U.S. patent application number 14/506457 was filed with the patent office on 2015-04-16 for compositions, biomarkers and their use in the treatment of cancer.
The applicant listed for this patent is Aptose Biosciences Inc.. Invention is credited to WILLIAM G. RICE.
Application Number | 20150104392 14/506457 |
Document ID | / |
Family ID | 52777444 |
Filed Date | 2015-04-16 |
United States Patent
Application |
20150104392 |
Kind Code |
A1 |
RICE; WILLIAM G. |
April 16, 2015 |
COMPOSITIONS, BIOMARKERS AND THEIR USE IN THE TREATMENT OF
CANCER
Abstract
The present invention relates to compositions, biomarkers, and
their use in treatment of cancer. In some embodiments, the
invention relates to the use of several biomarkers in methods for
determining the responsiveness of a human subject to a specific
compound, methods for monitoring the efficacy of the compound, or
methods for treating a human subject.
Inventors: |
RICE; WILLIAM G.; (Del Mar,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aptose Biosciences Inc. |
Toronto |
|
CA |
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|
Family ID: |
52777444 |
Appl. No.: |
14/506457 |
Filed: |
October 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62017505 |
Jun 26, 2014 |
|
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61919023 |
Dec 20, 2013 |
|
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61887285 |
Oct 4, 2013 |
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62037868 |
Aug 15, 2014 |
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Current U.S.
Class: |
424/9.2 ;
435/6.11; 435/6.12; 435/7.92; 436/501; 436/86; 506/7; 506/9;
514/287 |
Current CPC
Class: |
A61P 7/06 20180101; C07D
471/14 20130101; A61K 49/0004 20130101; A61P 35/02 20180101; A61K
45/06 20130101; C12Q 2600/158 20130101; A61P 35/00 20180101; C12Q
1/6886 20130101; A61K 31/706 20130101; A61K 31/4745 20130101; A61P
43/00 20180101; C12Q 2600/106 20130101; A61K 31/4745 20130101; A61K
2300/00 20130101; A61K 31/706 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/9.2 ;
514/287; 435/6.12; 506/9; 435/6.11; 436/501; 436/86; 435/7.92;
506/7 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 31/4745 20060101 A61K031/4745; A61K 49/00 20060101
A61K049/00 |
Claims
1. A method for determining the responsiveness of a human subject
comprising determining the level of activity of KLF4 in a
biological sample from a human subject, wherein a lower than normal
level of KLF4 activity is indicative that the human subject is
responsive to the compound of formula I: ##STR00006##
2. The method of claim 1, wherein a lower than normal level of KLF4
activity is indicative that the human subject is responsive to the
compound of formula I for treatment of acute myelogenous leukemia
(AML).
3. The method of claim 1, wherein a lower than normal level of KLF4
activity is indicative that the human subject is responsive to the
compound of formula I for treatment of myelodysplastic syndrome
(MDS), acute lymphocytic leukemia (ALL), chronic myelogenous
leukemia (CML), adult T-cell leukaemia (ATLL), lymphoma, gastric
cancer, or multiple myeloma.
4. The method of claim 1, wherein the human subject has AML.
5. The method of claim 1, wherein the human subject has MDS, ALL,
CML, ATLL, lymphoma, gastric cancer, or multiple myeloma.
6. A method for monitoring the efficacy of the compound of formula
I comprising determining the level of activity of KLF4 in a human
subject under treatment with the compound of formula I:
##STR00007## wherein an increase of the level of activity of KLF4
directly related to the treatment with the compound of formula I
indicates the efficacy of the compound of formula I and wherein a
decrease or unchanged level of activity of KLF4 during the
treatment with the compound of formula I indicates the lack of
efficacy of the compound of formula I.
7. The method of claim 6, wherein the human subject is treated for
AML.
8. The method of claim 6, wherein the human subject is treated for
MDS, ALL, CML, ATLL, lymphoma, gastric cancer, or multiple
myeloma.
9. A method for treating a human subject comprising determining the
level of activity of KLF4 in a human subject and administering to
the human subject an effective amount of a compound or a
pharmacologically acceptable salt or solvate thereof, the compound
having formula I: ##STR00008## if the level of KLF4 activity of the
human subject is lower than normal.
10. The method of claim 9, wherein the human subject has AML.
11. The method of claim 9, wherein the human subject has MDS, ALL,
CML, ATLL, lymphoma, gastric cancer, or multiple myeloma.
12. A method for determining the responsiveness of a human subject
comprising assaying for a genetic alteration of CDX2 or determining
the level of activity of CDX2 in a biological sample from a human
subject, wherein the presence of a genetic alteration of CDX2 or an
abnormal level of CDX2 activity is indicative that the human
subject is responsive to the compound of formula I:
##STR00009##
13. The method of claim 12, wherein the presence of a genetic
alteration of CDX2 or an abnormal level of CDX2 activity is
indicative that the human subject is responsive to the compound of
formula I for treatment of AML.
14. The method of claim 12, wherein the presence of a genetic
alteration of CDX2 or an abnormal level of CDX2 activity is
indicative that the human subject is responsive to the compound of
formula I for treatment of MDS, ALL, CML, ATLL, lymphoma, gastric
cancer, colorectal cancer or multiple myeloma.
15. The method of claim 12, wherein the human subject has AML.
16. The method of claim 1, wherein the human subject has MDS, ALL,
CML, ATLL, lymphoma, gastric cancer, colorectal cancer or multiple
myeloma.
17. A method for treating a human subject comprising assaying for a
genetic alteration of CDX2 or determining the level of activity of
CDX2 in a biological sample from a human subject and administering
to the human subject an effective amount of a compound or a
pharmacologically acceptable salt or solvate thereof, the compound
having formula I: ##STR00010## if the level of activity of CDX2 of
the human subject is higher than normal or in the presence of a
genetic alteration of CDX2 in the human subject.
18. The method of claim 17, wherein the human subject has AML.
19. The method of claim 17, wherein the human subject has MDS, ALL,
CML, ATLL, lymphoma, gastric cancer, colorectal cancer or multiple
myeloma.
20. The method of claim 1, further comprising assaying for a
genetic alteration of CDX2 or determining the level of activity of
CDX2 in the biological sample from the human subject, wherein a
lower than normal level of KLF4 activity in combination with the
presence of a genetic alteration of CDX2 or a higher than normal
level of CDX2 activity is indicative that the human subject is
responsive to the compound of formula I.
21. The method of claim 9, further comprising assaying for a
genetic alteration of CDX2 or determining the level of activity of
CDX2 in the biological sample from the human subject, and
administering to the human subject an effective amount of the
compound of formula I, or the pharmacologically acceptable salt or
solvate thereof, if the level of KLF4 activity of the human subject
is lower than normal in combination with the presence of a genetic
alteration of CDX2 or a higher than normal level of CDX2
activity.
22. A method for determining the responsiveness of a human subject
comprising determining the level of activity of at least two
biomarkers in a biological sample from a human subject, wherein at
least one biomarker is CDX2, and at least one other biomarker is
KLF4, wherein an abnormally high ratio of CDX2/KLF4 activity and/or
an abnormally low ratio of KLF4/CDX2 is indicative that the human
subject is responsive to the compound of formula I:
##STR00011##
23. The method of claim 22, wherein the activity is gene copy
number.
24. The method of claim 22, wherein an abnormally high ratio of
CDX2/KLF4 activity and/or an abnormally low ratio of KLF4/CDX2 is
indicative that the human subject is responsive to the compound of
formula I for treatment of AML.
25. The method of claim 22, wherein an abnormally high ratio of
CDX2/KLF4 activity and/or an abnormally low ratio of KLF4/CDX2 is
indicative that the human subject is responsive to the compound of
formula I for treatment of MDS, ALL, CML, ATLL, lymphoma, gastric
cancer, colorectal cancer or multiple myeloma.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/887,285, filed Oct. 4, 2013, U.S. Provisional
Application No. 61/919,023, filed Dec. 20, 2013 U.S. Provisional
Application No. 62/017,505 filed on Jun. 26, 2014, and U.S.
Provisional Application No. 62/037,868, filed Aug. 15, 2014, all of
which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to compositions,
biomarkers, and methods of using them. In particular, the invention
relates to the use of several biomarkers in methods for determining
the responsiveness of a human subject to a compound.
BACKGROUND OF THE INVENTION
[0003] Many drugs or drug candidates have been developed for the
treatment of various cancers, including some small molecule
compounds. However, not all patients are responsive to such drugs
or drug candidates. Certain biomarkers have been found correlative
to the responsiveness of some cancer patients to certain drugs.
However, there remain a large unmet medical need for determination
of the responsiveness of cancer patients to many cancer drugs or
drug candidates and more targeted treatment of specific cancers or
specific cancer patient populations.
[0004] In addition, patients with specific subsets of various
cancers may have specific genetic and/or phenotypic profiles, and
may respond differently from patients with other subsets of the
same types of cancers. Current treatments for many cancers are not
very effective in patients with specific subsets of cancers, or are
too toxic in such patients or in general. For example, acute
myelogenous leukemia (AML) is a subset of leukemia. It is the most
common form of adult leukemia (blood cancer) with a <20%
survival rate after 5 years and less than 5% if patients are age
>65. Current treatments for AML are blunt and harsh
chemotherapies (i.e., cytarabine, anthracycliine, etc.) that are
not targeted and have limiting off-target toxicities. Thus, there
is a severe unmet medical need for distinguishing patients who are
potentially sensitive to a certain treatment, and patients who are
not. The present invention meets this need and provides
compositions and methods for the effective treatment of cancer.
SUMMARY OF THE INVENTION
[0005] The present invention provides compositions and methods for
determining responsiveness of a human subject to treatment. In some
embodiments, biomarkers are provided. The present invention is
based, in part, on the discovery that a set of biomarkers in the
Caudal-related Homeobox protein CDX2--Kruppel-like factor 4 (KLF4)
signaling pathway are indicative of the responsiveness of human
subjects to a particular compound.
[0006] In one aspect, the invention provides methods for
determining the responsiveness of a human subject to a
pharmaceutical compound. In some embodiments, the methods comprise
determining the level of activity of one or more biomarkers in a
biological sample from the human subject. In some embodiments, at
least one biomarker is KLF4, CDX2, p21, p53, SP1, MYC, BCL3, CCND1,
and AATF, Caspase 3, or Annexin V.
[0007] In some embodiments, the pharmaceutical compound is a
2,4,5-trisubstituted imidazole compound described in US
2007/0123553A1, or 2-indolyl imidazo[4,5-d]phenanthroline compounds
described in U.S. Pat. No. 8,148,392, or functional derivatives
thereof. In some embodiments, the pharmaceutical compound is a
2-indolyl imidazo[4,5-d]phenanthroline derivative having the
structure of formula I:
##STR00001##
[0008] In some embodiments, a lower than a normal level of KLF4
activity is indicative that the human subject is responsive to the
compound of the present invention. In some embodiments, an
increased level of CDX2 activity than a normal level is indicative
that the human subject is responsive to the compound of the present
invention. In some embodiments, a lower than a normal level of p21
activity is indicative that the human subject is responsive to the
compound of the present invention. In some embodiments, for the
types of cancer in which KLF4 negatively regulates p53, a higher
than a normal level of p53 activity is indicative that the human
subject is responsive to the compound of the present invention. In
some other embodiments, for the types of cancer in which KLF4
positively regulates p53, a lower than a normal level of p53
activity is indicative that the human subject is responsive to the
compound of the present invention. In some embodiments, a lower
than a normal level of Caspase 3 activity is indicative that the
human subject is responsive to the compound of the present
invention. In some embodiments, a higher than a normal level of SP1
activity is indicative that the human subject is responsive to the
compound of the present invention. In some embodiments, a higher
than a normal level of MYC activity is indicative that the human
subject is responsive to the compound of the present invention. In
some embodiments, a higher than a normal level of BCL3 activity is
indicative that the human subject is responsive to the compound of
the present invention. In some embodiments, a higher than a normal
level of CCND1 activity is indicative that the human subject is
responsive to the compound of the present invention. In some
embodiments, a higher than a normal level of AATF activity is
indicative that the human subject is responsive to the compound of
the present invention. In some embodiments, the activity of at
least two biomarkers of the present invention is used to determine
the responsiveness. In some embodiments, the activity is gene copy
number. In some embodiments, the activity of KLF4 and CDX2 are used
to determine the responsiveness. In some embodiments, the ratio of
CDX2/KLF4 and/or KLF4/CDK2 gene copy numbers is used to determine
the responsiveness. In some embodiments, a lower KLF4/CDX2 ratio,
or a higher CDX2/KLF4 ratio than normal level is indicative that
the human subject is responsive to the compound of the present
invention. In some embodiments, a CDX2/KLF4 or KLF4/CDX2 ratio
similar to that of human subjects responsive to the treatment is
indicative that the human subject is responsive to the compound of
the present invention.
[0009] In some embodiments, the cancer is associated with abnormal
activity of the CDX2-KLF4 signaling pathway in the human subject.
In some embodiments, one or more components in the CDX2-KLF4
signaling pathway have abnormal activity when compared to the
activity of a control group. In some embodiments, the cancer is
leukemia/lymphoma. In some embodiments, the cancer is acute
myelogenous leukemia (AML). In some embodiments, the AML is elderly
AML. As used herein, elderly AML patients are those patients who
have, or may have AML, with an age above 60. In some embodiments,
elderly AML patients are in their first relapse. In some
embodiments, the AML is not elderly AML. As used herein,
non-elderly AML patients are those who have, or may have AML, with
an age equal to or below 60. In some embodiments, the non-elderly
AML patients are in their second relapse. In some embodiments, the
cancer is lymphoma, gastric cancer, multiple myeloma,
myelodysplastic syndromes, or combinations thereof.
[0010] In some embodiments, the method further comprises assaying
for a genetic alteration of CDX2 or determining the level of
activity of CDX2 in the biological sample from the human subject,
wherein the absence/presence of a genetic alteration of CDX2 or a
normal/abnormal level of CDX2 activity in the subject is used alone
or combined with the activity information of one or more biomarkers
described herein to determine if the human subject is responsive to
the compound of the present invention, such as formula I.
[0011] In another aspect, the invention provides methods for
monitoring the efficacy of an anti-cancer agent. In some
embodiments, the anti-cancer agent is the compound of formula I as
shown above. In some embodiments, the methods comprise determining
the level of activity of KLF4, CDX2, p21, p53, SP1, MYC, BCL3,
CCND1, AATF, Caspase 3, and/or Annexin V in a human subject under
treatment with the anti-cancer agent. In some embodiments, the
anti-cancer agent comprises the compound having formula I.
[0012] In some embodiments, an increased activity level of KLF4 in
a human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while a
decrease or unchanged activity level of KLF4 during the treatment
when compared to that in the same subject before the treatment
indicates the lack of efficacy of the anti-cancer agent. In some
embodiments, a lower activity level of KLF4 in a human subject
before treatment when compared to a predetermined standard level
indicates the efficacy of the anti-cancer agent. Alternatively, a
normalization or stabilization in the activity level of KLF4 toward
a predetermined standard level indicates efficacy of the
anti-cancer agent.
[0013] In some embodiments, a deceased activity level of CDX2 in a
human subject when compared to that of CDX2 in the subject before
the treatment indicates the efficacy of the anti-cancer agent,
while an increase or unchanged activity level of CDX2 during the
treatment when compared to that of CDX2 activity in the subject
before the treatment indicates the lack of efficacy of the
anti-cancer agent. In some embodiments, a higher activity level of
CDX2 in a human subject before treatment when compared to a
predetermined standard level indicates the efficacy of the
anti-cancer agent. Alternatively, a normalization or stabilization
in the activity level of CDX2 activity toward a predetermined
standard level indicates efficacy of the anti-cancer agent.
[0014] In some embodiments, an increased activity level of p21 in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while a
decrease or unchanged activity level of p21 during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
a lower activity level of p21 in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of p21 activity toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0015] In some embodiments, for the types of cancer in which KLF4
negatively regulates p53, a decreased activity level of p53 in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while an
increased or unchanged activity level of p53 during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
for the types of cancer in which KLF4 negatively regulates p53, a
higher activity level of p53 in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of p53 toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0016] In some embodiments, for the types of cancer in which KLF4
positively regulates p53, an increased or unchanged activity level
of p53 in a human subject when compared to that in the subject
before the treatment indicates the efficacy of the anti-cancer
agent, while a decreased activity level of p53 during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
for the types of cancer in which KLF4 positively regulates p53, a
lower activity level of p53 in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of p53 toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0017] In some embodiments, an increased activity level of Caspase
3 in a human subject when compared to that in the subject before
the treatment indicates the efficacy of the anti-cancer agent,
while a decreased or unchanged activity level of Caspase 3 during
the treatment when compared to that in the subject before the
treatment indicates the lack of efficacy of the anti-cancer agent.
In some embodiments, a lower activity level of Caspase 3 in a human
subject before treatment when compared to a predetermined standard
level indicates the efficacy of the anti-cancer agent.
Alternatively, a normalization or stabilization in the activity
level of Caspase 3 toward a predetermined standard level indicates
efficacy of the anti-cancer agent.
[0018] In some embodiments, an increased activity level of Annexin
V in a human subject when compared to that in the subject before
the treatment indicates the efficacy of the anti-cancer agent,
while a decreased or unchanged activity level of Annexin V during
the treatment when compared to that in the subject before the
treatment indicates the lack of efficacy of the anti-cancer agent.
In some embodiments, a lower activity level of Annexin V in a human
subject before treatment when compared to a predetermined standard
level indicates the efficacy of the anti-cancer agent.
Alternatively, a normalization or stabilization in the activity
level of Annexin V toward a predetermined standard level indicates
efficacy of the anti-cancer agent.
[0019] In some embodiments, a decreased activity level of SP1 in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while an
increased or unchanged activity level of SP1 during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
a higher activity level of SP1 in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of SP1 toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0020] In some embodiments, a decreased activity level of MYC in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while an
increased or unchanged activity level of MYC during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
a higher activity level of MYC in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of MYC toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0021] In some embodiments, a decreased activity level of BCL3 in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while an
increased or unchanged activity level of BCL3 during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
a higher activity level of BCL3 in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of BCL3 toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0022] In some embodiments, a decreased activity level of CCND1 in
a human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while an
increased or unchanged activity level of CCND1 during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
a higher activity level of CCND1 in a human subject before
treatment when compared to a predetermined standard level indicates
the efficacy of the anti-cancer agent. Alternatively, a
normalization or stabilization in the activity level of CCND1
toward a predetermined standard level indicates efficacy of the
anti-cancer agent.
[0023] In some embodiments, a decreased activity level of AATF in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while an
increased or unchanged activity level of AATF during the treatment
when compared to that in the subject before the treatment indicates
the lack of efficacy of the anti-cancer agent. In some embodiments,
a higher activity level of AATF in a human subject before treatment
when compared to a predetermined standard level indicates the
efficacy of the anti-cancer agent. Alternatively, a normalization
or stabilization in the activity level of AATF toward a
predetermined standard level indicates efficacy of the anti-cancer
agent.
[0024] In some embodiments, activities of at least two biomarkers
described herein are used to determine the responsiveness of a
human subject. In some embodiments, the activity is gene copy
number. In some embodiments, ratio of the activity of two
biomarkers (A/B, B/A, A/C, or C/A) is used to determine the
responsiveness. In some embodiments, at least one biomarker (i.e.,
A) is CDX2. In some embodiments, the other biomarker B is KLF4,
p21, Caspase 3, or Annexin V, while the other biomarker C is p53,
SP1, MYC, BCL3, CCND1, or AATF. In some embodiments, the ratio of
CDX2/B, B/CDX2, CDX2/C, or C/CDX2 copy numbers is used to determine
the responsiveness. In some embodiments, a lower B/CDX2 ratio, a
higher CDX2/B, a higher C/CDX2, or a lower CDX2/C ratio than a
predetermined standard is indicative that the human subject is
responsive to the compound of the present invention. In some
embodiments, an increased B/CDX2 ratio (or a decreased CDX2/B
ratio) or a decreased C/CDX2 (or an increased CDX2/C ratio) in a
human subject when compared to that in the subject before the
treatment indicates the efficacy of the anti-cancer agent, while
decreased or unchanged B/CDX2 ratio (or an increased or unchanged
CDX2/B ratio) or increased or unchanged C/CDX2 ratio (or a
decreased or unchanged CDX2/C ratio) during the treatment when
compared to that in the subject before the treatment indicates the
lack of efficacy of the anti-cancer agent. Alternatively, a
normalization or stabilization in the ratio of B/CDX2, CDX2/B,
C/CDX2, or CDX2/C toward a predetermined standard level indicates
efficacy of the anti-cancer agent. In some embodiments, the ratio
of CDX2/KLF4 and/or KLF4/CDX2 is used.
[0025] In certain embodiments of the method for monitoring the
efficacy of the anti-cancer agent, the human subject is treated for
AML. In certain other embodiments, the human subject is treated for
lymphoma, gastric cancer, multiple myeloma, myelodysplastic
syndromes, or combinations thereof.
[0026] In yet another aspect, the invention provides methods for
treating a human subject. In some embodiments, the human subject is
treated for AML. In certain other embodiments, the human subject is
treated for lymphoma, gastric cancer, multiple myeloma,
myelodysplastic syndromes, or combinations thereof. In some
embodiments, the methods comprise administering to the human
subject an effective amount of an anti-cancer agent or a
pharmacologically acceptable salt or solvate thereof of the present
invention. In some embodiments, the anti-cancer agent comprises a
compound that can modulate the activity of the CDX2-KLF4 signaling
pathway. In some embodiments, the anti-cancer agent comprises a
compound that has at least one function selected from (1)
inhibition of CDX2 activity; (2) induction of KLF4 activity; (3)
induction of p21 CDK inhibitor; (4) induction of G1/S Cell cycle
arrest; (5) induction of Caspase 3 enzyme; and (6) induction of
Apoptosis. In some embodiments, the methods further comprise
determining the level of activity of a biomarker. In some
embodiments, the biomarker is KLF4, CDX2, p21, p53, SP1, MYC, BCL3,
CCND1, AATF, Caspase 3, Annexin V, or any combination thereof. In
some embodiments, the biomarker activity in the human subject is
determined before, during, and/or after the treatment.
[0027] In certain embodiments, the methods for treating a human
subject comprise assaying for a genetic alteration of CDX2 or
determining the level of activity of CDX2 in the biological sample
from the human subject. In some embodiments, an effective amount of
the anti-cancer agent is administered to the human subject, if
there is a presence of a genetic alteration of CDX2 or a higher
than normal level of CDX2 activity. In some embodiments, the
anti-cancer agent is the compound of formula I, or the
pharmacologically acceptable salt or solvate thereof.
[0028] Additional aspects and embodiments of the invention will be
apparent from the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a diagram of non-limiting mechanism of how an
active agent of the present invention (e.g., formula I, a.k.a.
LOR-253 or APTO-253) treats cancer. Without wishing to be bound by
any particular theory, in certain cancer types, such as solid
tumors, KLF4 is down regulated, which is essential for accelerated
cell proliferation, epithelial-mesenchymal transition (EMT) and
metastasis; in certain hematologic cancers: KLF4 is down regulated,
which is essential for leukemogenesis. LOR-253 induces KLF4
expression which in turn inhibits cancer cell proliferation, EMT,
and metastasis, and/or triggers apoptosis.
[0030] FIG. 2 is a diagram that shows non-limiting mechanism of how
CDX2 functions in certain cancers and how LOR-253 treats such
cancers. Without wishing to be bound by any particular theory, in
normal cells of the hematopoietic system, the CDX2 gene is turned
off or expressed at a relatively low level. In certain cancers
including AML, CDX2 is turned on or increased, leading to
aberrantly expressed CDX2 transcription factor. CDX2 binds to the
promoter region of the KLF4 gene and inhibits KLF4 expression,
which is an essential step to promote leukemogenesis. LOR-253
induces KLF4 expression which in turn triggers apoptosis of the
cancerous leukocytes.
