, and was not
dependent upon enrichment of G-CIMP.sup.+ proneural patients.
TABLE-US-00004 TABLE 3 Survival data for the Support Vector Machine
(SVM) and Lasso models in the different GBM populations. Group
BLZ945 Vehicle Median Survival P value SVM Combined Neural 49 16
5.42 1.59E-01 SVM Combined Proneural 46 62 31.54 6.86E-04 SVM
Combined Mesenchymal 37 102 -2.25 8.92E-01 SVM Combined Classical
11 48 0.40 6.67E-01 SVM TCGA Proneural 45 88 7.64 7.27E-03 SVM TCGA
Proneural GCIMP 13 8 -40.60 2.01E-01 SVM TCGA Proneural non 22 44
-0.76 2.64E-01 GCIMP SVM TCGA GCIMP 14 8 -35.60 2.03E-01 SVM TCGA
non GCIMP 83 157 -1.06 7.27E-01 SVM TCGA Neural 23 30 2.84 7.73E-01
SVM TCGA Mesenchymal 53 99 0.30 7.62E-01 SVM TCGA Classical 31 66
-3.14 7.71E-01 Lasso Combined Neural 51 14 7.01 6.50E-02 Lasso
Combined Proneural 79 29 6.51 4.15E-02 Lasso Combined Mesenchymal
21 118 1.88 5.55E-01 Lasso Combined Clssical 28 31 0.33 9.68E-01
Lasso TCGA Proneural 84 49 9.98 5.41E-06 Lasso TCGA Proneural GCIMP
20 1 NA NA Lasso TCGA Proneural non GCIMP 40 26 10.84 1.04E-02
Lasso TCGA GCIMP 20 2 -16.13 7.21E-01 Lasso TCGA non GCIMP 100 140
0.10 4.14E-01 Lasso TCGA Neural 31 22 -5.19 2.77E-02 Lasso TCGA
Mesenchymal 23 129 0.40 8.35E-01 Lasso TCGA Classical 49 48 -1.42
6.34E-01
[0178] Analysis of associated hazard ratios demonstrated the
proneural-specific survival advantage in both TCGA and the combined
data sets (FIG. 8G). The proneural specificity is consistent with
the TAM signatures originally having been generated from the PDG
model of gliomagenesis, which most closely represents proneural
GBM. This suggests these gene signatures can provide useful
prognostic guidance for subjects undergoing treatment with
chemotherapeutics, particularly GBM patients treated with CSF-1R
inhibitors. As proneural GBM does not respond to aggressive chemo-
and radiotherapy compared to the other subtypes, the finding of
prognostic value associated with these signatures may have
important translational potential for this group of patients. Based
on the observed correlation, patients receiving chemotherapy who
exhibit a gene signature at least about 80% similar to either the
Lasso or the SVM gene signature are expected to respond positively
to that chemotherapeutic. In particular, this correlation is
expected to be useful with subjects treated with an inhibitor of
CSF-1R, particularly compounds of Formula (I) as described
herein.
TABLE-US-00005 TABLE 4 Hazard rations and associated 95% confidence
intervals for the Lasso regression model in different G-CIMP and
non-G-CIMP patient groups. G-CIMP corresponds to Glioma CpG Island
Methylator Phenotype. P values were obtained using Wald's test.
Patient Strata Population Model Hazard Ratio 95% CI P value
`BLZ945` Non-GCIMP Univariate 0.4921 (0.2766-0.8756) 0.0063 Lasso
Proneural* `BLZ945` All Proneural Univariate 0.3937 (0.2601-0.5961)
9.729e-06 Lasso G-CIMP All Proneural Univariate 0.3289
(0.1481-0.7304) 0.01367 G-CIMP All Proneural Multivariate* 0.4601
(0.1972-1.0733) 0.00783 `BLZ945` All Proneural Multivariate* 0.4295
(0.2304-0.8007) 0.07244 Lasso *Set of proneural patients with
methylation data that are definitively not G-CIMP positive (67/133
total Proneural TCGA patients.) ** Multivariate cox proportional
hazard model using both G-CIMP and `BLZ945` classification
strata.
