U.S. patent application number 15/524846 was filed with the patent office on 2019-07-11 for apilimod for use in the treatment of colorectal cancer.
The applicant listed for this patent is AI Therapeutics, Inc.. Invention is credited to Paul BECKETT, Neil BEEHARRY, Chris CONRAD, Sophia GAYLE, Marylens HERNANDEZ, Sean LANDRETTE, Henri LICHENSTEIN, Jonathan M. ROTHBERG, Tian XU.
Application Number | 20190209576 15/524846 |
Document ID | / |
Family ID | 54548301 |
Filed Date | 2019-07-11 |
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United States Patent
Application |
20190209576 |
Kind Code |
A1 |
BEEHARRY; Neil ; et
al. |
July 11, 2019 |
APILIMOD FOR USE IN THE TREATMENT OF COLORECTAL CANCER
Abstract
The present invention relates to methods for treating colorectal
cancer with apilimod and related compositions and methods.
Inventors: |
BEEHARRY; Neil; (Guilford,
CT) ; GAYLE; Sophia; (East Haven, CT) ;
LANDRETTE; Sean; (Meriden, CT) ; BECKETT; Paul;
(Yorktown Heights, NY) ; CONRAD; Chris; (Guilford,
CT) ; XU; Tian; (Cambridge, MA) ; HERNANDEZ;
Marylens; (Guilford, CT) ; ROTHBERG; Jonathan M.;
(Guilford, CT) ; LICHENSTEIN; Henri; (Guilford,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AI Therapeutics, Inc. |
Guilford |
CT |
US |
|
|
Family ID: |
54548301 |
Appl. No.: |
15/524846 |
Filed: |
November 6, 2015 |
PCT Filed: |
November 6, 2015 |
PCT NO: |
PCT/US2015/059526 |
371 Date: |
May 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62077127 |
Nov 7, 2014 |
|
|
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62115228 |
Feb 12, 2015 |
|
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62119540 |
Feb 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57419 20130101;
A61K 45/06 20130101; A61K 31/5377 20130101; A61K 9/0019 20130101;
A61K 9/0053 20130101; G01N 2800/52 20130101; G01N 33/5743 20130101;
A61K 31/437 20130101; A61P 35/04 20180101; A61K 31/44 20130101;
A61K 31/5377 20130101; A61K 2300/00 20130101; A61K 31/437 20130101;
A61K 2300/00 20130101; A61K 31/44 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; A61P 35/04 20060101 A61P035/04; A61K 45/06 20060101
A61K045/06; A61K 9/00 20060101 A61K009/00; A61K 31/437 20060101
A61K031/437 |
Claims
1. A method for treating colorectal cancer in a subject in need
thereof, the method comprising administering to the subject a
pharmaceutical composition comprising a therapeutically effective
amount of apilimod, or a pharmaceutically, acceptable salt
thereof.
2. The method of claim 1, wherein the apilimod is apilimod
dimesylate.
3. The method of claim 1, wherein the colorectal cancer is
refractory or metastatic.
4. The method of claim 1, wherein the composition is an oral dosage
form or a dosage form suitable for intravenous administration.
5. The method of claim 1, wherein the colorectal cancer is a stage
III or stage IV colorectal cancer, as defined by the TNM staging
system of the World Health Organization.
6. The method of claim 1, further comprising administering at least
one additional active agent.
7. The method of claim 1, wherein the at least one additional
active agent is a therapeutic agent or a non-therapeutic agent, or
a combination thereof.
8. The method of claim 7, wherein the therapeutic agent is selected
from the group consisting of a protein kinase inhibitor, a
PD-1/PDL-1 pathway inhibitor, a platinum based anti-neoplastic
agent, a topoisomerase inhibitor, a nucleoside metabolic inhibitor,
an alkylating agent, an intercalating agent, a tubulin binding
agent, a BRAT inhibitor, and combinations thereof.
9. The method of claim 8, wherein the therapeutic agent is selected
from the group consisting of vemurafenib, oxaliplatin, regorafenib,
irinotecan, 5-fluorouracil, pembrolizumab, avelumab, atezolizumab
(MPDL3280A), nivolumab (BMS-936558), pidilizumab (MK-3475),
MSB0010718C, MEDI4736, and combinations thereof.
10. The method of claim 9, wherein the therapeutic agent s
vemurafenib.
11. The method of claim 9, wherein the therapeutic agent s
regorafenib.
12. The method of claim 9, wherein the cancer is refractory or
metastatic colorectal cancer.
13. The method of claim 1 further comprising administering a
non-therapeutic agent selected to ameliorate one or more side
effects of the apilimod.
14. (canceled)
15. The method of claim 7, wherein the non-therapeutic agent is
selected from the group consisting of ondansetron, granisetron,
dolasetron and palonosetron.
16. The method of claim 7, wherein the non-therapeutic agent is
selected from the group consisting of pindolol and risperidone.
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. A method for inducing or potentiating autophagy or apoptosis in
a colorectal cancer cell, the method comprising contacting the cell
with a composition comprising apilimod dimesylate.
26. A method for identifying a human colorectal cancer patient for
treatment with a combination therapy comprising apilimod and
vemurafenib, the method comprising assaying a biological sample of
the subject's cancer for one or more of the V600E BRAF protein
mutation, the V600K BRAF protein mutation, or the genetic
equivalents thereof, wherein a subject having either of these
mutations is identified as a patient for treatment with a
combination therapy comprising apilimod and vemurafenib.
27. (canceled)
28. (canceled)
29. A method for identifying a colorectal cancer as sensitive to
apilimod treatment, the method comprising assaying the expression
of the SNX10 gene in a sample of the cancer, such as a biopsy
sample, wherein high SNX10 gene expression indicates that the
colorectal cancer is sensitive to apilimod treatment.
Description
RELATED APPLICATIONS
[0001] This application is a national stage entry, filed under 35
U.S.C. .sctn. 371, of International Application No.
PCT/US2015/059526, filed on Nov. 6, 2015, which claims priority to
U.S. Pat. App. Ser. No. 62/077,127, filed on Nov. 7, 2014, U.S.
Pat. App. Ser. No. 62/115,228, filed on Feb. 12, 2015, and U.S.
Pat. App. Ser. No. 62/119,540, filed on Feb. 23, 2015, the contents
of which are hereby fully incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions comprising
apilimod and methods of using same in the treatment of colorectal
cancer.
BACKGROUND OF THE INVENTION
[0003] Colorectal cancer remains a serious health concern. Globally
it is the third most common cancer, making up 10% of all cases. In
2012 there were 14 million new cases and 694,000 deaths from
colorectal cancer. Five year survival rates in the United States
are around 65%, highlighting the need for more effective
therapies.
[0004] Treatments for colorectal cancer may include some
combination of surgery, radiation therapy, chemotherapy, and
targeted therapy. Cancers that are confined within the wall of the
colon may be curable with surgery. But in many cases, it is not
possible to completely eliminate or cure the cancer once it has
metastasized. Depending upon where and how big the metastases are,
treatment may involve chemotherapy, surgery, gene therapy,
immunotherapy, radiation therapy, and combinations of these. There
remains a need for more effective treatment options for colorectal
cancer. The present invention addresses that need.
SUMMARY OF THE INVENTION
[0005] The present invention is based in part on the surprising
discovery that apilimod is a highly cytotoxic agent in colorectal
cancer cells, including colorectal cancer cells that are resistant
to other therapies, and further in combination with certain
chemotherapy agents.
[0006] In one aspect, the disclosure provides a composition for
treating colorectal cancer in a subject in need thereof, the
composition comprising a therapeutically effective amount of
apilimod, or a pharmaceutically acceptable salt, solvate,
clathrate, hydrate, polymorph, prodrug, analog or derivative
thereof. In embodiments, the apilimod is apilimod dimesylate. In
embodiments, the colorectal cancer is refractory or metastatic. In
embodiments, the colorectal cancer is a stage III or stage IV
colorectal cancer, as defined by the TNM staging system of the
World Health Organization. In embodiments, the composition is an
oral dosage form or a dosage form suitable for intravenous
administration.
[0007] In embodiments, the composition further comprises at least
one additional active agent. In embodiments, the at least one
additional active agent is a therapeutic agent or a non-therapeutic
agent, or a combination thereof. In embodiments, the at least one
additional active agent is a therapeutic agent selected from the
group consisting of a protein kinase inhibitor, a platinum based
anti-neoplastic agent, a topoisomerase inhibitor, a nucleoside
metabolic inhibitor, an alkylating agent, an intercalating agent, a
tubulin binding agent, a BRAF inhibitor, and combinations thereof.
In embodiments, the therapeutic agent is selected from the group
consisting of vemurafenib, oxaliplatin, regorafenib, irinotecan,
5-fluorouracil, and combinations thereof. In one embodiment, the
therapeutic agent is vemurafenib or regorafenib.
[0008] In embodiments, the composition further comprises a
non-therapeutic agent selected to ameliorate one or more side
effects of the apilimod. In embodiments, the non-therapeutic agent
is selected from the group consisting of ondansetron, granisetron,
dolasetron and palonosetron or from pindolol and risperidone.
[0009] In one aspect, the disclosure provides a method for treating
colorectal cancer in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of apilimod, or a pharmaceutically acceptable salt, solvate,
clathrate, hydrate, polymorph, prodrug, analog or derivative
thereof. In embodiments, the apilimod is apilimod dimesylate.