[0031] FIG. 3 is a graphical representation of the effect of
LOR-253 on proliferation of a number of cancer cell lines in vitro.
Cells of various cancer cell lines were incubated with LOR-253 as
described in the Examples, and the cell concentrations of various
cell lines for 50% of maximal inhibition of cell proliferation
(GI.sub.50) by LOR-253 were determined and shown in the figure.
[0032] FIG. 4 is a graphical representation of the effect of
LOR-253 on the expression levels of KLF4 in two cell lines, THP-1
and HL60.
[0033] FIG. 5 depicts BD FACSCalibur flow cytometer assay result
for THP1 and HL-60 AML cell lines treated with DMSO or LOR-253
(left panel). FIG. 5 also shows that treatment of LOR-253 results
in G1/S cell cycle arrest in THP1 and HL-60 cell lines (right
panel).
[0034] FIG. 6 depicts a BD FACSCanto flow cytometry acquisition
plots for THP-1 cells treated with DMSO, 0.5 or 1 uM of LOR-253.
THP-1 cells treated with LOR-253 showed elevated Annexin V
staining, indicating induction of apoptosis (Q3: Annexin
V+/PI-).
[0035] FIG. 7 depicts Caspase 3 expression level in THP1 and HL-60
AML cell lines treated with DMSO or LOR-253.
[0036] FIG. 8 depicts fold change in expression of BAX and BCL2 in
THP1 cells treated with DMSO or 0.5 .mu.M LOR-253
[0037] FIG. 9 depicts in vivo efficacy of LOR-253 HCL in H226
xenograft model mice. Tumor sizes of H226_xenograft mice treated
with LOR-253 HCL or negative control measured on the indicated days
are shown.
[0038] FIG. 10 depicts the pharmacokinetic (PK) in CD-1 nude mice
treated by LOR-253 at a dosage of 1, 5, or 15 mg/kg. The serum
level of LOR-253 has a dose related increase.
[0039] FIG. 11 depicts the pharmacodynamic (PD) responses in mice
treated for 5 consecutive days with 1, 5, and 15 mg/kg LOR-253. The
KLF4 protein level was measured 16 hours after the last dose.
[0040] FIG. 12 depicts tumor shrinkage in patient with NSCLC
(poorly differentiated adenocarcinoma) before the treatment (see
the upper panel) and after the treatment (see the lower panel)
[0041] FIG. 13 depicts standard curve generated by serially diluted
standards of known concentrations of CDX2 and KLF4 genes prepared
at 3.times.10.sup.2-3.times.10.sup.6 copies.
[0042] FIG. 14 depicts CDX2 expression levels in MM.1R, Eol-1,
Mino, HEL92.1.7, CA46, RL, THP1, and Toledo cell lines determined
as copy numbers using the standard curves.
[0043] FIG. 15 depicts KLF4 expression levels in MM.1R, Eol-1,
Mino, HEL92.1.7, CA46, RL, THP1, and Toledo cell lines determined
as copy numbers using the standard curves.
[0044] FIG. 16 depicts correlation of CDX2 mRNA copy number or KLF4
mRNA copy number to IC50 of LOR-253 in MM.1R, Eol-1, Mino,
HEL92.1.7, CA46, RL, THP1, and Toledo cell lines resulted from one
study.
[0045] FIG. 17 depicts correlation of CDX2/KLF4 or KLF4/CDX2 mRNA
ratio to IC50 of LOR-253 in MM.1R, Eol-1, Mino, HEL92.1.7, CA46,
RL, THP1, and Toledo cell lines resulted from one study.
[0046] FIG. 18 is a diagram that shows non-limiting mechanism of
how silencing KLF4 gene or gene product plays a central role in
various heme malignancies. For example, epigenetic methylation of
the KLF4 gene relates to adult T-cell lymphoma patients, mutations
in KLF4 genes or proteins relate to pediatric T-cell ALL patients,
elevated microRNA-2909 relates to pediatric B-cell ALL patients,
aberrant expression of CDX2 relates to AML, ALL, and
myelodysplastic syndrome (MDS) patients, which all lead to
silencing of KLF4 activity (including but not limited to
expressional and functional activities). Silencing of KLF4 has also
been observed in various lymphomas. The lower part of the diagram
illustrates that silenced KLF4 causes increased cancer cell
proliferation through various "cell fate genes".
[0047] FIG. 19 is a diagram that shows non-limiting mechanism of
how KLF4 gene activity can be silenced by genetic mutations or
epigenetic events and how LOR-253/APTO-253 can induce KLF4
expression. Such epigenetic events include, but are not limited to,
DNA hypomethylation or demethylation, aberrant/elevated expression
of CDX2 which can lead to increased presence of a demethylase KDM5B
in an upstream regulatory region of the klf4 gene, and elevated
amount of miR-2909. LOR-253/APTO-253 can induce KLF4 expression by
relieving gene silencing caused at least by CDX2/KDM5B and/or other
mechanisms
[0048] FIG. 20 depicts in vivo efficacy of LOR-253 HCL in Kasumi-1
xenograft model mice. Tumor sizes of Kasumi-1 xenograft mice
treated with LOR-253 HCL or negative control measured on the
indicated days are shown.
[0049] FIG. 21 depicts body weight measurements on the indicated
days of Kasumi-1 tumor-bearing Mice treated with LOR-253 HCL or
negative control.
[0050] FIG. 22 depicts in vivo efficacy of LOR-253 HCL as a single
agent or in combination with azacitidine in HL-60 xenograft model
mice. Tumor sizes of HL-60 xenograft mice treated with the
indicated conditions measured on the indicated days are shown.
[0051] FIG. 23 and FIG. 24 depict the tumor sizes of individual
animals at the beginning (Day 1) and end (Day 19) of the study of
FIG. 17, respectively.
[0052] FIG. 25 depicts in vivo efficacy of LOR-253 HCL in KG-1
xenograft model mice. Tumor sizes of KG-1 xenograft mice treated
with LOR-253 HCL or negative control measured on the indicated days
are shown.
[0053] FIG. 26 depicts in vivo efficacy of LOR-253 HCL as a single
agent or in combination with azacitidine in THP-1 xenograft model
mice. Tumor sizes of THP-1 xenograft mice treated with the
indicated conditions measured on the indicated days are shown.
[0054] FIG. 27 depicts correlation of CDX2 mRNA copy number or KLF4
mRNA copy number to IC50 of LOR-253/APTO-253 in MM.1R, Eol-1, Mino,
HEL92.1.7, CA46, RL, THP1, Toledo, U266B1, MV411, DB, HL60, CD47
LOW (or MOLM13 CD47-), CD47 HIGH (or MOLM13 CD47+), KG-1, and SKM-1
cell lines resulted from another study.
[0055] FIG. 28 depicts correlation of CDX2/KLF4 or KLF4/CDX2 mRNA
ratio to IC50 of LOR-253/APTO-253 in MM.1R, Eol-1, Mino, THP1,
Toledo, U266B1, MV411, DB, HL60, CD47 LOW (or MOLM13 CD47-), CD47
HIGH (or MOLM13 CD47+), KG-1, and SKM-1 cell lines resulted from
another study.
DETAILED DESCRIPTION
Definitions
[0056] The verb "comprise" as is used in this description and in
the claims and its conjugations are used in its non-limiting sense
to mean that items following the word are included, but items not
specifically mentioned are not excluded.
[0057] The term "a" or "an" refers to one or more of that entity;
for example, "a gene" refers to one or more genes or at least one
gene. As such, the terms "a" (or "an"), "one or more" and "at least
one" are used interchangeably herein. In addition, reference to "an
element" by the indefinite article "a" or "an" does not exclude the
possibility that more than one of the elements are present, unless
the context clearly requires that there is one and only one of the
elements.
[0058] The invention provides polynucleotides that can be used for
biomarker assays. In some embodiments the polynucleotides are
isolated, purified, chimeric, recombinant or synthetic. As used
herein, the terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", "nucleic acid fragment", and "isolated
nucleic acid fragment" are used interchangeably herein and
encompass DNA, RNA, cDNA, whether single stranded or double
stranded, as well as chemical modifications thereof. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. Nucleotides (usually found in their
5'-monophosphate form) are referred to by a single letter
designation as follows: "A" for adenylate or deoxyadenylate (for
RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate,
"G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for
deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C
or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and
"N" for any nucleotide. In some embodiments, the sequences of the
polynucleotides are derived from gene markers of the present
invention.
[0059] The invention also provides proteins or polypeptides that
can be used for biomarker assays. In some embodiments the proteins
or polypeptides are isolated, purified, chimeric, recombinant or
synthetic. As used herein, the term "polypeptide" or "protein"
refers to amino acid polymers of any length. The polymer may be
linear or branched, it may comprise modified amino acids, and it
may be interrupted by non-amino acids. The terms also encompass an
amino acid polymer that has been modified naturally or by
intervention; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other manipulation
or modification, such as conjugation with a labelling component.
Also included are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, eic), as well as other modifications known in the art.
Polypeptides can occur as single chains or associated chains.
Polypeptides of the invention can take various forms (e.g. native,
fusions, glycosylated, non-glycosylated, lipidated, non-lipidated,
phosphorylated, non-phosphorylated, myristoylated,
non-myristoylated, monomeric, multimeric, particulate, denatured,
etc). In some embodiments, the sequences of the proteins or
polypeptides are derived from gene markers of the present
invention.
[0060] Single letter amino acid abbreviations used herein have
their standard meaning in the art, and all peptide sequences
described herein are written according to convention, with the
N-terminal end to the left and the C-terminal end to the right.
[0061] The invention provides probes and primers that are derived
from the nucleic acid sequences of the biomarker genes. The term
"probe" as used herein refers to an oligonucleotide which is
capable of specific annealing to the amplification target. The term
"primer" as used herein refers to an oligonucleotide which is
capable of annealing to the amplification target allowing a DNA
polymerase to attach, thereby serving as a point of initiation of
DNA synthesis when placed under conditions in which synthesis of
primer extension product is induced, i.e., in the presence of
nucleotides and an agent for polymerization such as DNA polymerase
and at a suitable temperature and pH. The (amplification) primer is
preferably single stranded for maximum efficiency in amplification.
Preferably, the primer is an oligodeoxyribonucleotide. The primer
must be sufficiently long to prime the synthesis of extension
products in the presence of the agent for polymerization. The exact
lengths of the primers will depend on many factors, including
temperature and composition (A/T vs. G/C content) of primer. A pair
of bi-directional primers consists of one forward and one reverse
primer as commonly used in the art of DNA amplification such as in
PCR amplification.
[0062] The terms "array" or "matrix" refer to an arrangement of
addressable locations or "addresses" on a device. The locations can
be arranged in two-dimensional arrays, three-dimensional arrays, or
other matrix formats. The number of locations may range from
several to at least hundreds of thousands. Most importantly, each
location represents a totally independent reaction site. A "nucleic
acid array" refers to an array containing nucleic acid probes, such
as oligonucleotides or larger portions of genes. The nucleic acid
on the array is preferably single-stranded. Arrays wherein the
probes are oligonucleotides are referred to as "oligonucleotide
arrays" or "oligonucleotide chips." A "microarray," also referred
to herein as a "biochip" or "biological chip," is an array of
regions having a density of discrete regions of at least about
100/cm.sup.2, and preferably at least about 1000/cm.sup.2. The
regions in a microarray have typical dimensions, for example,
diameters, in the range of between about 10-250 .mu.m, and are
separated from other regions in the array by about the same
distance. None limiting examples of compositions and methods for
making and using arrays are described in U.S. Pat. Nos. 5,202,231,
5,695,940, 5,525,464, 5,445,934, 5,744,305, 5,677,195, 5,800,992,
5,871,928, 5,795,716, 5,700,637, 6,054,270, 5,807,522, and
6,110,426, each of which is incorporated by reference herein in its
entirety for all purposes. As used herein, when the level of a
biomarker goes toward the level of a predetermined standard level,
it is called normalization
[0063] As used herein, the term "marker" or "biomarker" encompasses
a broad range of intra- and extra-cellular events as well as
whole-organism physiological changes. A marker may represent
essentially any aspect of cell function, for example, but not
limited to, levels or rate of production of signaling molecules,
transcription factors, metabolites, gene transcripts as well as
post-translational modifications of proteins. Marker may include
partial and/or whole genome analysis of transcript levels, rates,
and/or stability, and partial and/or whole proteome analysis of
protein levels, activity and/or modifications. A signature may
refer to a gene or gene product which is up- or down-regulated in a
subject to be treated compared to clinically normal subjects. A
signature may also refer to a gene or gene product which is up- or
down-regulated in a treated subject having the disease compared to
an untreated subject. That is, the gene or gene product is
sufficiently specific to the treated cell that it may be used,
optionally with other genes or gene products, to identify, predict,
or detect efficacy of a small molecule. Thus, in some embodiments,
a signature is a gene or gene product that is characteristic of
efficacy of a compound in a diseased cell or the response of that
diseased cell to treatment by the compound.
[0064] As used herein, the term "sample" or "biological sample"
refers to a sample obtained from an organism or from components
(e.g., cells) of an organism. The sample may be of any biological
tissue or fluid. The sample may be a sample which is derived from a
patient. Such samples include, but are not limited to, sputum,
blood, blood cells (e.g., white blood cells), tissue or biopsy
samples (e.g., tumor biopsy), urine, peritoneal fluid, and pleural
fluid, patient derived xenografts (PDXs), or cells therefrom.
Biological samples may also include sections of tissues such as
frozen sections taken for histological purposes. Methods for
obtaining biological samples are well known in the art and any
standard methods for obtaining biological samples can be employed.
Biological samples that find use with the methods of the present
invention include but are not limited to blood (including but not
limited to serum, blood, plasma, whole blood and derivatives
thereof), skin, hair, hair follicles, saliva, oral mucous, vaginal
mucous, sweat, tears, epithelial tissues, urine, semen, seminal
fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid
(Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid,
lymph, and tissue extract sample or biopsy samples. (See, e.g.,
Clinical Proteomics: Methods and Protocols, Vol. 428 in Methods in
Molecular Biology, Ed. Antonia Vlahou (2008).) In some embodiments,
the biological sample is selected from blood (including but not
limited to serum, blood, plasma, whole blood and derivatives
thereof), skin, hair, hair follicles, saliva, oral mucous, vaginal
mucous, sweat, tears, epithelial tissues, urine, semen, seminal
fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid
(Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid,
lymph, and tissue extract sample or biopsy sample. In some
embodiments, the biological sample is a blood sample.
[0065] In some embodiments, a collection of activity profiles of a
panel of biomarkers is provided. As used herein, the term "activity
profile" refers to a set of data representing distinctive features
or characteristics of one or more biomarkers. Such features or
characteristics include, but are not limited to, transcript
abundance, transcript stability, transcription rate, translation
rate, post-translation modification, protein abundance, protein
stability, and/or protein enzymatic activity, etc. In some
embodiments, the activity profile comprises data related to gene
expression level of each biomarker. In some embodiments, the
collection comprising activity profiles is obtained from a specific
population of subjects. In some embodiments, the specific
population of subjects consists of clinically normal subjects. In
some embodiments, the population consists of patents responsive to
one or more anti-cancer agents of the present invention. In some
embodiments, the population consists of patients not responsive to
one or more anti-cancer agents of the present invention.
[0066] In some embodiments, the collection comprises activity
profiles that are statistically homogeneous in one or more aspects,
e.g., statistically homogeneous in one or more quantitative or
semi-quantitative parameters describing the features and
characteristics of the activity profiles. In some embodiments, the
quantitative parameters include, but are not limited to, transcript
abundance, transcript stability, transcription rate, translation
rate, post-translation modification, protein abundance, protein
stability, and/or protein enzymatic activity, etc. Whether a group
of activity profiles are statistically homogeneous or not in one or
more aspects can be determined by any suitable statistic test
and/or algorithm known to one skilled in the art.
[0067] In some embodiments, one or more of the biomarkers increase
its activity in response to the treatment. In some embodiments, one
or more of the biomarkers decrease its activity in response to the
treatment. In some embodiments, one or more of the biomarkers
remains its activity in response to the treatment. As used herein,
the activity of a biomarker can be a parameter at genomic DNA
level, transcriptional level, post-transcriptional level,
translational level, post-translational level, including, but not
limited to gene activity, RNA activity, and protein activity. The
gene activity can be gene copy number, gene amplification number,
or promoter activity, etc. RNA activity can be mRNA abundance,
synthesis rate, and/or stability, etc. Protein activity can be
protein abundance, synthesis rate, stability, enzymatic activity,
phosphorylation rate, modifications, binding activity, etc.
[0068] As used herein, when the level of a biomarker goes toward
the level of a predetermined standard level, it is called
normalization.
[0069] As used herein, when the level of a biomarker reduces its
speed of going away from the level of a predetermined standard
level, it is called stabilization.
[0070] The term "CDX2-KLF4 signaling pathway" as used herein refers
to a group of biological molecules that work together to control
one or more cellular functions through CDX2 and/or KLF4, or by
affecting the expression or activity of CDX2 or KLF4 as described
herein, directly or indirectly. Sometimes the expression level
and/or activity of CDX2 and/or KLF4 is also referred to as
"CDX2-KLF4 axis".
[0071] As used herein, the term "component in the CDX2-KLF4
signaling pathway", referrers to CDX2, KLF4, other genes, gene
products (including but not limited to RNA and protein), or other
biological molecules that can modulate the activity of CDX2 and/or
KLF4, directly or indirectly, or genes, gene products, or other
biological molecules that can be modulated by CDX2 and/or KLF4,
directly or indirectly. The modulation can either increase or
decrease the activity level of a given gene. Such components
include, but are not limited to those described in Lengerke et al.
("Caudal genes in blood development and leukemia", Ann NY Acad.
Sci. 2012 August; 1266:47-54.), Scholl et al. ("The homeobox gene
CDX2 is aberrantly expressed in most cases of acute myeloid
leukemia and promotes leukemogenesis", J. Clin. Invest.
117:1037-1048 (2007).), Yoon et al. ("Kruppel-like Factor 4
Mediates p53-dependent G1/S Cell Cycle Arrest in Response to DNA
Damage", J Biol Chem Vol. 278, No. 4, January 24, pp. 2101-2105,
2003), Faber et al. ("CDX2-driven leukemogenesis involves KLF4
repression and deregulated PPAR.gamma. signaling", J Clin Invest.
2013 Jan. 2; 123(1):299-314.), Rouhi et al. ("Deregulation of the
CDX2-KLF4 axis in acute myeloid leukemia and colon cancer",
Oncotarget. 2013 February; 4(2):174-175.), Lengerke et al. ("BMP
and Wnt specify hematopoietic fate by activation of the Cdx-Hox
pathway", Cell Stem Cell 2008 Jan. 10; 2(1):72-82.), Saandi et al.
("Regulation of the tumor suppressor homeogene Cdx2 by HNF4.alpha.
in intestinal cancer", Oncogene. 2013 Aug. 8; 32(32):3782-8.),
Malik et al., (miR-2909-mediated regulation of KLF4: a novel
molecular mechanism for differentiating between B-cell and T-cell
pediatric acute lymphoblastic leukemias. Mol Cancer. 13:175, 2014),
and Rowland et al. ("KLF4, p21 and context-dependent opposing
forces in cancer", Nat Rev Cancer. 2006 January; 6(1):11-23), each
of which is incorporated herein by reference in its entirety for
all purposes. In some embodiments, such components include, but are
not limited to CDX2, KLF4, KDM5B, miR-2909, p53, p21, Caspase-3,
Annexin V, BAX, BCL2, BCL3, BMP, Wnt, HNF4.alpha., Fgf, SP1, MYC,
CCND1, AATF, and Hox genes. KLF4 negatively regulates (or
suppresses) the activity of SP1, MYC, BCL3, CCND1, and AATF,
directly or indirectly, while positively regulates the activity of
p21. In addition, KLF4 negatively regulates the activity of p53 in
some cancer types (e.g. breast cancer, as described by Rowland et
al., The KLF4 tumour suppressor is a transcriptional repressor of
p53 that acts as a context-dependent oncogene. Nat Cell Biol. 2005.
7:1074-82), but positively regulates the activity of p53 in some
other cancer types (e.g., colon cancer and multiple myeloma, as
described by Ghaleb et al. (Kruppel-like factor 4 exhibits
antiapoptotic activity following gamma-radiation-induced DNA
damage. Oncogene. 2007. 26:2365-73), and Schoenhals et al.
(Kruppel-like factor 4 blocks tumor cell proliferation and promotes
drug resistance in multiple myeloma. Haematologica. 2013.
98:1442-9)). Many of the genes regulated by KLF4 are referred to as
"cell fate genes". An illustration of some possible effects of
silenced KLF4 gene expression or activity on some of the genes
modulated by KLF4 can be found in FIG. 13. Some other components in
the CDX2-KLF4 signaling pathway can modulate the expression of
KLF4. Examples of such components include, but are not limited to,
CDX2, KDM5B (a demethylase), and an miRNA "miR-2909". An
illustration of some possible mechanisms of how these KLF4
modulators can affect KLF4 gene expression or activity in various
cancer types can be found in FIG. 13 and FIG. 14. A component in
the CDX2-KLF4 signaling pathway can be used as a biomarker
according to the methods described herein for treating cancers,
especially for treating cancers by an anti-cancer agent of the
present invention, such as LOR-253/APTO-253.
[0072] As used herein, the term "modulate the CDX2-KLF4 signaling
pathway" refers to the process in which one or more component in
the CDX2-KLF4 signaling pathway is modulated by an agent or an
event (including a mutation). In some embodiments, such modulation
leads to increased, decreased, normalized, and/or stabilized
activity of one or more components in the CDX2-KLF4 signaling
pathway.
[0073] As used herein, the term "baseline level" of the activity of
a biomarker refers to the activity level of a given biomarker in a
human subject before any treatment.
[0074] The term lower alkyl refers to (C.sub.1-C.sub.6)alkyl. A
lower alkyl includes methyl, ethyl, propyl, isopropyl, butyl,
iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl,
(C.sub.3-C.sub.6)cycloalkyl (e.g., cyclopropyl, cyclobutyl,
cyclopentyl, or cyclohexyl),
(C.sub.3-C.sub.6)cycloalkyl(C.sub.1-C.sub.6)alkyl (e.g.,
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,
cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,
2-cyclopentylethyl, or 2-cyclohexylethyl), (C.sub.1-C.sub.6)alkoxy
(e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,
sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy)
(C.sub.2-C.sub.6)alkenyl (e.g., vinyl, allyl, 1-propenyl,
2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl,
3-hexenyl, 4-hexenyl, or 5-hexenyl), (C.sub.2-C.sub.6)alkynyl
(e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl,
3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,
1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl),
(C.sub.1-C.sub.6)alkanoyl (e.g., acetyl, propanoyl or butanoyl),
halo(C.sub.1-C.sub.6)alkyl (e.g., iodomethyl, bromomethyl,
chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,
2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl),
hydroxy(C.sub.1-C.sub.6)alkyl (e.g., hydroxymethyl, 1-hydroxyethyl,
2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,
1-hydroxy butyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl,
1-hydroxyhexyl, or 6-hydroxyhexyl), (C.sub.1-C.sub.6)alkoxycarbonyl
(e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or
hexyloxycarbonyl), (C.sub.1-C.sub.6)alkylthio (e.g., methylthio,
ethylthio, propylthio, isopropylthio, butylthio, isobutylthio,
pentylthio, or hexylthio), and/or (C.sub.2-C.sub.6)alkanoyloxy
(e.g., acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy,
pentanoyloxy, or hexanoyloxy).
