U.S. patent application number 15/395664 was filed with the patent office on 2017-11-23 for compositions and methods for treating cancers.
The applicant listed for this patent is Aptose Biosciences Inc.. Invention is credited to William G. RICE.
Application Number | 20170335400 15/395664 |
Document ID | / |
Family ID | 52777444 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170335400 |
Kind Code |
A1 |
RICE; William G. |
November 23, 2017 |
COMPOSITIONS AND METHODS FOR TREATING CANCERS
Abstract
The present invention relates to compositions and methods for
treating of cancer. In some embodiments, the invention relates to
the use of agents that can modulate a component in the CDX2-KLF4
signaling pathway to treat myelodysplastic syndromes (MDS), acute
myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), adult
T-cell leukaemia (ATLL), lymphoma, gastric cancer, multiple
myeloma, or combinations thereof, or a condition associated with
abnormal activity of the CDX2-KLF4 signaling pathway.
Inventors: |
RICE; William G.; (Del Mar,
CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Aptose Biosciences Inc. |
Toronto |
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CA |
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|
Family ID: |
52777444 |
Appl. No.: |
15/395664 |
Filed: |
December 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14506463 |
Oct 3, 2014 |
9567643 |
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15395664 |
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62037868 |
Aug 15, 2014 |
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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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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/06 20180101; A61P
35/02 20180101; C12Q 1/6886 20130101; A61P 35/00 20180101; A61K
45/06 20130101; C07D 471/14 20130101; A61K 31/706 20130101; A61P
43/00 20180101; A61K 49/0004 20130101; C12Q 2600/158 20130101; C12Q
2600/106 20130101; A61K 31/4745 20130101; A61K 31/4745 20130101;
A61K 2300/00 20130101; A61K 31/706 20130101; A61K 2300/00
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 45/06 20060101 A61K045/06; C07D 471/14 20060101
C07D471/14; A61K 31/4745 20060101 A61K031/4745; A61K 49/00 20060101
A61K049/00; A61K 31/706 20060101 A61K031/706 |
Claims
1. A method of treating myelodysplastic syndrome (MDS) in a human
subject, comprising administering to a human subject in need of
such treatment an effective amount of a compound or a
pharmacologically acceptable salt or solvate thereof, said compound
having formula I: ##STR00008## wherein R.sup.1 is C.sub.1-C.sub.4
alkyl; and R.sup.2 is halogen.
2. The method of claim 1, wherein the human subject has an
ineffective production of myeloid blood cells.
3. The method of claim 1, wherein the human subject has an
anemia.
4. The method of claim 1, wherein the human subject has low blood
counts caused by bone marrow failure.
5. The method of claim 1, wherein R.sup.1 is methyl, isopropyl, or
t-butyl.
6. The method of claim 5, wherein said compound has a formula
selected from the group consisting of ##STR00009##
7. The method of claim 5, wherein said compound has formula II:
##STR00010##
8. (canceled)
9. The method of claim 1, wherein said MDS is high-risk MDS.
10. A method of treating a human subject, comprising administering
to a human subject an effective amount of a compound or a
pharmacologically acceptable salt or solvate thereof, wherein said
human subject has a condition selected from the group consisting of
acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL),
adult T-cell leukaemia (ATLL), lymphoma, gastric cancer, and
multiple myeloma, and wherein said compound has formula II:
##STR00011##
11. The method of claim 10, wherein said condition is AML.
12. (canceled)
13. (canceled)
14. The method of claim 10, wherein said condition is ALL.
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. The method of claim 10, wherein said condition is gastric
cancer.
20. The method of claim 10, wherein said condition is multiple
myeloma.
21. The method of claim 1, wherein said compound is administered as
part of a combination therapy.
22. The method of claim 21, wherein the combination therapy
comprises radiotherapy.
23. The method of claim 21, wherein the combination therapy
comprises chemotherapy.
24. The method of claim 11, wherein said compound is administered
with a dosage of about 125 mg/m.sup.2.
25. The method of claim 11, wherein said compound is administered
two times per week for four weeks.
26. A method of treating a condition associated with lower than
normal level of KLF4 activity comprising administering to a human
subject in need of such treatment an effective amount of compound
has formula II: ##STR00012##
27. A method of treating a condition associated with abnormal level
of CDX2 activity comprising administering to a human subject in
need of such treatment an effective amount of compound has formula
II: ##STR00013##
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 and
methods for treating cancers.
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, 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.
[0004] Myelodysplastic syndromes (MDS) are a group of diseases that
affect the bone marrow and blood. Some types of MDS are mild and
easily managed, while other types are severe and life-threatening.
Myelodysplastic syndromes (MDS) are rarely cured; most patients
never actually complete treatment. The current treatment of MDS is
based on the stage and the mechanism of the disease that
predominates the particular phase of the disease process. Bone
marrow transplantation has been used in patients with poor
prognosis or late-stage MDS. Epstein and Slease, 1985, Surg. Ann.
17:125. This type of therapy, however, is both painful for donor
and recipient, because of the involvement of invasive procedures
and can cause severe and even fatal complications to the recipient,
particularly with allogeneic transplant and related Graft Versus
Host Disease (GVHD) results. Therefore, the risk of GVHD restricts
the use of bone marrow transplantation to patients with otherwise
fatal diseases. Further, as most patients are elderly and only a
few young MDS patients will have a matched donor, the use of bone
marrow transplantation is limited. There remains a need for more
effective methods for treating MDS and its related disorders.
[0005] Mild MDS can grow more severe over time. It can also develop
into a fast-growing, severe leukemia, acute myelogenous leukemia
(AML). 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.
[0006] The present invention meets this need and provides
compositions and methods for the effective treatment of
cancers.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions and methods for
treating cancers. In some embodiments, the present invention is
based, in part, on the discovery that the Caudal-related Homeobox
protein CDX2-Kruppel-like factor 4 (KLF4) signaling pathway ("the
CDX-KLF4 signaling pathway") is important for the pathogenesis of a
group of cancers, and that active agents with an ability of
modulating the CDX2-KLF4 signaling pathway can be used to treat
such cancers effectively.
[0008] In one aspect, the invention provides compositions for
treating cancers. In some embodiments, the compositions comprise at
least one anti-cancer active agent that can modulate the CDX2-KLF4
signaling pathway in a human subject. 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. As used herein, the term
"activity" of a component in the CDX2-KLF4 signaling pathway 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.
[0009] In some embodiments, the active agent is a small molecule
pharmaceutical compound. 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 has the structure of formula I:
##STR00001##
Wherein R1 is C1-C4 alkyl; and R2 is halogen.
[0010] In some embodiments, R.sup.1 is methyl, isopropyl, or
t-butyl.
[0011] In some embodiments, the pharmaceutical compound is selected
from the group consisting of:
##STR00002##
[0012] In some embodiments, the pharmaceutical compound has the
structure of formula II:
##STR00003##
[0013] 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/lymohoma. In some embodiments, the cancer is
myelodysplastic syndromes (MDS). In some embodiments, the MDS is
high-risk MDS. In some embodiments, the cancer is acute myelogenous
leukemia (AML). In some embodiments, the AML is refractory AML. In
some embodiments, the AML is elderly AML. In some other
embodiments, the cancer is acute lymphocytic leukemia (ALL). In
some embodiments, the ALL is a pediatric ALL. In some embodiments,
the pediatric ALL is pediatric T-cell ALL or pediatric B-cell ALL.
In some other embodiments, the cancer is chronic myelogenous
leukemia (CML). In some embodiments, the cancer is adult T-cell
leukaemia (ATLL). In some embodiments, the cancer is caused by
human T-lymphotrophic virus type 1. In some embodiments, the cancer
is lymphoma, gastric cancer, or multiple myeloma. In some
embodiments, the lymphoma is Hodgkin lymphoma, non-Hodgkin's
lymphoma (NHL), Burkitts lymphoma, or B-cell lymphoma. In some
embodiments, the cancer is a combination of any of the cancers
described herein.