TABLE-US-00006 TABLE 5 Hazard rations for the Lasso regression
model in different patient datasets. P values were obtained using
Wald's test. Only hazard ratios from the proneural subtypes are
statistically significant. Group Hazard Ratio 95% CI P value TCGA-
Proneural 0.29 (0.17-0.50) 6.32E-06 TCGA- Classical 1.28
(0.73-2.26) 3.89E-01 TCGA- Mesenchymal 0.93 (0.49-1.72) 8.07E-01
TCGA- Neural 1.93 (0.83-4.46) 1.25E-01 Combined- Proneural 0.44
(0.25-0.79) 5.97E-03 Combined- Classical 1.01 (0.47-2.17) 9.79E-01
Combined- Mesenchymal 1.02 (0.54-1.94) 9.43E-01 Combined- Neural
0.46 (0.22-1.01) 5.23E-02
Methods and Materials Used
[0179] Mice
[0180] All animal studies were approved by the Institutional Animal
Care and Use Committee of Memorial Sloan-Kettering Cancer Center.
The Nestin-Tv-a;Ink4a/Arf-/- mouse model (mixed strain background)
has been previously described (see E. Tchougounova et al., Oncogene
26, 6289 (2007)). Wild-type (WT) C57BL/6 mice and .beta.-actin-GFP
(C57BL/6) mice were purchased from Charles River Laboratories and
Jackson Laboratories respectively, and also bred within our animal
facility.
[0181] Intracranial Injections
[0182] The initiation of tumors with RCAS-PDGF-B-HA in adult mice
has been previously described (A. H. Shih et al., Cancer Res 64,
4783 (2004)). Briefly, mice were fully anesthetized with 10 mg/ml
ketamine/1 mg/ml xylazine and were subcutaneously injected with 50
.mu.l of the local anesthetic 0.25% bupivacaine at the surgical
site. Mice were intracranially injected with 1 .mu.l containing
2.times.105 DF-1:RCAS-PDGF-B-HA cells between 5-6 weeks of age
using a fixed stereotactic apparatus (Stoelting). Injections were
made to the right frontal cortex, approximately 1.5 mm lateral and
1 mm caudal from bregma, and at a depth of 2 mm.
[0183] To investigate the cell type specific expression of CSF-1
and CSF-1R in flow cytometric sorted cell populations, tumors were
initiated in mice with RCAS-PDGF-B-HA-SV40-eGFP (RCAS-PDGF-GFP) as
previously described (E. I. Fomchenko et al., PloS ONE 6, e20605
(2011).). Nestin-Tv-a;Ink4a/Arf-/- pups were injected with 1 .mu.l
of DF-1:RCAS-PDGF-B-GFP cells on post-natal day 2 into the left
cortex between the eye and ear.
[0184] BLZ945 Inhibitor and Treatment
[0185] The CSF-1R inhibitor BLZ945 was formulated in 20% captisol
at a concentration of 12.5 mg/ml. The vehicle control, 20%
captisol, was processed in the same manner. For BLZ945 studies,
mice were dosed with 200 mg/kg BLZ945 or vehicle (20% captisol) by
oral gavage once per day.
[0186] To determine if the drug was able to cross the blood-brain
barrier, tumor-bearing mice were treated with a single dose of
BLZ945 and sacrificed at different time points post treatment.
Plasma, and the left (contralateral) and right (tumor-bearing)
hemispheres of the brain were snap frozen in liquid nitrogen for
subsequent analysis of BLZ945 concentrations in the tissue. For
long-term survival studies, dosing was begun at 17 days/2.5 weeks
post-injection of RCAS-PDGF-B-HA. For the fixed time-point studies,
mice underwent MRI scans at 4-5 weeks post-injection of
RCAS-PDGF-B-HA, as previously described (Transl Oncol 2, 89
(2009)).
[0187] To determine tumor volume, regions of interest (ROI) were
circumscribed on T2 weighted images and their corresponding area in
mm.sup.2 was multiplied by the slice height of 0.7 mm. The total
tumor volume is the sum of the ROI volume in each slice, and the
volume for the first and last slice in which the tumor appear is
halved to approximate the volume of a trapezoid. When tumor volume
was in the range of 4.5-40 mm.sup.3, animals were randomly assigned
to treatment groups. A third cohort of mice with tumors larger than
40 mm.sup.3 was also treated with BLZ945 (denoted as BLZ945 Large).
A size-matched vehicle treated cohort was not included for this
cohort having the larger starting tumor burden because these mice
would not have been able to survive to the trial endpoint.
[0188] Mouse Sacrifice and Tissue Harvest
[0189] Mice were euthanized at defined time points as described in
the figure legends or when they became symptomatic from their
tumors, which included signs of poor grooming, lethargy, weight
loss, hunching, macrocephaly, or seizures.