[0075] A compound described herein or its function derivative can
be used according to the present invention. The term "derivative"
as used herein includes derivatives, analogs, prodrugs, and
unnatural precursors of a given compound.
[0076] The biomarkers of the present invention can be used to
determine treatment efficacy of the anti-cancer agents of the
present invention for a human subject. As used herein, the term
"treatment efficacy" and variants thereof are generally indicated
by alleviation of one or more signs or symptoms associated with the
disease and can be readily determined by one skilled in the art.
"Treatment efficacy" may also refer to the prevention or
amelioration of signs and symptoms of toxicities typically
associated with standard or non-standard treatments of a disease.
Determination of treatment efficacy is usually indication and
disease specific and can include any methods known or available in
the art for determining that a treatment is providing a beneficial
effect to a subject. For example, evidence of treatment efficacy
can include but is not limited to general improvements in the
overall health of the subject, such as but not limited to
enhancement of patient life quality, increase in predicted subject
survival rate, decrease in depression, decreasing the severity
and/or frequency one or more symptoms resulting from the disease,
diminishing the extent of the disease, stabilizing the disease
(e.g., preventing or delaying the worsening of the disease), delay
or slowing the progression of the disease, ameliorating the disease
state, etc. In some embodiments of the invention, the treatment
efficacy is clinical efficacy or statistically significant.
[0077] The terms "treating" and "treatment" as used herein refer to
an approach for obtaining beneficial or desired results including
clinical results, and may include even minimal changes or
improvements in one or more measurable markers of the disease or
condition being treated. A treatment is usually effective to reduce
at least one symptom of a condition, disease, disorder, injury or
damage. Exemplary markers of clinical improvement will be apparent
to persons skilled in the art. Examples include, but are not
limited to, one or more of the following: decreasing the severity
and/or frequency one or more symptoms resulting from the disease,
diminishing the extent of the disease, stabilizing the disease
(e.g., preventing or delaying the worsening of the disease), delay
or slowing the progression of the disease, ameliorating the disease
state, decreasing the dose of one or more other medications
required to treat the disease, and/or increasing the quality of
life, etc.
[0078] "Prophylaxis," "prophylactic treatment," or "preventive
treatment" refers to preventing or reducing the occurrence or
severity of one or more symptoms and/or their underlying cause, for
example, prevention of a disease or condition in a subject
susceptible to developing a disease or condition (e.g., at a higher
risk, as a result of genetic predisposition, environmental factors,
predisposing diseases or disorders, or the like).
[0079] The term "disorder" or "disease" used interchangeably
herein, refers to any alteration in the state of the body or one of
its organs and/or tissues, interrupting or disturbing the
performance of organ function and/or tissue function (e.g., causes
organ dysfunction) and/or causing a symptom such as discomfort,
dysfunction, distress, or even death to a subject afflicted with
the disease.
[0080] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be incorporated into a pharmaceutical composition administered to a
patient without causing any significant undesirable biological
effects or interacting in a deleterious manner with any of the
other components of the composition in which it is contained. When
the term "pharmaceutically acceptable" is used to refer to a
pharmaceutical carrier or excipient, it is implied that the carrier
or excipient has met the required standards of toxicological and
manufacturing testing or that it is included on the Inactive
Ingredient Guide prepared by the U.S. Food and Drug
administration.
[0081] The term "effective amount" refers to the amount of one or
more compounds that renders a desired treatment outcome. An
effective amount may be comprised within one or more doses, i.e., a
single dose or multiple doses may be required to achieve the
desired treatment endpoint.
[0082] The term "therapeutically effective amount" as used herein,
refers to the level or amount of one or more agents needed to treat
a condition, or reduce or prevent injury or damage, optionally
without causing significant negative or adverse side effects.
[0083] A "prophylactically effective amount" refers to an amount of
an agent sufficient to prevent or reduce severity of a future
disease or condition when administered to a subject who is
susceptible and/or who may develop a disease or condition.
[0084] According to the methods of the present invention, the term
"subject," and variants thereof as used herein, includes any
subject that has, is suspected of having, or is at risk for having
a disease or condition. Suitable subjects (or patients) include
mammals, such as laboratory animals (e.g., mouse, rat, rabbit,
guinea pig), farm animals, and domestic animals or pets (e.g., cat,
dog). Non-human primates and, preferably, human patients, are
included. A subject "at risk" may or may not have detectable
disease, and may or may not have displayed detectable disease prior
to the diagnostic or treatment methods described herein. "At risk"
denotes that a subject has one or more so-called risk factors,
which are measurable parameters that correlate with development of
a condition described herein, which are described herein. A subject
having one or more of these risk factors has a higher probability
of developing a condition described herein than a subject without
these risk factor(s). One example of such a risk factor is an
increase or decrease in a biomarker of the present invention as
compared to a clinically normal sample.
[0085] As used herein, the term "predetermined standard level" or
"predetermined activity profiles" refers to standardized data or
data set representing the average, representative features or
characteristics of one or more biomarkers in a specific population.
Such features or characteristics include, but are not limited to,
gene copy number, gene amplification, transcript abundance,
transcript stability, transcription rate, translation rate,
post-translation modification, protein abundance, protein
stability, and/or protein enzymatic activity, etc. In some
embodiments, the specific population of subjects are consisting of
about 5, about 10, about 20, about 50, about 100, about 200, about
300, about 400, about 500, about 1000, about 5000, about 10K, or
more individual subjects. The predetermined activity profile can be
a standardized data or data set collected before, during, or after
the specific population of subjects has been all exposed to a drug.
In some embodiments, the specific population is consisting of
subjects responsive to a given drug.
[0086] As used herein, a subject is "responsive" to a drug for
treating when the level of one or more of the biomarkers of the
present invention increases or decreases toward a pre-determined
standard level when the subject is exposed to a the drug, or when
the drug modifies the speed of level changes of one or more
biomarkers of the present invention compared to a placebo. For
methods related to detection, quantitation and comparison of
biomarker levels, see, e.g., Current Protocols in Molecular
Biology, Ed. Ausubel, Frederick M. (2010); Current Protocols in
Protein Science Last, Ed. Coligan, John E., et al. (2010); Current
Protocols in Nucleic Acid Chemistry, Ed. Egli, Martin (2010);
Current Protocols in Bioinformatics, Ed. Baxevanis, Andreas D.
(2010); and Molecular Cloning: A Laboratory Manual, Third Edition,
Sambrook, Joseph (2001), all of which are incorporated herein by
reference in their entirety.
[0087] In certain embodiments, when measuring biomarkers or other
indicators of treatment, an "increased" or "decreased" amount or
level may include a "statistically significant" amount. In some
embodiments, the administration of an anti-cancer agent such as
LOR-253 provides "statistically significant" therapeutic effect or
clinical efficacy for treating the cancer. In some embodiments,
such statistically significant therapeutic effect or clinical
efficacy includes slower cancer cell proliferation or tumor growth
caused by the anti-cancer agent as compared to a control vehicle. A
result is typically referred to as statistically significant if it
is unlikely to have occurred by chance. The significance level of a
test or result relates traditionally to the amount of evidence
required to accept that an event is unlikely to have arisen by
chance. In certain cases, statistical significance may be defined
as the probability of making a decision to reject the null
hypothesis when the null hypothesis is actually true (a decision
known as a Type I error, or "false positive determination"). This
decision is often made using the p-value: if the p-value is less
than the significance level, then the null hypothesis is rejected.
The smaller the p-value, the more significant the result. Bayes
factors may also be utilized to determine statistical significance
(see, e.g., Goodman S., Ann Intern Med. 130:1005-13, 1999). In some
embodiments, an "increased" or "decreased" amount or level is about
1.1.times., 1.2.times., 1.3.times., 1.4.times., 1.5.times.,
2.times., 2.5.times., 3.times., 3.5.times., 4.times., 4.5.times.,
5.times., 6.times., 7.times., 8.times., 9.times., 10.times.,
15.times., 20.times., 25.times., 30.times., 40.times., or 50.times.
more or less the amount of a predetermined standard, or the amount
of a determined time point relative to a previous or earlier
timepoint.
[0088] The phrase "pharmaceutically acceptable salt(s)", as used
herein, unless otherwise indicated, includes salts of acidic or
basic groups which may be present in a compound. Compounds that are
basic in nature are capable of forming a wide variety of salts with
various inorganic and organic acids. The acids that may be used to
prepare pharmaceutically acceptable acid addition salts of such
basic compounds are those that form non-toxic acid addition salts,
i.e., salts containing pharmacologically acceptable anions, such as
the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,
bistosylate, bitartrate, borate, bromide, calcium edetate,
camsylate, carbonate, chloride, clavulanate, citrate,
dihydrochloride, edetate, edislyate, estolate, esylate,
ethylsuccinate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, iodide, isothionate, lactate, lactobionate, laurate,
malate, maleate, mandelate, mesylate, methylsulfate, mucate,
napsylate, nitrate, oleate, oxalate, pamoate (embonate), palmitate,
pantothenate, phospate/diphosphate, polygalacturonate, salicylate,
stearate, subacetate, succinate, tannate, tartrate, teoclate,
tosylate, triethiodode, and valerate salts.
Cancers
[0089] The cancers which can be treated in accordance with one
embodiment of the present invention thus include, but are not
limited to, leukaemias; adenocarcinomas and carcinomas, including
squamous cell carcinomas. Carcinomas are also frequently referred
to as "solid tumours," as described above, and examples of commonly
occurring solid tumours that can be treated in accordance with the
present invention include, but are not limited to, anal cancer,
bladder cancer, colon cancer, colorectal cancer, duodenal cancer,
gastric (stomach) cancer, lung (non-small cell) cancer, oesophageal
cancer, prostate cancer, rectal cancer and small intestine cancer.
Accordingly, one embodiment of the present invention provides for
the use of a compound of Formula I in the treatment of a cancer
selected from the group of leukemia, bladder cancer, lung
(non-small cell) cancer, prostate cancer and a cancer of the GI
tract, wherein cancers of the GI tract include, but are not limited
to, anal cancer, colon cancer, colorectal cancer, duodenal cancer,
gastric (stomach) cancer, oesophageal cancer, rectal cancer and
small intestine cancer.
[0090] The term "leukaemia" or "leukemia" refers broadly to
progressive, malignant diseases of the blood-forming organs.
Leukaemia is typically characterized by a distorted proliferation
and development of leukocytes and their precursors in the blood and
bone marrow but can also refer to malignant diseases of other blood
cells such as erythroleukaemia, which affects immature red blood
cells. Leukaemia is generally clinically classified on the basis of
(1) the duration and character of the disease--acute or chronic;
(2) the type of cell involved--myeloid (myelogenous), lymphoid
(lymphogenous) or monocytic, and (3) the increase or non-increase
in the number of abnormal cells in the blood--leukaemic or
aleukaemic (subleukaemic). Leukaemia includes, for example, acute
leukaemia, chronic leukaemia, adult leukaemia, pediatric/child
leukaemia, lymphocytic leukemia, myeloid leukemia, acute
lymphocytic leukemia (ALL), acute nonlymphocytic leukaemia, chronic
lymphocytic leukaemia, acute granulocytic leukaemia, chronic
granulocytic leukaemia, acute promyelocytic leukaemia, chronic
myelogenous leukemia (CML), T-cell leukaemia, B-cell leukaemia,
adult T-cell leukaemia, pediatric T-cell ALL, pediatric B-cell ALL,
aleukaemic leukaemia, aleukocythemic leukaemia, basophylic
leukaemia, blast cell leukaemia, bovine leukaemia, chronic
myelocytic leukaemia, leukaemia cutis, embryonal leukaemia,
eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia,
hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic
leukaemia, stem cell leukaemia, acute monocytic leukaemia,
leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia,
lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia,
lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocytic
leukaemia, micromyeloblastic leukaemia, monocytic leukaemia,
myeloblastic leukaemia, myelocytic leukaemia, myeloid granulocytic
leukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cell
leukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder
cell leukaemia, Schilling's leukaemia, stem cell leukaemia,
subleukaemic leukaemia, and undifferentiated cell leukaemia.
[0091] The term "carcinoma" refers to a malignant new growth made
up of epithelial cells tending to infiltrate the surrounding
tissues and give rise to metastases. The term "carcinoma" also
encompasses adenocarcinomas. Adenocarcinomas are carcinomas that
originate in cells that make organs which have glandular
(secretory) properties or that originate in cells that line hollow
viscera, such as the gastrointestinal tract or bronchial epithelia,
and include adenocarcinomas of the lung and prostate.
[0092] Methods of the present invention can be applied in the
treatment of early stage cancers including early neoplasias that
may be small, slow growing, localized and/or nonaggressive, for
example, with the intent of curing the disease or causing
regression of the cancer, as well as in the treatment of
intermediate stage and in the treatment of late stage cancers
including advanced and/or metastatic and/or aggressive neoplasias,
for example, to slow the progression of the disease, to reduce
metastasis or to increase the survival of the patient. Similarly,
the combinations may be used in the treatment of low grade cancers,
intermediate grade cancers and or high grade cancers.
[0093] Methods of the present invention can also be used in the
treatment of indolent cancers, recurrent cancers including locally
recurrent, distantly recurrent and/or refractory cancers (i.e.
cancers that have not responded to treatment), metastatic cancers,
locally advanced cancers and aggressive cancers. Thus, an
"advanced" cancer includes locally advanced cancer and metastatic
cancer and refers to overt disease in a patient, wherein such overt
disease is not amenable to cure by local modalities of treatment,
such as surgery or radiotherapy. The term "metastatic cancer"
refers to cancer that has spread from one part of the body to
another. Advanced cancers may also be unresectable, that is, they
have spread to surrounding tissue and cannot be surgically
removed.
[0094] Methods of the present invention can also be used in the
treatment of drug resistant cancers, including multidrug resistant
tumours. As is known in the art, the resistance of cancer cells to
chemotherapy is one of the central problems in the management of
cancer.
[0095] One skilled in the art will appreciate that many of these
categories may overlap, for example, aggressive cancers are
typically also metastatic. "Aggressive cancer," as used herein,
refers to a rapidly growing cancer. One skilled in the art will
appreciate that for some cancers, such as breast cancer or prostate
cancer the term "aggressive cancer" will refer to an advanced
cancer that has relapsed within approximately the earlier
two-thirds of the spectrum of relapse times for a given cancer,
whereas for other types of cancer, nearly all cases present rapidly
growing cancers which are considered to be aggressive. The term can
thus cover a subsection of a certain cancer type or it may
encompass all of other cancer types.
[0096] In some embodiments, the cancer is leukemia/lymphoma. In
some embodiments, the cancer is acute myelogenous leukemia (AML).
In some embodiments, the cancer is lymphoma, gastric cancer,
multiple myeloma, myelodysplastic syndromes, or combinations
thereof. In some other embodiments, the cancer is T-cell leukemia,
e.g., adult T-cell leukemia associated with epigenetic methylation
of KLF4 gene. In some other embodiments, the cancer is ALL, e.g.,
pediatric ALL. In some other embodiments, the cancer is pediatric
T-cell ALL associated with one or more mutations in KLF4 gene or
protein. In some other embodiments, the cancer is pediatric B-cell
ALL associated with elevated miRNA-2902. In some other embodiments,
the cancer is AML, ALL or MDS, e.g., high risk MDS, all of which
associated with higher than normal CDX2 activity. In some other
embodiments, the cancer is Hodgkins, Burkitts, or B-cell lymphomas,
all of which associated with methylation of KLF4 gene.
[0097] Acute myeloid leukemia (AML): also known as acute
myelogenous leukemia or acute nonlymphocytic leukemia (ANLL), is a
cancer of the myeloid line of blood cells, characterized by the
rapid growth of abnormal white blood cells that accumulate in the
bone marrow and interfere with the production of normal blood
cells. The symptoms of AML are caused by replacement of normal bone
marrow with leukemic cells, which causes a drop in red blood cells,
platelets, and normal white blood cells. These symptoms include
fatigue, shortness of breath, easy bruising and bleeding, and
increased risk of infection. Several risk factors and chromosomal
abnormalities have been identified, but the specific cause is not
clear. As an acute leukemia, AML progresses rapidly and is
typically fatal within weeks or months if left untreated. Several
risk factors for developing AML include, but are not limited to,
preleukemic blood disorders, such as myelodysplastic syndrome or
myeloproliferative disease; exposure to anticancer chemotherapy;
radiation, such as high amounts of ionizing radiation exposure; and
genetic reasons, such as those described in Taylor et al. ("The
hereditary basis of human leukemia". In Henderson E S, Lister T A,
Greaves M F. Leukemia (6th ed.). Philadelphia: W B Saunders. p.
210. ISBN 0-7216-5381-2), Horwitz et al. ("Anticipation in familial
leukemia". Am. J. Hum. Genet. 59 (5): 990-8. PMC 1914843. PMID
8900225), Crittenden ("An interpretation of familial aggregation
based on multiple genetic and environmental factors". Ann. N.Y.
Acad. Sci. 91 (3): 769-80), and Horwitz ("The genetics of familial
leukemia". Leukemia 11 (8): 1347-59). The World Health Organization
(WHO) classification of acute myeloid leukemia attempts to be more
clinically useful and to produce more meaningful prognostic
information than the FAB criteria. Each of the WHO categories
contains numerous descriptive subcategories of interest to the
hematopathologist and oncologist; however, most of the clinically
significant information in the WHO schema is communicated via
categorization into one of the subtypes listed below:
TABLE-US-00001 ICD-O (International Classification of Diseases Name
Description for Oncology) Acute myeloid Includes: Multiple leukemia
with AML with translocations between chromosome 8 recurrent genetic
and 21 [t(8; 21)] (ICD-O abnormalities 9896/3); RUNX1/RUNX1T1 AML
with inversions in chromosome 16 [inv(16)] (ICD-O 9871/3);
CBFB/MYH11 APL with translocations between chromosome 15 and 17
[t(15; 17)] (ICD-O 9866/3); RARA; PML AML with translocations in
chromosomes 9 and 11 [t(9; 11)]; MLLT3-MLL Patients with AML in
this category generally have a high rate of remission and a better
prognosis compared to other types of AML. AML with multilineage
This category includes patients who have had a prior M9895/3
dysplasia myelodysplastic syndrome (MDS) or myeloproliferative
disease (MPD) that transforms into AML. This category of AML occurs
most often in elderly patients and often has a worse prognosis. AML
and MDS, This category includes patients who have had prior M9920/3
therapy-related chemotherapy and/or radiation and subsequently
develop AML or MDS. These leukemias may be characterized by
specific chromosomal abnormalities, and often carry a worse
prognosis. AML not otherwise Includes subtypes of AML that do not
fall into the above M9861/3 categorized categories.
[0098] The French-American-British (FAB) classification system
divides AML into eight subtypes, M0 through to M7, based on the
type of cell from which the leukemia developed and its degree of
maturity. Although the WHO classification (see above) may be more
useful, the FAB system is still widely used.
TABLE-US-00002 Percentage of adult AML Type Name Cytogenetics
patients M0 acute myeloblastic leukemia, minimally differentiated
5% M1 acute myeloblastic leukemia, without maturation 15% M2 acute
myeloblastic leukemia, with granulocytic maturation t(8; 21)(q22;
q22), 25% t(6; 9) M3 promyelocytic, or acute promyelocytic leukemia
(APL) t(15; 17) 10% M4 acute myelomonocytic leukemia
inv(16)(p13q22), 20% del(16q) M4eo myelomonocytic together with
bone marrow eosinophilia inv(16), t(16; 16) 5% M5 acute monoblastic
leukemia (M5a) or acute monocytic leukemia del (11q), t(9; 11), 10%
(M5b) t(11; 19) M6 acute erythroid leukemias, including
erythroleukemia (M6a) and 5% very rare pure erythroid leukemia
(M6b) M7 acute megakaryoblastic leukemia t(1; 22) 5%
[0099] Previous methods for treating AML are described in Bishop J
("The treatment of adult acute myeloid leukemia". Semin Oncol 24
(1): 57-69. 1997), Weick et al. ("A randomized investigation of
high-dose versus standard-dose cytosine arabinoside with
daunorubicin in patients with previously untreated acute myeloid
leukemia: a Southwest Oncology Group study" (PDF). Blood 88 (8):
2841-51, 1996), Bishop et al. ("A randomized study of high-dose
cytarabine in induction in acute myeloid leukemia" Blood 87 (5):
1710-7, 1996), Huang et al. ("Use of all-trans retinoic acid in the
treatment of acute promyelocytic leukemia". Blood 72 (2): 567-72,
1988), Tallman et al. ("All-trans-retinoic acid in acute
promyelocytic leukemia". N. Engl. J. Med. 337 (15): 1021-8, 1997),
Fenaux et al. ("A randomized comparison of all transretinoic acid
(ATRA) followed by chemotherapy and ATRA plus chemotherapy and the
role of maintenance therapy in newly diagnosed acute promyelocytic
leukemia. The European APL Group". Blood 94 (4): 1192-200, 1999),
Estey E ("Treatment of acute myelogenous leukemia". Oncology
(Williston Park) 16 (3): 343-52, 355-6; discussion 357, 362, 365-6,
2002), and Cassileth et al. ("Maintenance chemotherapy prolongs
remission duration in adult acute nonlymphocytic leukemia". J Clin
Oncol 6 (4): 583-7, 1988).
[0100] Acute lymphocytic leukemia (ALL): also known as acute
lymphoblastic leukemia, is an acute leukemia that is characterized
by dysregulated proliferation and accumulation of leukemic
lymphocytes/lymphoblasts (immature white blood cells such as early
B- and T-lymphocyte progenitors) in the bone marrow and various
extramedullary sites. ALL is the most common type of cancer in
children, and it is a relatively uncommon cancer in adults. Risk
factors for developing ALL include, but are not limited to, genetic
disorders/mutations and various epigenetic modifications such as
those described by Iacobucci et al. ("Cytogenetic and molecular
predictors of outcome in acute lymphocytic leukemia: recent
developments", Curr Hematol Malig Rep. 2012 June; 7(2):133-43.) and
Florean et al. ("Epigenomics of leukemia: from mechanisms to
therapeutic applications". Epigenomics. 2011 October;
3(5):581-609).
[0101] Chronic myelogenous leukemia (CML): also known as chronic
myeloid leukemia, is a chronic leukemia that is characterized by
dysregulated/increased proliferation of various blood cells,
predominantly myeloid cells in peripheral blood, and their
precursors in the bone marrow, resulting in their accumulation in
the blood. It is less frequent than chronic lymphocytic leukemia
(CLL) in adults in the Western world, and the median age of CML
onset is 50-60 years. Risk factors for developing CML also include,
but are not limited to, genetic disorders/mutations and various
epigenetic modifications such as those described by Florean et al.
The disease course is triphasic, starting with an early phase, also
known as chronic phase (CP) disease. Then, leukemia stem cells can
acquire additional genetic defects.
[0102] Adult T-cell leukaemia (ATLL): also known as adult T-cell
lymphoma, is an uncommon lymphoproliferative disorder of mature
CD4+ T cells that is caused by the retrovirus human T-lymphotrophic
virus type 1 (HTLV-1) as reviewed by Qayyum et al. ("Adult T-cell
leukemia/lymphoma". Arch Pathol Lab Med. 2014 February;
138(2):282-6). Presently, about 20 million people worldwide are
HTLV-1 carriers, with most infected individuals residing in endemic
areas such as southern Japan, Africa, the Caribbean basin, and
Latin America. The lifelong viral carrier state and long latency
(20-40 years) are common after HTLV-1 infection, therefore this
type of leukemia/lymphoma is almost exclusively found in adults and
is extremely rare in children. The lifetime risk of progression to
ATLL in an HTLV-1-positive patient is 2.1% for women and 6.6% for
men. The mean age of onset is 60 years old (range, 20-80 years
old). The overwhelming majority of ATLL cases occur in patients
infected during the early years of life, presumably because of a
less efficient immune response in this age group. In addition, the
prolonged infection may increase chances of accruing subsequent
mutations, and ultimately malignant transformation. Major paths of
viral transmission are breast feeding, blood exposure, and
unprotected sex. The World Health Organization Classification of
Tumors of Hematopoietic and Lymphoid Tissues in 2008 subclassified
ATLL into 4 distinct variants according to the Shimoyama
classification: acute (60%), lymphomatous (20%), chronic (15%), and
smoldering (5%). There are no absolutely necessary features for
each variant, and overlap is seen. The acute variant manifests as
marked leukocytosis with atypical lymphocytes and eosinophilia.