[0010] In embodiments, the method further comprises administering
at least one additional active agent to the subject. The at least
one additional active agent may be a therapeutic agent or a
non-therapeutic agent, or a combination thereof. The at least one
additional active agent may be administered in a single dosage form
with the apilimod or in a separate dosage form.
[0011] In embodiments, the at least one additional active agent is
a therapeutic agent. In embodiments, the therapeutic agent is a
B-Raf enzyme inhibitor. In embodiments, the therapeutic agent is
vemurafenib, oxaliplatin, regorafenib, irinotecan,
5-fluorouracil.
[0012] In embodiments, the method comprises administering apilimod
and vemurafenib together in a combination therapy regimen for the
treatment of colorectal cancer. In embodiments, the colorectal
cancer is a late-stage colorectal cancer. In one embodiment, the
late-stage colorectal cancer is resistant to vemurafenib alone.
[0013] In embodiments, the at least one additional active agent is
selected from an alkylating agent, an intercalating agent, a
tubulin binding agent, a PD-1/PDL-1 pathway inhibitor, and
combinations thereof. In embodiments, the at least one additional
active agent is a therapeutic agent selected from vemurafenib,
5-fluorouracil (Fluoroplex.RTM., irinotecan (Camptostar.RTM.),
capecitabine (Xeloda.RTM.), oxaliplatin (Eloxatin.RTM.) and
regorafenib (Stivarga.RTM.), and combinations thereof. In
embodiments, the at least one additional active agent is a
therapeutic agent selected from bevacizumab (Avastin.RTM.),
ramucirumab (Cyramza.RTM.), cetuximab (Erbitex.RTM.), panitumumab
(Vecibix.RTM.), ipilimumab (Yervoy.RTM.), pembrolizumab
(Keytruda.TM.), dabrafenib (Tafinlar.TM.), vemurafenib
(Zelboraf.TM.), trametinib (Mekinist.TM.), Zviv-Aflibercept
(Zaltrap.RTM.), nivolumab (Opdivo.RTM.) and combinations thereof.
In embodiments, the at least one additional active agent is a
PD-1/PDL-1 pathway inhibitor. In embodiments, the PD-1/PDL-1
pathway inhibitor is selected from pembrolizumab (Keytruda),
avelumab, atezolizumab (MPDL3280A), nivolumab (BMS-936558),
pidilizumab (MK-3475), MSB0010718C, and MEDI4736.
[0014] In embodiments, the at least one additional active agent is
a non-therapeutic agent selected to ameliorate one or more side
effects of apilimod. In one embodiment, the non-therapeutic agent
is selected from the group consisting of ondansetron, granisetron,
dolasetron and palonosetron. In one embodiment, the non-therapeutic
agent is selected from the group consisting of pindolol and
risperidone.
[0015] In embodiments, the apilimod may be administered in any
suitable dosage form. In one embodiment, the apilimod is
administered in an oral dosage form. In another embodiment, the
dosage form is suitable for intravenous administration. In one
embodiment, where the dosage form is suitable for intravenous
administration, administration is by a single injection or by a
drip bag.
[0016] In embodiments of the methods described here, the subject is
a human colorectal cancer patient. In one embodiment, the human
colorectal cancer patient in need of treatment according to the
methods described here is one having late-stage, malignant or
metastatic colorectal cancer. In embodiments, the human colorectal
cancer patient in need of treatment is one whose cancer is
refractory to a standard chemotherapy regimen. In embodiments, the
human colorectal cancer patient in need of treatment is one whose
colorectal cancer has recurred following treatment with a standard
chemotherapy regimen.
[0017] In embodiments, the standard chemotherapy regimen comprises
an immunotherapy regimen or a targeted therapy regimen. In
embodiments, the immunotherapy regimen comprises one or more
therapeutic agents selected from the group consisting of anti-CTLA4
antibodies (e.g., ipilimumab), PD-1/PDL-1 pathway inhibitors (e.g.,
pembrolizumab (Keytruda), MSB0010718C, MEDI4736, MPDL3280A,
BMS-936559), nivolumab (Opdivo), pidilizumab, AMP-224, and
Interleukin-2 (IL-2, aldesleukin, Proleukin). In embodiments, the
targeted therapy regimen comprises one or more therapeutic agents
selected from the group consisting of BRAF inhibitors (e.g.,
dabrafenib (Tafinlar), sorafenib (Nexavar) and vemurafenib
(Zelboraf), MEK inhibitors (e.g., trametinib (Mekinist)) and KIT
inhibitors (e.g., dasatinib (Sprycel), imatinib (Gleevec), and
nilotinib (Tasigna)). In embodiments, the PD-1/PDL-1 pathway
inhibitor is selected from pembrolizumab (Keytruda), avelumab,
atezolizumab (MPDL3280A), nivolumab (BMS-936558), pidilizumab
(MK-3475), MSB0010718C, and MEDI4736.
[0018] In embodiments, the method is a method of treating a
colorectal cancer using a combination therapy comprising apilimod
and a chemotherapy regimen for the treatment of the colorectal
cancer. In one embodiment, the apilimod is administered as an
adjunctive therapy to the chemotherapy regimen. In embodiments, the
chemotherapy regimen comprises one or more of dacarbazine,
temozolomide, Nab-paclitaxel, carmustine, cisplatin, carboplatin,
and vinblastine for the treatment of malignant or metastatic
colorectal cancer. In embodiments, the chemotherapy regimen
comprises one or more of vemurafenib, dabrafenib and trametinib for
metastatic colorectal cancer. In one embodiment, the chemotherapy
regimen comprises one or more of high dose interleukin-2 and
ipilimumab.
[0019] In one embodiment, the disclosure provides methods for
inducing or potentiating autophagy or apoptosis in a colorectal
cancer cell. In accordance with this embodiment, the colorectal
cancer cell may be in vitro or in vivo. In one embodiment, the
colorectal cancer cell is in vitro. In one embodiment, the
colorectal cancer cell is in vivo in a mammalian subject. In one
embodiment, the colorectal cancer cell is a late stage colorectal
cancer. In one embodiment, the colorectal cancer cell is a
metastatic cell or a cell that has metastasized. In embodiments,
the colorectal cancer cell has a BRAF mutant phenotype. In
embodiments, the BRAF mutant phenotype is characterized by the
V600E or V600K mutation of human BRAF.
[0020] The disclosure also provides a method for inducing or
potentiating autophagy or apoptosis in a colorectal cancer cell,
the method comprising contacting the cell with a composition
comprising apilimod, or a pharmaceutically acceptable salt,
solvate, clathrate, hydrate, polymorph, prodrug, analog or
derivative thereof. In embodiments, the apilimod is apilimod
dimesylate.
[0021] The disclosure also provides a method for identifying a
human colorectal cancer patient for treatment with a combination
therapy comprising an apilimod composition and vemurafenib, the
method comprising assaying a biological sample of the subject's
cancer for one or more of the V600E BRAF protein mutation, the
V600K BRAF protein mutation, or the genetic equivalents thereof,
wherein a subject having either of these mutations is identified as
a patient for treatment with a combination therapy comprising an
apilimod composition and vemurafenib.
[0022] In embodiments, the disclosure also provides a method for
identifying a colorectal cancer that is sensitive to apilimod, the
method comprising assaying a sample of cancer cells from the
cancer, for example a biopsy, for expression of the SNX10 gene,
wherein high expression levels of SNX10 in the sample of cancer
cells indicates that the cells are sensitive to apilimod. In
embodiments, the SNX10 gene expression level is called as `high`
relative to the SNX10 gene expression level of a reference cell
line. For example, an SNX10 gene expression level in a biopsy
sample that is higher than that of COL0205 cells, may be considered
"high" for purposes of indicating sensitivity to apilimod.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1: LAM-002 (apilimod dimesylate)+oxaliplatin
combination in HCT116 cells (5 day assay). A, bar graph showing
cell viability (%). B, combination index (CI) vs fractional effect
graph showing synergy determinations at ED.sub.50, ED.sub.75 and
ED.sub.90.
[0024] FIG. 2: LAM-002+regorafenib combination in HCT116 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0025] FIG. 3: LAM-002+regorafenib combination in RKO cells (5 day
assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0026] FIG. 4: LAM-002+irinotecan combination in HCT116 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0027] FIG. 5: LAM-002+5-fluorouracil combination in HCT116 cells
(5 day assay). A, bar graph showing cell viability (%). B,
combination index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0028] FIG. 6: LAM-002+vemurafenib combination in HCT116 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0029] FIG. 7: LAM-002+vemurafenib combination in RKO cells (5 day
assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0030] FIG. 8: LAM-002+vemurafenib combination in HT-29 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0031] FIG. 9: LAM-002+vemurafenib combination in HCT-15 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0032] FIG. 10: LAM-002+vemurafenib combination in SW1116 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0033] FIG. 11: LAM-002+vemurafenib combination in SW480 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
[0034] FIG. 12: LAM-002+vemurafenib combination in SW620 cells (5
day assay). A, bar graph showing cell viability (%). B, combination
index (CI) vs fractional effect graph showing synergy
determinations at ED.sub.50, ED.sub.75 and ED.sub.90.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention provides compositions and methods
related to the use of apilimod for treating colorectal cancer in a
subject, preferably a human subject, in need of such treatment. The
invention generally relates to new uses of apilimod based upon the
surprising discovery of apilimod's cytotoxic activity against a
range of cancer cells including numerous colorectal cancer cell
lines. In addition, the present invention provides novel
therapeutic approaches to colorectal cancer treatment based upon
combination therapy utilizing apilimod and at least one additional
therapeutic agent. The combination therapies described herein
exploit the unique cytotoxic activity of apilimod which can provide
a synergistic effect when combined with other therapeutic agents,
including for example, anti-cancer agents.