[0190] To isolate tissues for snap freezing in liquid nitrogen,
mice were euthanized by carbon dioxide asphyxiation or fully
anesthetized with avertin (2,2,2-tribromoethanol, Sigma) and
cervically dislocated prior to tissue harvest. For flow cytometry,
mice were fully anesthetized with avertin and transcardially
perfused with 20 ml of PBS. The brain was then isolated and the
tumor macrodissected from the surrounding normal tissue. For
proliferation analysis, mice were injected intraperitoneally with
100 mg/g of bromodeoxyuridine (BrdU; Sigma) 2 hours prior to
sacrifice. To isolate tissues for frozen histology, mice were fully
anesthetized with avertin, transcardially perfused with 10 ml of
PBS, followed by 10 ml of 4% paraformaldehyde in PBS (PFA). The
brain was postfixed in PFA overnight at 4.degree. C. while other
tissues were cryopreserved in 30% sucrose at 4.degree. C. After
post-fixation, the brain was then transferred to 30% sucrose and
incubated at 4.degree. C. until the brain was fully equilibrated
and sank to the bottom of the tube (typically 2 to 3 days). All
tissues were then embedded in OCT (Tissue-Tek) and 10 .mu.m
cryostat tissue sections were used for all subsequent analysis.
[0191] Histology, Immunohistochemistry, and Analysis
[0192] For grading of tumor malignancy, hematoxylin and eosin
(H&E) staining was performed, and the tissues blindly scored by
an independent neuropathologist.
[0193] For immunofluorescence, 10 .mu.m thick frozen sections were
thawed and dried at room temperature and then washed in PBS. For
the standard staining protocol, tissue sections were blocked in
0.5% PNB in PBS for at least 1 hour at room temperature or up to
overnight at 4.degree. C., followed by incubation in primary
antibody in 0.25% PNB for 2 hours at room temperature or overnight
at 4.degree. C. Primary antibody information and dilutions are
listed in Table 6. Sections were then washed in PBS and incubated
with the appropriate fluorophore-conjugated secondary antibody
(Molecular Probes) at a dilution 1:500 in 0.25% PNB for 1 hour at
room temperature. After washing in PBS, tissue sections were
counterstained with DAPI (5 mg/ml stock diluted 1:5000 in PBS) for
5 minutes prior to mounting with ProLong Gold Antifade mounting
media (Invitrogen).
[0194] For angiogenesis and proliferation analysis, tissue sections
were first subjected to citrate buffer based antigen retrieval by
submerging in antigen unmasking solution (0.94% v/v in distilled
water; Vector Laboratories) and microwaving for 10 minutes on half
power, followed by cooling to room temperature for at least 30
minutes. For angiogenesis analysis, tissues were then washed in PBS
and blocked with mouse Ig blocking reagent (Vector Laboratories)
according to the manufacturer's instructions for 1 hour at room
temperature. For proliferation analysis, after antigen retrieval,
tissue sections were incubated with 2M HCl for 15 minutes at room
temperature to denature DNA and then in neutralizing 0.1M sodium
borate buffer (pH 8.5) for 5 minutes. After PBS washes, the rest of
the staining was performed according to the standard protocol.
[0195] For staining for phagocytosis analysis, 10 .mu.m thick
frozen sections were thawed and dried at room temperature and then
washed in PBS. Tissue sections were blocked in 0.5% PNB in PBS for
at least 1 hour at room temperature, followed by incubation in
rabbit anti-cleaved caspase-3 primary antibody diluted 1:500 in
0.5% PNB overnight at 4.degree. C. The next day, slides were washed
6 times for 5 minutes in PBS prior to incubation with
goat-anti-rabbit Alexa568 secondary antibody (1:500 in 0.5% PNB)
for 1 hour at room temperature. Tissue sections were then washed 6
times for 5 minutes in PBS and blocked overnight at 4.degree. C. in
a new buffer of 5% donkey serum, 3% bovine serum albumin, and 0.5%
PNB in PBS. The following day, slides were incubated for 2 hours at
room temperature with the next set of primary antibodies: rabbit
anti-Olig2 (1:200) and rat anti-CD11b (1:200) diluted in 5% donkey
serum, 3% bovine serum albumin, and 0.5% PNB in PBS. Slides were
washed 6 times for 5 minutes in PBS prior to incubation with
donkey-anti-rabbit Alexa647 (1:500) and donkey-anti-rat Alexa488
(1:500) secondary antibodies in 0.5% PNB for 1 hour at room
temperature. Tissue sections were then washed 4 times for 5 minutes
in PBS prior to staining with DAPI (5 mg/mL stock diluted 1:5000 in
PBS) for 5 minutes, washed twice more in PBS for 5 minutes, and
mounted with ProLong Gold Antifade mounting media (Invitrogen).
Co-staining for CSF-1R (first primary antibody) and Iba1 (second
primary antibody) was also performed in series in the same manner,
with the addition of citrate buffer based antigen retrieval at the
outset.