Symptoms include hypercalcemia with or without osteolytic lesions,
renal dysfunction and neuropsychiatric disturbances, elevated
lactate dehydrogenase level, central nervous ring-enhancing
lesions, and secondary respiratory complications. The lymphomatous
variant is an aggressive advanced disease resembling acute-onset
subtype, and marked lymphadenopathy without leukemia is a prominent
feature of this variant. The chronic variant typically presents
with skin rash, leukocytosis with absolute lymphocytosis, mild
lymphadenopathy, and hypercalcemia. The smoldering variant is
asymptomatic and is characterized by normal white blood cell count
with less than 5% circulating atypical lymphoid cells and without
associated hypercalcemia or organomegaly, although skin and
pulmonary involvement often occur. Progression from the smoldering
variant to the acute variant can occur.
[0103] Lymphoma: Lymphoma is a type of blood cancer that occurs
when B or T lymphocytes, the white blood cells that form a part of
the immune system and help protect the body from infection and
disease, divide faster than normal cells or live longer than they
are supposed to. Typically, lymphoma presents as a solid tumor of
lymphoid cells. The current WHO classification, published in 2001
and updated in 2008, is the latest classification of lymphoma and
is based upon the foundations laid within the "Revised
European-American Lymphoma classification" (REAL):
[0104] A. Mature B-cell neoplasms: [0105] Chronic lymphocytic
leukemia/Small lymphocytic lymphoma [0106] B-cell prolymphocytic
leukemia [0107] Lymphoplasmacytic lymphoma (such as Waldenstrom
macroglobulinemia) [0108] Splenic marginal zone lymphoma [0109]
Plasma cell neoplasms: [0110] Plasma cell myeloma [0111]
Plasmacytoma [0112] Monoclonal immunoglobulin deposition diseases
[0113] Heavy chain diseases [0114] Extranodal marginal zone B cell
lymphoma, also called MALT lymphoma [0115] Nodal marginal zone B
cell lymphoma (NMZL) [0116] Follicular lymphoma [0117] Mantle cell
lymphoma [0118] Diffuse large B cell lymphoma [0119] Mediastinal
(thymic) large B cell lymphoma [0120] Intravascular large B cell
lymphoma [0121] Primary effusion lymphoma [0122] Burkitt
lymphoma/leukemia
[0123] B. Mature T cell and natural killer (NK) cell neoplasms
[0124] T cell prolymphocytic leukemia [0125] T cell large granular
lymphocytic leukemia [0126] Aggressive NK cell leukemia [0127]
Adult T cell leukemia/lymphoma [0128] Extranodal NK/T cell
lymphoma, nasal type [0129] Enteropathy-type T cell lymphoma [0130]
Hepatosplenic T cell lymphoma [0131] Blastic NK cell lymphoma
[0132] Mycosis fungoides/Sezary syndrome [0133] Primary cutaneous
CD30-positive T cell lymphoproliferative disorders [0134] Primary
cutaneous anaplastic large cell lymphoma [0135] Lymphomatoid
papulosis [0136] Angioimmunoblastic T cell lymphoma [0137]
Peripheral T cell lymphoma, unspecified [0138] Anaplastic large
cell lymphoma
[0139] C. Hodgkin Lymphoma [0140] Classical Hodgkin lymphomas:
[0141] Nodular sclerosis [0142] Mixed cellularity [0143]
Lymphocyte-rich [0144] Lymphocyte depleted or not depleted [0145]
Nodular lymphocyte-predominant Hodgkin lymphoma
[0146] D. Immunodeficiency-associated lymphoproliferative disorders
[0147] Associated with a primary immune disorder [0148] Associated
with the Human Immunodeficiency Virus (HIV) [0149] Post-transplant
[0150] Associated with methotrexate therapy [0151] Primary central
nervous system lymphoma occurs most often in immuno-compromised
patients, in particular those with AIDS, but it can occur in the
immunocompetent as well. It has a poor prognosis, particularly in
those with AIDS. Treatment can consist of corticosteroids,
radiotherapy, and chemotherapy, often with methotrexate. Subtypes
of lymphoma with relative incidence, histopathology,
immunophenotype, overall t-year survival are show below (Robbins
basic pathology (8th ed.). Philadelphia: Saunders/Elsevier. 2007.
pp. Table 12-8.):
TABLE-US-00003 [0151] Overall Relative 5-year Lymphoma type
incidence.sup.[13] Histopathology.sup.[13] Immunophenotype survival
Other comments Precursor T-cell 40% of Lymphoblasts TdT, CD2, CD7
It often presents as a leukemia/lymphoma lymphomas with irregular
mediastinal mass in nuclear contours, because of childhood.
condensed involvement of the chromatin, small thymus. It is highly
nucleoli and scant associated with cytoplasm NOTCH1 mutations.
without granules. Most common in adolescent males. Follicular 40%
of Small "cleaved" CD10, surface Ig 72-77% Occurs in older lymphoma
lymphomas cells (centrocytes) adults. Usually in adults mixed with
large involves lymph activated cells nodes, bone marrow
(centroblasts). and spleen. Usually nodular Associated with
("follicular") t(14; 18) translocation growth pattern
overexpressing Bcl-2. Indolent Diffuse large B cell 40 to 50%
Variable. Most Variable expression 60% Occurs in all ages, but
lymphoma of resemble B cells of CD10 and most commonly in lymphomas
of large germinal surface Ig older adults. Often in adults centers.
Diffuse occurs outside lymph growth pattern. nodes. Aggressive.
Mantle cell 3 to 4% of Lymphocytes of CD5 50% to Occurs mainly in
lymphoma lymphomas small to 70% adult males. Usually in adults
intermediate size involves lymph growing in nodes, bone marrow,
diffuse pattern spleen and GI tract. Associated with t(11; 14)
translocation overexpressing cyclin D1. Moderately aggressive.
B-cell chronic 3 to 4% of Small resting CD5, surface 50% Occurs in
older lymphocytic lymphomas lymphocytes immunoglobulin adults.
Usually leukemia/lymphoma in adults mixed with involves lymph
variable number nodes, bone marrow of large activated and spleen.
Most cells. Lymph patients have nodes are peripheral blood
diffusely effaced involvement. Indolent. MALT lymphoma ~5% of
Variable cell size CD5, CD10, Frequently occurs lymphomas and
surface Ig outside lymph nodes. in adults differentiation. Very
indolent. May 40% show plasma be cured by local cell excision.
differentiation. Homing of B cells to epithelium creates
lymphoepithelial lesions. Burkitt's lymphoma <1% of Round
lymphoid CD10, surface Ig 50% Endemic in Africa, lymphomas cells of
sporadic elsewhere. in the intermediate size More common in United
with several immunocompromised States nucleoli. Starry- and in
children. Often sky appearance visceral involvement. by diffuse
spread Highly aggressive. with interspersed apoptosis. Mycosis
fungoides Most Usually small CD4 75% Localized or more common
lymphoid cells generalized skin cutaneous with convoluted symptoms.
Generally lymphoid nuclei that often indolent. In a more malignancy
infiltrate the aggressive variant, epidermis, Sezary's disease,
there creating Pautrier is skin erythema and microabscesseses.
peripheral blood involvement. Peripheral T-cell Most Variable.
Usually CD3 Probably consists of lymphoma-Not- common T a mix small
to several rare tumor Otherwise-Specified cell large lymphoid
types. It is often lymphoma cells with disseminated and irregular
nuclear generally aggressive. contours. Nodular sclerosis Most
Reed-Sternberg CD15, CD30 Most common in form of Hodgkin common
cell variants and young adults. It often lymphoma type of
inflammation. arises in the Hodgkin's usually broad mediastinum or
lymphoma sclerotic bands cervical lymph nodes. that consists of
collagen. Mixed-cellularity Second Many classic CD15, CD30 Most
common in subtype of Hodgkin most Reed-Sternberg men. More likely
to lymphoma common cells and be diagnosed at form of inflammation
advanced stages than Hodgkin's the nodular sclerosis lymphoma form.
Epstein-Barr virus involved in 70% of cases.
[0152] Gastric cancer: a.k.a. stomach cancer, which refers to
cancer arising from any part of the stomach. Stomach cancer is
often either asymptomatic (producing no noticeable symptoms) or it
may cause only nonspecific symptoms (symptoms which are not
specific to just stomach cancer, but also to other related or
unrelated disorders) in its early stages. It can be diagnosed by
gastroscopic exam, upper GI series, or computed tomography or CT
scanning. It is previously treated by surgery, chemotherapy, and
radiation.
[0153] Colorectal cancer: a.k.a. colon cancer, rectal cancer, bowel
cancer or colorectal adenocarcinoma, is a cancer from uncontrolled
cell growth in the colon or rectum (parts of the large intestine),
or in the appendix. Greater than 75-95% of colon cancer occurs in
people with little or no genetic risk. Other risk factors include
older age, male gender, high intake of fat, alcohol or red meat,
obesity, smoking and a lack of physical exercise. Approximately 10%
of cases are linked to insufficient activity. The risk for alcohol
appears to increase at greater than one drink per day. Colorectal
cancer is a disease originating from the epithelial cells lining
the colon or rectum of the gastrointestinal tract, most frequently
as a result of mutations in the Wnt signaling pathway that
artificially increase signaling activity. The mutations can be
inherited or are acquired, and most probably occur in the
intestinal crypt stem cell. Genes in the Wnt signaling pathway that
are related to colorectal cancer include, but are not limited to,
APC, .beta.-catenin, AXIN1, AXIN2, TCF7L2, or NKD1. Beyond the
defects in the Wnt-APC-beta-catenin signaling pathway, other
mutations must occur for the cell to become cancerous. The p53
protein, produced by the TP53 gene, normally monitors cell division
and kills cells if they have Wnt pathway defects. Eventually, a
cell line acquires a mutation in the TP53 gene and transforms the
tissue from an adenoma into an invasive carcinoma. Other apoptotic
proteins commonly deactivated in colorectal cancers are TGF-.beta.
and DCC. Other oncogenes overexpressed in colorectal cancer
include, genes encoding the proteins KRAS, RAF, and PI3K, which
normally stimulate the cell to divide in response to growth
factors, can acquire mutations that result in over-activation of
cell proliferation. In addition to the oncogenic and inactivating
imutations described for the genes above, non-hypermutated samples
also contain mutated CTNNB1, FAM123B, SOX9, ATM, and ARID1A.
Progressing through a distinct set of genetic events, hypermutated
tumors display mutated forms of ACVR2A, TGFBR2, MSH3, MSH6, SLC9A9,
TCF7L2, and BRAF. The common theme among these genes, across both
tumor types, is their involvement in WNT and TGF-.beta. signaling
pathways, which in turn results in increased activity of MYC, a
central player in colorectal cancer.
[0154] Multiple myeloma: a.k.a. plasma cell myeloma or Kahler's
disease is a cancer of plasma cells, a type of white blood cell
normally responsible for producing antibodies. It can be
symptomatic myeloma, asymptomatic myeloma and MGUS (monoclonal
gammopathy of undetermined significance). Myeloma is diagnosed with
blood tests (serum protein electrophoresis, serum free kappa/lambda
light chain assay), bone marrow examination, urine protein
electrophoresis, and X-rays of commonly involved bones. It is
previously treated by steroids, chemotherapy, proteasome
inhibitors, immunomodulatory drugs (IMiDs) such as thalidomide or
lenalidomide, and stem cell transplants.
[0155] Myelodysplastic syndromes (MDS): Myelodysplastic syndrome
("MDS") refers to a diverse group of hematopoietic stem cell
disorders, which are hematological (blood-related) medical
conditions with ineffective production (or dysplasia) of the
myeloid class of blood cells. MDS is characterized by a cellular
marrow with impaired morphology and maturation (dysmyelopoiesis),
peripheral blood cytopenias, and a variable risk of progression to
acute leukemia, resulting from ineffective blood cell production.
The Merck Manual 953 (17.sup.th ed. 1999) and List et al., 1990, J.
Clin. Oncol. 8:1424. Some types of MDS, referred to as "low-risk
MDS", progress slowly and may cause mild to moderate anemia, or
decrements to other types of cells. Some other types of MDS are
called "high-risk MDS" and may cause severe problems. In patients
with high-risk MDS, immature cells called blast cells make up more
than five percent of the cells in the marrow and do not develop
into normal red cells, white cells and platelets, often causing
more severe deficiency in those cells/platelets. When MDS patients
develop more than 20 percent blast cells, they are reclassified as
having AML with trilineage dysplasia (AML-TLD).
[0156] The initial hematopoietic stem cell injury can be from
causes such as, but not limited to, cytotoxic chemotherapy,
radiation, virus, chemical exposure, and genetic predisposition. A
clonal mutation predominates over bone marrow, suppressing healthy
stem cells. In the early stages of MDS, the main cause of
cytopenias is increased programmed cell death (apoptosis). As the
disease progresses and converts into leukemia, gene mutation rarely
occurs and a proliferation of leukemic cells overwhelms the healthy
marrow. The disease course differs, with some cases behaving as an
indolent disease and others behaving aggressively with a very short
clinical course that converts into an acute form of leukemia.
Patients with MDS can develop severe anemia and require blood
transfusions. In some cases, the disease worsens and the patient
develops cytopenias (low blood counts) caused by progressive bone
marrow failure.
[0157] According to French-American-British classification
published in 1976, which was revised in 1982, cases were classified
into five categories:
TABLE-US-00004 ICD-O Name Description M9980/3 Refractory anemia
(RA) characterized by less than 5% primitive blood cells
(myeloblasts) in the bone marrow and pathological abnormalities
primarily seen in red cell precursors M9982/3 Refractory anemia
with ring also characterized by less than 5% myeloblasts in the
bone sideroblasts (RARS) marrow, but distinguished by the presence
of 15% or greater red cell precursors in the marrow being abnormal
iron-stuffed cells called "ringed sideroblasts" M9983/3 Refractory
anemia with excess blasts characterized by 5-20% myeloblasts in the
marrow (RAEB) M9984/3 Refractory anemia with excess blasts
characterized by 21-30% myeloblasts in the marrow (>30% in
transformation (RAEB-T) blasts is defined as acute myeloid
leukemia) M9945/3 Chronic myelomonocytic leukemia characterized by
less than 20% myeloblasts in the bone (CMML), not to be confused
with marrow and greater than 1*109/L monocytes (a type of white
chronic myelogenous leukemia or blood cell) circulating in the
peripheral blood. CML
[0158] The World Health Organization (WHO) modified this
classification, introducing several new disease categories and
eliminating others. Most recently the WHO has evolved a new
classification scheme (2008) which is based more on genetic
findings:
TABLE-US-00005 Old system New system Refractory anemia (RA)
Refractory cytopenia with unilineage dysplasia (Refractory anemia,
Refractory neutropenia, and Refractory thrombocytopenia) Refractory
anemia with Refractory anemia with ring sideroblasts (RARS) ringed
sideroblasts Refractory anemia with ring sideroblasts -
thrombocytosis (RARS-t) (provisional entity) (RARS) which is in
essence a myelodysplastic/myeloproliferative disorder and usually
has a JAK2 mutation (janus kinase) - New WHO classification 2008
Refractory cytopenia with multilineage dysplasia (RCMD) includes
the subset Refractory cytopenia with multilineage dysplasia and
ring sideroblasts (RCMD-RS). RCMD includes patients with
pathological changes not restricted to red cells (i.e., prominent
white cell precursor and platelet precursor (megakaryocyte)
dysplasia. Refractory anemia with Refractory anemia with excess
blasts I and II. RAEB was divided into *RAEB-I (5-9% excess blasts
(RAEB) blasts) and RAEB-II (10-19%) blasts, which has a poorer
prognosis than RAEB-I. Auer rods may be seen in RAEB-II which may
be difficult to distinguish from acute myeloid leukemia. Refractory
anemia with The category of RAEB-T was eliminated; such patients
are now considered to have excess blasts in acute leukemia.
5q-syndrome, typically seen in older women with normal or high
transformation (RAEB-T) platelet counts and isolated deletions of
the long arm of chromosome 5 in bone marrow cells, was added to the
classification. Chronic myelomonocytic CMML was removed from the
myelodysplastic syndromes and put in a new category of leukemia
(CMML) myelodysplastic-myeloproliferative overlap syndromes.
5q-syndrome Myelodysplasia unclassifiable (seen in those cases of
megakaryocyte dysplasia with fibrosis and others) Refractory
cytopenia of childhood (dysplasia in childhood) - New WHO
classification 2008
[0159] Signs and symptoms of MDS include, but are not limited to,
anemia (low red blood cell count or reduced hemoglobin), with
chronic tiredness, shortness of breath, chilled sensation,
sometimes chest pain; neutropenia (low neutrophil count), with
increased susceptibility to infection; thrombocytopenia (low
platelet count), with increased susceptibility to bleeding and
ecchymosis (bruising), as well as subcutaneous hemorrhaging
resulting in purpura or petechia; splenomegaly or rarely
hepatomegaly; abnormal granules in cells, abnormal nuclear shape
and size; and/or chromosomal abnormalities, including chromosomal
translocations and abnormal chromosome number.
[0160] Previous treatment methods for MDS include, but are not
limited to, bone marrow transplantation, use of hematopoietic
growth factors or cytokines to stimulate blood cell development in
a recipient, such as erythropoietin (EPO), granulocyte macrophage
colony stimulating factor (GM-CSF), and granulocyte colony
stimulating factor (G-CSF) (Metcalf, 1985, Science 229:16; Dexter,
1987, J. Cell Sci. 88:1; Golde and Gasson, 1988, Scientific
American, July: 62; Tabbara and Robinson, 1991, Anti-Cancer Res.
11:81; Ogawa, 1989, Environ. Health Presp. 80:199; and Dexter,
1989, Br. Med. Bull. 45:337.). Unfortunately, bone transplantation
is painful for donor and recipient, and hematopoietic growth
factors have not proven effective in many clinical settings.
[0161] In some embodiments, the histology of the cancer is
determined before, during or after the treatment. Any suitable test
can be used to determine the histology of the cancer. Such test and
examination include, but are not limited to, common signs and
symptoms of esophaeal cancer, including but not limited to,
backwards movement of food through the esophagus and possibly mouth
(regurgitation), chest pain not related to eating, difficulty
swallowing solids or liquids, heartburn, vomiting blood,
hoarseness, chronic cough, hiccups, pneumonia, bone pain, bleeding
into the esophagus, and weight loss, medical history and physical
exam, imaging tests, chest X-ray, computed tomography (CT) scan,
magnetic resonance imaging (MRI) scan, positron emission tomography
(PET) scan, bone scan, sputum cytology, needle biopsy,
bronchoscopy, endobronchial ultrasound, endoscopic esophageal
ultrasound, mediastinoscopy and mediastinotomy, thoracentesis,
thoracoscopy, immunohistochemistry, molecular tests, blood tests,
barium swallow, endoscopic ultrasound, esophaogastroduodenoscopy
(EGD) and biopsy, or any suitable methods derived from thereof.
Biomarkers
[0162] The present invention provides biomarkers associated with
cancers, which can be used in many ways to help the cancer
treatment. In some embodiments, the biomarkers are those components
in the CDX2-KLF4 signaling pathway. In some embodiments, the
activity level of one or more components in the CDX2-KLF4 signaling
pathway is measured, and compared to a control. In some
embodiments, the activity level is measured before, during, or
after the treatment by an anti-cancer agent of the present
invention. In some embodiments, when the activity level is measured
before, during, or after the treatment, the control is the normal
activity level of a biomarker in a patient or a group of patients
who are responsive to an anti-cancer agent of the present
invention. In some embodiments, when the activity level is measured
during or after the treatment, the control is activity level of a
biomarker in the same patient subjected to the treatment, before
the patient receives the treatment.
[0163] CDX2, a.k.a. Caudal Type Homeobox 2, CDX3, Caudal Type
HomeoBox Transcription Factor 2, Caudal-Type Homeobox Protein 2, or
Homeobox Protein CDX-2, is a member of the caudal-related homeobox
transcription factor gene family. The encoded protein is a major
regulator of intestine-specific genes involved in cell growth and
differentiation. This protein also plays a role in early embryonic
development of the intestinal tract. Aberrant expression of this
gene is associated with intestinal inflammation and tumorigenesis.
Diseases associated with CDX2 include atrophic gastritis, and
signet ring cell adenocarcinoma, and among its related
super-pathways are Transcription Role of VDR in regulation of genes
involved in osteoporosis and Cytoskeleton remodeling Regulation of
actin cytoskeleton by Rho GTPases. GO annotations related to this
gene include transcription regulatory region sequence-specific DNA
binding and sequence-specific DNA binding transcription factor
activity. An important paralog of this gene is CDX1. It is involved
in the transcriptional regulation of multiple genes expressed in
the intestinal epithelium, and important in broad range of
functions from early differentiation to maintenance of the
intestinal epithelial lining of both the small and large intestine.
DNA and protein sequences of human CDX2 were previously reported,
see GenBank Nos. NC.sub.--000013.10, NC.sub.--018924.2,
NT.sub.--024524.14, NP.sub.--001256.3, ENSP00000370408, and Uniprot
No. Q99626, each of which is incorporated herein by reference in
its entirety for all purposes. Such sequences can be utilized to
design procedures for detection and analysis of the level of CDX2
activity by ways known to one skilled in the art. CDX2 is
aberrantly expressed in most cases of acute myeloid leukemia and
promotes leukemogenesis (Scholl et al., The Journal of Clinical
Investigation, 17(4): 1037-1048), with an mRNA copy numbers varied
between about 27 copies to about 89,000 copies. As used herein, the
phrase "CDX2 gene is turned on" or "CDX2 activity is on" refers to
that the mRNA copies of CDX2 in a human subject is at least about
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies. Otherwise,
the CDX2 gene is considered as being turned off.
[0164] Kruppel-Like Factor 4 (KLF4), a.k.a. Gut, EZF, GKLF,
Epithelial Zinc Finger Protein EZF, Gut-Enriched Krueppel-Like
Factor, Endothelial Kruppel-Like Zinc Finger Protein, or
Krueppel-Like Factor 4, is associated with diseases including, but
are not limited to, leukemia, skin squamous cell carcinoma, and
familial adenomatous polyposis. An important paralog of this gene
is KLF1. KLF4 can act both as activator and as repressor. It binds
the 5'-CACCC-3' core sequence, such as the promoter region of its
own gene. It regulates the expression of key transcription factors
during embryonic development, and plays an important role in
maintaining embryonic stem cells, and in preventing their
differentiation. It is required for establishing the barrier
function of the skin and for postnatal maturation and maintenance
of the ocular surface. It is also involved in the differentiation
of epithelial cells and may also function in skeletal and kidney
development. It further contributes to the down-regulation of
p53/TP53 transcription, and induction of p21. DNA and protein
sequences of human KLF4 were previously reported, see GenBank Nos.
NC.sub.--000009.11, NT.sub.--008470.19, NC.sub.--018920.2, and
Uniprot No. O43474, each of which is incorporated herein by
reference in its entirety for all purposes. Such sequences can be
utilized to design procedures for detection and analysis of the
level of KLF4 activity by ways known to one skilled in the art.