[0036] As used throughout the present disclosure, the term
"apilimod" may refer to apilimod itself (free base), or may
encompass pharmaceutically acceptable salts, solvates, clathrates,
hydrates, polymorphs, prodrugs, analogs or derivatives of apilimod,
as described below. In embodiments of the methods for treating
colorectal cancer described here, the apilimod is apilimod
dimesylate. The structure of apilimod free base is shown in Formula
I:
##STR00001##
[0037] The IUPAC name of apilimod is:
(E)-4-(6-(2-(3-methylbenzylidene)hydrazinyl)-2-(2-(pyridin-2-yl)ethoxy)py-
rimidin-4-yl)morpholine) and the CAS number is 541550-19-0.
[0038] Apilimod can be prepared, for example, according to the
methods described in U.S. Pat. Nos. 7,923,557, and 7,863,270, and
WO 2006/128129.
[0039] As used herein, the term "pharmaceutically acceptable salt,"
is a salt formed from, for example, an acid and a basic group of an
apilimod composition. Illustrative salts include, but are not
limited, to sulfate, citrate, acetate, oxalate, chloride, bromide,
iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
besylate, gentisinate, fumarate, gluconate, glucaronate,
saccharate, formate, benzoate, glutamate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate
(e.g., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts.
[0040] The term "pharmaceutically acceptable salt" also refers to a
salt prepared from an apilimod composition having an acidic
functional group, such as a carboxylic acid functional group, and a
pharmaceutically acceptable inorganic or organic base.
[0041] The term "pharmaceutically acceptable salt" also refers to a
salt prepared from an apilimod composition having a basic
functional group, such as an amino functional group, and a
pharmaceutically acceptable inorganic or organic acid.
[0042] The salts of the compounds described herein can be
synthesized from the parent compound by conventional chemical
methods such as methods described in Pharmaceutical Salts:
Properties, Selection, and Use, P. Hemrich Stahl (Editor), Camille
G. Wermuth (Editor), ISBN: 3-90639-026-8, August 2002. Generally,
such salts can be prepared by reacting the parent compound with the
appropriate acid in water or in an organic solvent, or in a mixture
of the two.
[0043] One salt form of a compound described herein can be
converted to the free base and optionally to another salt form by
methods well known to the skilled person. For example, the free
base can be formed by passing the salt solution through a column
containing an amine stationary phase (e.g. a Strata-NH.sub.2
column). Alternatively, a solution of the salt in water can be
treated with sodium bicarbonate to decompose the salt and
precipitate out the free base. The free base may then be combined
with another acid using routine methods.
[0044] As used herein, the term "polymorph" means solid crystalline
forms of a compound of the present invention (e.g., apilimod) or
complex thereof. Different polymorphs of the same compound can
exhibit different physical, chemical and/or spectroscopic
properties. Different physical properties include, but are not
limited to stability (e.g., to heat or light), compressibility and
density (important in formulation and product manufacturing), and
dissolution rates (which can affect bioavailability). Differences
in stability can result from changes in chemical reactivity (e.g.,
differential oxidation, such that a dosage form discolors more
rapidly when comprised of one polymorph than when comprised of
another polymorph) or mechanical characteristics (e.g., tablets
crumble on storage as a kinetically favored polymorph converts to
thermodynamically more stable polymorph) or both (e.g., tablets of
one polymorph are more susceptible to breakdown at high humidity).
Different physical properties of polymorphs can affect their
processing. For example, one polymorph might be more likely to form
solvates or might be more difficult to filter or wash free of
impurities than another due to, for example, the shape or size
distribution of particles of it.
[0045] As used herein, the term "hydrate" means a compound of the
present invention (e.g., apilimod) or a salt thereof, which further
includes a stoichiometric or non-stoichiometric amount of water
bound by non-covalent intermolecular forces.
[0046] As used herein, the term "clathrate" means a compound of the
present invention (e.g., apilimod) or a salt thereof in the form of
a crystal lattice that contains spaces (e.g., channels) that have a
guest molecule (e.g., a solvent or water) trapped within.
[0047] As used herein, the term "prodrug" means a derivative of a
compound described herein (e.g., apilimod) that can hydrolyze,
oxidize, or otherwise react under biological conditions (in vitro
or in vivo) to provide a compound of the invention. Prodrugs may
only become active upon such reaction under biological conditions,
or they may have activity in their unreacted forms. Examples of
prodrugs contemplated in this invention include, but are not
limited to, analogs or derivatives of a compound described herein
(e.g., apilimod) that comprise biohydrolyzable moieties such as
biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable
carbamates, biohydrolyzable carbonates, biohydrolyzable ureides,
and biohydrolyzable phosphate analogues. Other examples of prodrugs
include derivatives of compounds of any one of the formulae
disclosed herein that comprise --NO, --NO.sub.2, --ONO, or
--ONO.sub.2 moieties. Prodrugs can typically be prepared using
well-known methods, such as those described by Burger's Medicinal
Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E.
Wolff ed., 5th ed).
[0048] As used herein, the term "solvate" or "pharmaceutically
acceptable solvate," is a solvate formed from the association of
one or more solvent molecules to one of the compounds disclosed
herein (e.g., apilimod). The term solvate includes hydrates (e.g.,
hemi-hydrate, mono-hydrate, dihydrate, trihydrate, tetrahydrate,
and the like).
[0049] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another but differs
slightly in composition (as in the replacement of one atom by an
atom of a different element or in the presence of a particular
functional group, or the replacement of one functional group by
another functional group). Thus, an analog is a compound that is
similar or comparable in function and appearance, but not in
structure or origin to the reference compound. As used herein, the
term "derivative" refers to compounds that have a common core
structure, and are substituted with various groups as described
herein.
Colorectal Cancer
[0050] Colorectal cancer (also known as colon cancer, rectal
cancer, or bowel cancer) is the development of cancer in the colon
or rectum. Colon cancer is staged according to the TNM staging
system. The TNM system is one of the most widely used cancer
staging systems and has been adopted by the Union for International
Cancer Control (UICC) and the American Joint Committee on Cancer
(AJCC). The TNM system is based on the size and/or extent (reach)
of the primary tumor (T), the amount of spread to nearby lymph
nodes (N), and the presence of metastasis (M) or secondary tumors
formed by the spread of cancer cells to other parts of the body. A
number is added to each letter to indicate the size and/or extent
of the primary tumor and the degree of cancer spread.
[0051] In both cancer of the colon and rectum, chemotherapy may be
used in addition to surgery in certain cases. The decision to add
chemotherapy in management of colon and rectal cancer depends on
the stage of the disease and is more common for later stages, e.g.,
stages III and IV, compared to early stages (I and II) where
surgery alone is the standard treatment. For stage III and IV colon
cancer (cancer in which the cancer has spread to the lymph nodes or
distant organs), fluorouracil, capecitabine or oxaliplatin
chemotherapy is standard treatment.
[0052] If the cancer is widely metastatic or unresectable,
treatment is then palliative. Typical chemotherapy medications used
may include capecitabine, fluorouracil, irinotecan, and
oxaliplatin. Anti-angiogenic drugs such as bevacizumab or epidermal
growth factor receptor inhibitors, such as cetuximab and
panitumumab, may also be used.
Methods of Treatment
[0053] The present invention provides methods for the treatment of
colorectal cancer in a subject in need thereof by administering to
the subject a therapeutically effective amount of an apilimod
composition of the invention, said composition comprising apilimod,
or a pharmaceutically acceptable salt, solvate, clathrate, hydrate,
polymorph, prodrug, analog or derivative thereof. In one
embodiment, the apilimod composition comprises apilimod free base
or apilimod dimesylate. The present invention further provides the
use of an apilimod composition for the preparation of a medicament
useful for the treatment of colorectal cancer.
[0054] In the context of the methods described herein, the amount
of an apilimod composition administered to the subject is a
therapeutically effective amount. The term "therapeutically
effective amount" refers to an amount sufficient to treat,
ameliorate a symptom of, reduce the severity of, or stabilize or
cause the regression of colorectal cancer in the subject being
treated, or to enhance or improve the therapeutic effect of another
therapy, such as vemurafenib.
[0055] In accordance with the methods described herein, a "subject
in need of" is a subject having colorectal cancer. In one aspect,
the subject is a human patient having malignant colorectal cancer
or late-stage colorectal cancer. In this context, "stage" refers to
the clinical stage of the cancer. For example, stage 0 to 2
colorectal cancer or stage 3 or stage 4 colorectal cancer. In
embodiments, the subject is a human patient having stage 3 or stage
4 colorectal cancer. The subject in need of treatment can also be
one that is "non-responsive" or "refractory" to a currently
available therapy. For example the subject's cancer may be
resistant or refractory to treatment with vemurafenib or one or
more of oxaliplatin, regorafenib, irinotecan, 5-fluorouracil. In
embodiments, the subject in need of treatment is one having a
cancer characterized by the V600E or V600K mutation of human BRAF.
In the context of the present disclosure, the terms
"non-responsive" and "refractory" refer to the subject's response
to therapy as not clinically significant according to the
definition for a clinical response in standard medical
practice.