[0196] Tissue sections were visualized under a Carl Zeiss
Axioimager Z1 microscope equipped with an Apotome. The analysis of
immunofluorescence staining, cell number, proliferation, apoptosis,
and colocalization studies were performed using TissueQuest
analysis software (TissueGnostics) as previously described (Journal
Immunol Methods 237, 39 (2000)).
[0197] Overviews of tissue sections from gliomas stained for
angiogenesis analysis were generated by TissueGnostics acquisition
software by stitching together individual 200.times. images. All
parameters of angiogenesis were quantitated using MetaMorph
(Molecular Devices), as previously described (V. Gocheva, et al.,
Biol Chem 391, 937 (2010)).
[0198] For analysis of phagocytosis, 15 randomly selected fields of
view from within the tumor were acquired using the 63.times. oil
immersion objective (total magnification 630.times.) and the
Apotome to ensure cells were in the same optical section. Positive
cells were counted manually using Volocity (PerkinElmer) and were
discriminated by the presence of a DAPI+ nucleus. Apoptotic cells
were counted as those that had cytoplasmic cleaved caspase-3 (CC3)+
staining and condensed nuclei. A cell was considered to have been
engulfed by a macrophage when it was surrounded by a contiguous
CD11b+ ring that encircled at least two-thirds of the cell border.
The numbers of mice analyzed are specified in the figure
legends.
Protein Isolation and Western Blotting
[0199] Mice were treated with BLZ945 or vehicle and sacrificed 1
hour following the final dose and tumors were harvested. Samples
were biochemically fractionated as described previously.
Synaptosomal membrane fractions were lysed in NP-40 lysis buffer
(0.5% NP-40, 50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1.times. complete
Mini protease inhibitor cocktail (Roche), 1.times. PhosSTOP
phosphatase inhibitor cocktail (Roche)) and protein quantified
using the BCA assay (Pierce). Protein lysates were loaded (90
.mu.g/lane) onto SDS-PAGE gels and transferred to PVDF membranes
for immunoblotting.
[0200] Membranes were probed with antibodies against phospho-CSF-1R
Y721 (1:1000; Cell Signaling Technology), CSF-1R (1:1000; Santa
Cruz Biotechnology), or GAPDH (1:1000; Cell Signaling Technology)
and detected using HRP-conjugated anti-rabbit (Jackson
Immunoresearch) antibodies using chemiluminescence detection
(Millipore). Bands from western blots were quantified in the
dynamic range using the Gel analysis module in ImageJ software.
[0201] Primary bone marrow derived macrophages (BMDMs) were
cultured in the absence of CSF-1 for 12 hours prior to stimulation
with CSF-1 (10 ng/ml) for the time points indicated in FIG. S2, in
the presence or absence of 67 nM BLZ945. Whole protein lysates were
isolated with NP40 lysis buffer and detected by western blot as
described above.
Preparation of Single Cell Suspensions and Flow Cytometry
[0202] For investigation of brain macrophage populations by flow
cytometric analysis or sorting, the tumor was digested to a single
cell suspension by incubation with 5 ml of papain digestion
solution (0.94 mg/ml papain [Worthington], 0.48 mM EDTA, 0.18 mg/ml
NAcety-L-cysteine [Sigma], 0.06 mg/ml DNase I [Sigma], diluted in
Earl's Balanced Salt Solution and allowed to activate at room
temperature for at least 30 minutes). Following digestion, the
enzyme was inactivated by the addition of 2 ml of 0.71 mg/ml
ovomucoid (Worthington). The cell suspension was then passed
through a 40 .mu.m mesh to remove undigested tissue, washed with
FACS buffer (1% IgG Free BSA in PBS [Jackson Immunoresearch]), and
centrifuged at a low speed of 750 rpm (Sorvall Legend RT), to
remove debris and obtain the cell pellet. As many immune cell
epitopes are papain-sensitive, for investigation of immune cell
infiltration by flow cytometric analysis, tumors were digested to a
single cell suspension by incubation for 10 minutes at 37.degree.
C. with 5 mL of 1.5 mg/ml collagenase Ill (Worthington) and 0.06
mg/mL DNase I in 1.times. Hanks Balanced Salt Solution (HBSS) with
calcium and magnesium.