[0165] p21, a.k.a. Cyclin-Dependent Kinase Inhibitor 1A, Cip1,
CDJN1, CIP1, WAFT, CAP20, MDA-6, SDI1, CDK-interacting protein 1,
CDK-interaction protein 1, cyclin-dependent kinase inhibitor 1, DNA
synthesis inhibitor, Melanoma Differentiation Associated Protein,
p21CIP, wild type P53-activated fragment, mDA6, or PCI1, encodes a
potent cyclin-dependent kinase inhibitor. The encoded protein binds
to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and
thus functions as a regulator of cell cycle progression at G1. The
expression of this gene is tightly controlled by the tumor
suppressor protein p53, through which this protein mediates the
p53-dependent cell cycle G1 phase arrest in response to a variety
of stress stimuli. This protein can interact with proliferating
cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and
plays a regulatory role in S phase DNA replication and DNA damage
repair. This protein was reported to be specifically cleaved by
CASP3-like spases, which thus leads to a dramatic activation of
CDK2, and may be instrumental in the execution of apoptosis
following caspase activation. Multiple alternatively spliced
variants have been found for this gene. DNA and protein sequences
of human p21 were previously reported, see GenBank Nos.
NC.sub.--000006.11, NT.sub.--007592.15, NC.sub.--018917.2 and
Uniprot No. P38936, each of which is incorporated herein by
reference in its entirety for all purposes. Such sequences can be
utilized to design procedures for detection and analysis of the
level of p21 activity by ways known to one skilled in the art.
[0166] Some other biomarkers provided by the present invention are
H3K4 demethylase Jarid1b (KDM5B, a.k.a. Plu-1 or Rbp2-h1), microRNA
miR-2909, tumor suppressor p53 (a.k.a TP53 or tumor protein
(EC:2.7.1.37)), cysteine-aspartic acid protease Caspase-3, Annexin
V, B-cell CLL/Lymphoma 2 (BCL2), B-cell CLL/Lymphoma 3 (BCL3),
BCL2-associated X protein (BAX), bone morphogenetic proteins
(BMPs), Wnt (a.k.a. murine int-1), hepatocyte nuclear factor 4a
(HNF4.alpha.), fibroblast growth factors (Fgf), homeobox (Hox),
transcription factor SP1, transcription factor MYC, cyclin D1
(CCND1, a.k.a. PRAD1), and apoptosis-antagonizing transcription
factor (AATF).
[0167] In some embodiments, the activity level of one or more
biomarkers of the present invention is determined, for example, by
a method known to skill in the art, including but not limited to
those described herein. Additionally, the levels of more than one
biomarker can be determined in order to generate a composite of the
level of more than one biomarker. In some embodiments, the methods
of the present invention comprise determining a composite level of
a panel of selected biomarkers. The composite can contain any of
the biomarkers described by the present application.
[0168] The biomarkers can be any in vitro or in vivo information
for the activity of genes described herein, including, but not
limited to gene amplification, gene expression, RNA levels, protein
activity, pathway activation or pathway signaling. The information
can reflect increased, decreased, or unchanged level of the
activity of a selected biomarker when compared to a control group.
In one embodiment, biomarkers include any form of mutations at the
DNA, RNA, or protein level that are associated with gene activity.
In another embodiment, biomarkers include any measurement directly
or indirectly associated with activity. Such information is
collectively referred as biomarker profile herein.
[0169] In some embodiments the gene copy number of a given
biomarker in a human subject is at least 1.5.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., 10.times., 100.times., 1000.times., 10000.times. or more
of the normal copy number in a control group. In some embodiments,
the increase is determined by comparing to one or more standard
levels or by comparing to levels known in the art as standard
levels.
[0170] The biomarker profile can be determined by any suitable
methods known to one skilled in the art. In some embodiments, a
biological sample is taken from a subject and analyzed. In some
embodiments, the biological sample is then assayed for activity of
one or more biomarkers, such as gene amplification number, RNA,
mRNA, cDNA, cRNA, protein, etc.
[0171] In some embodiments, mRNA from a biological sample is
directly used in determining the level of activity of one or more
biomarkers. In some embodiments, the level is determined by
hybridization. In some embodiments, the RNA is transformed into
cDNA (complementary DNA) copy using methods known in the art. In
some particular embodiments, the cDNA is labeled with a fluorescent
label or other detectable label. The cDNA is then hybridized to a
substrate containing a plurality of probes of interest. A probe of
interest typically hybridizes under stringent hybridization
conditions to at least one DNA sequence of a gene signature. In
certain embodiments, the plurality of probes are capable of
hybridizing to the sequences derived from the gene biomarkers under
the hybridization conditions. In some embodiments, the conditions
comprise using 6.times.SSC (0.9 M NaCl, 0.09 M sodium citrate, pH
7.4) at 65.degree. C. The probes may comprise nucleic acids. The
term "nucleic acid" encompasses known nucleotide analogs or
modified backbone residues or linkages, which are synthetic,
naturally occurring, and non-naturally occurring, which have
similar binding properties as the reference nucleic acid, and which
are metabolized in a manner similar to the reference nucleotides.
Examples of such analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, peptide-nucleic acids (PNAs). Methods
for detecting can include but are not limited to RT-PCR, northern
blot analyses, gene expression analyses, microarray analyses, gene
expression chip analyses, hybridization techniques (including
FISH), expression beadchip arrays, and chromatography as well as
any other techniques known in the art. Methods for detecting DNA
can include but are not limited to PCR, real-time PCR, digital PCR,
hybridization (including FISH), microarray analyses, SNP detection
assays, SNP genotyping assays and chromatography as well as any
other techniques known in the art
[0172] In some embodiments, the method comprises detecting the
protein expression level of a biomarker. The protein expression
level can be determined by any suitable methods known to one
skilled in the art. Any suitable methods of protein detection,
quantization and comparison can be used, such as those described in
Tschesche (Methods in Protein Biochemistry, ISBN Walter de Gruyter,
2011, ISBN 3110252368, 9783110252361), Goluch et al. (Chip-based
detection of protein cancer markers, ProQuest, 2007, ISBN
0549463453, 9780549463450), Speicher (Proteome Analysis:
Interpreting the Genome, Elsevier, 2004, ISBN 0080515304,
9780080515304), Albala et al. (Protein Arrays, Biochips and
Proteomics, CRC Press, 2003, ISBN 0203911121, 9780203911129),
Walker (The Protein Protocols Handbook, Springer, 2002, ISBN
0896039404, 9780896039407), Fung (Protein Arrays: Methods and
Protocols, Springer, 2004, ISBN 1592597599, 9781592597598), and
Bienvenut (Acceleration and Improvement of Protein Identification
by Mass Spectrometry, Springer, 2005, ISBN 1402033184,
9781402033186), each of which is incorporated by reference in its
entirety for all purposes. In some embodiments, the protein
expression level of biomarkers are detected and measured by
immunohistochemistry (IHC), western blot, protein immunestaining,
protein immuneprecipitation, immuneeletrophoresis, immunoblotting,
BCA assay, spectrophotometry, mass spectrometry or enzyme assay, or
combinations thereof. For additional methods related to detection,
quantitation and comparison of biomarker levels, see, e.g., Current
Protocols in Molecular Biology, Ed. Ausubel, Frederick M. (2010);
Current Protocols in Protein Science Last, Ed. Coligan, John E., et
al. (2010); Current Protocols in Nucleic Acid Chemistry, Ed. Egli,
Martin (2010); Current Protocols in Bioinformatics, Ed. Baxevanis,
Andreas D. (2010); and Molecular Cloning: A Laboratory Manual,
Third Edition, Sambrook, Joseph (2001), all of which are
incorporated herein by reference in their entirety.
[0173] In some embodiments, antibody to a given biomarker is used.
In some embodiments, a kit for detection is used. Such antibodies
and kits are available from EMD Millipore, OriGene Custom Assay
Services, R&D Systems for biochemical assays, GenScript Custom
Assay Services, Enzo Life Sciences for kits & assays,
Cloud-Clone Corp. ELISAs, or Cloud-Clone Corp. CLIAs. The term
"antibody" as used herein is intended to include monoclonal
antibodies, polyclonal antibodies, and chimeric antibodies. The
antibody may be from recombinant sources and/or produced in
transgenic animals. The term "antibody fragment" as used herein is
intended to include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv,
dimers, minibodies, diabodies, and multimers thereof and
biospecific antibody fragments. Antibodies can be fragmented using
conventional techniques. For example, F(ab')2 fragments can be
generated by treating the antibody with pepsin. The resulting
F(ab')2 fragment can be treated to reduce disulfide bridges to
produce Fab' fragments. Papain digestion can lead to the formation
of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv,
dimers, minibodies, diabodies, biospecific antibody fragments and
other fragments can also be synthesized by recombinant
techniques.
[0174] Immunoassays carried out in accordance with the present
invention may be homogeneous assays or heterogeneous assays. In a
homogeneous assay the immunological reaction usually involves the
specific antibody, a labeled analyte, and the sample of interest.
The signal arising from the label is modified, directly or
indirectly, upon the binding of the antibody to the labeled
analyte. Both the immunological reaction and detection of the
extent thereof can be carried out in a homogeneous solution.
Immunochemical labels which may be employed include free radicals,
radioisotopes, fluorescent dyes, enzymes, bacteriophages, or
coenzymes.
[0175] In a heterogeneous assay approach, the reagents are usually
the sample, the antibody, and means for producing a detectable
signal. Samples as described above may be used. The antibody can be
immobilized on a support, such as a bead (such as protein A and
protein G agarose beads), plate or slide, and contacted with the
specimen suspected of containing the antigen in a liquid phase. The
support is then separated from the liquid phase and either the
support phase or the liquid phase is examined for a detectable
signal employing means for producing such signal. The signal is
related to the presence of the analyte in the sample. Means for
producing a detectable signal include the use of radioactive
labels, fluorescent labels, or enzyme labels. For example, if the
antigen to be detected contains a second binding site, an antibody
which binds to that site can be conjugated to a detectable group
and added to the liquid phase reaction solution before the
separation step. The presence of the detectable group on the solid
support indicates the presence of the antigen in the test sample.
Examples of suitable immunoassays include, but are not limited to
immunoblotting, immunoprecipitation, immunofluorescence methods,
chemiluminescence methods, electrochemiluminescence (ECL) or
enzyme-linked immunoassays.
[0176] Those skilled in the art will be familiar with numerous
specific immunoassay formats and variations thereof which may be
useful for carrying out the method disclosed herein. See generally
E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton,
Fla.); see also U.S. Pat. No. 4,727,022, U.S. Pat. No. 4,659,678,
U.S. Pat. No. 4,376,110, U.S. Pat. No. 4,275,149, U.S. Pat. No.
4,233,402,U.S. Pat. No. 4,230,767, each of which is herein
incorporated by reference in its entirety for all purposes.
[0177] Antibodies can be conjugated to a solid support suitable for
a diagnostic assay (e.g., beads such as protein A or protein G
agarose, microspheres, plates, slides or wells formed from
materials such as latex or polystyrene) in accordance with known
techniques, such as passive binding. Antibodies as described herein
may likewise be conjugated to detectable labels or groups such as
radiolabels (e.g., 35S, 125I, 131I), enzyme labels (e.g.,
horseradish peroxidase, alkaline phosphatase), and fluorescent
labels (e.g., fluorescein, Alexa, green fluorescent protein,
rhodamine) in accordance with known techniques.
[0178] Antibodies can also be useful for detecting
post-translational modifications of proteins, polypeptides,
mutations, and polymorphisms, such as tyrosine phosphorylation,
threonine phosphorylation, serine phosphorylation, glycosylation
(e.g., O-GlcNAc). Such antibodies specifically detect the
phosphorylated amino acids in a protein or proteins of interest,
and can be used in immunoblotting, immunofluorescence, and ELISA
assays described herein. These antibodies are well-known to those
skilled in the art, and commercially available. Post-translational
modifications can also be determined using metastable ions in
reflector matrix-assisted laser desorption ionization-time of
flight mass spectrometry (MALDI-TOF) (Wirth, U. et al. (2002)
Proteomics 2(10): 1445-51).
[0179] In some embodiments, detection reagents can be immobilized
on a solid matrix such as a porous strip to form at least one
detection site. In some embodiments, polynucleotide or polypeptide
arrays or microarrays containing a plurality of detection agents
that hybridize to nucleotide or polypeptide of the biomarkers are
utilized. Alternatively, the substrate array can be on, e.g., a
solid substrate, e.g., a "chip" as described in U.S. Pat. No.
5,744,305. Alternatively, the substrate array can be a solution
array.
[0180] Non-limiting examples for compositions and methods for
detecting the components in the CDX2-KLF4 signaling pathway are
described in U.S. Pat. Nos. 4,762,706, 5,081,230, 5,300,631,
5,443,956, 7,695,926, 7,785,817, 7,479,376, 7,364,868 and U.S.
Patent Publication Nos. 20050196793, 20110281277, 20120251509,
20050186642, 20140011279, 20110171221, 20040235073, 20130011411,
and 20130034862, each of which is incorporated herein by reference
in its entirety for all purposes.
[0181] The present invention also provides kits for detecting
and/or measuring activity level of one or more biomarkers of the
present invention. In some embodiments, the kits are manufactured
based on the detecting/measuring methods described herein or
methods known to one skilled in the art. In some embodiments, the
kits comprise packaging material and at least one vial comprising
agent for detecting/measuring. In some embodiments, the kits
comprise packaging material and at least one vial comprising agent
for detecting/measuring the activity of at least one component in
the CDX2-KLF4 signaling pathway. In some embodiments, the kits
comprise packaging material and at least one vial comprising agent
for detecting/measuring the activity of CDX2, KLF4, Caspase-3,
Annexin V, p53, and/or p21. Optionally, the kits further comprise
additional components selected from buffers, preservatives, aqueous
diluent. In some embodiments, the kits comprise a sample that can
be used as a control, such as a sample obtained from one or more
human subjects who are responsive to a treatment by an anti-cancer
agent of the present invention. In some embodiments, control is a
sample obtained from one or more human subjects who are not
responsive to a treatment by an anti-cancer agent of the present
invention. Alternatively, the kits comprise an instruction, which
provides standard activity level of human subjects who are
responsive to a treatment by an anti-cancer agent of the present
invention (e.g., a predetermined range of activity level of one or
more biomarkers of the present invention in the responsive human
subjects), and/or the standard activity level of human subjects who
are not responsive to a treatment by an anti-cancer agent of the
present invention (e.g., a predetermined range of activity level of
one or more biomarkers of the present invention in the
non-responsive human subjects). By using the kits of the present
invention, one can make a determination regarding if the activity
level of a given biomarker in the human subject being tested is
statistically distinguishable from the standard activity level.
Such determination can be used in combination with methods
described herein.
Anti-Cancer Compositions
[0182] Anti-cancer compositions that can be utilized in the present
invention comprise at least one active agent. In some embodiments,
the active agent can modulate the activity of the CDX2-KLF4
signaling pathway. As used herein, the term "modulate" refers to
that the compositions can increase, decrease, eliminate, enhance,
delay, reduce, or block the activity of a component in the
CDX2-KLF4 signaling pathway. In some embodiments, the compounds can
decrease the CDX2 activity, and/or increase the KLF4 activity in a
human subject. In some embodiments, the compounds can increase or
decrease the activity of one or more upstream or downstream
components in the signaling pathway. In some embodiments, the
compounds can increase the activity of one or more downstream
components that are positively regulated by KLF4 (e.g., p21), or
decrease the activity of one or more downstream components in the
signaling pathway that are negatively regulated by KLF4 (e.g.,
SP1). In some embodiments, the compounds can decrease the activity
of one or more downstream components that are positively regulated
by CDX2, or increase the activity of one or more downstream
components in the signaling pathway that are negatively regulated
by CDX2.
[0183] The active agents can be chemical compounds or compositions,
biological molecules, or combinations thereof. In some embodiments,
the active agents are small molecules. As used herein, the term
"small molecule" refers to a molecule having a molecular weight of
less than 500 MW, wherein the drug is a non-peptidyl or peptide
agent. In some embodiments, the active agents are antibodies. In
some embodiments, the active agents are antibodies. In some
embodiments, the active agents are polynucleotides, such as
siRNA.
[0184] In some embodiments, the active agents contain one or more
entities that can inhibit or decrease the activity of CDX2, e.g.,
at DNA, RNA, protein level, or combinations thereof. In one
embodiment, the anti-CDX2 agent is an anti-CDX2 antibody. According
to the present invention, an anti-CDX2 antibody at least comprises
one or more anti-CDX2 CDRs. In some embodiments, the anti-CDX2
agents are small interference RNA molecules, such as those
disclosed by Wang et al. ("siRNA targeting of Cdx2 inhibits growth
of human gastric cancer MGC-803 cells", World J Gastroenterol. Apr.
28, 2012; 18(16): 1903-1914.)
[0185] In some embodiments, the active agents contain one or more
entities that can increase the activity of KLF4, or increase the
activity of an entity in the CDX2-KLF4 signaling pathway that is
positively regulated by KLF4, e.g., at DNA, RNA, protein level, or
combinations thereof. In some embodiments, the active agent of the
present invention is a 2-indolyl imidazo[4,5-d]phenanthroline
derivative, such as those described in U.S. Pat. No. 8,148,392 or
U.S. Patent Publication No. 2007/0123553A1, each of which is herein
incorporated by reference in its entirety for all purposes.
[0186] In some embodiments, the compound has the structure of
formula I:
##STR00002##
[0187] In some embodiments, the compound has the structural formula
(II), or a salt thereof:
##STR00003##
wherein: R1, R2, R3, R4, R6 and R7 are independently selected from
hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted lower
alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,
substituted lower alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,
amido, carboxyl, aryl, substituted aryl, heterocycle, substituted
heterocycle, heteroalkyl, substituted heteroalkyl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl, nitro,
or cyano or --S(O)1-2R wherein R is alkyl, substituted alkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heterocycle,
or substituted heteroaryl; and wherein R5 is H, alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,
--CH2-aryl, --CH2-- heteroaryl.
[0188] In some embodiments, R1, R2, R3, R4 are independently
hydrogen; halogen; C1-C4 alkyl; C1-C4 alkoxy; or C6-C14 aryl; R5 is
hydrogen; C1-C4 alkyl; C1-C4 alkyl substituted with C6-C14 aryl; or
C4-C6 cycloalkyl; R6 is hydrogen; halogen; C1-C4 alkyl; C1-C4 alkyl
substituted with C5-C6 heterocycloalkyl wherein the heteroatom is
N; C6-C14 aryl; C6-C14 aryl substituted with C1-C4 alkyl or halo;
C5-C6 cycloalkyl; C5-C6 heterocycloalkyl; or polycycloalkyl. In
some other embodiments, R1, R2, R3, R4 are independently hydrogen;
halogen; C1-C4 alkyl; C1-C4 alkoxy; or phenyl; R5 is hydrogen;
C1-C4 alkyl; C1-C4 alkyl substituted with phenyl; or cyclopentyl;
R6 is hydrogen; halogen; C1-C4 alkyl; C1-C4 alkyl substituted with
C5-C6 heterocycloalkyl wherein the heteroatom is N; phenyl; phenyl
substituted with C1-C4 alkyl or halo; C5-C6 cycloalkyl; C5-C6
heterocycloalkyl; or adamantane; and R7 is H.
[0189] In some embodiments, the compound has the structural formula
(III), or a salt thereof:
##STR00004##
[0190] Wherein R1 is C1-C4 alkyl; and R2 is halogen. In some
embodiments, R1 is methyl, isopropyl, or t-butyl.
[0191] In some embodiments, said compound has a formula selected
from the group consisting of
##STR00005##
[0192] The active agents of the present invention are typically
formulated prior to administration. The present invention thus
provides pharmaceutical compositions comprising one or more active
agents of the present invention. In some embodiments, the
pharmaceutical compositions comprise a pharmaceutically acceptable
carrier, diluent, or excipient. The pharmaceutical compositions are
prepared by known procedures using well-known and readily available
ingredients.
[0193] Active agents of the present invention or pharmaceutical
compositions comprising the active agents may be administered via
any suitable methods, including but not limited to, orally,
topically, parenterally, by inhalation or spray, or rectally in
dosage unit formulations. In some embodiments, the dosage unit
formulations contain conventional non-toxic pharmaceutically
acceptable carriers, adjuvants and vehicles. In the usual course of
therapy, the active agent is incorporated into an acceptable
vehicle to form a composition for topical administration to the
affected area, such as hydrophobic or hydrophilic creams or
lotions, or into a form suitable for oral, rectal or parenteral
administration, such as syrups, elixirs, tablets, troches,
lozenges, hard or soft capsules, pills, suppositories, oily or
aqueous suspensions, dispersible powders or granules, emulsions,
injectables, or solutions. The term parenteral as used herein
includes, but are not limited to, subcutaneous injections,
intradermal, intra-articular, intravenous, intramuscular,
intravascular, intrastemal, intrathecal injection or infusion
techniques.
[0194] The present invention also provides for pharmaceutical
compositions comprising one or more of the active agents of the
present invention and a vehicle, such as an artificial membrane
vesicle (including a liposome, lipid micelle and the like),
microparticle or microcapsule.
[0195] Compositions intended for oral use may be prepared in either
solid or fluid unit dosage forms. Fluid unit dosage form can be
prepared according to procedures known in the art for the
manufacture of pharmaceutical compositions and such compositions
may contain one or more agents selected from the group consisting
of sweetening agents, flavouring agents, colouring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. An elixir is prepared by using a
hydroalcoholic (e.g., ethanol) vehicle with suitable sweeteners
such as sugar and saccharin, together with an aromatic flavoring
agent. Suspensions can be prepared with an aqueous vehicle with the
aid of a suspending agent such as acacia, tragacanth,
methylcellulose and the like.
[0196] Solid formulations such as tablets contain the active
ingredient in admixture with non-toxic pharmaceutically acceptable
excipients that are suitable for the manufacture of tablets. These
excipients may be 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 and other conventional ingredients
such as dicalcium phosphate, magnesium aluminum silicate, calcium
sulfate, starch, lactose, methylcellulose, and functionally similar
materials. The tablets may be uncoated or they may be 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 may be employed.
[0197] Formulations for oral use may also 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. Soft gelatin capsules are
prepared by machine encapsulation of a slurry of the compound with
an acceptable vegetable oil, light liquid petrolatum or other inert
oil.
[0198] Aqueous suspensions contain active materials in admixture
with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxylmethylcellulose, methyl cellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia: dispersing or wetting agents may be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
hepta-decaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl-p-hydroxy benzoate,
one or more colouring agents, one or more flavouring agents or one
or more sweetening agents, such as sucrose or saccharin.
[0199] Oily suspensions may be formulated by suspending the active
ingredients in a vegetable oil, for example peanut oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavouring agents may be added
to provide palatable oral preparations. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0200] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavouring and colouring agents, may also be
present.
[0201] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oil phase may be a
vegetable oil, for example olive oil or peanut oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0202] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to known art using those
suitable dispersing or wetting agents and suspending agents that
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or a suspension in a
non-toxic parentally acceptable diluent or solvent, for example as
a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables. Adjuvants
such as local anaesthetics, preservatives and buffering agents can
also be included in the injectable solution or suspension.
[0203] The pharmaceutical compositions of the present invention may
be administered, together or separately, in the form of
suppositories for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures
but liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols.
[0204] Other pharmaceutical compositions and methods of preparing
pharmaceutical compositions are known in the art and are described,
for example, in "Remington: The Science and Practice of Pharmacy"
(formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A.,
Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000).
[0205] The pharmaceutical compositions of the present invention may
be administered to a subject by a variety of routes depending on
the cancer to be treated, for example, the compositions may be
administered orally, topically, parenterally, by inhalation or
spray, or rectally in dosage unit formulations. In one embodiment,
the compounds are administered systemically to a subject, for
example, by bolus injection or infusion into a subject's
bloodstream or by oral administration. When used in conjunction
with one or more known chemotherapeutic agents, the compounds can
be administered prior to, or after, administration of the
chemotherapeutic agents, or they can be administered concomitantly.