Combination Therapy
[0056] The present invention also provides methods comprising
combination therapy. As used herein, "combination therapy" or
"co-therapy" includes the administration of a therapeutically
effective amount of an apilimod composition with at least one
additional active agent, as part of a specific treatment regimen
intended to provide a beneficial effect from the co-action of the
apilimod composition and the additional active agent. "Combination
therapy" is not intended to encompass the administration of two or
more therapeutic compounds as part of separate monotherapy regimens
that incidentally and arbitrarily result in a beneficial effect
that was not intended or predicted.
[0057] In one embodiment, the method is a method of treating
colorectal cancer using a combination therapy comprising apilimod
and a chemotherapy regimen for the treatment of colorectal cancer.
In embodiments, the chemotherapy regimen comprises one or more of
vemurafenib, oxaliplatin, regorafenib, irinotecan, or
5-fluorouracil.
[0058] The at least one additional active agent may be a
therapeutic agent, for example an anti-cancer agent or a cancer
chemotherapeutic agent, or a non-therapeutic agent, and
combinations thereof. With respect to therapeutic agents, the
beneficial effect of the combination includes, but is not limited
to, pharmacokinetic or pharmacodynamic co-action resulting from the
combination of therapeutically active compounds. With respect to
non-therapeutic agents, the beneficial effect of the combination
may relate to the mitigation of a toxicity, side effect, or adverse
event associated with a therapeutically active agent in the
combination.
[0059] In one embodiment, the at least one additional agent is a
non-therapeutic agent which mitigates one or more side effects of
an apilimod composition, the one or more side effects selected from
any of nausea, vomiting, headache, dizziness, lightheadedness,
drowsiness and stress. In one aspect of this embodiment, the
non-therapeutic agent is an antagonist of a serotonin receptor,
also known as 5-hydroxytryptamine receptors or 5-HT receptors. In
one aspect, the non-therapeutic agent is an antagonist of a
5-HT.sub.3 or 5-HT.sub.1a receptor. In one aspect, the
non-therapeutic agent is selected from the group consisting of
ondansetron, granisetron, dolasetron and palonosetron. In another
aspect, the non-therapeutic agent is selected from the group
consisting of pindolol and risperidone.
[0060] In one embodiment, the at least one additional active agent
is a therapeutic agent. In one embodiment, the therapeutic agent is
an anti-cancer agent. In embodiments, the anti-cancer agent is
vemurafenib, oxaliplatin, regorafenib, irinotecan, or
5-fluorouracil, and combinations thereof. In embodiments, the
anti-cancer agent is vemurafenib or regoragenib.
[0061] In embodiments, a composition comprising apilimod is
administered along with the at least one additional active agent in
a single dosage form or in separate dosage forms. In one
embodiment, the dosage form is an oral dosage form. In another
embodiment, the dosage form is suitable for intravenous
administration.
[0062] In one embodiment, the anti-cancer agent is a drug that is
approved for use in treating colorectal cancer. Non-limiting
examples of such drugs include oxaliplatin, regorafenib,
irinotecan, and 5-fluorouracil.
[0063] In embodiments, the anti-cancer agent is selected from
vermurafenib, oxaliplatin, regorafenib, irinotecan, and
5-fluorouracil.
[0064] In embodiments, the at least one additional agent is a
monoclonal antibody such as, for example, alemtuzumab, bevacizumab,
catumaxomab, cetuximab, edrecolomab, gemtuzumab, ofatumumab,
panitumumab, rituximab, trastuzumab, eculizumab, efalizumab,
muromab-CD3, natalizumab, adalimumab, afelimomab, certolizumab
pegol, golimumab, infliximab, basiliximab, canakinumab, daclizumab,
mepolizumab, tocilizumab, ustekinumab, ibritumomab tiuxetan,
tositumomab, abagovomab, adecatumumab, alemtuzumab, anti-CD30
monoclonal antibody Xmab2513, anti-MET monoclonal antibody MetMab,
apolizumab, apomab, arcitumomab, basiliximab, bispecific antibody
2B1, blinatumomab, brentuximab vedotin, capromab pendetide,
cixutumumab, claudiximab, conatumumab, dacetuzumab, denosumab,
eculizumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab,
fresolimumab, galiximab, ganitumab, gemtuzumab ozogamicin,
glembatumumab, ibritumomab, inotuzumab ozogamicin, ipilimumab,
lexatumumab, lintuzumab, lintuzumab, lucatumumab, mapatumumab,
matuzumab, milatuzumab, monoclonal antibody CC49, necitumumab,
nimotuzumab, ofatumumab, oregovomab, pertuzumab, ramacurimab,
ranibizumab, siplizumab, sonepcizumab, tanezumab, tositumomab,
trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab,
visilizumab, volociximab, and zalutumumab.
[0065] In embodiments, the at least one additional inhibitor is a
BRAF inhibitor, a MEK inhibitor, PD-1/PDL-1 pathway inhibitor, or a
check point inhibitor. In embodiments, the PD-1/PDL-1 pathway
inhibitor is selected from pembrolizumab (Keytruda), avelumab,
atezolizumab (MPDL3280A), nivolumab (BMS-936558), pidilizumab
(MK-3475), MSB0010718C, and MEDI4736.
[0066] In the context of combination therapy, administration of the
apilimod may be simultaneous with or sequential to the
administration of the one or more additional active agents. In
another embodiment, administration of the different components of a
combination therapy may be at different frequencies. The one or
more additional agents may be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a compound of the present
invention.
[0067] The one or more additional active agents can be formulated
for co-administration with apilimod in a single dosage form, as
described in greater detail herein. The one or more additional
active agents can be administered separately from the dosage form
that comprises the compound of the present invention. When the
additional active agent is administered separately from the
apilimod composition, it can be by the same or a different route of
administration as the apilimod composition.
[0068] Preferably, the administration of apilimod in combination
with one or more additional agents provides a synergistic response
in the subject being treated. In this context, the term
"synergistic" refers to the efficacy of the combination being more
effective than the additive effects of either single therapy alone.
The synergistic effect of a combination therapy according to the
invention can permit the use of lower dosages and/or less frequent
administration of at least one agent in the combination compared to
its dose and/or frequency outside of the combination. Additional
beneficial effects of the combination can be manifested in the
avoidance or reduction of adverse or unwanted side effects
associated with the use of either therapy in the combination alone
(also referred to as monotherapy).
[0069] "Combination therapy" also embraces the administration of
the compounds of the present invention in further combination with
non-drug therapies (e.g., surgery or radiation treatment). Where
the combination therapy further comprises a non-drug treatment, the
non-drug treatment may be conducted at any suitable time so long as
a beneficial effect from the co-action of the combination of the
therapeutic compounds and non-drug treatment is achieved. For
example, in appropriate cases, the beneficial effect is still
achieved when the non-drug treatment is temporally removed from the
administration of the therapeutic compounds, perhaps by days or
even weeks.
[0070] In accordance with any of the methods described herein, a
therapeutically effective amount of apilimod, e.g., apilimod
dimesylate, can range from about 0.001 mg/kg to about 1000 mg/kg,
about 0.01 mg/kg to about 100 mg/kg, about 10 mg/kg to about 250
mg/kg, about 0.1 mg/kg to about 15 mg/kg; or any range in which the
low end of the range is any amount between 0.001 mg/kg and 900
mg/kg and the upper end of the range is any amount between 0.1
mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg
and 20 mg/kg). Effective doses will also vary, as recognized by
those skilled in the art, depending on the diseases treated, route
of administration, excipient usage, and the possibility of co-usage
with other therapeutic treatments such as use of other agents. See,
e.g., U.S. Pat. No. 7,863,270, incorporated herein by
reference.
[0071] In more specific aspects, apilimod, e.g., apilimod
dimesylate, is administered at a dosage regimen of 30-1000 mg/day
(e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
125, 150, 175, 200, 225, 250, 275, or 300 mg/day) for at least 1
week (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 36, 48, or more
weeks). Preferably, apilimod is administered at a dosage regimen of
100-1000 mg/day for 4 or 16 weeks. Alternatively or subsequently,
apilimod is administered at a dosage regimen of 100 mg-300 mg twice
a day for 8 weeks, or optionally, for 52 weeks. Alternatively or
subsequently, apilimod is administered at a dosage regimen of 50
mg-1000 mg twice a day for 8 weeks, or optionally, for 52
weeks.
[0072] A therapeutically effective amount of the apilimod can be
administered once daily, from two to five times daily, up to two
times or up to three times daily, or up to eight times daily. In
one embodiment, the apilimod is administered thrice daily, twice
daily, once daily, fourteen days on (four times daily, thrice daily
or twice daily, or once daily) and 7 days off in a 3-week cycle, up
to five or seven days on (four times daily, thrice daily or twice
daily, or once daily) and 14-16 days off in 3 week cycle, or once
every two days, or once a week, or once every 2 weeks, or once
every 3 weeks.
[0073] A "subject" as used in the context of the methods described
herein is preferably a human subject but may also include other
mammals. The mammal can be e.g., any mammal, e.g., a human,
primate, vertebrate, bird, mouse, rat, fowl, dog, cat, cow, horse,
goat, camel, sheep or a pig. The term "patient" refers to a human
subject.
[0074] The present invention also provides a monotherapy for the
treatment of colorectal cancer as described herein. As used herein,
"monotherapy" refers to the administration of a single active agent
(also referred to as the therapeutic agent), e.g., apilimod, and in
embodiments, apilimod dimesylate, to a subject in need thereof.
[0075] As used herein, "treatment", "treating" or "treat" describes
the management and care of a patient for the purpose of combating
the colorectal cancer and includes alleviating one or more symptoms
or complications of the cancer, including for example slowing the
growth of the cancer, slowing or preventing the occurrence of
metastases, or further metastases, and promoting regression of one
or more tumors in the subject being treated.