[0203] The cell suspension was then washed with PBS and passed
through a 40 .mu.m mesh to remove undigested tissue. To remove
myelin debris, the cell pellet was resuspended in 15 ml of room
temperature 25% Percoll prepared from stock isotonic Percoll (90%
Percoll [Sigma], 10% 10.times.HBSS), and then spun for 15 minutes
at 1500 rpm (Sorvall Legend RT) with accelerator and brake set to
1. The cell pellet was then washed with 1.times.HBSS prior to being
resuspended in FACS buffer. After counting, cells were incubated
with 1 .mu.l of Fc Block for every million cells for at least 15
minutes at 4.degree. C. Cells were then stained with the
appropriate antibodies for 10 minutes at 4.degree. C., washed with
FACS buffer, and resuspended in FACS buffer containing DAPI (5
mg/ml diluted 1:5000) for live/dead cell exclusion. Antibodies used
for flow cytometry are listed in Table 6.
TABLE-US-00007 TABLE 6 List of Antibodies and sources. Anitbody
Clone Vendor Fluorophore(s) Dilu-tion CD45 30-F11 BD Pharmingen
FITC, APC, 1:100-1:200 PE-Cy7 CD3e 145-2C11 BD Pharrningen PE-Cy7
1:250 Gr-1 RB6-8C5 BD Pharmingen FITC 1:200 CD4 GK1.5 BD Pharmingen
PE 1:1000 CD11b Ml/70 BD Pharmingen A488, APC, 1:200 PE Ly6G 1A8 BD
Pharmingen PE-Cy7 1:2000 F4/80 Cl:A3-1 Serotec PE 1:50 CD8a 53-6.7
Biolegend A488 1:1000 CD19 6D5 Biolegend PE 1:2000 NK1.1 PK136
Biolegend APC 1:1000 CD206 MR5D3 Biolegend A488 1:50
[0204] For analysis, samples were run on a BD LSR II (Becton
Dickstein), and all subsequent compensation and gating performed
with FlowJo analysis software (TreeStar). For sorting, samples were
run on a BD FACSAria (Becton Dickstein) cell sorter and cells were
collected into FACS buffer. Cells were then centrifuged and
resuspended in 500 .mu.l Trizol (Invitrogen) before snap freezing
in liquid nitrogen and storage at -80.degree. C.
Derivation of Mouse Primary Glioma Cultures, Neurospheres and
Glioma Cell Lines
[0205] Macrodissected tumors were digested to a single cell
suspension by incubation for 8-12 minutes at 37.degree. C. as
described above. The cell suspension was washed with Neural Stem
Cell (NSC) Basal Media (Stem Cell Technologies), and centrifuged at
low speed (750 rpm Sorvall Legend RT), to remove debris. To derive
mouse primary glioma cultures the cell pellet was resuspended in
DMEM containing 10% FBS (Gibco). These primary cultures were used
at early passage (P2-P3), and contain a mixture of different cell
types found in gliomas including tumor cells, macrophages, and
astrocytes as determined by immunofluorescence staining. Primary
glioma cultures were grown for 24 hours on poly-L-lysine coated
coverslips (BD Biocoat). Cells were then fixed with 4% PFA in 0.1M
phosphate buffer overnight at 4.degree. C., permeabilized with 0.1%
Triton-X for 5 minutes and blocked with 0.5% PNB for at least one
hour. The presence of macrophages, tumor cells and astrocytes were
examined by immunofluorescent staining of CD11b (1:200), Nestin
(1:500) and GFAP (1:1000), respectively (Table 7).
TABLE-US-00008 TABLE 7 List of antibodies used for staining.
Antibody Clone Vendor Dilution Goat anti-mouse CD31 -- R&D
Systems 1:100 Mouse anti-human smooth 1A4 DakoCytomation 1:100
muscle actin (SMA) Rabbit anti-cleaved caspase 3 -- Cell Signaling
1:500 (Asp175) (CC3) Technology Rabbit anti-human CSF-1R C-20 Santa
Cruz 1:200 Rabbit anti-Iba1 -- Wako 1.1000 Rabbit anti-green
fluorescent -- Molecular Probes 1:200 protein (GFP) Rabbit
anti-Olig2 -- Millipore/ 1:200 Chemicon Mouse anti-rat Nestin -- BD
Pharmingen 1:500 Rat anti-mouse CD11b M1/70 BD Pharmingen 1:200 Rat
anti-BrdU BU1/ Serotec 1:200 75(ICR1) Rat anti-mouse CD68 FA-11
Serotec 1:1000 Chicken anti-GFAP -- Abeam 1:1000
[0206] For neurosphere formation the cell pellet was resuspended in
neurosphere media consisting of mouse NSC Basal Media, NSC
proliferation supplements, 10 ng/ml EGF, 20 ng/ml basic-FGF and 1
mg/ml Heparin (Stem Cell Technologies). Fresh media was added every
72 hours for 2 weeks. Primary neurospheres were collected,
mechanically disaggregated to a single cell suspension and
propagated by serial passaging. To generate glioma cell lines,
secondary neurospheres were dissociated to single cell suspensions
and cultivated in DMEM+10% FBS as a monolayer. Multiple glioma cell
lines were derived from independent mice, denoted GBM1-4 herein.