The one or more chemotherapeutic may also be administered
systemically, for example, by bolus injection, infusion, or oral
administration.
[0206] The pharmaceutical compositions of the present invention may
be used as part of a neo-adjuvant therapy (complement to primary
therapy), or as part of an adjuvant therapy regimen. The present
invention contemplates the use of the pharmaceutical compositions
of the present invention at various stages in tumor development and
progression, including, but not limited to, in the treatment of
advanced and/or aggressive neoplasias (i.e. overt disease in a
subject that is not amenable to cure by local modalities of
treatment, such as surgery or radiotherapy), metastatic disease,
locally advanced disease and/or refractory tumors (i.e. a cancer or
tumor that has not responded to treatment). As used herein, the
term "primary therapy" refers to a first line of treatment upon the
initial diagnosis of cancer in a subject. Exemplary primary
therapies may involve surgery, a wide range of chemotherapies and
radiotherapy. "Adjuvant therapy" refers to a therapy that follows a
primary therapy and that is administered to subjects at risk of
relapsing. Adjuvant systemic therapy is usually begun soon after
primary therapy to delay recurrence, prolong survival or cure a
subject.
[0207] The pharmaceutical compositions of the present invention can
be used alone or in combination with one or more other anti-cancer
agents, such as chemotherapeutic agents as part of a primary
therapy or an adjuvant therapy. Combinations of the pharmaceutical
compositions of the present invention and standard
chemotherapeutics may act to improve the efficacy of the
chemotherapeutic and, therefore, can be used to improve standard
cancer therapies. This application can be important in the
treatment of drug-resistant cancers which are not responsive to
standard treatment. Drug-resistant cancers can arise, for example,
from heterogeneity of tumor cell populations, alterations in
response to chemotherapy and increased malignant potential. Such
changes are often more pronounced at advanced stages of
disease.
[0208] The pharmaceutical compositions of the present invention can
be used alone or in combination with radiation therapeutic. In some
embodiments, the radiation therapeutic is administered at a dosage
of about 40 Gy to about 80 Gy. In some embodiments the dosage is
about 50 Gy to about 70 Gy, in some embodiments, the dosage is
about 50 Gy to about 65 Gy. In some embodiments, the radiation
therapy is administered at a dosage of about 50 Gy, about 55 Gy,
about 60 Gy or about 65 Gy.
[0209] The dosage to be administered is not subject to defined
limits, but it will usually be an effective amount. It will usually
be the equivalent, on a molar basis of the pharmacologically active
free form produced from a dosage formulation upon the metabolic
release of the active free drug to achieve its desired
pharmacological and physiological effects. The compositions may be
formulated in a unit dosage form. The term "unit dosage form"
refers to physically discrete units suitable as unitary dosages for
human subjects and other mammals, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable
pharmaceutical excipient.
[0210] In some embodiments, the active agents are compounds having
the structure of formulas described herein. Examples of ranges for
the compound(s) in each dosage unit are from about 0.05 to about
100 mg, or more usually, from about 1.0 to about 50 mg. Daily
dosages of the compounds of the present invention will typically
fall within the range of about 0.01 to about 100 mg/kg of body
weight, or within the range of about 20 mg/m.sup.2 to about 300
mg/m.sup.2 in single or divided dose. However, it will be
understood that the actual amount of the compound(s) to be
administered will be determined by a physician, in the light of the
relevant circumstances, including the condition to be treated, the
chosen route of administration, the actual compound administered,
the age, weight, and response of the individual patient, and the
severity of the patient's symptoms. The above dosage range is given
by way of example only and is not intended to limit the scope of
the invention in any way. In some instances dosage levels below the
lower limit of the aforesaid range may be more than adequate, while
in other cases still larger doses may be employed without causing
harmful side effects, for example, by first dividing the larger
dose into several smaller doses for administration throughout the
day.
[0211] Pharmaceutical compositions comprising one or more compounds
described herein in combination with one or more other anti-cancer
agents can also be used.
[0212] In some embodiments, the anti-cancer agents are
chemotherapeutics. In some embodiments, the chemotherapeutics are
selected from alkylating agents, anti-metabolites, anti-microtubule
agents, Topoisomerase inhibitors, and Cytotoxic antibiotics.
Examples of chemotherapeutic agents include, but are not limited
to, paclitaxel (Taxol.RTM.), docetaxel (Taxotere.RTM.), cisplatin,
carboplatin (Paraplatin.RTM.), gemcitabine hydrochloride
(Gemzar.RTM.), doxorubicin, etoposide (Etopophos.RTM.,
Vepesid.RTM.), pemetrexed (Alimta.RTM.), topotecan (Hycamtin.RTM.),
vinblastine (Velbe.RTM.), Vindesine (Eldisine.RTM.), vinorelbine
(Navelbine.RTM.), ifosfamide (Mitoxana.RTM.), Mitomycin, and
gemcitabine. These agents may be given in combination, for example,
vinorelbine and cisplatin or carboplatin; gemcitabine with
cisplatin or carboplatin or paclitaxel; MIC (mitomycin, ifosfamide
and cisplatin); MVP (mitomycin, vinblastine and cisplatin); and EC
(etoposide and carboplatin). Examples of useful chemotherapeutic
drugs include, but are not limited to, hydroxyurea, busulphan,
cisplatin, carboplatin, chlorambucil, melphalan, cyclophosphamide,
Ifosphamide, danorubicin, doxorubicin, epirubicin, mitoxantrone,
vincristine, vinblastine, Navelbine.RTM. (vinorelbine), etoposide,
teniposide, paclitaxel, docetaxel, gemcitabine, cytosine,
arabinoside, bleomycin, neocarcinostatin, suramin, taxol, mitomycin
C and the like.
[0213] As used herein, the term "alkylating agents" refers to
agents that have the ability to alhylate molecules in a subject,
including proteins, RNA and DNA. Non-limiting examples of
alkylating agents include nitrogen mustards, nitrosoureas,
tetrazines, aziridines, cisplatins and derivatives, and
non-classical alkylating agents. Nitrogen mustards include
mechlorethamine, cyclophosphamide, melphalan, chlorambucil,
ifosfamide and busulfan. Nitrosoureas include
N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU)
and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines
include dacarbazine, mitozolomide and temozolomide. Aziridines
include thiotepa, mytomycin and diaziquone (AZQ). Cisplatin and
derivatives include cisplatin, carboplatin and oxaliplatin. They
impair cell function by forming covalent bonds with the amino,
carboxyl, sulfhydryl, and phosphate groups in biologically
important molecules.[20] Non-classical alkylating agents include
procarbazine and hexamethylmelamine.
[0214] As used herein, the term "anti-metabolites" refers to
molecule that impedes DNA, RNA, or protein synthesis. In some
embodiments, anti-metabolites resemble either nucleobases or
nucleosides (a nucleotide without the phosphate group), but have
altered chemical groups. These drugs exert their effect by either
blocking the enzymes required for DNA synthesis or becoming
incorporated into DNA or RNA. By inhibiting the enzymes involved in
DNA synthesis, they prevent mitosis because the DNA cannot
duplicate itself. Also, after misincorporation of the molecules
into DNA, DNA damage can occur and programmed cell death
(apoptosis) is induced. In some embodiments, the anti-metabolites
are anti-folates, fluoropyrimidines, deoxynucleoside analogues and
thiopurines. In some embodiments, the anti-metabolites are selected
from methotrexate, pemetrexed, fluorouracil, capecitabine,
cytarabine, gemcitabine, decitabine, Vidaza, fludarabine,
nelarabine, cladribine, clofarabine, pentostatin, thioguanine and
mercaptopurine.
[0215] As used herein, the term "anti-microtubule agents" refers to
chemicals that block cell division by preventing microtubule
function. Representative examples of such agents include taxanes
(e.g., paclitaxel (discussed in more detail below) and docetaxel)
(Schiff et al., Nature 277: 665-667, 1979; Long and Fairchild,
Cancer Research 54: 4355-4361, 1994; Ringel and Horwitz, J. Natl.
Cancer Inst. 83(4): 288-291, 1991; Pazdur et al, Cancer Treat. Rev.
19(4): 351-386, 1993), campothecin, mitoxantrone, eleutherobin
(e.g., U.S. Pat. No. 5,473,057), sarcodictyins (including
sarcodictyin A), epothilones A and B (Bollag et al., Cancer
Research 55: 2325-2333, 1995), discodermolide (ter Haar et al.,
Biochemistry 35: 243-250, 1996), deuterium oxide (D2O) (James and
Lefebvre, Genetics 130(2): 305-314, 1992; Sollott et al., J. Clin.
Invest. 95: 1869-1876, 1995), hexylene glycol
(2-methyl-2,4-pentanediol) (Oka et al., Cell Struct. Funct. 16(2):
125-134, 1991), tubercidin (7-deazaadenosine) (Mooberry et al.,
Cancer Lett. 96(2): 261-266, 1995), LY290181
(2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-cardonitrile)
(Panda et al., J. Biol. Chem. 272(12): 7681-7687, 1997; Wood et
al., Mol. Pharmacol. 52(3): 437-444, 1997), aluminum fluoride (Song
et al., J. Cell. Sci. Suppl. 14: 147-150, 1991), ethylene glycol
bis-(succinimidylsuccinate) (Caplow and Shanks, J. Biol. Chem.
265(15): 8935-8941, 1990), glycine ethyl ester (Mejillano et al.,
Biochemistry 31(13): 3478-3483, 1992), nocodazole (Ding et al., J.
Exp. Med 171(3): 715-727, 1990; Dotti et al., J. Cell Sci. Suppl.
15: 75-84, 1991; Oka et al., Cell Struct. Funct. 16(2): 125-134,
1991; Weimer et al., J. Cell Biol. 136(1), 71-80, 1997),
cytochalasin B (Illinger et al., Biol. Cell 73(2-3): 131-138,
1991), colchicine and CI-980 (Allen et al., Am. J. Physiol. 261(4
Pt. 1): L315-L321, 1991; Ding et al., J. Exp. Med. 171(3): 715-727,
1990; Gonzalez et al., Exp. Cell. Res. 192(1): 10-15, 1991;
Stargell et al., Mol. Cell. Biol. 12(4): 1443-1450, 1992; Garcia et
al., Antican. Drugs 6(4): 533-544, 1995), colcemid (Barlow et al.,
Cell. Motil. Cytoskeleton 19(1): 9-17, 1991; Meschini et al., J.
Microsc. 176(Pt. 3): 204-210, 1994; Oka et al., Cell Struct. Funct.
16(2): 125-134, 1991), podophyllotoxin (Ding et al., J. Exp. Med.
171(3): 715-727, 1990), benomyl (Hardwick et al., J. Cell. Biol.
131(3): 709-720, 1995; Shero et al., Genes Dev. 5(4): 549-560,
1991), oryzalin (Stargell et al., Mol. Cell. Biol. 12(4):
1443-1450, 1992), majusculamide C (Moore, J. Ind. Microbiol. 16(2):
134-143, 1996), demecolcine (Van Dolah and Ramsdell, J. Cell.
Physiol. 166(1): 49-56, 1996; Wiemer et al., J. Cell. Biol. 136(1):
71-80, 1997), methyl-2-benzimidazolecarbamate (MBC) (Brown et al.,
J. Cell. Biol. 123(2): 387-403, 1993), LY195448 (Barlow &
Cabral, Cell Motil. Cytoskel. 19: 9-17, 1991), subtilisin (Saoudi
et al., J. Cell Sci. 108: 357-367, 1995), 1069C85 (Raynaud et al.,
Cancer Chemother Pharmacol. 35: 169-173, 1994), steganacin (Hamel,
Med. Res. Rev. 16(2): 207-231, 1996), combretastatins (Hamel, Med
Res. Rev. 16(2): 207-231, 1996), curacins (Hamel, Med. Res. Rev.
16(2): 207-231, 1996), estradiol (Aizu-Yokata et al., Carcinogen.
15(9): 1875-1879, 1994), 2-methoxyestradiol (Hamel, Med. Res. Rev.
16(2): 207-231, 1996), flavanols (Hamel, Med. Res. Rev. 16(2):
207-231, 1996), rotenone (Hamel, Med. Res. Rev. 16(2): 207-231,
1996), griseofulvin (Hamel, Med. Res. Rev. 16(2): 207-231, 1996),
vinca alkaloids, including vinblastine and vincristine (Ding et
al., J. Exp. Med 171(3): 715-727, 1990; Dirk et al., Neurochem.
Res. 15(11): 1135-1139, 1990; Hamel, Med. Res. Rev. 16(2): 207-231,
1996; Illinger et al., Biol. Cell 73(2-3): 131-138, 1991; Wiemer et
al., J. Cell. Biol. 136(1): 71-80, 1997), maytansinoids and
ansamitocins (Hamel, Med Res. Rev. 16(2): 207-231, 1996), rhizoxin
(Hamel, Med. Res. Rev. 16(2): 207-231, 1996), phomopsin A (Hamel,
Med. Res. Rev. 16(2): 207-231, 1996), ustiloxins (Hamel, Med. Res.
Rev. 16(2): 207-231, 1996), dolastatin 10 (Hamel, Med Res. Rev.
16(2): 207-231, 1996), dolastatin 15 (Hamel, Med. Res. Rev. 16(2):
207-231, 1996), halichondrins and halistatins (Hamel, Med. Res.
Rev. 16(2): 207-231, 1996), spongistatins (Hamel, Med. Res. Rev.
16(2): 207-231, 1996), cryptophycins (Hamel, Med. Res. Rev. 16(2):
207-231, 1996), rhazinilam (Hamel, Med. Res. Rev. 16(2): 207-231,
1996), betaine (Hashimoto et al., Zool. Sci. 1: 195-204, 1984),
taurine (Hashimoto et al., Zool. Sci. 1: 195-204, 1984),
isethionate (Hashimoto et al., Zool. Sci. 1: 195-204, 1984), HO-221
(Ando et al., Cancer Chemother Pharmacol. 37: 63-69, 1995),
adociasulfate-2 (Sakowicz et al., Science 280: 292-295, 1998),
estramustine (Panda et al., Proc. Natl. Acad. Sci. USA 94:
10560-10564, 1997), monoclonal anti-idiotypic antibodies (Leu et
al., Proc. Natl. Acad. Sci. USA 91(22): 10690-10694, 1994),
microtubule assembly promoting protein (taxol-like protein, TALP)
(Hwang et al., Biochem. Biophys. Res. Commun. 208(3): 1174-1180,
1995), cell swelling induced by hypotonic (190 mosmol/L)
conditions, insulin (100 mmol/L) or glutamine (10 mmol/L)
(Haussinger et al., Biochem. Cell. Biol. 72(1-2): 12-19, 1994),
dynein binding (Ohba et al., Biochim. Biophys. Acta 1158(3):
323-332, 1993), gibberelin (Mita and Shibaoka, Protoplasma
119(1/2): 100-109, 1984), XCHOI (kinesin-like protein) (Yonetani et
al., Mol. Biol. Cell 7(suppl): 211 A, 1996), lysophosphatidic acid
(Cook et al., Mol. Biol. Cell 6(suppl): 260A, 1995), lithium ion
(Bhattacharyya and Wolff, Biochem. Biophys. Res. Commun. 73(2):
383-390, 1976), plant cell wall components (e.g., poly-L-lysine and
extensin) (Akashi et al., Planta 182(3): 363-369, 1990), glycerol
buffers (Schilstra et al., Biochem. J. 277(Pt. 3): 839-847, 1991;
Farrell and Keates, Biochem. Cell. Biol. 68(11): 1256-1261, 1990;
Lopez et al., J. Cell. Biochem. 43(3): 281-291, 1990), Triton X-100
microtubule stabilizing buffer (Brown et al., J. Cell Sci. 104(Pt.
2): 339-352, 1993; Safiejko-Mroczka and Bell, J. Histochem.
Cytochem. 44(6): 641-656, 1996), microtubule associated proteins
(e.g., MAP2, MAP4, tau, big tau, ensconsin, elongation
factor-1-alpha (EF-1.alpha.) and E-MAP-115) (Burgess et al., Cell
Motil. Cytoskeleton 20(4): 289-300, 1991; Saoudi et al., J. Cell.
Sci. 108(Pt. 1): 357-367, 1995; Bulinski and Bossler, J. Cell. Sci.
107(Pt. 10): 2839-2849, 1994; Ookata et al., J. Cell Biol. 128(5):
849-862, 1995; Boyne et al., J. Comp. Neurol 358(2): 279-293, 1995;
Ferreira and Caceres, J. Neurosci. 11(2): 392-400, 1991; Thurston
et al., Chromosoma 105(1):20-30, 1996; Wang et al., Brain Res. Mol.
Brain Res. 38(2): 200-208, 1996; Moore and Cyr, Mol. Biol. Cell
7(suppl): 221-A, 1996; Masson and Kreis, J. Cell Biol. 123(2),
357-371, 1993), cellular entities (e.g., histone H1, myelin basic
protein and kinetochores) (Saoudi et al., J. Cell. Sci. 108(Pt. 1):
357-367, 1995; Simerly et al., J. Cell Biol. 111(4): 1491-1504,
1990), endogenous microtubular structures (e.g., axonemal
structures, plugs and GTP caps) (Dye et al., Cell Motil.
Cytoskeleton 21(3): 171-186, 1992; Azhar and Murphy, Cell Motil.
Cytoskeleton 15(3): 156-161, 1990; Walker et al., J. Cell Biol.
114(1): 73-81, 1991; Drechsel and Kirschner, Curr. Biol. 4(12):
1053-1061, 1994), stable tubule only polypeptide (e.g., STOP145 and
STOP220) (Pirollet et al., Biochim. Biophys. Acta 1160(1): 113-119,
1992; Pirollet et al., Biochemistry 31(37): 8849-8855, 1992; Bosc
et al., Proc. Natl. Acad. Sci. USA 93(5): 2125-2130, 1996; Margolis
et al., EMBO J. 9(12): 4095-4102, 1990) and tension from mitotic
forces (Nicklas and Ward, J. Cell Biol. 126(5): 1241-1253, 1994),
as well as any analogues and derivatives of any of the above. Such
compounds can act by either depolymerizing microtubules (e.g.,
colchicine and vinblastine), or by stabilizing microtubule
formation (e.g., paclitaxel).
[0216] In some embodiments, an anti-tumor agent include mitotic
inhibitors, for example vinca alkaloid derivatives such as
vinblastine vinorelbine, vindescine and vincristine; colchines
allochochine, halichondrine, N-benzoyltrimethyl-methyl ether
colchicinic acid, dolastatin 10, maystansine, rhizoxine, taxanes
such as taxol (paclitaxel), docetaxel (Taxotere),
2'-N-[3-(dimethylamino)propyl]glutaramate (taxol derivative),
thiocholchicine, trityl cysteine, teniposide, methotrexate,
azathioprine, fluorouricil, cytocine arabinoside,
2'2'-difluorodeoxycytidine (gemcitabine), adriamycin and mitamycin.
Alkylating agents, for example cis-platin, carboplatin oxiplatin,
iproplatin, Ethyl ester of N-acetyl-DL-sarcosyl-L-leucine (Asaley
or Asalex), 1,4-cyclohexadiene-1,4-dicarbamic acid,
2,5-bis(1-azirdinyl)-3,6-dioxo-, diethyl ester (diaziquone),
1,4-bis(methanesulfonyloxy)butane (bisulfan or leucosulfan)
chlorozotocin, clomesone, cyanomorpholinodoxorubicin, cyclodisone,
dianhydroglactitol, fluorodopan, hepsulfam, mitomycin C,
hycantheonemitomycin C, mitozolamide,
1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride,
piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard,
teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil
nitrogen mustard, bis(3-mesyloxypropyl)amine hydrochloride,
mitomycin, nitrosoureas agents such as
cyclohexyl-chloroethylnitrosourea,
methylcyclohexyl-chloroethylnitrosourea
1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitroso-urea,
bis(2-chloroethyl)nitrosourea, procarbazine, dacarbazine, nitrogen
mustard-related compounds such as mechloroethamine,
cyclophosphamide, ifosamide, melphalan, chlorambucil, estramustine
sodium phosphate, strptozoin, and temozolamide. DNA
anti-metabolites, for example 5-fluorouracil, cytosine arabinoside,
hydroxyurea,
2-[(3hydroxy-2-pyrinodinyl)methylene]-hydrazinecarbothioamide,
deoxyfluorouridine, 5-hydroxy-2-formylpyridine thiosemicarbazone,
alpha-2'-deoxy-6-thioguanosine, aphidicolin glycinate,
5-azadeoxycytidine, beta-thioguanine deoxyriboside, cyclocytidine,
guanazole, inosine glycodialdehyde, macbecin II, pyrazolimidazole,
cladribine, pentostatin, thioguanine, mercaptopurine, bleomycin,
2-chlorodeoxyadenosine, inhibitors of thymidylate synthase such as
raltitrexed and pemetrexed disodium, clofarabine, floxuridine and
fludarabine. DNA/RNA antimetabolites, for example, L-alanosine,
5-azacytidine, acivicin, aminopterin and derivatives thereof such
as
N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl-
]-L-aspartic acid,
N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]-L-aspart-
ic acid,
N-[2-chloro-4-[[(2,4-diaminopteridinyl)methyl]amino]benzoyl]-L-as-
partic acid, soluble Baker's antifol, dichloroallyl lawsone,
brequinar, ftoraf, dihydro-5-azacytidine, methotrexate,
N-(phosphonoacetyl)-L-aspartic acid tetrasodium salt, pyrazofuran,
trimetrexate, plicamycin, actinomycin D, cryptophycin, and analogs
such as cryptophycin-52 or, for example, one of the preferred
anti-metabolites disclosed in European Patent Application No.
239362 such as
N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]--
2-thenoyl)-L-glutamic acid; growth factor inhibitors; cell cycle
inhibitors; intercalating antibiotics, for example adriamycin and
bleomycin; proteins, for example interferon; and anti-hormones, for
example anti-estrogens such as Nolvadex.TM. (tamoxifen) or, for
example anti-androgens such as Casodex.TM.
(4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluoromet-
hyl)propionanilide).
[0217] As used herein, the term "topoisomerase inhibitors" refers
to agents that can modulcate the activity of topoisomerase I and/or
topoisomerase II. In some embodiments, the topoisomerase inhibitor
of this invention can be a topoisomerase I inhibitor, which can be,
in some embodiments, a camptothecin derivative. A camptothecin
derivative of this invention can be, but is not limited to
Belotecan (CKD602), Camptothecin, 7-Ethyl-10-Hydroxy-CPT,
10-Hydroxy-CPT, Rubitecan (9-Nitro-CPT), 7-Ethyl-CPT, Topotecan,
Irinotecan, Silatecan (DB67) and any combinations thereof. In some
embodiments of this invention, the topoisomerase I inhibitor can be
an indenoisoquinoline derivative, which can be but is not limited
to NSC706744, NSC725776, NSC724998 and any combinations thereof. In
further embodiments of this invention, the topoisomerase inhibitor
is a topoisomerase II inhibitor, which in some embodiments can be
an acridine derivative, which can be but is not limited to
Amsacrine, in some embodiments the topoisomerase II inhibitor can
be a podophyllotoxin derivative, which can be but is not limited to
etoposide, and in some embodiments the topoisomerase II inhibitor
can be a bisdioxopiperazine derivative, which can be but is not
limited to ICRF-193, dexrazoxane (ICRF-187) and any combinations
thereof. In yet further embodiments of this invention, the
topoisomerase inhibitor can be Resveratrol (PMID: 20304553; PMID:
15796584)41, Epigallocatechin gallate (PMID: 18293940; PMID:
11594758; PMID: 11558576; PMID: 1313232)42,43, Genistein (PMID:
17458941)44, Daidzein (PMID: 17458941)45. Quercetin (PMID: 1313232;
PMID: 16950806; PMID: 15312049), natural flavones related to
quercetin that inhibit topoisomerase, such as acacetin, apigenin,
kaempferol and morin (PMID: 8567688)46-48, Luteolin (PMID:
12027807; PMID: 16950806; PMID: 15312049)46; Myricetin (PMID:
20025993)49 and any combinations thereof. In certain embodiments,
the topoisomerase inhibitor can be an interfering RNA (RNAi)
molecule that targets topoisomerase I, topoisomerase II or both.