[0076] In embodiments, the administration of a composition as
described herein leads to the elimination of a symptom or
complication of the cancer being treated, however, elimination is
not required. In one embodiment, the severity of the symptom is
decreased. In the context of cancer, such symptoms may include
clinical markers of severity or progression including the degree to
which a tumor secrets growth factors, degrades the extracellular
matrix, becomes vascularized, loses adhesion to juxtaposed tissues,
or metastasizes, as well as the number of metastases.
[0077] Treating colorectal cancer according to the methods
described herein can result in a reduction in size of a tumor. A
reduction in size of a tumor may also be referred to as "tumor
regression". Preferably, after treatment, tumor size is reduced by
5% or greater relative to its size prior to treatment; more
preferably, tumor size is reduced by 10% or greater; more
preferably, reduced by 20% or greater; more preferably, reduced by
30% or greater; more preferably, reduced by 40% or greater; even
more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75% or greater. Size of a tumor may be
measured by any reproducible means of measurement. The size of a
tumor may be measured as a diameter of the tumor.
[0078] Treating colorectal cancer according to the methods
described herein can result in a reduction in tumor volume.
Preferably, after treatment, tumor volume is reduced by 5% or
greater relative to its size prior to treatment; more preferably,
tumor volume is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75% or greater. Tumor volume may be measured by any
reproducible means of measurement.
[0079] Treating colorectal cancer according to the methods
described herein can result in a decrease in number of tumors.
Preferably, after treatment, tumor number is reduced by 5% or
greater relative to number prior to treatment; more preferably,
tumor number is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. Number of tumors may be measured by any reproducible
means of measurement. The number of tumors may be measured by
counting tumors visible to the naked eye or at a specified
magnification. Preferably, the specified magnification is 2.times.,
3.times., 4.times., 5.times., 10.times., or 50.times..
[0080] Treating colorectal cancer according to the methods
described herein can result in a decrease in number of metastatic
lesions in other tissues or organs distant from the primary tumor
site. Preferably, after treatment, the number of metastatic lesions
is reduced by 5% or greater relative to number prior to treatment;
more preferably, the number of metastatic lesions is reduced by 10%
or greater; more preferably, reduced by 20% or greater; more
preferably, reduced by 30% or greater; more preferably, reduced by
40% or greater; even more preferably, reduced by 50% or greater;
and most preferably, reduced by greater than 75%. The number of
metastatic lesions may be measured by any reproducible means of
measurement. The number of metastatic lesions may be measured by
counting metastatic lesions visible to the naked eye or at a
specified magnification. Preferably, the specified magnification is
2.times., 3.times., 4.times., 5.times., 10.times., or
50.times..
[0081] Treating colorectal cancer according to the methods
described herein can result in an increase in average survival time
of a population of treated subjects in comparison to a population
receiving carrier alone. Preferably, the average survival time is
increased by more than 30 days; more preferably, by more than 60
days; more preferably, by more than 90 days; and most preferably,
by more than 120 days. An increase in average survival time of a
population may be measured by any reproducible means. An increase
in average survival time of a population may be measured, for
example, by calculating for a population the average length of
survival following initiation of treatment with an active compound.
An increase in average survival time of a population may also be
measured, for example, by calculating for a population the average
length of survival following completion of a first round of
treatment with an active compound.
[0082] Treating colorectal cancer according to the methods
described herein can result in increase in average survival time of
a population of treated subjects in comparison to a population
receiving monotherapy with a drug that is not an apilimod
composition as described herein. Preferably, the average survival
time is increased by more than 30 days; more preferably, by more
than 60 days; more preferably, by more than 90 days; and most
preferably, by more than 120 days. An increase in average survival
time of a population may be measured by any reproducible means. An
increase in average survival time of a population may be measured,
for example, by calculating for a population the average length of
survival following initiation of treatment with an active compound.
An increase in average survival time of a population may also be
measured, for example, by calculating for a population the average
length of survival following completion of a first round of
treatment with an active compound.
[0083] Treating colorectal cancer according to the methods
described herein can result in a decrease in the mortality rate of
a population of treated subjects in comparison to a population
receiving carrier alone. Treating colorectal cancer according to
the methods described herein can result in a decrease in the
mortality rate of a population of treated subjects in comparison to
an untreated population. Treating colorectal cancer according to
the methods described herein can result in a decrease in the
mortality rate of a population of treated subjects in comparison to
a population receiving monotherapy with a drug that is not an
apilimod composition. Preferably, the mortality rate is decreased
by more than 2%; more preferably, by more than 5%; more preferably,
by more than 10%; and most preferably, by more than 25%. A decrease
in the mortality rate of a population of treated subjects may be
measured by any reproducible means. A decrease in the mortality
rate of a population may be measured, for example, by calculating
for a population the average number of disease-related deaths per
unit time following initiation of treatment with an active
compound. A decrease in the mortality rate of a population may also
be measured, for example, by calculating for a population the
average number of disease-related deaths per unit time following
completion of a first round of treatment with an active
compound.
[0084] Treating colorectal cancer according to the methods
described herein can result in a decrease in tumor growth rate.
Preferably, after treatment, tumor growth rate is reduced by at
least 5% relative to number prior to treatment; more preferably,
tumor growth rate is reduced by at least 10%; more preferably,
reduced by at least 20%; more preferably, reduced by at least 30%;
more preferably, reduced by at least 40%; more preferably, reduced
by at least 50%; even more preferably, reduced by at least 50%; and
most preferably, reduced by at least 75%. Tumor growth rate may be
measured by any reproducible means of measurement. Tumor growth
rate can be measured according to a change in tumor diameter per
unit time. In one embodiment, after treatment the tumor growth rate
may be about zero and is determined to maintain the same size,
e.g., has stopped growing.
[0085] Treating a colorectal cancer according to the methods
described herein can result in a decrease in tumor regrowth.
Preferably, after treatment, tumor regrowth is less than 5%; more
preferably, tumor regrowth is less than 10%; more preferably, less
than 20%; more preferably, less than 30%; more preferably, less
than 40%; more preferably, less than 50%; even more preferably,
less than 50%; and most preferably, less than 75%. Tumor regrowth
may be measured by any reproducible means of measurement. Tumor
regrowth is measured, for example, by measuring an increase in the
diameter of a tumor after a prior tumor shrinkage that followed
treatment. A decrease in tumor regrowth is indicated by failure of
tumors to reoccur after treatment has stopped.
[0086] As used herein, the term "selectively" means tending to
occur at a higher frequency in one population than in another
population. The compared populations can be cell populations.
Preferably, an apilimod composition as described herein acts
selectively on hyper-proliferating cells or abnormally
proliferating cells, compared to normal cells. As used herein, a
"normal cell" is a cell that cannot be classified as part of a
"cell proliferative disorder". A normal cell lacks unregulated or
abnormal growth, or both, that can lead to the development of an
unwanted condition or disease. Preferably, a normal cell possesses
normally functioning cell cycle checkpoint control mechanisms.
Preferably, an apilimod composition acts selectively to modulate
one molecular target (e.g., a target kinase) but does not
significantly modulate another molecular target (e.g., a non-target
kinase). The invention also provides a method for selectively
inhibiting the activity of an enzyme, such as a kinase. Preferably,
an event occurs selectively in population A relative to population
B if it occurs greater than two times more frequently in population
A as compared to population B. An event occurs selectively if it
occurs greater than five times more frequently in population A. An
event occurs selectively if it occurs greater than ten times more
frequently in population A; more preferably, greater than fifty
times; even more preferably, greater than 100 times; and most
preferably, greater than 1000 times more frequently in population A
as compared to population B. For example, cell death would be said
to occur selectively in diseased or hyper-proliferating cells if it
occurred greater than twice as frequently in diseased or
hyper-proliferating cells as compared to normal cells.
Pharmaceutical Compositions and Formulations
[0087] The present invention provides apilimod compositions that
are preferably pharmaceutically acceptable compositions suitable
for use in a mammal, preferably a human. In this context, the
compositions may further comprise at least one pharmaceutically
acceptable excipient or carrier, wherein the amount is effective
for the treatment of colorectal cancer.
[0088] In one embodiment, the apilimod composition comprises
apilimod free base or apilimod dimesylate.
[0089] In one embodiment, the apilimod composition is combined with
at least one additional active agent in a single dosage form. In
one embodiment, the composition further comprises an
antioxidant.
[0090] In one embodiment, the at least one additional active agent
is selected from the group consisting of an alkylating agent, an
intercalating agent, a tubulin binding agent, a PD-1/PDL-1 pathway
inhibitor, a corticosteroid, and combinations thereof. In
embodiments, the at least one additional active agent is a
therapeutic agent selected from the group consisting of
vemurafenib, ibrutinib, rituximab, doxorubicin, prednisolone,
vincristine, velcade, and everolimus, and combinations thereof. In
embodiments, the at least one additional active agent is a
therapeutic agent selected from the group consisting of
vemurafenib, oxaliplatin, regorafenib, irinotecan, and
5-fluorouracil. In embodiments, the at least one additional active
agent is a therapeutic agent selected from the group consisting of
pembrolizumab (Keytruda), avelumab, atezolizumab (MPDL3280A),
nivolumab (BMS-936558), pidilizumab (MK-3475), MSB0010718C, and
MEDI4736.