[0014] In some embodiments, the human subject has one or more
symptoms of MDS. In some embodiments, the human subject to be
treated has an ineffective production of blood cells, such as
myeloid blood cells. In some embodiments, the human subject has an
anemia. In some embodiments, the human subject has low blood counts
caused by bone marrow failure.
[0015] In some embodiments, the cancer is acute myelogenous
leukemia (AML). In some embodiments, the AML is refractory 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.
[0016] In some embodiments, the cancer is acute lymphocytic
leukemia (ALL). In some embodiments, the ALL is a pediatric or
child ALL. As used herein, pediatric or child ALL patients are
those patients who have, or may have ALL, with an age under 21. In
some embodiments, the ALL is not pediatric or child ALL. As used
herein, non-pediatric ALL patients are those who have, or may have
ALL, with an age equal to or above 21.
[0017] In some embodiments, an anti-cancer compound of the present
invention is administered to a human subject in need as part of a
combination therapy. In some embodiments, the combination therapy
comprises radiotherapy. In some embodiments, the combination
therapy comprises chemotherapy.
[0018] In some embodiments, an effective amount of an anti-cancer
agent or a pharmacologically acceptable salt or solvate thereof of
the present invention is administered to the human subject. In some
embodiments, an effective amount of the anti-cancer agent is
effective to treat the cancer or symptoms of the cancer, effective
to inhibit cancer cell proliferation, or effective to prevent or
reduce severity of a future occurrence of cancer when administered
to a subject who is susceptible and/or who may develop a cancer or
symptoms of the cancer. In some embodiments, the administration of
the anti-cancer agent provides statistically significant
therapeutic effect or clinical efficacy for treating the cancer. In
some embodiments, an anti-cancer compound of the present invention
is administered with a dosage of about 0.01 to about 200 mg. In
some embodiments, an anti-cancer compound of the present invention
is administered with a dosage of about 0.05 to about 100 mg. In
some embodiments, an anti-cancer compound of the present invention
is administered with a dosage of about 1.0 to about 50 mg. In some
embodiments, the daily dosages of the compounds of the present
invention 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/m2 to
about 400 mg/m2 in single or divided dose. In some embodiments, an
anti-cancer compound of the present invention is administered once,
twice, three times or more per day.
[0019] In some embodiments, the treatment last 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more cycles. In some embodiments, each cycle lasts at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some
embodiments, the gap between two cycles is at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or more days.
[0020] In some embodiments, the anti-cancer agent is unit dosage
form.
[0021] In some embodiments, the cancer to be treated is AML, the
compound is LOR-253 (a. k. a. APTO-253), and the compound is
administered with a dosage of about 125 mg/m2. In certain
embodiments, the compound is administered two times per week for
four weeks.
[0022] The invention provides methods for treating a condition
associated with abnormal activity of the CDX2-KLF4 signaling
pathway. In some embodiments, the condition is associated with
abnormal activity of one or more component of the CDX2-KLF4
signaling pathway, such as CDX2, KLF4, p21, and/or p53 in a human
subject. In some embodiments, the condition is associated with
abnormal (e.g., lower than normal) level of KLF4 activity. In some
embodiments, the condition is associated with abnormal (e.g.,
higher than normal) level of CDX2 activity. In some embodiments,
the abnormal level of CDX2 is due to a genetic alteration of
CDX2.
[0023] In some embodiments, the methods of the present invention
further comprise determining the activity of a component in the
CDX2-KLF4 signaling pathway, such as the KLF4 activity and/or CDX2
activity in the human subject before, during, and/or after the
treatment. In some embodiments, the methods of the present
invention further comprise determining the presence or absence of a
genetic alteration of CDX2.
[0024] Additional aspects and embodiments of the invention will be
apparent from the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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).
[0030] 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-).
[0031] FIG. 7 depicts Caspase 3 expression level in THP1 and HL-60
AML cell lines treated with DMSO or LOR-253.
[0032] FIG. 8 depicts fold change in expression of BAX and BCL2 in
THP1 cells treated with DMSO or 0.5 .mu.M LOR-253
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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).
[0037] FIG. 13 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 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 lymphoma. The lower part of
the diagram illustrates that silenced KLF4 causes increased cancer
cell proliferation through various "cell fate genes".
[0038] FIG. 14 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
[0039] FIG. 15 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.
[0040] FIG. 16 depicts body weight measurements on the indicated
days of Kasumi-1 tumor-bearing Mice treated with LOR-253 HCL or
negative control.
[0041] FIG. 17 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.
[0042] FIG. 18 and FIG. 19 depict the tumor sizes of individual
animals at the beginning (Day 1) and end (Day 19) of the study of
FIG. 17, respectively.
[0043] FIG. 20 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.
[0044] FIG. 21 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.
DETAILED DESCRIPTION
Definitions
[0045] 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.
[0046] 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.
[0047] The invention provides isolated, chimeric, recombinant or
synthetic polynucleotide sequences. 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 isolated,
chimeric, recombinant or synthetic polynucleotide sequences are
derived from gene markers of the present invention.
[0048] The invention also provides proteins or polypeptides. 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.
[0049] 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.
[0050] As used herein, the term "component in the CDX2-KLF4
signaling pathway", refers to CDX2, KLF4, or 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 CDX2, KLF4, KDM5B, miR-2909, p53,
p21, Caspase-3, Annexin V, BAX, BCL2, BCL3, BMP, Wnt, HNF4.alpha.,
Fgf, Hox, SP1, MYC, CCND1, AATF, and those described in 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, Vol. 278, No. 4, Issue of January 24, pp. 2101-2105, 2003),
Faber et al. (CDX2-driven leukemogenesis involves KLF4 repression
and deregulated PPAR.gamma. signaling, J Clin Invest.
doi:10.1172/JCI64745.), 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. 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 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 a 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.
[0051] 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.
[0052] 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).
[0053] 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.
[0054] 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.
[0055] 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.
[0056] "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).
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] In certain embodiments, when measuring the activity level of
a component in the CDX2-KLF4 signaling pathway 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.
[0064] According to some embodiments of the present invention,
administering of anti-cancer agents such as LOR-253 according to
the methods of the present invention provides statistically
significant therapeutic effect. In one embodiment, the
statistically significant therapeutic effect is determined based on
one or more standards or criteria provided by one or more
regulatory agencies in the United States, e.g., FDA or other
countries. In other embodiments, the statistically significant
therapeutic effect is determined based on results obtained from
regulatory agency approved clinical trial set up and/or
procedure.
[0065] In some embodiments, the statistically significant
therapeutic effect is determined based on a patient population of
at least 300, 400, 500, 600, 700, 800, 900, 1000 or 2000. In some
embodiments, the statistically significant therapeutic effect is
determined based on data obtained from randomized and double
blinded clinical trial set up. In some embodiments, the
statistically significant therapeutic effect is determined based on
data with a p value of less than or equal to about 0.05, 0.04,
0.03, 0.02 or 0.01. In some embodiments, the statistically
significant therapeutic effect is determined based on data with a
confidence interval greater than or equal to 95%, 96%, 97%, 98% or
99%. In some embodiments, the statistically significant therapeutic
effect is determined on approval of Phase III clinical trial of the
methods provided by the present invention, e.g., by FDA in the
US.
[0066] In some embodiments, the statistically significant
therapeutic effect is determined by a randomized double blind
clinical trial of a patient population of at least 300 or 350,
treated with anti-cancer agents such as LOR-253 in combination with
standard care. In some embodiment, the statistically significant
therapeutic effect is determined by a randomized clinical trial of
a patient population of at least 300 or 350 and using 28 day
mortality rate, in-hospital mortality rate, ICU mortality rate, ICU
duration, ICU free days, sequential organ failure assessment score
(SOFA), relative risk of death, ICU frequency, duration of
ventilation, frequency of ventilation, ventilation free days or any
combination thereof or any other commonly accepted criteria for
sepsis assessment.