Glioma cells were infected with a pBabe-H2B-mCherry construct as
described previously (O. Florey, et al., Nat Cell Biol 13, 1335
(2011)).
Isolation of Bone Marrow-Derived Macrophages (BMDMs)
[0207] For bone marrow isolation, followed by macrophage
derivation, C57BL/6 WT, C57BL/6 .beta.-actin-GFP or Nestin-Tv-a;
Ink4a/Arf-/- mice were anesthetized with Avertin (Sigma) and then
sacrificed via cervical dislocation. Femurs and tibiae were
harvested under sterile conditions from both legs and flushed. The
marrow was passed through a 40 .mu.m strainer and cultured in 30 ml
Teflon.RTM. bags (PermaLife PL-30) with 10 ng/ml recombinant mouse
CSF-1 (R&D Systems). Bone marrow cells were cultured in
Teflon.RTM. bags for 7 days, with fresh CSF-1-containing media
replacing old media every other day to induce macrophage
differentiation.
[0208] Additional cell lines U-87 MG (HTB-14) glioma and CRL-2467
microglia cell lines were purchased from the ATCC. The U-87 MG cell
line was cultured in DMEM+10% FBS. The CRL-2467 cell line was
cultured in DMEM+10% FBS with 30 ng/ml recombinant mouse CSF-1.
[0209] Glioma cell-conditioned media (GCM) experiments Media that
had been conditioned by glioma tumor cell lines grown in serum free
media for 24 hours was passed through 0.22 .mu.m filters to remove
cellular debris, and is referred to herein as glioma
cell-conditioned media (GCM). GCM was used to stimulate
differentiated C57BL/6 WT or .beta.-actin-GFP+ BMDMs. Control
macrophages received fresh media containing 10% FBS and 10 ng/ml
recombinant mouse CSF-1. When indicated, differentiated BMDMs were
cultivated in GCM containing either DMSO as vehicle, or 67 nM
BLZ945, 670 nM BLZ945, or in regular media containing 10 ng/ml
mouse recombinant CSF-1 and 10 ng/ml IL-4 (R&D Systems) for 24
hours or 48 hours prior to experimental analysis.
Analysis of Mrc1/CD206 Expression by Flow Cytometry
[0210] For mouse primary glioma cultures (containing a mixed
population of tumor cells, TAMs, astrocytes etc.), 1.times.106
cells were cultivated in DMEM+10% FBS in the presence of BLZ945 or
DMSO as vehicle. For BMDMs, 1.times.10.sup.6 cells were cultivated
in DMEM supplemented with recombinant mouse CSF-1 or GCM in the
presence of BLZ945 or DMSO as vehicle. After 48 hours, cells were
scraped and washed with FACS buffer. Cells were counted and
incubated with 1 .mu.l of Fc Block (BD Pharmingen) per 106 cells
for at least 15 minutes at 4.degree. C. Cells were then stained
with CD45 and CD11b antibodies for 10 minutes at 4.degree. C. and
washed with FACS buffer. Cells were fixed and permeabilized using
the BD Cytofix/Cytoperm.TM. kit (BD Biosciences) according to the
manufacturer's instructions. Subsequently cells were stained with
anti-CD206 antibody. For analysis, samples were run on a BD LSR II
(Becton Dickstein), and all subsequent compensation and gating
performed with FlowJo analysis software (TreeStar).
Cell Cycle Analysis
[0211] Control or GCM pre-stimulated macrophages derived from
.beta.-actin-GFP+ mice were cocultured in a 1:1 ratio with
1.times.105 serum starved mCherry-positive glioma cells (from the
cell lines derived above) for 48 hours in the presence of 670 nM
BLZ945 or DMSO as vehicle. Following collection of trypsinized
co-cultured cells, wells were rinsed in additional media and this
volume was collected to ensure harvesting of all macrophages, which
adhered tightly to cell culture dishes. Samples were then washed
once with FACS buffer, followed by incubation for 10 minutes at
room temperature in permeabilizing buffer (10 mM PIPES, 0.1 M NaCl,
2 mM MgCl2, 0.1% Triton X-100, pH 6.8) containing 0.1 mg DAPI
(Invitrogen). After acquisition on an LSR II flow cytometer (BD)
using a UV laser (350-360 nm), cell cycle status of glioma tumor
cells was analyzed using the Flow Jo Dean-Jett-Fox program for cell
cycle analysis.