[0091] In embodiments, the at least one additional active agent is
a therapeutic agent selected from the group consisting of
dacarbazine, temozolomide, Nab-paclitaxel, carmustine, cisplatin,
carboplatin, vinblastine, ipilimumab, Interleukin-2 (IL-2,
Proleukin), pembrolizumab (Keytruda), dabrafenib (Tafinlar),
vemurafenib (Zelboraf), trametinib (Mekinist), dasatinib (Sprycel),
imatinib (Gleevec), and nilotinib (Tasigna) and combinations
thereof.
[0092] In embodiments, the at least one additional active agent is
a non-therapeutic agent selected to ameliorate one or more side
effects of the apilimod composition. In one embodiment, the
non-therapeutic agent is selected from the group consisting of
ondansetron, granisetron, dolasetron and palonosetron. In one
embodiment, the non-therapeutic agent is selected from the group
consisting of pindolol and risperidone.
[0093] In embodiments, the at least one additional active agent is
selected from an inhibitor of BRAF, an inhibitor of the Raf/MEK/ERK
pathway, an inhibitor of the mTOR pathway, a PI3K inhibitor, a dual
PI3K/mTOR inhibitor, a SRC inhibitor, a VEGF inhibitor, a Janus
kinase (JAK) inhibitor, a Raf inhibitor, an Erk inhibitor, a
farnesyltransferase inhibitor, a histone deacetylase inhibitor, an
anti-mitotic agent, a multi-drug resistance efflux inhibitor, an
antibiotic, and a therapeutic antibody. In one embodiment, the at
least one additional active agent is selected from a
farnesyltransferase inhibitor (e.g., tipifarnib), an anti-mitotic
agent (e.g., docetaxel), a histone deacetylase inhibitor (e.g.,
vorinostat), and a multi-drug resistance efflux inhibitor.
[0094] In one embodiment, the mTOR inhibitor is selected from the
group consisting of rapamycin (also referred to as sirolimus),
everolimus, temsirolimus, ridaforolimus, umirolimus, zotarolimus,
AZD8055, INK128, WYE-132, Torin-1, pyrazolopyrimidine analogs
PP242, PP30, PP487, PP121, KU0063794, KU-BMCL-200908069-1,
Wyeth-BMCL-200910075-9b, INK-128, XL388, AZD8055, P2281, and P529.
See, e.g., Liu et al. Drug Disc. Today Ther. Strateg., 6(2): 47-55
(2009).
[0095] In one embodiment, the mTOR inhibitor is
trans-4-[4-amino-5-(7-methoxy-1H-indol-2-yeimidazo[5,1-f][1,2,4]triazin-7-
-yl]cyclohexane carboxylic acid (also known as OSI-027), and any
salts, solvates, hydrates, and other physical forms, crystalline or
amorphous, thereof. See US 2007/0112005. OSI-027 can be prepared
according to US 2007/0112005, incorporated herein by reference. In
one embodiment, the mTOR inhibitor is OXA-01. See e.g., WO
2013152342 A1.
[0096] In one embodiment, the PI3K inhibitor is selected from the
group consisting of GS-1101 (Idelalisib), GDC0941 (Pictilisib),
LY294002, BKM120 (Buparlisib), PI-103, TGX-221, IC-87114, XL 147,
ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, PIK-93,
PIK-90, AZD6482, IPI-145 (Duvelisib), TG100-115, AS-252424, PIK294,
AS-604850, GSK2636771, BAY 80-6946 (Copanlisib), CH5132799,
CAY10505, PIK-293, TG100713, CZC24832 and HS-173.
[0097] In one embodiment, the dual PI3K/mTOR inhibitor is selected
from the group consisting of, GDC-094, WAY-001, WYE-354, WAY-600,
WYE-687, Wyeth-BMCL-200910075-16b, Wyeth-BMCL-200910096-27,
KU0063794 and KUBMCL-200908069-5, NVP-BEZ235, XL-765, PF-04691502,
GDC-0980 (Apitolisib), GSK1059615, PF-05212384, BGT226, PKI-402,
VS-558 and GSK2126458. See, e.g., Liu et al. Drug Disc. Today Ther.
Strateg., 6(2): 47-55 (2009), incorporated herein by reference.
[0098] In one embodiment, the mTOR pathway inhibitor is a
polypeptide (e.g., an antibody or fragment thereof) or a nucleic
acid (e.g., a double-stranded small interfering RNA, a short
hairpin RNA, a micro-RNA, an antisense oligonucleotide, a locked
nucleic acid, or an aptamer) that binds to and inhibits the
expression level or activity or a protein (or nucleic acid encoding
the protein) in the mTOR pathway. For example, the polypeptide or
nucleic acid inhibits mTOR Complex 1 (mTORC1),
regulatory-associated protein of mTOR (Raptor), mammalian lethal
with SEC13 protein 8 (MLST8), proline-rich Akt substrate of 40 kDa
(PRAS40), DEP domain-containing mTOR-interacting protein (DEPTOR),
mTOR Complex 2 (mTORC2), rapamycin-insensitive companion of mTOR
(RICTOR), G protein beta subunit-like (G.beta.L), mammalian
stress-activated protein kinase interacting protein 1 (mSIN1),
paxillin, RhoA, Ras-related C3 botulinum toxin substrate 1 (Rac1),
Cell division control protein 42 homolog (Cdc42), protein kinase C
.alpha. (PKC.alpha.), the serine/threonine protein kinase Akt,
phosphoinositide 3-kinase (PI3K), p70S6K, Ras, and/or eukaryotic
translation initiation factor 4E (eIF4E)-binding proteins (4EBPs),
or the nucleic acid encoding one of these proteins.
[0099] In one embodiment, the SRC inhibitor is selected from the
group consisting of bosutinib, saracatinib, dasatinib, ponatinib,
KX2-391, XL-228, TG100435/TG100855, and DCC2036. See, e.g., Puls et
al. Oncologist. 2011 May; 16(5): 566-578. In one embodiment, the
SRC inhibitor is a polypeptide (e.g., an antibody or fragment
thereof) or nucleic acid (e.g., a double-stranded small interfering
RNA, a short hairpin RNA, a micro-RNA, an antisense
oligonucleotide, a locked nucleic acid, or an aptamer) that binds
to and inhibits the expression level or activity of the SRC protein
or a nucleic acid encoding the SRC protein.
[0100] In one embodiment, the VEGF inhibitor is selected from
bevacizumab, sunitinib, pazopanib, axitinib, sorafenib,
regorafenib, lenvatinib, and motesanib. In one embodiment, the VEGF
inhibitor is a polypeptide (e.g., an antibody or fragment thereof)
or nucleic acid (e.g., a double-stranded small interfering RNA, a
short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a
morpholino, a locked nucleic acid, or an aptamer) that binds to and
inhibits the expression level or activity of a VEGF protein, a VEGF
receptor protein, or a nucleic acid encoding one of these proteins.
For example, the VEGF inhibitor is a soluble VEGF receptor (e.g., a
soluble VEGF-C/D receptor (sVEGFR-3)).
[0101] In one embodiment, the JAK inhibitor is selected from
facitinib, ruxolitinib, baricitinib, CYT387 (CAS number
1056634-68-4), lestaurtinib, pacritinib, and TG101348 (CAS number
936091-26-8). In one embodiment, the JAK inhibitor is a polypeptide
(e.g., an antibody or fragment thereof) or nucleic acid (e.g., a
double-stranded small interfering RNA, a short hairpin RNA, a
micro-RNA, an antisense oligonucleotide, a morpholino, a locked
nucleic acid, or an aptamer) that binds to and inhibits the
expression level or activity of a JAK (e.g., JAK1, JAK2, JAK3, or
TYK2) or a nucleic acid encoding the JAK protein.
[0102] In one embodiment, the Raf inhibitor is selected from
PLX4032 (vemurafenib), sorafenib, PLX-4720, GSK2118436
(dabrafenib), GDC-0879, RAF265, AZ 628, NVP-BHG712, SB90885, ZM
336372, GW5074, TAK-632, CEP-32496 and LGX818 (Encorafenib). In one
embodiment, the Raf inhibitor is a polypeptide (e.g., an antibody
or fragment thereof) or nucleic acid (e.g., a double-stranded small
interfering RNA, a short hairpin RNA, a micro-RNA, an antisense
oligonucleotide, a morpholino, a locked nucleic acid, or an
aptamer) that binds to and inhibits the expression level or
activity of a Raf (e.g., A-Raf, B-Raf, C-Raf) or a nucleic acid
encoding the Raf protein. In one embodiment, the MEK inhibitor is
selected from AZD6244 (Selumetinib), PD0325901, GSK1120212
(Trametinib), U0126-EtOH, PD184352, RDEA119 (Rafametinib), PD98059,
BIX 02189, MEK162 (Binimetinib), AS-703026 (Pimasertib), SL-327,
BIX02188, AZD8330, TAK-733 and PD318088. In one embodiment, the MEK
inhibitor is a polypeptide (e.g., an antibody or fragment thereof)
or nucleic acid (e.g., a double-stranded small interfering RNA, a
short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a
morpholino, a locked nucleic acid, or an aptamer) that binds to and
inhibits the expression level or activity of a MEK (e.g., MEK-1,
MEK-2) or a nucleic acid encoding the MEK protein.