[0067] In general, statistical analysis can include any suitable
method permitted by a regulatory agency, e.g., FDA in the US or
China or any other country. In some embodiments, statistical
analysis includes non-stratified analysis, log-rank analysis, e.g.,
from Kaplan-Meier, Jacobson-Truax, Gulliken-Lord-Novick,
Edwards-Nunnally, Hageman-Arrindel and Hierarchical Linear Modeling
(HLM) and Cox regression analysis.
[0068] As used herein, the phrase "per day" describes an amount
administered on those days when an agent is administered. The
phrase "per day" does not indicate that an amount is administered
every day.
[0069] 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.
[0070] The term "prodrug", as used herein, unless otherwise
indicated, means compounds that are drug precursors, which
following administration, release the drug in vivo via some
chemical or physiological process (e.g., a prodrug on being brought
to the physiological pH is converted to the desired drug form).
[0071] "Continuous dosing schedule", as used herein, unless
otherwise indicated, refers to a dosing schedule wherein compound
of present invention, or a dosage form comprising said compound, is
administered during a treatment period without a rest period.
Throughout the treatment period of a continuous dosing schedule,
the compound, or a dosage form comprising the compound, can be
administered, for example, daily, every other day, every third day,
every forth day, every fifth day, etc. On a day when compound, or a
dosage form comprising the compound is administered, it can be
administered in a single dose, or in multiple doses throughout the
day.
[0072] "Intermittent dosing schedule", as used herein, unless
otherwise indicated, refers to a dosing schedule that comprises a
treatment period and a rest period. Throughout the treatment period
of an intermittent dosing schedule, compound of present invention,
or a dosage form comprising said compound, can be administered, for
example, daily, or every other day, every third day, every forth
day, every fifth day, etc. On a day when compound or a dosage form
comprising the compound is administered, it can be administered in
a single dose, or in multiple doses throughout the day. During the
rest period, a compound or a dosage form comprising the compound is
not administered. In some embodiments, the rest period lasts at
least one day, at least two days, at least three days, at least
four days, at least five days, at least six days, at least a week,
at least 1.5 week, at least 2 weeks, at least 3 weeks, at least 4
weeks, at least one month, at least two months, at least three
months, at least four months, at least five months, at least half
year, at least one year, at least two years, or more. In some
intermittent dosing regimens, the treatment period is typically
from about 1 day to 30 days, such as about 10 days to 30 days, for
example, about 2, 3 or 4 weeks, and the rest period is typically
from 1 to 30 days, such as 3 to 15 days, for example, 1 or 2 weeks.
The combination of any treatment period from 10 to 30 days with any
rest period from 3 to 15 days is contemplated. Intermittent dosing
regimens can be expressed as treatment period in weeks/rest period
in weeks. For example, a 4/1 intermittent dosing schedule refers to
an intermittent dosing schedule wherein the treatment period is
four weeks and the rest period is one week. A 4/2 intermittent
dosing schedule refers to an intermittent dosing schedule wherein
the treatment period is four weeks and the rest period is two
weeks. Similarly, a 3/1 intermittent dosing schedule refers to an
intermittent dosing schedule wherein the treatment period is three
weeks and the rest period is one week.
[0073] The human subject treated by the anti-cancer agent of the
present invention may show complete response or partial response.
Complete Response (CR), as used herein, unless otherwise indicated,
refers to disappearance of all measurable and nonmeasurable
symptoms and no appearance of new symptoms in a patient under the
treatment. Partial Response (PR), as used herein, unless other wise
indicated, refers to at least one measurable and nonmeasurable
symptom is significantly reduced, or without appearance of new
symptom in a patient under treatment.
[0074] It should be further appreciated that dosing regimens can be
adjusted by one skilled in the art to more conveniently accommodate
coordination of the dosing regimens, and additional therapeutic
agents, if such adjustments are therapeutically acceptable.
[0075] As used herein, "C.sub.max" refers to the maximum plasma
concentration; t.sub.max refers to the time when the C.sub.max
occurs following administering the dosage; AUC refers to area under
the plasma concentration-time curve from time zero to infinity;
t.sub.1/2 refers to plasma elimination half-life; % CV refers to
percent coefficient of variation; C.sub.(trough 24 h) refers to
trough plasma concentration at 24 hours after dosing; and QD
indicates once daily.
Cancers
[0076] 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. As used herein, "C.sub.max" refers to the
maximum plasma concentration; t.sub.max refers to the time when the
C.sub.max occurs following administering the dosage; AUC refers to
area under the plasma concentration-time curve from time zero to
infinity; t.sub.1/2 refers to plasma elimination half-life; % CV
refers to percent coefficient of variation; C.sub.(trough 24 h)
refers to trough plasma concentration at 24 hours after dosing; and
QD indicates once daily.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] In some embodiments, the cancer is leukemia/lymohoma. 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.
[0084] Acute Myeloid Leukemia (AML):
[0085] 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: WB 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.
[0086] The French-American-British (FAB) classification system
divides AML into eight subtypes, MO 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%
[0087] 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).
[0088] Acute Lymphocytic Leukemia (ALL):
[0089] 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).
[0090] Chronic Myelogenous Leukemia (CML):
[0091] 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.
[0092] Adult T-Cell Leukaemia (ATLL):
[0093] 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.
[0094] Lymphoma:
[0095] 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): [0096] A. Mature B-cell neoplasms: [0097]
Chronic lymphocytic leukemia/Small lymphocytic lymphoma [0098]
B-cell prolymphocytic leukemia [0099] Lymphoplasmacytic lymphoma
(such as Waldenstrom macroglobulinemia) [0100] Splenic marginal
zone lymphoma [0101] Plasma cell neoplasms: [0102] Plasma cell
myeloma [0103] Plasmacytoma [0104] Monoclonal immunoglobulin
deposition diseases [0105] Heavy chain diseases [0106] Extranodal
marginal zone B cell lymphoma, also called MALT lymphoma [0107]
Nodal marginal zone B cell lymphoma (NMZL) [0108] Follicular
lymphoma [0109] Mantle cell lymphoma [0110] Diffuse large B cell
lymphoma [0111] Mediastinal (thymic) large B cell lymphoma [0112]
Intravascular large B cell lymphoma [0113] Primary effusion
lymphoma [0114] Burkitt lymphoma/leukemia [0115] B. Mature T cell
and natural killer (NK) cell neoplasms [0116] T cell prolymphocytic
leukemia [0117] T cell large granular lymphocytic leukemia [0118]
Aggressive NK cell leukemia [0119] Adult T cell leukemia/lymphoma
[0120] Extranodal NK/T cell lymphoma, nasal type [0121]
Enteropathy-type T cell lymphoma [0122] Hepatosplenic T cell
lymphoma [0123] Blastic NK cell lymphoma [0124] Mycosis
fungoides/Sezary syndrome [0125] Primary cutaneous CD30-positive T
cell lymphoproliferative disorders [0126] Primary cutaneous
anaplastic large cell lymphoma [0127] Lymphomatoid papulosis [0128]
Angioimmunoblastic T cell lymphoma [0129] Peripheral T cell
lymphoma, unspecified [0130] Anaplastic large cell lymphoma [0131]
C. Hodgkin Lymphoma [0132] Classical Hodgkin lymphomas: [0133]
Nodular sclerosis [0134] Mixed cellularity [0135] Lymphocyte-rich
[0136] Lymphocyte depleted or not depleted [0137] Nodular
lymphocyte-predominant Hodgkin lymphoma [0138] D.
Immunodeficiency-associated lymphoproliferative disorders [0139]
Associated with a primary immune disorder [0140] Associated with
the Human Immunodeficiency Virus (HIV) [0141] Post-transplant
[0142] Associated with methotrexate therapy [0143] 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 [0143] 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.
[0144] Gastric Cancer:
[0145] 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.
[0146] Colorectal Cancer:
[0147] 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 mutations described for
the genes above, non-hypermutated samples also contain mutated
CTNNB1, FAM123B, SOX9, ATM, and ARIDIA. 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.