Nonlimiting examples of RNAi molecules include small interfering
RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense
nucleic acid molecules, and the like as are well known in the art.
Nonlimiting examples of siRNAs and shRNAs of this invention are
provided in Table 2. In some embodiments, a zinc finger nuclease,
an antibody and/or a ribozyme can be employed to inhibit
topoisomerase activity in the methods of this invention.
[0218] As used herein, the term "cytotoxic antibiotics" cytotoxic
antibiotics include, but are not limited to, antinomycin,
bleomycin, mitomycin, plicamycin and the like. Examples of tyrosine
kinase inhibitors include, but are not limited to, nilotinib,
imatinib, gefitinib, erlotinib, cetuximab, panitumumab,
zalutumumab, nimotuzumab, matuzuman and the like.
[0219] In some embodiments, the other anti-cancer agents are
monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab,
gemtuzumab, rituximab, and trastuzumab; photosensitizers, such as
aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and
verteporfin; and other agents, such as alitretinoin, altretamine,
amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene,
bortezomib, celecoxib, denileukin diftitox, erlotinib,
estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin,
masoprocol, mitotane, pegaspargase, and tretinoin.
[0220] In some embodiments, the other anti-cancer agents are those
that can be used to treat leukemia, such as drugs for Acute
Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic
Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), and
Meningeal Leukemia, include but are not limited to, Abitrexate
(Methotrexate), Adriamycin PFS (Doxorubicin Hydrochloride),
Adriamycin RDF (Doxorubicin Hydrochloride), Arranon (Nelarabine),
Asparaginase Erwinia chrysanthemi, Cerubidine (Daunorubicin
Hydrochloride), Clafen (Cyclophosphamide), Clofarabine, Clofarex
(Clofarabine), Clolar (Clofarabine), Cyclophosphamide, Cytarabine,
Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dasatinib,
Daunorubicin Hydrochloride, Doxorubicin Hydrochloride, Erwinaze
(Asparaginase Erwinia Chrysanthemi), Folex (Methotrexate), Folex
PFS (Methotrexate), Gleevec (Imatinib Mesylate), Iclusig (Ponatinib
Hydrochloride), Imatinib Mesylate, Marqibo (Vincristine Sulfate
Liposome), Mercaptopurine, Methotrexate, Methotrexate LPF
(Methorexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate),
Nelarabine, Neosar (Cyclophosphamide), Oncaspar (Pegaspargase),
Pegaspargase, Purinethol (Mercaptopurine), Rubidomycin
(Daunorubicin Hydrochloride), Sprycel (Dasatinib), Tarabine PFS
(Cytarabine), Vincasar PFS, incristine Sulfate), Vincristine
Sulfate, Vincristine Sulfate Liposome, Hyper-CVAD, Adriamycin PFS
(Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin
Hydrochloride), Arsenic Trioxide, Cerubidine (Daunorubicin
Hydrochloride), Clafen (Cyclophosphamide), Cyclophosphamide,
Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide),
Daunorubicin Hydrochloride, Doxorubicin Hydrochloride, Neosar
(Cyclophosphamide), Rubidomycin (Daunorubicin Hydrochloride),
Tarabine PFS (Cytarabine), Trisenox (Arsenic Trioxide), Vincasar
PFS (Vincristine Sulfate), Vincristine Sulfate, ADE, Alemtuzumab,
Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Arzerra
(Ofatumumab), Bendamustine, Hydrochloride, Campath (Alemtuzumab),
Chlorambucil, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan
(Cyclophosphamide), Fludara (Fludarabine Phosphate), Fludarabine
Phosphate, Gazyva (Obinutuzumab), Ibrutinib, Imbruvica (Ibrutinib),
Leukeran (Chlorambucil), Linfolizin (Chlorambucil), Neosar
(Cyclophosphamide), Obinutuzumab, Ofatumumab, Treanda (Bendamustine
Hydrochloride), CHLORAMBUCIL-PREDNISONE, CVP, Bosulif (Bosutinib),
Bosutinib, Busulfan, Busulfex (Busulfan), Clafen
(Cyclophosphamide), Cyclophosphamide, Cytarabine, Cytosar-U
(Cytarabine), Cytoxan (Cyclophosphamide), Dasatinib, Gleevec
(Imatinib Mesylate), Iclusig (Ponatinib Hydrochloride), Imatinib
Mesylate, Myleran (Busulfan), Neosar (Cyclophosphamide), Nilotinib,
Omacetaxine Mepesuccinate, Ponatinib Hydrochloride, Sprycel
(Dasatinib), Synribo (Omacetaxine Mepesuccinate), Tarabine PFS
(Cytarabine), Tasigna (Nilotinib), Cytarabine, Cytosar-U
(Cytarabine), and Tarabine PFS (Cytarabine)
[0221] In some embodiments, additional anti-cancer agents that can
be used together with the compounds of the present invention
include agents that can inhibit EGFR (epidermal growth factor
receptor) responses, such as EGFR antibodies, EGF antibodies, and
molecules that are EGFR inhibitors; VEGF (vascular endothelial
growth factor) inhibitors; and erbB2 receptor inhibitors, such as
organic molecules or antibodies that bind to the erbB2 receptor.
EGFR inhibitors are described in, for example in WO 95/19970, WO
98/14451, WO 98/02434 and U.S. Pat. No. 5,747,498. EGFR-inhibiting
agents include, but are not limited to, the monoclonal antibodies
C225 and anti-EGFR 22Mab (ImClone Systems Incorporated of New York,
N.Y., USA), the compounds ZD-1839 (AstraZeneca), BIBX-1382
(Boehringer Ingelheim), MDX-447 (Medarex Inc. of Annandale, N.J.,
USA), and OLX-103 (Merck & Co. of Whitehouse Station, N.J.,
USA), VRCTC-310 (Ventech Research) and EGF fusion toxin (Seragen
Inc. of Hopkinton, Mass.). VEGF inhibitors, for example AG-13736
(Pfizer, Inc.), can also be combined or co-administered with the
composition. VEGF inhibitors are described in, for example in WO
99/24440, WO 95/21613, WO 99/61422, U.S. Pat. No. 5,834,504, WO
98/50356 (published Nov. 12, 1998), U.S. Pat. No. 5,883,113, U.S.
Pat. No. 5,886,020, U.S. Pat. No. 5,792,783, U.S. Pat. No.
6,534,524, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, and
WO 98/02437, all of which are herein incorporated by reference in
their entirety. Other examples of some specific VEGF inhibitors are
IM862 (Cytran Inc. of Kirkland, Wash., USA); Avastin.TM. or
bevacizumab, an anti-VEGF monoclonal antibody (Genentech, Inc. of
South San Francisco, Calif.); and angiozyme, a synthetic ribozyme
from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.).
ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome plc),
and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.
of The Woodlands, Tex., USA) and 2B-1 (Chiron), may be administered
in combination with the composition. Such erbB2 inhibitors include
those described in WO 98/02434, WO 99/35146, WO 99/35132, WO
98/02437, WO 97/13760, WO 95/19970, U.S. Pat. No. 5,587,458, and
U.S. Pat. No. 5,877,305, each of which is herein incorporated by
reference in its entirety.
[0222] Without wishing to be bound by any particular theory, the
anti-cancer agents of the present invention can act through one or
more mechanisms. Such mechanisms include, but are not limited to:
(1) inhibition of CDX2 activity; (2) induction of KLF4 activity;
(3) induction of p21 CDK inhibitor; (4) induction of G1/S Cell
cycle arrest; (5) induction of Caspase 3 enzyme; and (6) induction
of apoptosis.
[0223] The present invention provides methods of using the
biomarkers and active agents of the present invention.
[0224] In some embodiments, provided are methods for determining
the responsiveness of a human subject to a cancer treatment. The
cancer can be Acute Myeloid leukemia (AML). The cancer also can be
acute lymphocytic leukemia (ALL), chronic myelogenous leukemia
(CML), adult T-cell leukaemia (ATLL), lymphoma, gastric cancer,
multiple myeloma, myelodysplastic syndromes, or combinations
thereof. In some embodiments, the treatment involves using an
active agent that can modulate one or more components in the
CDX2-KLF4 signaling pathway, including but not limited to, CDX2,
KLF4, Caspase-3, Annexin V, p53, KDM5B, miR-2909, BAX, BCL2, BCL3,
BMP, Wnt, HNF4.alpha., Fgf, Hox, SP1, MYC, CCND1, AATF, and p21. In
some embodiments, the treatment involves using an active agent that
can induce G1/S cell cycle arrest. In some embodiments, the
treatment involves using an active agent that can induce Capase 3
enzyme. In some embodiments, the treatment involves using an active
agent that can induce apoptosis. In some embodiments, the active
agent is selected from 2,4,5-trisubstituted imidazole compounds
described in US 2007/0123553A1, or 2-indolyl
imidazo[4,5-d]phenanthroline compounds described in U.S. Pat. No.
8,148,392, or functional derivatives thereof. In some embodiments,
the active agent comprises the compound of formula I. (i.e.,
LOR-253).
[0225] In some embodiments, the human subject is determined to be
responsive to the treatment if meeting one or more selection
criteria described below: [0226] The human subject has a genetic
alteration of CDX2 locus when compared to that of a human subject
or the loci of a group of human subjects who are responsive to a
treatment of the present invention. Such alterations can be
epigenetic and/or genetic modifications, including but not limited
to those described in Costa et al. (Oncogene, 18(35):5010-5014,
1999), Abdel-Rahman et al. (Oncogene, 24:1542-1551, 2005), Yagi et
al. (Br. J. Cancer., 79(3-4): 440-444, 1999), Chawengsaksophak et
al. (Nature, 386(6620):84-7, 1997), Wood et al. (Science, 2007;
318(5853):1108-1113), Wicking et al. (Oncogene, 1998;
17(5):657-659), and da Costa L T, et al. (Oncogene, 1999;
18(35):5010-5014), each of which is incorporated by reference in
its entirety for all purposes; [0227] The human subject has a
higher level of CDX2 activity than normal level, or a level similar
to the level of human subjects who are responsive to a treatment of
the present invention; [0228] The human subject has a lower level
of KLF4 activity than normal level, or a level similar to the level
of human subjects who are responsive to a treatment of the present
invention; [0229] The human subject has a lower level of p21
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention; [0230] The human subject has a higher level of p53
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention, wherein said human has a cancer type in which KLF4
negatively regulates p53; [0231] The human subject has a lower
level of p53 activity than normal level, or a level similar to the
level of human subjects who are responsive to a treatment of the
present invention, wherein said human has a cancer type in which
KLF4 positively regulates p53; [0232] The human subject has a
higher level of BCL3 activity than normal level, or a level similar
to the level of human subjects who are responsive to a treatment of
the present invention; [0233] The human subject has a higher level
of SP1 activity than normal level, or a level similar to the level
of human subjects who are responsive to a treatment of the present
invention; [0234] The human subject has a higher level of MYC
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention; [0235] The human subject has a higher level of CCND1
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention; [0236] The human subject has a higher level of AATF
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention; [0237] The human subject has a lower level of Caspase 3
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention; [0238] The human subject has a lower level of Annexin V
activity than normal level, or a level similar to the level of
human subjects who are responsive to a treatment of the present
invention; [0239] The human subject has a higher ratio of CDX2/B
activity (wherein B is KLF4, p21, Caspase 3, or Annexin V; or
wherein B is p53 if the human subject has a cancer type in which
KLF4 positively regulates p53) than normal level, or a level
similar to the level of human subjects who are responsive to a
treatment of the present invention; [0240] The human subject has a
lower ratio of B/CDX2 activity (wherein B is KLF4, p21, Caspase 3,
or Annexin V; or wherein B is p53 if the human subject has a cancer
type in which KLF4 positively regulates p53) than normal level, or
a level similar to the level of human subjects who are responsive
to a treatment of the present invention; [0241] The human subject
has a lower ratio of CDX2/C activity (wherein C is BCL3, SP1, MYC,
CCND1, or AATF; or wherein C is p53 if the human subject has a
cancer type in which KLF4 negatively regulates p53) than normal
level, or a level similar to the level of human subjects who are
responsive to a treatment of the present invention; [0242] The
human subject has a higher ratio of C/CDX2 activity (wherein C is
BCL3, SP1, MYC, CCND1, or AATF; or wherein C is p53 if the human
subject has a cancer type in which KLF4 negatively regulates p53)
than normal level, or a level similar to the level of human
subjects who are responsive to a treatment of the present
invention. Otherwise, the human subject is determined to be not
responsive to the treatment.
[0243] In some embodiments, the normal level is the activity of a
biomarker in healthy human subjects, or human subjects without or
with only minor detectable symptoms of disorders.
[0244] An activity level is similar to a predetermined standard
activity level (e.g., activity level of human subjects who are
responsive to a treatment of the present invention) if the
difference between the tested activity level and the predetermined
standard activity level is statistically insignificant, or the
difference is least than about 20%, about 19%, about 18%, about
17%, about 16%, about 15%, about 14%, about 13%, about 12%, about
11%, about 10%, about 9%, about 8%, about 7%, about 6% about 5%,
about 4%, about 3%, about 2%, about 1%, or less of the
predetermined standard activity level.
[0245] Also provided are methods for monitoring the efficacy of an
active agent in the treatment of cancer. These methods include
determining the activity of one or more biomarkers of the present
invention in a biological sample from a patient and providing that
information regarding the biomarkers to an entity that provides a
determination or evaluation of the treatment or efficacy based on
biomarker information. In some embodiments, the biomarker activity
is determined during or after taking at least one dosage of the
active agent of the present invention. In some embodiments, the
entity can provide a determination that treatment with the active
agent should be used or should be continued, if the human subject
meets one or more selection criteria described below: [0246] The
human subject has a genetic alteration of CDX2 locus when compared
to that of a human subject or the loci of a group of human subjects
who are responsive to a treatment of the present invention; [0247]
The human subject has an decreased level of CDX2 activity when
compared to that of the same human subject before the treatment,
and/or has a normalization or stabilization of the CDX2 activity
when compared that of a human subject or a group of human subjects
who are responsive to a treatment of the present invention; [0248]
The human subject has an increased level of KLF4 activity when
compared to that of the same human subject before the treatment,
and/or has a normalization or stabilization of the KLF4 activity
when compared that of a human subject or a group of human subjects
who are responsive to a treatment of the present invention; [0249]
The human subject has an increased level of p21 activity when
compared to that of the same human subject before the treatment,
and/or has a normalization or stabilization of the p21 activity
when compared that of a human subject or a group of human subjects
who are responsive to a treatment of the present invention; [0250]
The human subject has a decreased level of p53 activity when
compared to that of the same human subject before the treatment,
and/or has a normalization or stabilization of the p53 activity
when compared that of a human subject or a group of human subjects
who are responsive to a treatment of the present invention, wherein
said human has a cancer type in which KLF4 negatively regulates
p53; [0251] The human subject has an increased level of p53
activity when compared to that of the same human subject before the
treatment, and/or has a normalization or stabilization of the p53
activity when compared that of a human subject or a group of human
subjects who are responsive to a treatment of the present
invention, wherein said human has a cancer type in which KLF4
positively regulates p53; [0252] The human subject has a decreased
level of BCL3 activity when compared to that of the same human
subject before the treatment, and/or has a normalization or
stabilization of the BCL3 activity when compared that of a human
subject or a group of human subjects who are responsive to a
treatment of the present invention; [0253] The human subject has a
decreased level of SP1 activity when compared to that of the same
human subject before the treatment, and/or has a normalization or
stabilization of the SP1 activity when compared that of a human
subject or a group of human subjects who are responsive to a
treatment of the present invention; [0254] The human subject has a
decreased level of MYC activity when compared to that of the same
human subject before the treatment, and/or has a normalization or
stabilization of the MYC activity when compared that of a human
subject or a group of human subjects who are responsive to a
treatment of the present invention; [0255] The human subject has a
decreased level of CCND1 activity when compared to that of the same
human subject before the treatment, and/or has a normalization or
stabilization of the CCND1 activity when compared that of a human
subject or a group of human subjects who are responsive to a
treatment of the present invention; [0256] The human subject has a
decreased level of AATF activity when compared to that of the same
human subject before the treatment, and/or has a normalization or
stabilization of the AATF activity when compared that of a human
subject or a group of human subjects who are responsive to a
treatment of the present invention; [0257] The human subject has an
increased level of Caspase 3 activity when compared to that of the
same human subject before the treatment, and/or has a normalization
or stabilization of the Caspase 3 activity when compared that of a
human subject or a group of human subjects who are responsive to a
treatment of the present invention; [0258] The human subject has an
increased level of Annexin V activity when compared to that of the
same human subject before the treatment, and/or has a normalization
or stabilization of the Annexin V activity when compared that of a
human subject or a group of human subjects who are responsive to a
treatment of the present invention; [0259] The human subject has a
decreased ratio of CDX2/B activity (wherein B is KLF4, p21, Caspase
3, or Annexin V; or wherein B is p53 if the human subject has a
cancer type in which KLF4 positively regulates p53) when compared
to that of a human subject or a group of human subjects who are
responsive to a treatment of the present invention; [0260] The
human subject has an increased ratio of B/CDX2 activity (wherein B
is KLF4, p21, Caspase 3, or Annexin V; or wherein B is p53 if the
human subject has a cancer type in which KLF4 positively regulates
p53) when compared to that of a human subject or a group of human
subjects who are responsive to a treatment of the present
invention; [0261] The human subject has an increased ratio of
CDX2/C activity (wherein C is BCL3, SP1, MYC, CCND1, or AATF; or
wherein C is p53 if the human subject has a cancer type in which
KLF4 negatively regulates p53) when compared to that of a human
subject or a group of human subjects who are responsive to a
treatment of the present invention; [0262] The human subject has a
decreased ratio of C/CDX2 activity (wherein C is BCL3, SP1, MYC,
CCND1, or AATF; or wherein C is p53 if the human subject has a
cancer type in which KLF4 negatively regulates p53) when compared
to that of a human subject or a group of human subjects who are
responsive to a treatment of the present invention. Otherwise, the
entity can provide a determination that treatment with the active
agent should not be used or should be continued.
[0263] In some embodiments, the normal level of CDX2/KLF4 or
KLF4/CDX2 activity ratio is used to determine if a human subject is
responsive to the treatment. In some embodiments, the normal level
of CDX2/KLF4 activity ratio, or CDX2/KLF4 activity ratio in human
subjects responsive to the treatment is about 0.01, about 0.02,
about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about
0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about
0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about
2.0, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about
8.0, about 9.0, about 10.0, about 20.0, about 30.0, about 40.0,
about 50.0, about 60.0, about 70.0, about 80.0, about 90.0, about
100, about 200, about 300, about 400, about 500, about 600, about
700, about 800, about 900, about 1000, or more. In some
embodiments, the normal level of KLF4/CDX2 activity ratio, or
KLF4/CDX2 activity ratio in human subjects responsive to the
treatment is about 100, about 90, about 80, about 70, about 60,
about 50, about 40, about 30, about 20, about 10, about 9, about 8,
about 7, about 6, about 5, about 4, about 3, about 2, about 1,
about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4,
about 0.3, about 0.2, about 0.1, about 0.09, about 0.08, about
0.07, about 0.06, about 0.05, about 0.04, about 0.03, about 0.02,
about 0.01, or less.
[0264] Also provided are methods for treating a human subject. In
some embodiments, the treatment is a cancer treatment. In some
embodiments, the cancer can be Acute Myeloid leukemia (AML),
leukemia, ALL, pediatric T-cell ALL or pediatric B-cell ALL, CML,
ATLL, lymphoma, Hodgkin lymphoma, non-Hodgkin's lymphoma (NHL),
Burkitts lymphoma, or B-cell lymphoma, gastric cancer, multiple
myeloma, myelodysplastic syndromes, or combinations thereof. In
some embodiments, the methods comprise modulating the activity of
at least one component in the CDX2-KLF4 signaling pathway. In some
embodiments, the methods comprise administering an active agent to
the human subject. In some embodiments, the active agent can
modulate the activity of CDX2, KLF4, Caspase 3, Annexin V, p53,
BCL3, SP1, MYC, CCND1, AATF, and/or p21. In some embodiments, the
methods comprise administering an active agent to induce G1/S Cell
cycle arrest; In some embodiments, the methods comprise
administering an active agent to induce Caspase 3 enzyme. In some
embodiments, the methods comprise administering an active agent to
induce apoptosis. In some embodiments, the active agent is selected
from 2,4,5-trisubstituted imidazole compounds described in US
2007/0123553A1, or 2-indolyl imidazo[4,5-d]phenanthroline compounds
described in U.S. Pat. No. 8,148,392, or functional derivatives
thereof. In some embodiments, the active agent comprises the
compound of formula I. (i.e., LOR-253/APTO-253).
[0265] In some embodiments, the methods comprise administering
therapeutically effective amount of an active agent of the present
invention to a human subject. In some embodiments, the methods
further comprise determining the activity of at least one biomarker
described herein in a sample obtained from the human subject. In
some embodiments, the determination can be made before, during,
after the treatment. In some embodiments, the determination can be
made after the human subject takes at least one, two, three, four,
five, six, seven, eight, nine, ten, ore more dosages of the active
agent.
[0266] Further provided are methods for providing useful
information for predicting, determining, evaluating or monitoring
the treatment or efficacy of treatment of cancer with the active
agent of the present invention. These methods include determining
activity level of one or more biomarkers described herein in a
biological sample from a patient and providing that information
regarding the biomarkers to an entity that provides a determination
or evaluation of the treatment or efficacy based on the biomarker
information. If the activity of one or more biomarkers fit the
selection criteria described herein, the entity can provide a
determination that treatment with an active agent treatment of the
present invention should be used or should be continued. If the
activity of one or more biomarkers does not fit the selection
criteria described herein, the entity can provide a determination
that treatment with an active agent treatment of the present
invention should not be used or should be discontinued.
[0267] The following examples illustrate various aspects of the
invention. The examples should, of course, be understood to be
merely illustrative of only certain embodiments of the invention
and not to constitute limitations upon the scope of the
invention.
EXAMPLES
Example 1
In Vitro Antiproliferative Activity of LOR-253 in Leukemia and
Lymphoma Cell Lines
[0268] Antiproliferative activity of LOR-253 was determined by XTT
assay as follows. Adherent cells (2.times.10.sup.3/well) in 100
.mu.L of growth medium were seeded in 96-well cell culture plates
and incubated overnight at 37.degree. C. The medium was removed and
replaced with a total volume of 100 .mu.L growth medium containing
LOR-253 (or 0.1% DMSO vehicle control), as described in the
respective experiments. Suspension cells were plated
(4-6.times.10.sup.3/well) in 50 .mu.L growth medium, and 50 .mu.L
growth medium containing LOR-253 (or 0.1% DMSO vehicle control) was
added to each well. After incubation of the cells at 37.degree. C.
for 5 days, cell viability was quantitated with the use of sodium
3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benze-
ne sulfonic acid hydrate (XTT) colorimetric assay (Roche). XTT
labeling reagent (1 mg/mL) was mixed with electron-coupling
reagent, following the manufacturer's instructions, and 50 .mu.L of
the mixture was added directly to the cells. The plates were
further incubated at 37.degree. C. for 4 h, and the absorbance of
each well was measured at 490 nm with a multiwell spectrophotometer
(Bio-Tek Instruments Inc.). The data were adjusted relative to the
blank and expressed as a percentage of cell growth compared with
the vehicle control. (Ref. Huesca et al., Mol Cancer Ther. 2009.