[0103] In one embodiment, the Akt inhibitor is selected from
MK-2206, KRX-0401 (perifosine), GSK690693, GDC-0068 (Ipatasertib),
AZD5363, CCT128930, A-674563, PHT-427. In one embodiment, the Akt
inhibitor is a polypeptide (e.g., an antibody or fragment thereof)
or nucleic acid (e.g., a double-stranded small interfering RNA, a
short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a
morpholino, a locked nucleic acid, or an aptamer) that binds to and
inhibits the expression level or activity of a Akt (e.g., Akt-1,
Akt-2, Akt-3) or a nucleic acid encoding the Akt protein.
[0104] In one embodiment, the farnesyltransferase inhibitor is
selected from LB42708 or tipifarnib. In one embodiment, the
farnesyltransferase inhibitor is a polypeptide (e.g., an antibody
or fragment thereof) or nucleic acid (e.g., a double-stranded small
interfering RNA, a short hairpin RNA, a micro-RNA, an antisense
oligonucleotide, a morpholino, a locked nucleic acid, or an
aptamer) that binds to and inhibits the expression level or
activity of farnesyltransferase or a nucleic acid encoding the
farnesyltransferase protein. In one embodiment, the histone
modulating inhibitor is selected from anacardic acid, C646, MG149
(histone acetyltransferase), GSK J4 Hcl (histone demethylase),
GSK343 (active against EZH2), BIX 01294 (histone
methyltransferase), MK0683 (Vorinostat), MS275 (Entinostat), LBH589
(Panobinostat), Trichostatin A, MGCD0103 (Mocetinostat),
Tasquinimod, TMP269, Nexturastat A, RG2833, PDX101
(Belinostat).
[0105] In one embodiment, the anti-mitotic agent is selected from
Griseofulvin, vinorelbine tartrate, paclitaxel, docetaxel,
vincristine, vinblastine, Epothilone A, Epothilone B, ABT-751,
CYT997 (Lexibulin), vinflunine tartrate, Fosbretabulin, GSK461364,
ON-01910 (Rigosertib), Ro3280, BI2536, NMS-P937, BI 6727
(Volasertib), HMN-214 and MLN0905.
[0106] In one embodiment, the polyether antibiotic is selected from
sodium monensin, nigericin, valinomycin, salinomycin.
[0107] A "pharmaceutical composition" is a formulation containing
the compounds described herein in a pharmaceutically acceptable
form suitable for administration to a subject. As used herein, the
phrase "pharmaceutically acceptable" refers to those compounds,
materials, compositions, carriers, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0108] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes excipient that is acceptable for
veterinary use as well as human pharmaceutical use. Examples of
pharmaceutically acceptable excipients include, without limitation,
sterile liquids, water, buffered saline, ethanol, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like), oils, detergents, suspending agents, carbohydrates
(e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g.,
ascorbic acid or glutathione), chelating agents, low molecular
weight proteins, or suitable mixtures thereof.
[0109] A pharmaceutical composition can be provided in bulk or in
dosage unit form. It is especially advantageous to formulate
pharmaceutical compositions in dosage unit form for ease of
administration and uniformity of dosage. The term "dosage unit
form" as used herein refers to physically discrete units suited as
unitary dosages for the subject to be treated; each unit containing
a predetermined quantity of active compound calculated to produce
the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved. A dosage unit form can be an
ampoule, a vial, a suppository, a dragee, a tablet, a capsule, an
IV bag, or a single pump on an aerosol inhaler.
[0110] In therapeutic applications, the dosages vary depending on
the agent, the age, weight, and clinical condition of the recipient
patient, and the experience and judgment of the clinician or
practitioner administering the therapy, among other factors
affecting the selected dosage. Generally, the dose should be a
therapeutically effective amount. Dosages can be provided in
mg/kg/day units of measurement (which dose may be adjusted for the
patient's weight in kg, body surface area in m.sup.2, and age in
years). An effective amount of a pharmaceutical composition is that
which provides an objectively identifiable improvement as noted by
the clinician or other qualified observer. For example, alleviating
a symptom of a disorder, disease or condition. As used herein, the
term "dosage effective manner" refers to amount of a pharmaceutical
composition to produce the desired biological effect in a subject
or cell.
[0111] For example, the dosage unit form can comprise 1 nanogram to
2 milligrams, or 0.1 milligrams to 2 grams; or from 10 milligrams
to 1 gram, or from 50 milligrams to 500 milligrams or from 1
microgram to 20 milligrams; or from 1 microgram to 10 milligrams;
or from 0.1 milligrams to 2 milligrams.
[0112] The pharmaceutical compositions can take any suitable form
(e.g, liquids, aerosols, solutions, inhalants, mists, sprays; or
solids, powders, ointments, pastes, creams, lotions, gels, patches
and the like) for administration by any desired route (e.g,
pulmonary, inhalation, intranasal, oral, buccal, sublingual,
parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, intrapleural, intrathecal, transdermal,
transmucosal, rectal, and the like). For example, a pharmaceutical
composition of the invention may be in the form of an aqueous
solution or powder for aerosol administration by inhalation or
insufflation (either through the mouth or the nose), in the form of
a tablet or capsule for oral administration; in the form of a
sterile aqueous solution or dispersion suitable for administration
by either direct injection or by addition to sterile infusion
fluids for intravenous infusion; or in the form of a lotion, cream,
foam, patch, suspension, solution, or suppository for transdermal
or transmucosal administration.
[0113] A pharmaceutical composition can be in the form of an orally
acceptable dosage form including, but not limited to, capsules,
tablets, buccal forms, troches, lozenges, and oral liquids in the
form of emulsions, aqueous suspensions, dispersions or solutions.
Capsules may contain mixtures of a compound of the present
invention with inert fillers and/or diluents such as the
pharmaceutically acceptable starches (e.g., corn, potato or tapioca
starch), sugars, artificial sweetening agents, powdered celluloses,
such as crystalline and microcrystalline celluloses, flours,
gelatins, gums, etc. In the case of tablets for oral use, carriers
which are commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, can also be added.
For oral administration in a capsule form, useful diluents include
lactose and dried corn starch. When aqueous suspensions and/or
emulsions are administered orally, the compound of the present
invention may be suspended or dissolved in an oily phase is
combined with emulsifying and/or suspending agents. If desired,
certain sweetening and/or flavoring and/or coloring agents may be
added.
[0114] A pharmaceutical composition can be in the form of a tablet.
The tablet can comprise a unit dosage of a compound of the present
invention together with an inert diluent or carrier such as a sugar
or sugar alcohol, for example lactose, sucrose, sorbitol or
mannitol. The tablet can further comprise a non-sugar derived
diluent such as sodium carbonate, calcium phosphate, calcium
carbonate, or a cellulose or derivative thereof such as methyl
cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and
starches such as corn starch. The tablet can further comprise
binding and granulating agents such as polyvinylpyrrolidone,
disintegrants (e.g. swellable crosslinked polymers such as
crosslinked carboxymethylcellulose), lubricating agents (e.g.
stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT),
buffering agents (for example phosphate or citrate buffers), and
effervescent agents such as citrate/bicarbonate mixtures.
[0115] The tablet can be a coated tablet. The coating can be a
protective film coating (e.g. a wax or varnish) or a coating
designed to control the release of the active agent, for example a
delayed release (release of the active after a predetermined lag
time following ingestion) or release at a particular location in
the gastrointestinal tract. The latter can be achieved, for
example, using enteric film coatings such as those sold under the
brand name Eudragit.RTM..
[0116] Tablet formulations may be made by conventional compression,
wet granulation or dry granulation methods and utilize
pharmaceutically acceptable diluents, binding agents, lubricants,
disintegrants, surface modifying agents (including surfactants),
suspending or stabilizing agents, including, but not limited to,
magnesium stearate, stearic acid, talc, sodium lauryl sulfate,
microcrystalline cellulose, carboxymethylcellulose calcium,
polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan
gum, sodium citrate, complex silicates, calcium carbonate, glycine,
dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate,
lactose, kaolin, mannitol, sodium chloride, talc, dry starches and
powdered sugar. Preferred surface modifying agents include nonionic
and anionic surface modifying agents. Representative examples of
surface modifying agents include, but are not limited to, poloxamer
188, benzalkonium chloride, calcium stearate, cetostearyl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon
dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum
silicate, and triethanolamine.
[0117] A pharmaceutical composition can be in the form of a hard or
soft gelatin capsule. In accordance with this formulation, the
compound of the present invention may be in a solid, semi-solid, or
liquid form.
[0118] A pharmaceutical composition can be in the form of a sterile
aqueous solution or dispersion suitable for parenteral
administration. The term parenteral as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intraarterial, intrasynovial, intrasternal,
intrathecal, intralesional and intracranial injection or infusion
techniques.
[0119] A pharmaceutical composition can be in the form of a sterile
aqueous solution or dispersion suitable for administration by
either direct injection or by addition to sterile infusion fluids
for intravenous infusion, and comprises a solvent or dispersion
medium containing, water, ethanol, a polyol (e.g., glycerol,
propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof, or one or more vegetable oils. Solutions or suspensions of
the compound of the present invention as a free base or
pharmacologically acceptable salt can be prepared in water suitably
mixed with a surfactant. Examples of suitable surfactants are given
below. Dispersions can also be prepared, for example, in glycerol,
liquid polyethylene glycols and mixtures of the same in oils.