[0148] Multiple Myeloma:
[0149] 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.
[0150] Myelodysplastic Syndromes (MDS):
[0151] 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).
[0152] 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.
[0153] 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
[0154] 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- platelet counts and isolated deletions of the
long arm of chromosome 5 in bone marrow T) 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
[0155] 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; weakness or feeling tired, paler skin, easy
bruising or bleeding, petechiae, fever, 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.
[0156] Many factors can increase risk of MDS, which include, but
are not limited to, being male or white, being older than 60 years,
past treatment with chemotherapy or radiation therapy, being
exposed to certain chemicals, including tobacco smoke, pesticides,
and solvents such as benzene, and being exposed to heavy metals,
such as mercury or lead.
[0157] Previous treatment methods for MDS include 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. Other methods include
5-azacytidine, decitabine, lenalidomide, immunosuppression,
leukemia therapy, and investigational approaches.
[0158] 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.
CDX2-KLF4 Signaling Pathway
[0159] 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".
[0160] 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_000013.10, NC_018924.2, NT_024524.14,
NP_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 30 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 30 copies. Otherwise, the CDX2
gene is considered as being turned off.
[0161] 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_000009.11, NT_008470.19, NC_018920.2, and Uniprot No. 043474,
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.
[0162] p21, a.k.a. Cyclin-Dependent Kinase Inhibitor 1A, Cip1,
CDJN1, CIP1, WAF1, 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_000006.11, NT_007592.15, NC_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.
[0163] Some other components of the CDX2-KLF4 signaling pathway are
H3K4 demethylase Jaridlb (KDM5B, a.k.a. Plu-1 or Rbp2-hl), 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
4.alpha. (HNF4.alpha.), fibroblast growth factors (Fgf), homeobox
(Hox), transcription factor SP1, transcription factor MYC, cyclin
Dl (CCND1, a.k.a. PRAD1), and apoptosis-antagonizing transcription
factor (AATF).
[0164] An active agent of the present invention can modulate the
activity of one or more component in the CDX2-KLF4 signaling
pathway. In some embodiments, the active agent is a small molecule.
In some embodiments, the active agent is a polypeptide, such as an
antibody. In some embodiments, the active agent is a
polynucleotide, such as siRNA.
[0165] In some embodiments, the active agent modulates the gene
copy number of a component in the CDX2-KLF4 signaling pathway. In
some embodiments, the active agent can increase or decrease the
gene copy number by 0.5.times., 1.0.times., 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
when compared to the gene copy number before treatment.
[0166] In some embodiments, the active agent modulates the mRNA
abundance of a component in the CDX2-KLF4 signaling pathway. In
some embodiments, the active agent can increase or decrease the
mRNA abundance by 0.5.times., 1.0.times., 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
when compared to the mRNA abundance before treatment.
[0167] In some embodiments, the active agent modulates the protein
level of a component in the CDX2-KLF4 signaling pathway. In some
embodiments, the active agent can increase or decrease the protein
level by 0.5.times., 1.0.times., 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 when
compared to the protein level before treatment.
[0168] In some embodiments, the active agent modulates the mRNA
and/or protein stability of a component in the CDX2-KLF4 signaling
pathway. In some embodiments, the active agent can increase or
decrease the stability when compared to the stability before
treatment.
[0169] In some embodiments, the active agent modulates the
enzymatic activity of a component in the CDX2-KLF4 signaling
pathway. In some embodiments, the active agent can increase or
decrease the enzymatic activity when compared to the stability
before treatment.
[0170] The activity of a component in the CDX2-KLF4 signaling
pathway 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 a component in
the CDX2-KLF4 signaling pathway, 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. 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 protein expression level is used in
determining the level of activity. The protein expression level of
a component in the CDX2-KLF4 signaling pathway 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 of a component in the
CDX2-KLF4 signaling pathway can be used as an active agent. In some
embodiments, antibody of a component in the CDX2-KLF4 signaling
pathway that negatively control KLF4 and/or p21, or a component in
the CDX2-KLF4 signaling pathway that are negatively controlled by
KLF4 and/or p21 can be used as an active agent. For example,
antibodies of CDX2 can be used to treat the cancers of the present
invention.
[0174] In some embodiments, antibody of a component in the
CDX2-KLF4 signaling pathway can be used to detect the protein level
of the component. 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.
[0175] 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.
[0176] 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
oligonucleotides, immunoblotting, immunoprecipitation,
immunofluorescence methods, chemiluminescence methods,
electrochemiluminescence (ECL) or enzyme-linked immunoassays.
[0177] 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.
[0178] 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.
[0179] 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).
[0180] 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.
[0181] 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.
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] 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.
[0184] 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.
[0185] 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.
[0186] In some embodiments, the active agents contain one or more
antibodies that can reduce, inhibit or delay the activity of a
component in the CDX2-KLF4 signaling pathway that negatively
regulates KLF4, or negatively regulated by KLF4. In some
embodiments, the component can be CDX2, p53, p21, Caspase-3,
Annexin V, BAX, BCL2, BCL3, BMP, Wnt, HNF4.alpha., Fgf, Hox, SP1,
MYC, CCND1, or AATF. In some embodiments, the active agents are
antibodies. In some embodiments, the active agents are siRNA. For
example, 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.
[0187] For example, antisense RNA, ribozyme, dsRNAi, RNA
interference (RNAi) technologies can be used in the present
invention to target RNA transcripts of one or more component in the
CDX2-KLF4 signaling pathway. Antisense RNA technology involves
expressing in, or introducing into, a cell an RNA molecule (or RNA
derivative) that is complementary to, or antisense to, sequences
found in a particular mRNA in a cell. By associating with the mRNA,
the antisense RNA can inhibit translation of the encoded gene
product. For example, the anti-CDX2 agents can be 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.)
[0188] RNA interference (RNAi) is the process of sequence-specific,
post-transcriptional gene silencing or transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene. The
RNAi technique is discussed, for example, in Elibashir, et al.,
Methods Enzymol. 26:199 (2002); McManus & Sharp, Nature Rev.
Genetics 3:737 (2002); PCT application WO 01/75164; Martinez et
al., Cell 110:563 (2002); Elbashir et al., supra; Lagos-Quintana et
al., Curr. Biol. 12:735 (2002); Tuschl et al., Nature Biotechnol.
20:446 (2002); Tuschl, Chembiochem. 2:239 (2001); Harborth et al.,
J. Cell Sci. 114:4557 (2001); et al., EMBO J. 20:6877 (2001);
Lagos-Quintana et al., Science 294:8538 (2001); Hutvagner et al.,
loc cit, 834; Elbashir et al., Nature 411:494 (2001).
[0189] The term "dsRNA" or "dsRNA molecule" or "double-strand RNA
effector molecule" refers to an at least partially double-strand
ribonucleic acid molecule containing a region of at least about 19
or more nucleotides that are in a double-strand conformation. The
double-stranded RNA effector molecule may be a duplex
double-stranded RNA formed from two separate RNA strands or it may
be a single RNA strand with regions of self-complementarity capable
of assuming an at least partially double-stranded hairpin
conformation (i.e., a hairpin dsRNA or stem-loop dsRNA). In various
embodiments, the dsRNA consists entirely of ribonucleotides or
consists of a mixture of ribonucleotides and deoxynucleotides, such
as RNA/DNA hybrids. The dsRNA may be a single molecule with regions
of self-complementarity such that nucleotides in one segment of the
molecule base pair with nucleotides in another segment of the
molecule. In one aspect, the regions of self-complementarity are
linked by a region of at least about 3-4 nucleotides, or about 5,
6, 7, 9 to 15 nucleotides or more, which lacks complementarity to
another part of the molecule and thus remains single-stranded
(i.e., the "loop region"). Such a molecule will assume a partially
double-stranded stem-loop structure, optionally, with short single
stranded 5' and/or 3' ends. In one aspect the regions of
self-complementarity of the hairpin dsRNA or the double-stranded
region of a duplex dsRNA will comprise an Effector Sequence and an
Effector Complement (e.g., linked by a single-stranded loop region
in a hairpin dsRNA). The Effector Sequence or Effector Strand is
that strand of the double-stranded region or duplex which is
incorporated in or associates with RISC. In one aspect the
double-stranded RNA effector molecule will comprise an at least 19
contiguous nucleotide effector sequence, preferably 19 to 29, 19 to
27, or 19 to 21 nucleotides, which is a reverse complement to the
RNA of a target gene, or an opposite strand replication
intermediate, or the anti-genomic plus strand or non-mRNA plus
strand sequences of the target gene.