Proliferation Assays
[0212] Cell growth rate was determined using the MTT cell
proliferation kit (Roche). Briefly, cells were plated in triplicate
in 96-well plates (1.times.10.sup.3 cells/well for glioma cell
lines and 5.times.10.sup.3 cells/well for BMDM and CRL-2467 cells)
in the presence or absence of 6.7-6700 nM of BLZ945. Media was
changed every 48 hours. BMDM and CRL-2467 cells were supplemented
with 10 ng/ml and 30 ng/ml recombinant mouse CSF-1 respectively
unless otherwise indicated. 10 .mu.l of MTT labeling reagent was
added to each well and then incubated for 4 hours at 37.degree. C.,
followed by the addition of 100 .mu.l MTT solubilization reagent
overnight. The mixture was gently resuspended and absorbance was
measured at 595 nm and 750 nm on a spectraMax 340 pc plate reader
(Molecular Devices).
Secondary Neurosphere Formation Assay
[0213] Primary neurospheres were disaggregated to a single cell
suspension and 5.times.10.sup.3 cells were plated in a 6 well plate
in neurosphere media in the presence of BLZ945 or DMSO as vehicle.
Media was changed every 48 hours. Secondary neurosphere formation
was assayed by counting the number of neurospheres obtained after 2
weeks.
RNA Isolation, cDNA Synthesis and Quantitative Real Time PCR
[0214] RNA was isolated with Trizol, DNase treated, and 0.5 .mu.g
of RNA was used for cDNA synthesis. Taqman probes (Applied
Biosystems) for Cd11b (Mm00434455_m1), Cd68 (Mm03047343_m1), Csf-1
(Mm00432688_m1), Csf-1r (Mm00432689_m1), II34 (Mm00712774_m1), Mrc1
(Mm00485148_m1), and Tv-a (custom), were used for qPCR. Assays were
run in triplicate and expression was normalized to ubiquitin C
(Mm01201237_m1) for each sample.
Microarrays and Gene Expression Profiling
[0215] All samples were prepared and processed by the genomics core
facility at MSKCC. RNA was isolated using Trizol and the quality
was assessed by running on an Agilent Bioanalyzer. 75 ng of total
RNA was reverse transcribed and labeled using the Genechip 3' IVT
Express Kit (Affymetrix). The resulting cRNA was hybridized to
Affymetrix MOE 430A 2.0 chips. Raw expression data were analyzed
using GCOS 1.4 (Affymetrix). Data were normalized to a target
intensity of 500 to account for differences in global chip
intensity.
Microarray Analysis
[0216] All bioinformatic analyses were completed in R using the
Bioconductor Suite of packages. Robust Multi-Array Average (RMA)
expression values were generated using the `ally` package and
quantile normalized (R. A. Irizarry et al., Nucleic Acids Res 31,
e15 (2003); L. Gautier, et al., Bioinformatics 20, 307 (2004). The
`limma` package (G. K. Smyth, Statistical Applications in Genetics
and Molecular Biology 3, Article 3 (2004)) was used to identify
differentially expressed genes between the vehicle and BLZ945
treated samples. Differential expression was considered significant
at a fold change of +/-2 with a false discovery rate of 10%. Gene
set enrichment analysis (GSEA) was used as described previously
(15). For subsequent analysis and comparison to human datasets,
mouse expression values were mean centered across all samples.
Lasso Regression Method for Gene Signature Identification
[0217] Mouse expression data was normalized and mean centered as
described above. Differentially expressed genes were used for
further analysis. A Lasso regression model was trained to
differentiate between Vehicle and BLZ945 treated samples using the
`glmnet` package (J. Friedman, et al., Journal of Statistical
Software 33, 1 (2010).). The regularization parameter for Lasso
regression was chosen by 4-fold cross validation.
Patient Datasets
[0218] TCGA expression data was downloaded from the TCGA data
portal and all clinical data was downloaded from the data portal
<http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp>. Clinical
and expression data for the Rembrandt data set was downloaded from
<https://caintegrator.nci.nih.gov/rembrandt/>. The Freije
(GSE4412), Murat (GSE7696), and Phillips (GSE4271) datasets were
downloaded from the NCBI <http://www.ncbi.nlm.nih.gov/geo/>.
For the Freije datasets, only samples that were run on the HGU133A
platform were considered, as samples on the HGU133B platform
contained minimal overlap with the remaining datasets. Each data
set was imported separately using the `Affy` package and RMA
expression values were generated. All data sets were quantile
normalized and each gene was mean centered across all patients.