[0120] The pharmaceutical compositions for use in the methods of
the present invention can further comprise one or more additives in
addition to any carrier or diluent (such as lactose or mannitol)
that is present in the formulation. The one or more additives can
comprise or consist of one or more surfactants. Surfactants
typically have one or more long aliphatic chains such as fatty
acids which enables them to insert directly into the lipid
structures of cells to enhance drug penetration and absorption. An
empirical parameter commonly used to characterize the relative
hydrophilicity and hydrophobicity of surfactants is the
hydrophilic-lipophilic balance ("HLB" value). Surfactants with
lower HLB values are more hydrophobic, and have greater solubility
in oils, while surfactants with higher HLB values are more
hydrophilic, and have greater solubility in aqueous solutions.
Thus, hydrophilic surfactants are generally considered to be those
compounds having an HLB value greater than about 10, and
hydrophobic surfactants are generally those having an HLB value
less than about 10. However, these HLB values are merely a guide
since for many surfactants, the HLB values can differ by as much as
about 8 HLB units, depending upon the empirical method chosen to
determine the HLB value.
[0121] Among the surfactants for use in the compositions of the
invention are polyethylene glycol (PEG)-fatty acids and PEG-fatty
acid mono and diesters, PEG glycerol esters, alcohol-oil
transesterification products, polyglyceryl fatty acids, propylene
glycol fatty acid esters, sterol and sterol derivatives,
polyethylene glycol sorbitan fatty acid esters, polyethylene glycol
alkyl ethers, sugar and its derivatives, polyethylene glycol alkyl
phenols, polyoxyethylene-polyoxypropylene (POE-POP) block
copolymers, sorbitan fatty acid esters, ionic surfactants,
fat-soluble vitamins and their salts, water-soluble vitamins and
their amphiphilic derivatives, amino acids and their salts, and
organic acids and their esters and anhydrides.
[0122] The present invention also provides packaging and kits
comprising pharmaceutical compositions for use in the methods of
the present invention. The kit can comprise one or more containers
selected from the group consisting of a bottle, a vial, an ampoule,
a blister pack, and a syringe. The kit can further include one or
more of instructions for use in treating and/or preventing a
disease, condition or disorder of the present invention, one or
more syringes, one or more applicators, or a sterile solution
suitable for reconstituting a pharmaceutical composition of the
present invention.
[0123] All percentages and ratios used herein, unless otherwise
indicated, are by weight. Other features and advantages of the
present invention are apparent from the different examples. The
provided examples illustrate different components and methodology
useful in practicing the present invention. The examples do not
limit the claimed invention. Based on the present disclosure the
skilled artisan can identify and employ other components and
methodology useful for practicing the present invention.
EXAMPLES
Example 1: Apilimod Inhibits Proliferation of Diverse Colon Cancer
Cell Lines
[0124] The colon cancer cell lines HCT116 (BRAF wild-type) and
HT-29 (BRAF mutated, V600E) cells were grown in DMEM (Corning), RKO
(BRAF mutated, V600E) were grown in MEM (Corning), and HCT-15 (BRAF
wild-type), SW1116 (BRAF wild-type), SW480 (BRAF wild-type) and
SW620 (BRAF wild-type) were grown in RPMI-1640 (Corning) and
supplemented with 10% FBS (Sigma Aldrich F2442-500ML, Lot 12D370)
and Penicillin/Streptomycin (100.times.) (CellGro Ref 30-002). For
drug studies, HCT116, HT-29, RKO, HCT-15, SW1116, SW480 and SW620
cells were seeded at a density of 750, 8000, 300, 480, 1600, 2000,
and 2000 cells per well, respectively, into 96 well plates in a
final volume of 50 .mu.L.
[0125] The apilimod used in these studies was apilimod dimesylate.
The term "LAM-002" refers to apilimod dimesylate.
[0126] For single treatment studies, 24 h after seeding, cells were
treated with apilimod alone (final concentration 0.5-10000 nM;
3-fold dilutions and a total of 10 dilutions), or with one of
vemurafenib (final concentration 56.6-30000 nM; 2-fold dilutions
and a total of 10 dilutions), regorafenib, oxaliplatin,
5-fluorouracil or irinotecan (all drugs screened at final
concentration 2.5-50000 nM; 3-fold dilutions and a total of 10
dilutions). All drug dilutions were made up as a 2.times. stock and
50 .mu.L added to appropriate wells. Cells were treated for 120 h
before viability was assessed using CellTiterGlo.RTM. (Promega)
where the relative luminescence of untreated cells was set to 100%
viability and each drug concentration expressed as a percentage of
untreated cells. EC.sub.50 values were determined using GraphPad
Prism (GraphPad Software, Inc). Briefly, raw data was log
transformed and then analyzed using nonlinear regression (curve
fit) where the data were constrained (bottom=0, top=100).
[0127] For determination of synergy between apilimod and each of
vemurafenib, regorafenib, oxaliplatin, 5-fluorouracil or
irinotecan, HCT116, HT-29, RKO, HCT-15, SW1116, SW480, and SW620
cells were seeded as outlined above. 24 h later, cells were treated
with apilimod dimesylate alone (final concentration 2-250 nM;
2-fold dilutions and a total of 8 dilutions), with vemurafenib
alone (final concentration 234-30000 nM; 2-fold dilutions and a
total of 8 dilutions), with oxaliplatin, irinotecan, 5-fluorouracil
or regorafenib alone (for all, final concentration 195-25000 nM;
2-fold dilutions and a total of 8 dilutions) or the combination of
each concentration of apilimod mesylate with each concentration of
vemurafenib, oxaliplatin, irinotecan, 5-fluorouracil or regorafenib
(8.times.8 matrix). Cells were treated for 120 h before viability
was assessed using CellTiterGlo.RTM. (Promega) where the relative
luminescence of untreated cells was set to 100% viability and each
drug concentration expressed as a percentage of untreated
cells.
[0128] Bar graphs show the effect of a single concentration of
apilimod, a single concentration of vemurafenib and the effect of
the combination of drugs (at the single agent concentrations) on
cell viability. The expected value was calculated (fraction of
viability for apilimod multiplied by fraction of viability for
vemurafenib) and is shown.
[0129] For calculation of synergy, CalcuSyn (version 2.11, Biosoft)
was used to determine the combination index (CI) as defined by Chou
et al. (Chou T C, Talalay P. Quantitative analysis of dose-effect
relationships: the combined effects of multiple drugs or enzyme
inhibitors. Adv Enzyme Regul 1984; 22:27-55). We constrained the
analysis to assess CI values which were within a clinically
achievable concentration and also where the fraction effect (Fa)
was greater than 0.75 (ie a greater than 75% reduction in cell
viability with the combination of drugs). CI versus fractional
effect graphs are shown (datapoints denoted by `x`, lines show 95%
confidence interval), where drug combinations producing CI values
>1 are antagonistic, CI=1 are additive and CI<1 are
synergistic. In addition, the CI value at the ED50, ED75 and ED90
are shown for apilimod and vemurafenib. The same methodology was
applied to cells treated with apilimod in combination with
oxaliplatin, irinotecan, regorafenib, and 5-fluorouracil.
[0130] Using this approach, apilimod was found to act
synergistically in HCT116 cells with each of oxaliplatin,
irinotecan, regorafenib, and 5-fluorouracil. Apilimod also
demonstrated synergistic activity with both regorafenib in RKO
cells. Apilimod was also found to be synergistic with vemurafenib
in HCT116, HT-29, RKO, HCT-15, SW1116, SW480, and SW620 cells.
These data demonstrate that apilimod can act both alone and
synergistically with other anti-cancer agents against colon cancer
cells. In addition, the data show that the combination of apilimod
with vemurafenib shows synergistic activity in cells with wild-type
BRAF as well as cells harboring mutated BRAF (V600E), which are
often resistant to standard therapy.
TABLE-US-00001 TABLE 1 Apilimod and vemurafenib synergy in
colorectal cancer cells (5 day assays). Cell Line BRAF status EC50
(nM) HCT116 WT 10 HCT-15 WT 15 SW1116 WT 12 SW480 WT 24 SW620 WT 9
RKO Mutant (V600E) 52 HT-29 Mutant (V600E) 19
Example 2: SNX10 Expression as a Biomarker for LAM-002 Response in
Colon Cancer Lines
[0131] SNX10 is a gene involved in intracellular trafficking that
was identified in a high-throughput screen conducted to identify
genes that confer resistance to LAM-002. Genetic ablation of SNX10
conferred resistance to LAM-002 treatment. This suggested that
over-expression of SNX10 would induce sensitivity to LAM-002. To
validate this hypothesis, the expression level of SNX10 was
correlated with LAM-002 sensitivity in various colon cancer
lines.
[0132] Expression profiles were obtained from the public databases
CCLE (Barretina, Caponigro, Stransky et al. The Cancer Cell Line
Encyclopedia enables predictive modelling of anticancer drug
sensitivity. Nature. 2012 Mar. 28; 483(7391):603-7) and COSMIC
(Forbes SA1, Beare D2, Gunasekaran P2, et al. COSMIC: exploring the
world's knowledge of somatic mutations in human cancer. Nucleic
Acids Res. 2015 January; 43 (Database issue):D805-11). R
statistical package (R Development Core Team (2008). R: A language
and environment for statistical computing. R Foundation for
Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0) was
used for the normalization of expression and the correlation
analysis.
[0133] To determine LAM-002 sensitivity in colon cancer cell lines,
the EC90 was calculated using a 10 point dose response as described
above for EC50 determination. The results revealed a correlation
between SNX10 expression and LAM-002 sensitivity whereby colon
cancer cells expressing higher levels of SNX10 are the most
sensitive to LAM-002 (Table 2). These findings support SNX10
expression as a predictive biomarker for LAM-002 sensitivity in
colon cancer cells.
* * * * *