[0190] In some embodiments, the dsRNA effector molecule of the
invention is a "hairpin dsRNA", a "dsRNA hairpin", "short-hairpin
RNA" or "shRNA", i.e., an RNA molecule of less than approximately
400 to 500 nucleotides (nt), or less than 100 to 200 nt, in which
at least one stretch of at least 15 to 100 nucleotides (e.g., 17 to
50 nt, 19 to 29 nt) is based paired with a complementary sequence
located on the same RNA molecule (single RNA strand), and where
said sequence and complementary sequence are separated by an
unpaired region of at least about 4 to 7 nucleotides (or about 9 to
about 15 nt, about 15 to about 100 nt, about 100 to about 1000 nt)
which forms a single-stranded loop above the stem structure created
by the two regions of base complementarity. The shRNA molecules
comprise at least one stem-loop structure comprising a
double-stranded stem region of about 17 to about 100 bp; about 17
to about 50 bp; about 40 to about 100 bp; about 18 to about 40 bp;
or from about 19 to about 29 bp; homologous and complementary to a
target sequence to be inhibited; and an unpaired loop region of at
least about 4 to 7 nucleotides, or about 9 to about 15 nucleotides,
about 15 to about 100 nt, about 100 to about 1000 nt, which forms a
single-stranded loop above the stem structure created by the two
regions of base complementarity. It will be recognized, however,
that it is not strictly necessary to include a "loop region" or
"loop sequence" because an RNA molecule comprising a sequence
followed immediately by its reverse complement will tend to assume
a stem-loop conformation even when not separated by an irrelevant
"stuffer" sequence.
[0191] 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, or increase the activity of an entity
that can positively regulate 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.
[0192] In some embodiments, the compound has the structure of
formula I, or a salt thereof:
##STR00004##
Wherein R1 is C.sub.1-C.sub.4 alkyl; and R2 is halogen. In some
embodiments, R.sup.1 is methyl, isopropyl, or t-butyl.
[0193] In some embodiments, the compound has the structure of
formula II, or a salt thereof:
##STR00005##
[0194] In some embodiments, the compound has the structural formula
(III), or a salt thereof:
##STR00006##
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.
[0195] In some embodiments, R1, R2, R3, R4 are independently
hydrogen; halogen; C.sub.1-C.sub.4 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.
[0196] In some embodiments, said compound has a formula selected
from the group consisting of
##STR00007##
[0197] 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.
[0198] 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 one embodiment the pharmaceutical
composition is administered parenterally, paracancerally,
transmucosally, tansdermally, intramuscularly, intravenously,
intradermally, subcutaneously, intraperitonealy,
intraventricularly, intracranially and intratumorally. 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] In some embodiments, the delivery systems suitable include
time-release, delayed release, sustained release, or controlled
release delivery systems. In some embodiments, a composition of the
present invention can be delivered in a controlled release system,
such as sustained-release matrices. Non-limiting examples of
sustained-release matrices include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al.,
1981, J. Biomed. Mater. Res., 15:167-277 and Langer, 1982, Chem.
Tech., 12:98-105), or poly(vinylalcohol)], polylactides (U.S. Pat.
No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., 1983, Biopolymers, 22:547-556),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). In some embodiments,
the composition may be administered using intravenous infusion, an
implantable osmotic pump, a transdermal patch, liposomes, or other
modes of administration. In one embodiment, a pump may be used (see
Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);
Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J.
Med. 321:574 (1989). In another embodiment, polymeric materials can
be used. In yet another embodiment, a controlled release system can
be placed in proximity to the therapeutic target, for example
liver, thus requiring only a fraction of the systemic dose (see,
e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984). Other controlled release systems
are discussed in the review by Langer (Science 249:1527-1533
(1990).
[0209] In some embodiments, the release of the composition occurs
in bursts. Examples of systems in which release occurs in bursts
includes, e.g., systems in which the composition is entrapped in
liposomes which are encapsulated in a polymer matrix, the liposomes
being sensitive to specific stimuli, e.g., temperature, pH, light
or a degrading enzyme and systems in which the composition is
encapsulated by an ionically-coated microcapsule with a
microcapsule core degrading enzyme.
[0210] In some embodiments, the release of the composition is
gradual/continuous. Examples of systems in which release of the
inhibitor is gradual and continuous include, e.g., erosional
systems in which the composition is contained in a form within a
matrix and effusional systems in which the composition penneates at
a controlled rate, e.g., through a polymer. Such sustained release
systems can be e.g., in the form of pellets, or capsules.
[0211] Other embodiments of the compositions administered according
to the invention incorporate particulate forms, protective
coatings, protease inhibitors or permeation enhancers for various
routes of administration, such as parenteral, pulmonary, nasal and
oral.
[0212] 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.
[0213] 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, Philidelphia, Pa. (2000).
[0214] 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.
[0215] The pharmaceutical compositions of the present invention may
be used as part of a neo-adjuvant therapy (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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] In some embodiments, the active agents are compounds having
the structure of formulas described herein. 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.
[0220] Dosages for a particular individual can be determined by one
of ordinary skill in the art using conventional considerations,
(e.g. by means of an appropriate, conventional pharmacological
protocol). A physician may, for example, prescribe a relatively low
dose at first, subsequently increasing the dose until an
appropriate response is obtained. The dose administered to an
individual is sufficient to effect a beneficial therapeutic
response in the individual over time, or, e.g., to reduce symptoms,
or other appropriate activity, depending on the application. The
dose is determined by the efficacy of the particular formulation,
and the activity, stability or serum half-life of the composition
and the condition of the individual, as well as the body weight or
surface area of the individual to be treated. The size of the dose
is also determined by the existence, nature, and extent of any
adverse side-effects that accompany the administration of a
particular vector, formulation, or the like in a particular
individual.
[0221] In some embodiments, dosage unit for a compound of the
present invention can be about 0.001 mg, about 0.002 mg, about
0.003 mg, about 0.004 mg, about 0.005 mg, about 0.006 mg, about
0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02
mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg,
about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about
0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg,
about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg,
about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8
mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg,
about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg,
about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 200 mg,
about 300 mg, about 400 mg, about 500 mg or more.
[0222] In some embodiments, daily dosages of the compounds of the
present invention will typically be about 0.001 mg/kg of body
weight, about 0.002 mg/kg of body weight, about 0.003 mg/kg of body
weight, about 0.004 mg/kg of body weight, about 0.005 mg/kg of body
weight, about 0.006 mg/kg of body weight, about 0.007 mg/kg of body
weight, about 0.008 mg/kg of body weight, about 0.009 mg/kg of body
weight, about 0.01 mg/kg of body weight, about 0.02 mg/kg of body
weight, about 0.03 mg/kg of body weight, about 0.04 mg/kg of body
weight, about 0.05 mg/kg of body weight, about 0.06 mg/kg of body
weight, about 0.07 mg/kg of body weight, about 0.08 mg/kg of body
weight, about 0.09 mg/kg of body weight, about 0.1 mg/kg of body
weight, about 0.2 mg/kg of body weight, about 0.3 mg/kg of body
weight, about 0.4 mg/kg of body weight, about 0.5 mg/kg of body
weight, about 0.6 mg/kg of body weight, about 0.7 mg/kg of body
weight, about 0.8 mg/kg of body weight, about 0.9 mg/kg of body
weight, about 1 mg/kg of body weight, about 2 mg/kg of body weight,
about 3 mg/kg of body weight, about 4 mg/kg of body weight, about 5
mg/kg of body weight, about 6 mg/kg of body weight, about 7 mg/kg
of body weight, about 8 mg/kg of body weight, about 9 mg/kg of body
weight, about 10 mg/kg of body weight, about 15 mg/kg of body
weight, about 20 mg/kg of body weight, about 30 mg/kg of body
weight, about 35 mg/kg of body weight, about 40 mg/kg of body
weight, about 45 mg/kg of body weight, about 50 mg/kg of body
weight, about 60 mg/kg of body weight, about 70 mg/kg of body
weight, about 80 mg/kg of body weight, about 90 mg/kg of body
weight, about 100 mg/kg of body weight, about 200 mg/kg of body
weight, about 300 mg/kg of body weight, about 400 mg/kg of body
weight, about 500 mg/kg of body weight or more. Either single or
divided dose can be used.