Subtyping of Non TCGA Patients
[0219] To investigate subtype specific survival differences in all
publically available datasets, a subtype classifier described
previously (R. G. Verhaak et al., Cancer Cell 17, 98 (2010)) was
utilized to train a support vector machine (SVM). The 840 genes
used by Verhaak and colleagues for the ClacNc analysis were used to
subset the dataset. Subsequently, data sets were subsetted for
genes that were called present across all patient data sets
described above. The remaining 776 genes were used to train a
multiclass SVM on the Core samples from the TCGA dataset. The SVM
was completed using a Gaussian radial basis kernel function using
the `kernlab` package (A. Karatzoglou, et al., J. Statistical
Software, 11, 9 (2004)). This SVM was then used to predict the
subtype of the remainder of the TCGA patients and public
datasets.
Patient Classification
[0220] A SVM was trained on mouse expression data to classify
patients into "Vehicle" classification or "BLZ945" classification.
Patient expression data was subsetted for common genes across all
data sets and genes that have known mouse homologues. Similarly,
mouse expression data was subsetted for genes with human homologues
that were common across all patient samples. Subsequently, mouse
data was subsetted for differentially expressed genes identified
using the `limma` package. Human data was subsetted for the human
homologues of these differentially expressed genes. This led to a
feature reduction from 257 differentially expressed genes to 206
differentially expressed genes with known human homologues across
all patient datasets. The `kernlab` package was then used to train
a SVM on the mouse expression data using a vanilla kernel function.
This SVM was then used to predict patients into either "Vehicle"
classifier or "BLZ945" classifier.
[0221] A similar approach was used to classify patients with a
Lasso regression model. The subsetting of patient and mouse data
was identical to that described above. Instead of using the
`kernlab` package, the Lasso regression model was trained using the
`glmnet` package. This model was then used to predict patient
classification into either "Vehicle" classifier or "BLZ945"
classifier. G-CIMP patient status was determined by hierarchical
clustering of patient methylation data (H. Noushmehr et al., Cancer
Cell 17, 510 (2010)) as described below.
Stratification of Patients by G-CIMP Status
[0222] Experimentally, it appears that the survival advantage
offered by the "BLZ945" treatment signature was not due to an
enrichment of Glioma CpG Island Methylator Phenotype (GCIMP)
patients, which have previously been shown to be associated with
improved overall survival (Noushmehr). Of the 453 GBMs analyzed
from the TCGA dataset, 263 also had genomic methylation data and
were classified into the methylation clusters as described
previously. Of the 21 G-CIMP patients, 20 (95%) were classified
into the "BLZ945" classification, showing a strong enrichment of
BLZ945 samples in the G-CIMP patients. Despite this enrichment,
survival analysis of Proneural patients known to be GCIMP negative
(67/133 total Proneural patients) revealed that the "BLZ945"
classification group still showed an increase in survival of
.about.10.8 months (P=0.014).
[0223] Moreover, cox proportional hazard models demonstrated that
the increase in survival demonstrated by "BLZ945" classification
was not dependent upon G-CIMP patients. The hazard ratio associated
with the BLZ945 signature was significant with and without G-CIMP
patients. Also, the hazard ratio for G-CIMP strata was not
significant when the BLZ945 signature was also considered in a
mixed model. Thus, although the G-CIMP patients are clearly
enriched for mock "BLZ945" classification samples, the survival
benefit offered by this classification is not dependent upon GCIMP
status.
Survival Analysis
[0224] Survival analysis was completed using the `survival` package
in R (T. Therneau, in R package version 2.36-12. (2012)). Hazard
ratios were determined utilizing the `coxph` function from the
`survival` package. Patients were stratified based on the
probability of the Lasso regression classification model, G-CIMP
status, or both as indicated. P values were generated using Wald's
test.
Plots for Patient Analyses
[0225] All Kaplan-Meier survival curves, heatmaps and volcano plots
were generated in R v 2.14.1 using the `gplots` package (G. R.
Warnes et al., R package version 2.10.1, (2011).). Hazard ratio
forest plots were generated in GraphPad Prism Pro5.
Data Presentation and Statistical Analysis
[0226] Data are presented as means with their respective standard
error (SEM) or as statistical scatter plots using GraphPad Prism
Pro5. Numeric data were analyzed by unpaired twotailed Student's
t-test unless otherwise noted. For survival curves, P values were
obtained using Log Rank (Mantel-Cox) test, and Fisher's exact test
was used for histological tumor grading. P=0.05 was considered as
statistically significant.
* * * * *
References