8:2586-96; Lorus publication on LOR-133)
[0269] The in vitro assay results (FIG. 3) indicate that
leukemia/lymphoma, including AML cell lines, such as HL-60, MV 411,
THP-1, HEL92.1.7, CCRF-CEM, MOLT-4, Jurkat, K-562, Ramos, and Raji,
are the cell lines most sensitive to LOR-253, while other cell
lines of lung cancer, bladder cancer, colon cancer, prostate
cancer, melanoma, and breast cancer, are less sensitive to LOR-253.
The variability in sensitivity to LOR-253 may be attributable to
the extent of innate KLF4 suppression. The IC.sub.50 values of
LOR-253 for each leukemia and lymphoma cell lines are shown
below.
TABLE-US-00006 Cancer Type Cell Line IC.sub.50 (.mu.M) Acute
myeloid leukemia Kasumi-1 0.0069 KG-1 0.033 HL-60 0.046 EOL-1 0.058
NB4 0.06 MV-4-11 0.069 OCI-AML2 0.076 THP-1 0.077 MOLM13 (CD47+)
0.13 NOMO-1 0.121 SKM-1 0.145 HEL92.1.7 0.305 Acute lymphoblastic
leukemia CCRF-CEM 0.039 MOLT-4 0.07 Jurkat 0.071 Chronic
Myelogenous Leukemia K562 0.25 Non-Hodgkin's Lymphoma Ramos 0.125
Raji 0.011 CA46 0.19 Toledo 0.077 DB 0.099 Mino 0.14 RL 0.19
Multiple myeloma MM.1R 0.15 U266B1 0.18 HuNS1 0.072
Example 2
KLF4/p21 Induction in AML Cell Lines In Vitro
[0270] To determine if KLF4 and/or p21 expressions are induced by
LOR-253, AML cells (THP1, HL-60) were treated with DMSO (vehicle
control) or 0.5 .mu.M LOR-253 for 16 hours. Total RNA was extracted
using the TRIzol Plus RNA Purification kit (Ambion, Life
Technologies), according to the manufacturer's instructions. First
strand cDNA was synthesized from 1-2 ug total RNA using random
hexamer primers (Invitrogen) and the SuperScript II Reverse
Transcriptase kit (Invitrogen). Quantitative RT-PCR was performed
in the ABI Prism 7000 Sequence Detection System using cDNAs and
human TaqMan Gene Expression Assay primer/probe sets for
Kruppel-like factor 4 (KLF4), cyclin-dependent kinase inhibitor 1A
(p21) and the ABI TaqMan Universal PCR master mix protocol. Gene
expression was normalized with .beta.-actin gene expression in the
same sample, and fold changes in KLF4 or p21 were expressed
relative to the corresponding expression level in the DMSO treated
samples using the comparative CT method. Treatment of THP1 and
HL-60 AML cell lines with LOR-253 results in increased expression
KLF4 and p21 (See FIG. 4).
Example 3
Treatment with LOR-253 Results in G1/S Cell Cycle Arrest in AML
Cell Lines
[0271] THP1 and HL-60 AML cell lines were treated with DMSO
(vehicle control) or 0.5 .mu.M LOR-253 for 16 hours. Cells were
washed once in PBS+1% FCS and fixed using ice cold 70% ethanol
overnight. Fixed cells were washed twice, resuspended in PI/RNaseA
solution, containing 20 .mu.g/mL propidium iodide and 250 .mu.g/mL
RNaseA, and stained for 30 minutes at 37.degree. C. Stained cells
were analyzed using a BD FACSCalibur flow cytometer. The results
indicate treatment with LOR-253 results in G1/S cell cycle arrest
in AML cell lines (FIG. 5).
Example 4
Treatment with LOR-253 Induces Apotosis in Cell Lines
[0272] THP-1 cells were treated with DMSO, 0.5 or 1 uM of LOR-253
for 16 hours. Cells were washed twice with cold PBS and resuspended
Annexin V binding buffer. 1.times.10.sup.5 cells in 100 .mu.L were
stained with FITC-Annexin V and propidium iodide, and incubated for
15 minutes at room temperature. After staining, 400 ul of binding
buffer was added and the cells were kept on ice until analyzed on a
BD FACSCanto flow cytometer. THP-1 cells treated with LOR-253
showed elevated Annexin V staining, indicating induction of
apoptosis. Increasing concentration of LOR-253 used to treat the
cells resulted in an increase in the early apoptotic population
(Q3: Annexin V+/PI-). The results indicate treatment with LOR-253
induces apotosis in AML cell lines (FIG. 6).
[0273] THP1 and HL-60 cells were treated with DMSO (vehicle
control) or 0.5 .mu.M LOR-253 for 48 hours. Cell lysates were
collected using a 1% Triton-X100 lysis. Caspase 3 activity was
measured using EnzChek Caspase-3 Assay Kit #1 (Life Technologies)
with 5 .mu.g of cell lysastes according to manufacturer's protocol.
Treatment of THP1 and HL-60 cells with LOR-253 results in elevated
Caspase 3 activity, indicating induction of apoptosis (FIG. 7).
[0274] THP1 cells were treated with DMSO (vehicle control) or 0.5
.mu.M LOR-253 for 48 hours. Total RNA was extracted using the
TRIzol Plus RNA Purification kit (Ambion, Life Technologies),
according to the manufacturer's instructions. First strand cDNA was
synthesized from 1-2 ug total RNA using random hexamer primers
(Invitrogen) and the SuperScript II Reverse Transcriptase kit
(Invitrogen). Quantitative RT-PCR was performed in the ABI Prism
7000 Sequence Detection System using cDNAs and human TaqMan Gene
Expression Assay primer/probe sets for BCL2-associated X protein
(BAX), B-cell CLL/Lymphoma 2 (BCL2) and the ABI TaqMan Universal
PCR master mix protocol. Gene expression was normalized with
.beta.-actin gene expression in the same sample, and fold changes
in BAX or BCL2 were expressed relative to the corresponding
expression level in the DMSO treated samples using the comparative
CT method. Elevation of BAX and repression of BCL2 upon treatment
of THP1 cells with LOR-253 indicates induction of apoptosis (FIG.
8).
Example 5
In Vivo Efficacy of LOR-253 HCL in H226 Xenograft Model--Dose and
Schedule
[0275] H226 model mice were treated according to several
administration schedules as shown in the table below.
TABLE-US-00007 Administration Treatment Group Dose level Schedule
(days)/cycle (1 cycle = 28 days) 1 n = 8 10 mg/kg-iv 2T-12B-2T 2
cycles 2 n = 8 10 mg/kg-iv 3T-12B-3T 2 cycles 3 n = 8 10 mg/kg-iv
2T-5B-2T 2 cycles 4 n = 8 10 mg/kg-iv 3T-5B-3T 2 cycles 5 n = 8
Vehicle alone 3T-5B-3T 2 cycles T = consecutive days treatment B =
break without treatment
[0276] The results show that groups 2, 3, and 4 are effectively
treated by LOR-253 (FIG. 9). The results indicates that schedule
with one week gap is superior to two-week gap, and suggests weekly
administration schedule is preferred.
Example 6
In Vivo Dose-Dependent Pharmacokinetic (PK) and Pharmacodynamic
(PD) Responses in Xenograft CD-1 Nude Mice Treated by LOR-253
HCL
[0277] To study the pharmacokinetic (PK) in tumor cells treated by
LOR-253, CD-1 nude mice were treated by i.v. bolus injections of
LOR-253HCl at 1, 5, and 15 mg/kg. The serum level of LOR-253 was
measured. The result indicates that the serum level of LOR-253 has
a dose related increase (FIG. 10).
[0278] To study the pharmacodynamic (PD) responses, mice were
treated for 5 consecutive days with 1, 5, and 15 mg/kg LOR-253.
Tumors were measured 16 hours after the last dose, and the KLF-4
protein level was measured. Average KLF4 protein levels increased
in a dose-dependent manner, correlating with tumor biodistribution
and dose-response antitumor activity (FIG. 11).
Example 7
LOR-253 for Treating AML, Phase I Clinical Trial
[0279] Preclinical testing established a broad therapeutic index
for LOR-253: efficacy of LOR-253 was demonstrated in xenograft
models against multiple tumor types; extensive GLP safety and PK
studies were performed in rats and dogs; no treatment-related
cardiovascular effects were observed in repeat-dose toxicity study
of dogs at highest dose tested (data not shown); no significant
inhibition of hERG tail current density or CYP450 enzymes; and GLP
blood compatibility studies confirmed the suitability of IV
formulation.
[0280] Phase I: This was an open-label, phase 1 study to determine
the maximum tolerated dose (MTD) or appropriate target dose if MTD
not reached to identify the recommended phase 2 dose of LOR-253HCl
in patients with advanced or metastatic solid tumours. In the first
set of doses, LOR-253HCl was given in ascending doses until the
maximum administered dose or appropriate target dose is reached. In
the other set of doses, LOR-253HCl was given in ascending doses
starting from 20 mg/m.sup.2 until the maximum administered dose or
appropriate target dose is reached. Patient as treated on
LOR-253HCl for 2 cycles for the evaluation. [0281] Other Name: No
other names are used. [0282] Study Type: Interventional [0283]
Study Design: Endpoint Classification: Safety Study [0284]
Intervention Model: Single Group Assignment [0285] Masking: Open
Label [0286] Primary Purpose: Treatment [0287] Official Title:
Open-Label, Phase 1 Study of LOR-253HCl in Patients With Advanced
or Metastatic Solid Tumours
Primary Outcome Measures:
[0287] [0288] To determine the maximum tolerated dose (MTD) or
appropriate target dose if MTD not reached to identify the
recommended phase 2 dose of LOR-253HCl in patients with advanced or
metastatic solid tumours. [Time Frame: 8 weeks]
Secondary Outcome Measures:
[0288] [0289] To characterize the safety profile of LOR-253HCl when
administered to patients with advanced or metastatic solid tumours.
[Time Frame: 8 weeks]
Inclusion Criteria:
[0289] [0290] 1. Male or female 18 years of age or older. [0291] 2.
Histologically confirmed diagnosis of solid tumor for which no
effective therapy is available or that is unresponsive to
conventional therapy. [0292] 3. Meet laboratory parameter
requirements at study entry.
Exclusion Criteria:
[0292] [0293] 1. Chemotherapy, radiotherapy, biologic therapy,
immunotherapy or any other investigational drugs within 21 days of
beginning study treatment with LOR-253HCl. [0294] 2. A hematologic
malignancy. [0295] 3. A history of brain or other central nervous
system metastases. [0296] 4. Have a presence of a significant
infection. [0297] 5. Clinically significant autoimmune disease.
[0298] 6. Uncontrolled intercurrent illness. [0299] 7. With iron or
copper overload syndromes. [0300] 8. Pregnancy or breast
feeding.
[0301] Safety and Antitumor Activity Demonstrated in Phase I Trial:
[0302] Dose-Escalating Study in Patients with Advanced Solid Tumors
[0303] Excellent Safety Profile (N=27 Patients) across 7 dose
levels [0304] Most common AE and SAE: Fatigue and 1 case of
reversible hypophosphatemia [0305] Stable Disease achieved in 41%
of evaluable patients; No RECIST PR [0306] Tumor shrinkage (FIG.
12) in patient with NSCLC (poorly differentiated adenocarcinoma)
and extensive metastases refractory to prior standard and
investigational therapies
[0307] The phase I trial results demonstrated that LOR-253 is a
safe and active drug for treating solid tumors.
Example 8
Absolute Quantification of CDX2 and KLF4
[0308] Absolute quantitation was performed using serially diluted
standards of known concentrations of CDX2 and KLF4 genes to
generate a standard curve. Plasmids containing CDX2 or KLF4 were
prepared at 3.times.10.sup.2-3.times.10.sup.6 copies (FIG. 13).
Total RNA was extracted from cells using the TRIzol Plus RNA
Purification kit (Ambion, Life Technologies), according to the
manufacturer's instructions. First strand cDNA was synthesized from
1-2 ug total RNA using random hexamer primers (Invitrogen) and the
SuperScript II Reverse Transcriptase kit (Invitrogen). Quantitative
RT-PCR was performed in the ABI Prism 7000 Sequence Detection
System using cDNAs and human TaqMan Gene Expression Assay
primer/probe sets for Kruppel-like factor 4 (KLF4), caudal type
homeobox 2 (CDX2), and the ABI TaqMan Universal PCR master mix
protocol. KLF4 and CDX2 expression levels in MM.1R, Eol-1, Mino,
HEL92.1.7, CA46, RL, THP1, and Toledo cell lines were determined as
copy numbers using the standard curves (FIG. 14 and FIG. 15).
Ratios of CDX2/KLF4 and KLF4/CDX2 copy numbers were calculated and
plotted on a logarithmic scale against IC.sub.50 values. The
results indicate that CDX2/KLF4 ratios inversely correlate with
IC.sub.50 values in AML cell lines i.e. cells with higher CDX2/KLF4
ratios have lower IC.sub.50 values. Conversely, KLF4/CDX2 ratios
directly correlate with IC50 i.e. cells with low KLF4/CDX2 ratios
have low IC.sub.50 values. See table below and FIG. 16 and FIG.
17.
TABLE-US-00008 CDX2 and KLF4 expression levels and ratios in heme
cancers Cell line CDX2 KLF4 CDX2/KLF4 KLF4/CDX2 MM.1R 3.99 537.74
0.0074 134.77 EOL-1 21729.06 2104.58 10.32 0.0968 Mino 1.84 11.12
0.165 6.04 THP-1 216.68 8730.42 0.025 40.3 Toledo 10.01 8.87 1.13
0.886 RL 11.56 UD -- -- CA46 33.18 UD -- -- HEL92.1.7 4.56 UD -- --
UD, undetectable
[0309] Additional experiments of absolute quantitation were
performed using the same method on more cell lines including MM.1R,
Eol-1, Mino, HEL92.1.7, CA46, RL, THP1, Toledo, U266B1, MV411, DB,
HL60, CD47 LOW (or MOLM13 CD47-), CD47 HIGH (or MOLM13 CD47+),
KG-1, and SKM-1 cell lines. The results are shown in FIG. 27 and
FIG. 28, which indicate the same findings as described above.
Example 9
In Vivo Efficacy of LOR-253 HCL in a Kasumi-1 AML Xenograft
Model
[0310] The anti-tumor activity of LOR-253HCl was evaluated in
another in vivo animal model of human AML. The human AML cell line
Kasumi-1 was implanted subcutaneously into athymic nude mice. Some
tumor-bearing mice were treated with LOR-253HCl at 30 mg/kg (15
mg/kg twice per day) for two consecutive days per week for four
weeks (the "LOR-253 treatment group"). LOR-253 was administered by
intravenous (i.v.) bolus injection as the hydrochloric salt form
(LOR-253-HCl) formulated in 20% Polyethylene Glycol 400, 10%
Propylene Glycol, and 10% Solutol HS in 60% water. Other
tumor-bearing mice were treated with a formulation that did not
have LOR-253 (the "vehicle control group").
[0311] The major endpoint was to observe for tumor growth
inhibition after treatment with LOR-253HCl and compare the
anti-tumor effects of different dosing schedules. Tumor sizes were
measured three times per week from day 10 after the tumor cell
inoculation. Tumors were measured in three dimensions using
calipers and the volume was expressed in millimeters cubed using
the formula: V=0.5 a.times.b.times.c, where a, b, and c are the
length, width, and height of the tumor, respectively. Mean tumor
volumes+/-standard error (SE) were calculated from each measurement
and then plotted in a standard graph to compare the anti-tumor
efficacy of drug treatment to that of control (FIG. 20). Toxicity
was assessed by clinical observations and by measurement of mouse
body weight in grams twice per week over the course of the study.
Mean body weights were calculated from each measurement and then
plotted to compare body weight changes in the drug treatment group
to that of control (FIG. 21).
[0312] The tumor inhibition results as shown in FIG. 20 demonstrate
that the LOR-253 treatment group produced a statistically
significantly increased inhibitory effect in this model compared to
the vehicle control group (p=0.028 by Student's t-test). In
addition, the toxicity results as shown in FIG. 21 demonstrate that
the mice in the treatment group did not show body weight loss. The
mice in the treatment group also did not show other overt signs of
toxicity. These results indicate that this treatment schedule was
well tolerated. LOR-253HCl showed significant tumor growth
inhibition as a single agent at twice per week dosing without
obvious signs of toxicity, suggesting that LOR-253HCl has a
sufficient therapeutic window and that this agent is a potential
chemotherapeutic agent for the treatment of AML.
Example 10
In Vivo Efficacy of LOR-253 HCL in an HL-60 AML Xenograft Model
[0313] The anti-tumor activity of LOR-253HCl, as a single agent and
in combination with azacitidine, was evaluated in another in vivo
animal model of human AML. The human AML cell line HL-60 was
implanted subcutaneously into athymic nude mice. Tumor-bearing mice
were treated with LOR-253HCl alone or in combination with
azacitidine, with azacitidine alone, or with a negative control
vehicle. The detailed treatment conditions are as follows. [0314]
Group 1: Negative control group. LOR-253HCl control vehicle,
2.times./day, 3 cycles of 2 consecutive days, 5 days between
cycles, i.v., plus 1% D-mannitol every four days by s.c. injection
[0315] Group 2: Azacitidine (in 1% D-mannitol) at 10 mg/kg 1.times.
on days 1, 4, 8, 11, 15 and 18 by subcutaneous (s.c.) injection
[0316] Group 3: LOR-253HCl at 15 mg/kg bid for 3 cycles, each cycle
is 2 consecutive days of dosing per week with 5 days of non-dosing,
i.v., n=9 (2T-5B) [0317] Group 4: LOR-253HCl at 15 mg/kg
2.times./day (bid) for 3 cycles, each cycle is 1 day of dosing per
week with 6 days of non-dosing, i.v., n=9 (1T-6B) [0318] Group 5:
Combination of LOR-253 (2T-5B) and azacitidine. LOR-253HCl at 15
mg/kg bid for 3 cycles, each cycle is 2 consecutive days of dosing
per week with 5 days of non-dosing, i.v., n=9 (2T-5B) plus
azacitidine at 10 mg/kg 1.times. every four days by s.c. injection
[0319] Group 6: Combination of LOR-253 (1T-6B) and azacitidine.
LOR-253HCl at 15 mg/kg bid for 3 cycles, each cycle is 1 day of
dosing per week with 6 days of non-dosing, i.v., n=9 (1T-6B) plus
azacitidine at 10 mg/kg 1.times. every four days by s.c.
injection
[0320] The major endpoint was to observe for tumor growth
inhibition after treatment with LOR-253HCl in combination with
azacitidine. Tumor sizes were measured three times per week. Tumors
were measured in three dimensions using calipers and the volume was
expressed in millimeters cubed using the formula: V=0.5
a.times.b.times.c, where a, b, and c are the length, width, and
height of the tumor, respectively. Mean tumor volumes+/-standard
error (SE) were calculated from each measurement and then plotted
in a standard graph to compare the anti-tumor efficacy of drug
treatment to that of control (FIG. 22).
[0321] The tumor inhibition results as shown in FIG. 22 demonstrate
that LOR-253HCl administered alone at 15 mg/kg twice per day for
either one (Group 4) or two (Group 3) consecutive days per week
inhibited growth of HL-60 tumors to approximately the same extent
as or slightly more than azacitidine alone. Both once and twice
weekly dosing of LOR-253HCl in combination with azacitadine (Group
6 and Group 5, respectively) resulted in significantly higher
levels of tumor growth inhibition compared to either single agent
alone (p=0.0002 and p=0.0006 for 1.times. and 2.times.LOR-253HCl
treatment, respectively; compared to control, as determined by
Student's t-test). FIG. 23 and FIG. 24 show the tumor size data
from individual animals at the beginning (Day 1) and end (Day 19)
of this study.
[0322] Because LOR-253HCl in combination with azacitadine resulted
in even higher levels of tumor growth inhibition than either single
agent alone, LOR-253HCl may also provide additive anticancer
efficacy to a standard of care chemotherapeutic for hematological
malignancies.
Example 11
In Vivo Efficacy of LOR-253 HCL in a KG-1 AML Xenograft Model
[0323] The anti-tumor activity of LOR-253HCl was evaluated in yet
another in vivo animal model of human AML. Xenograft model mice for
AML cell line KG-1 was generated with the same method as in
Examples 9 and 10, and were treated with LOR-253HCl or control
according to the following regime.
[0324] Control-IV-Day 1
[0325] LOR-253-15 mg/kg-iv, bid, 2T/wk-Day 1
[0326] Control-IV-Day 8
[0327] LOR-253-15 mg/kg-iv, bid, 2T/wk-Day 8
[0328] Control-IV-Day 13
[0329] LOR-253-15 mg/kg-iv, bid, 2T/wk-Day 13
[0330] Control-IV-Day 16
[0331] LOR-253-15 mg/kg-iv, bid, 2T/wk-Day 16
[0332] Control-IV-Day 20
[0333] LOR-253-15 mg/kg-iv, bid, 2T/wk-Day 20
[0334] Control-IV-Day 26
[0335] LOR-253-15 mg/kg-iv, bid, 2T/wk-Day 26
[0336] Tumor sizes were measure as described in Examples 9 and 10,
and the results are shown in FIG. 25. LOR-253HCl showed significant
tumor growth inhibition as a single agent in this AML animal model
as well.
Example 12
In Vivo Efficacy of LOR-253 HCL in a THP-1 AML Xenograft Model
[0337] The anti-tumor activity of LOR-253HCl, as a single agent and
in combination with azacitidine, was evaluated in yet another in
vivo animal model of human AML. Xenograft model mice for AML cell
line THP-1 was generated with the same method as in Examples 9 and
10, and were treated with LOR-253HCl alone or in combination with
azacitidine, with azacitidine alone, or with a negative control
vehicle, and tumor sizes were measured as described in Examples 9
and 10. The detailed treatment conditions are shown as follows, and
the results are shown in FIG. 26. [0338] Group 1 Control: received
i.v. treatments with LOR-253 control vehicle (CV) on Days 1, 2, 8,
9, 15 &16; received subcutaneous (SC) treatments with
Azacitidine control vehicle on Days 1, 4, 8, 11, 15, 18, 22, 25, 29
&32; and received intraperitoneal (IP) treatments with
LOR-253-CV on Days 22, 23, 24, 25, 29&30. [0339]
Group-2-LOR-253: received i.v. treatments on Days 1, 2, 8, 9, 15
&16; received IP treatments on Days 22, 23, 24, 25, 29&30
[0340] Group-3-Azacitidine: received SC treatments on Days 1, 4, 8,
11, 15, 18, 22, 25, 29 &32. [0341] Group-4-Combination:
received i.v. treatments with LOR-253 on Days 1, 2, 8, 9, 15
&16; received IP treatments with LOR-253 on Days 22, 23, 24
&25; and received SC-treatment with Azacitidine on Days 1, 4,
8, 11, 15, 18, 22 &25
[0342] The tumor inhibition results of this Example demonstrate
that LOR-253HCl administered alone inhibited growth of THP-1 tumors
to about the same extent as or slightly more than azacitidine
alone. When used in combination with azacitadine, LOR-253HCl again
resulted in significantly higher levels of tumor growth
inhibition.
[0343] The disclosures, including the claims, figures and/or
drawings, of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by
reference in their entireties.
[0344] Unless defined otherwise, all technical and scientific terms
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials, similar or equivalent to those described
herein, can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein. All publications, patents, and patent publications cited
are incorporated by reference herein in their entirety for all
purposes.
[0345] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
[0346] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
* * * * *