[0223] In some embodiments, the dosage form of the present
invention is effective to provide a maximum total plasma
concentration in said human subject of no more than about 1 pg/mL,
about 5 pg/mL, about 10 pg/mL, about 50 pg/mL, about 100 pg/mL,
about 500 pg/mL, about 1 ng/mL, about 5 ng/mL, about 10 ng/mL,
about 50 ng/mL, about 100 ng/mL, about 500 ng/mL, about 1 .mu.g/mL,
about 5 .mu.g/mL, about 10 .mu.g/mL, about 50 .mu.g/mL, about 100
.mu.g/mL, about 500 .mu.g/mL, about 1 mg/mL or more of free base
equivalent of the compound.
[0224] In a further aspect of any of dosage forms as described
herein, the dosage form is an oral dosage form. In a still further
aspect, the dosage form is an intravenous dosage form. The dosage
form is suitable for administration to a human subject particularly
for use in the treatment of any of the disorders described herein.
In some embodiments, the dosage form is a subcutaneous dosage
form.
[0225] Pharmaceutical compositions comprising one or more compounds
described herein in combination with one or more other anti-cancer
agents can also be used. Such conjoint treatment may be achieved by
way of the simultaneous, sequential or separate dosing of the
individual components of the treatment.
[0226] 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.
[0227] 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.
[0228] 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 misincorperation 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.
[0229] 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 (D20) (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).
[0230] 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-aspartic 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).
[0231] 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.
[0232] 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.
[0233] 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.
[0234] In some embodiments, the other anti-cancer agents are those
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).
[0235] 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.
[0236] In some embodiments, a continuous dosing schedule is used
for the treatment. In some embodiments, a compound of a dosage form
comprising the compound of the present invention is administered
once per day, twice per day, three times per day, or more times per
day, or continuously during a period of a day, or continuously
through out a day, on a daily schedule, or every other day, every
third day, every forth day, every fifth day, every sixth day, or
every week. The treatment can last a period of time determined by
doctors, or until the cancer cells are completely or significantly
inhibited.
[0237] In some embodiments, an intermittent dosing schedule is used
for the treatment. Such treatment is especially useful if there is
toxicity concern. In some embodiments,
[0238] 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, Lymphoma and Multiple Myeloma Cell Lines
[0239] Antiproliferative activity of LOR-253 was determined by XTT
assay as follows. Cells (4.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) benzene 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)
[0240] The in vitro essay 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
[0241] 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
[0242] 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 minuets 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 Apoptosis in Cell
Lines
[0243] 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 apoptosis in AML cell lines (FIG. 6).
[0244] 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).
[0245] 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
[0246] H226 model mice were treated according to several
administration schedules as shown in the table below.
TABLE-US-00007 Administration Schedule/cycle Treatment Group Dose
level (days) (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 for one
[0247] 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
[0248] 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-253 HCl 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).
[0249] 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
[0250] 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.
[0251] 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-253 HCl
in patients with advanced or metastatic solid tumours. In the first
set of doses, LOR-253 HCl was given in ascending doses until the
maximum administered dose or appropriate target dose is reached. In
the other set of doses, LOR-253 HCl was given in ascending doses
starting from 20 mg/m2 until the maximum administered dose or
appropriate target dose is reached. Patient as treated on LOR-253
HCl for 2 cycles for the evaluation.
Other Name: No other names are used.
Study Type: Interventional
Study Design: Endpoint Classification: Safety Study
[0252] Intervention Model: Single Group Assignment [0253] Masking:
Open Label [0254] Primary Purpose: Treatment
Official Title: Open-Label, Phase 1 Study of LOR-253 HCl in
Patients With Advanced or Metastatic Solid Tumours
Primary Outcome Measures:
[0254] [0255] To determine the maximum tolerated dose (MTD) or
appropriate target dose if MTD not reached to identify the
recommended phase 2 dose of LOR-253 HCl in patients with advanced
or metastatic solid tumours. [Time Frame: 8 weeks]
Secondary Outcome Measures:
[0255] [0256] To characterize the safety profile of LOR-253 HCl
when administered to patients with advanced or metastatic solid
tumours. [Time Frame: 8 weeks]
Inclusion Criteria:
[0256] [0257] 1. Male or female 18 years of age or older. [0258] 2.
Histologically confirmed diagnosis of solid tumor for which no
effective therapy is available or that is unresponsive to
conventional therapy. [0259] 3. Meet laboratory parameter
requirements at study entry.
Exclusion Criteria:
[0259] [0260] 1. Chemotherapy, radiotherapy, biologic therapy,
immunotherapy or any other investigational drugs within 21 days of
beginning study treatment with LOR-253 HCl. [0261] 2. A hematologic
malignancy. [0262] 3. A history of brain or other central nervous
system metastases. [0263] 4. Have a presence of a significant
infection. [0264] 5. Clinically significant autoimmune disease.
[0265] 6. Uncontrolled intercurrent illness. [0266] 7. With iron or
copper overload syndromes. [0267] 8. Pregnancy or breast
feeding.
[0268] Safety and Antitumor Activity Demonstrated in Phase I Trial:
[0269] Dose-Escalating Study in Patients with Advanced Solid Tumors
[0270] Excellent Safety Profile (N=27 Patients) across 7 dose
levels [0271] Most common AE and SAE: Fatigue and 1 case of
reversible hypophosphatemia [0272] Stable Disease achieved in 41%
of evaluable patients; No RECIST PR [0273] Tumor shrinkage (FIG.
12) in patient with NSCLC (poorly differentiated adenocarcinoma)
and extensive metastases refractory to prior standard and
investigational therapies
[0274] The phase I trial results demonstrated that LOR-253 is a
safe and active drug for treating solid tumors.
Example 8--In Vitro Antiproliferative Activity of LOR-253 in
Myelodysplastic Syndromes (MDS), Acute Lymphocytic Leukemia (ALL),
Chronic Myelogenous Leukemia (CML), Adult T-Cell Leukaemia (ATLL),
Lymphoma, Gastric Cancer, and Multiple Myeloma Cell Lines
[0275] Antiproliferative activity of LOR-253 in MDS, ALL, CML,
ATLL, lymphoma, gastric cancer, and multiple myeloma cell lines are
determined by XTT assay as described in Example 1.
[0276] MDS cell lines tested in the assay include, but are not
limited to, TER-3 (Mishima et al., New human myelodysplastic cell
line, TER-3: G-CSF specific downregulation of
Ca2+/calmodulin-dependent protein kinase IV, J Cell Physiol. 2002
May; 191(2):183-90), MDS92 (Tohyama et al., A novel
factor-dependent human myelodysplastic cell line, MDS92, contains
haemopoietic cells of several lineages, Br J Haematol. 1995
December; 91(4):795-9), and SKM-1 (Kimura et al., Antiproliferative
and Antitumor Effects of Azacitidine Against the Human
Myelodysplastic Syndrome Cell Line SKM-1, Anticancer Research
32:795-798, 2002), each of which is herein incorporated by
reference in its entirety.
[0277] ALL cell lines tested in the assay include, but are not
limited to, CCRF-CEM (Foley et al., Continuous culture of human
lymphoblasts from peripheral blood of a child with acute leukemia.
Cancer 1965 18:522-529), MOLT-4 (Minowada et al., Rosette-forming
human lymphoid cell lines. I. Establishment and evidence for origin
of thymus-derived lymphocytes. Journal of the National Cancer
Institute 1972 49 (3): 891-895), and Jurkat (Schneider et al.,
Characterization of EBV-genome negative "null" and "T" cell lines
derived from children with acute lymphoblastic leukemia and
leukemic transformed non-Hodgkin lymphoma. Int J Cancer. 1977 May
15; 19(5):621-6).
[0278] CML cell lines tested in the assay include, but are not
limited to, K562 (Drexler, Leukemia cell lines: in vitro models for
the study of chronic myeloid leukemia. Leuk Res. 1994 December;
18(12):919-27).
[0279] Lymphoma cell lines tested in the assay include, but are not
limited to, Ramos, Raji, CA46, Toledo, DB, Mino, and RL.
[0280] Gastric cancer cell lines tested in the assay include, but
are not limited to, AGS (ATCC Accession No. CRL-1739TM), SNU-1
(ATCC Accession No. CRL-5971TM), SNU-5 (ATCC Accession No.
CRL-5973TM), SNU-16 (ATCC Accession No. CRL-5974TM), Hs 746T (ATCC
Accession No. HTB-135TM), NCI-N87 (N87; ATCC Accession No.
CRL-5822TM), KATO III (ATCC Accession No. HTB-103TM), SNU-520,
SNU-719, NUGC-4, STKM-2, MKN-45, MKN-74, 20M, AKG, ECC4, G42LATE,
GCIY, GCIY, GT3TKB, H-111, H-162, H-30, H-55, HGC-27, HSC-39,
HUG-1N, JR1, KWS, MKN-1, MKN-28, MKN-7, MKN-74, MKN-74, MKN-74,
NCI-N87, NUGC-3, OKAJIMA, SK-GT-1, SK-GT-2, SK-GT-5, SNU-16,
SNU-216, SNU-484, SNU-55, SNU-601, SNU-638, SNU-668, TGBC11TKB,
TGBC11TKB, TMK-1, and YCC-3
[0281] Multiple myeloma cell lines tested in the assay include, but
are not limited to, HMCLs described in Lombardi et al. (Molecular
characterization of human multiple myeloma cell lines by
integrative genomics: insights into the biology of the disease.
Genes Chromosomes Cancer. 2007 March; 46(3):226-38.), XGs, NANs,
BCN, MDN, SBN HMCLs, U266, RPMI8226, RPMI1640, ANBL-6, KMS-11,
KMS12-BM, KMS12-PE, KMM1, LP1, L363, OPM2, NCI-H929, JIM3, MO-1,
LP1, L363, NCIH929, OPM2, UHKT-89, SKMM2, U266B1 (Nilsson et al.,
Established immunoglobulin producing myeloma (IgE) and
lymphoblastoid (IgG) cell lines from an IgE myeloma patient. Clin
Exp Immunol. 1970 October; 7(4):477-89), MM.1R (Greenstein et al.,
Characterization of the MM.1 human multiple myeloma (MM) cell
lines: a model system to elucidate the characteristics, behavior,
and signaling of steroid-sensitive and -resistant MM cells. Exp
Hematol. 2003 April; 31(4):271-82), and HuNS1 (Winkelhake, Myelomas
for producing human/human hybridomas. U.S. Pat. No. 4,720,459 dated
Jan. 19, 1988).
[0282] The in vitro essay results indicate that one or more cell
lines of MDS, ALL, CML, ATLL, lymphoma, gastric cancer, or multiple
myeloma cell lines are sensitive to LOR-253.
Example 9--KLF4/p21 Induction in Myelodysplastic Syndromes (MDS),
ALL, CML, ATLL, Lymphoma, Gastric Cancer, and Multiple Myeloma Cell
Lines In Vitro
[0283] To determine if KLF4 and/or p21 expressions are induced by
LOR-253, myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma,
gastric cancer, and multiple myeloma cell lines described in
Example 8 are treated with DMSO (vehicle control) or 0.1, 0.2, 0.5
or 1 .mu.M LOR-253 for 16 hours. Total RNA is extracted using the
TRIzol Plus RNA Purification kit (Ambion, Life Technologies),
according to the manufacturer's instructions. First strand cDNA is
synthesized from 1-2 ug total RNA using random hexamer primers
(Invitrogen) and the SuperScript II Reverse Transcriptase kit
(Invitrogen). Quantitative RT-PCR is 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 is
normalized with .beta.-actin gene expression in the same sample,
and fold changes in KLF4 or p21 are expressed relative to the
corresponding expression level in the DMSO treated samples using
the comparative CT method. Treatment of one or more myelodysplastic
syndromes (MDS), ALL, CML, ATLL, lymphoma, gastric cancer, or
multiple myeloma cell lines with LOR-253 results in increased
expression KLF4 and/or p21.
Example 10--Treatment with LOR-253 Results in G1/S Cell Cycle
Arrest in Myelodysplastic Syndromes (MDS), ALL, CML, ATLL,
Lymphoma, Gastric Cancer, and Multiple Myeloma Cell Lines
[0284] Myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma,
gastric cancer, and multiple myeloma cell lines described in
Example 8 are treated with DMSO (vehicle control) or 0.1, 0.2, 0.5
or 1 .mu.M LOR-253 for 16 hours. Cells are washed once in PBS+1%
FCS and fixed using ice cold 70% ethanol overnight. Fixed cells are
washed twice, resuspended in PI/RNaseA solution, containing 20
.mu.g/mL propidium iodide and 250 .mu.g/mL RNaseA, and stained for
30 minuets at 37.degree. C. Stained cells are analyzed using a BD
FACSCalibur flow cytometer. The results indicate treatment with
LOR-253 results in G1/S cell cycle arrest in one or more
myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma, gastric
cancer, or multiple myeloma cell lines.
Example 11--Treatment with LOR-253 Induces Apoptosis in
Myelodysplastic Syndromes (MDS), ALL, CML, ATLL, Lymphoma, Gastric
Cancer, and Multiple Myeloma Cell Lines
[0285] Myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma,
gastric cancer, and multiple myeloma cell lines described in
Example 8 are treated with DMSO, 0.1, 0.2, 0.5 or 1 .mu.M of
LOR-253 for 16 hours. Cells are washed twice with cold PBS and
resuspended Annexin V binding buffer. 1.times.10.sup.5 cells in 100
.mu.L are stained with FITC-Annexin V and propidium iodide, and
incubated for 15 minutes at room temperature. After staining, 400
ul of binding buffer is added and the cells are kept on ice until
analyzed on a BD FACSCanto flow cytometer. One or more cell lines
treated with LOR-253 show elevated Annexin V staining, indicating
induction of apoptosis. Increasing concentration of LOR-253 used to
treat the cells results in an increase in the early apoptotic
population (Q3: Annexin V+/PI-).
[0286] Cell lines are also treated with DMSO (vehicle control) or
0.1, 0.2, 0.5 or 1 .mu.M LOR-253 for 48 hours. Cell lysates are
collected using a 1% Triton-X100 lysis. Caspase 3 activity is
measured using EnzChek Caspase-3 Assay Kit #1 (Life Technologies)
with 5 .mu.g of cell lysastes according to manufacturer's protocol.
Treatment of one or more cell lines with LOR-253 results in
elevated Caspase 3 activity, indicating induction of apoptosis.
[0287] Total RNA of treated cell lines are extracted using the
TRIzol Plus RNA Purification kit (Ambion, Life Technologies),
according to the manufacturer's instructions. First strand cDNA is
synthesized from 1-2 .mu.g total RNA using random hexamer primers
(Invitrogen) and the SuperScript II Reverse Transcriptase kit
(Invitrogen). Quantitative RT-PCR is 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 is normalized with
.beta.-actin gene expression in the same sample, and fold changes
in BAX or BCL2 are 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 one or more myelodysplastic syndromes (MDS), ALL, CML, ATLL,
lymphoma, gastric cancer, or multiple myeloma cell lines with
LOR-253 indicates induction of apoptosis.
Example 12--In Vivo Efficacy of LOR-253 HCL in Xenograft Model of
Myelodysplastic Syndromes (MDS), ALL, CML, ATLL, Lymphoma, Gastric
Cancer, and Multiple Myeloma
[0288] Model mice of myelodysplastic syndromes (MDS), ALL, CML,
ATLL, lymphoma, gastric cancer, and multiple myeloma are created
according to standard procedures. The model mice are treated
according to several administration schedules as shown in the table
below.
TABLE-US-00008 Treatment Administration (1 cycle = 7, 14, 21, or
Group Dose level Schedule/cycle 28 days) 1 n = 8 1 mg/kg-iv
2T-12B-2T 1, 2, 3, or 4 cycles 2 n = 8 1 mg/kg-iv 3T-12B-3T 1, 2,
3, or 4 cycles 3 n = 8 1 mg/kg-iv 2T-5B-2T 1, 2, 3, or 4 cycles 4 n
= 8 1 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles 5 n = 8 5 mg/kg-iv
2T-12B-2T 1, 2, 3, or 4 cycles 6 n = 8 5 mg/kg-iv 3T-12B-3T 1, 2,
3, or 4 cycles 7 n = 8 5 mg/kg-iv 2T-5B-2T 1, 2, 3, or 4 cycles 8 n
= 8 5 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles 9 n = 8 10 mg/kg-iv
2T-12B-2T 1, 2, 3, or 4 cycles 10 n = 8 10 mg/kg-iv 3T-12B-3T 1, 2,
3, or 4 cycles 11 n = 8 10 mg/kg-iv 2T-5B-2T 1, 2, 3, or 4 cycles
12 n = 8 10 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles 13 n = 8 20
mg/kg-iv 2T-12B-2T 1, 2, 3, or 4 cycles 14 n = 8 20 mg/kg-iv
3T-12B-3T 1, 2, 3, or 4 cycles 15 n = 8 20 mg/kg-iv 2T-5B-2T 1, 2,
3, or 4 cycles 16 n = 8 20 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles
17 n = 8 Vehicle alone 3T-5B-3T 1, 2, 3, or 4 cycles T =
consecutive days treatment B = break for one
[0289] The results show that one or more groups are effectively
treated by LOR-253.
Example 13--In Vivo Dose-Dependent Pharmacokinetic (PK) and
Pharmacodynamic (PD) Responses in Xenograft Model Mice Treated by
LOR-253 HCL with Myelodysplastic Syndromes (MDS), ALL, CML, ATLL,
Lymphoma, Gastric Cancer, and Multiple Myeloma
[0290] To study the pharmacokinetic (PK) in myelodysplastic
syndromes (MDS), ALL, CML, ATLL, lymphoma, gastric cancer, and
multiple myeloma tumor cells treated by LOR-253, CD-1 nude mice are
treated by i.v. bolus injections of LOR-253 HCl at 1, 5, and 15
mg/kg. The serum level of LOR-253 in each treatment is
measured.
[0291] To study the pharmacodynamic (PD) responses, mice are
treated for 5 consecutive days with 1, 5, and 15 mg/kg LOR-253.
Tumors are measured 16 hours after the last dose, and the KLF-4
protein level is measured. This results show that average KLF4
protein levels in one or more treatment increase in a
dose-dependent manner, correlating with tumor biodistribution and
dose-response antitumor activity.
Example 14--In Vivo Efficacy of LOR-253 HCL in a Kasumi-1 AML
Xenograft Model
[0292] The anti-tumor activity of LOR-253 HCl 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-253 HCl 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").
[0293] The major endpoint was to observe for tumor growth
inhibition after treatment with LOR-253 HCl 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. 15).
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. 16).
[0294] The tumor inhibition results as shown in FIG. 15 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. 16 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-253 HCl showed significant tumor growth
inhibition as a single agent at twice per week dosing without
obvious signs of toxicity, suggesting that LOR-253 HCl has a
sufficient therapeutic window and that this agent is a potential
chemotherapeutic agent for the treatment of AML.
Example 15--In Vivo Efficacy of LOR-253 HCL in an HL-60 AML
Xenograft Model
[0295] The anti-tumor activity of LOR-253 HCl, 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-253 HCl alone or in combination with
azacitidine, with azacitidine alone, or with a negative control
vehicle. The detailed treatment conditions are as follows. [0296]
Group 1: Negative control group. LOR-253 HCl control vehicle,
2.lamda./day, 3 cycles of 2 consecutive days, 5 days between
cycles, i.v., plus 1% D-mannitol every four days by s.c. injection
[0297] 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
[0298] Group 3: LOR-253 HCl 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) [0299] Group 4: LOR-253 HCl 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) [0300]
Group 5: Combination of LOR-253 (2T-5B) and azacitidine. LOR-253
HCl 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 [0301] Group 6: Combination of LOR-253 (1T-6B) and
azacitidine. LOR-253 HCl 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
[0302] The major endpoint was to observe for tumor growth
inhibition after treatment with LOR-253 HCl 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. 17).
[0303] The tumor inhibition results as shown in FIG. 17 demonstrate
that LOR-253 HCl 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-253 HCl 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-253 HCl
treatment, respectively; compared to control, as determined by
Student's t-test). FIG. 18 and FIG. 19 show the tumor size data
from individual animals at the beginning (Day 1) and end (Day 19)
of this study.
[0304] Because LOR-253 HCl in combination with azacitadine resulted
in even higher levels of tumor growth inhibition than either single
agent alone, LOR-253 HCl may also provide additive anticancer
efficacy to a standard of care chemotherapeutic for hematological
malignancies.
Example 16--In Vivo Efficacy of LOR-253 HCL in a KG-1 AML Xenograft
Model
[0305] The anti-tumor activity of LOR-253 HCl 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 14 and 15, and were treated with LOR-253 HCl or control
according to the following regime. [0306] Control-IV-Day 1 [0307]
LOR-253-15 mg/kg-iv,bid,2T/wk-Day 1 [0308] Control-IV-Day 8 [0309]
LOR-253-15 mg/kg-iv,bid,2T/wk-Day 8 [0310] Control-IV-Day 13 [0311]
LOR-253-15 mg/kg-iv,bid,2T/wk-Day 13 [0312] Control-IV-Day 16
[0313] LOR-253-15 mg/kg-iv,bid,2T/wk-Day 16 [0314] Control-IV-Day
20 [0315] LOR-253-15 mg/kg-iv,bid,2T/wk-Day 20 [0316]
Control-IV-Day 26 [0317] LOR-253-15 mg/kg-iv,bid,2T/wk-Day 26
[0318] Tumor sizes were measure as described in Examples 14 and 15,
and the results are shown in FIG. 20. LOR-253 HCl showed
significant tumor growth inhibition as a single agent in this AML
animal model as well.
Example 17--In Vivo Efficacy of LOR-253 HCL in a THP-1 AML
Xenograft Model
[0319] The anti-tumor activity of LOR-253 HCl, 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
14 and 15, and were treated with LOR-253 HCl alone or in
combination with azacitidine, with azacitidine alone, or with a
negative control vehicle, and tumor sizes were measured as
described in Examples 14 and 15. The detailed treatment conditions
are shown as follows, and the results are shown in FIG. 21. [0320]
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.
[0321] 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 [0322] Group-3-Azacitidine: received SC treatments on
Days 1, 4, 8, 11, 15, 18, 22, 25, 29 &32. [0323]
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
[0324] The tumor inhibition results of this Example demonstrate
that LOR-253 HCl 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-253 HCl again resulted in significantly higher levels of tumor
growth inhibition.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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.
* * * * *