U.S. patent application number 15/365794 was filed with the patent office on 2017-06-08 for methods and compositions for treating cancer.
The applicant listed for this patent is GEORGETOWN UNIVERSITY. Invention is credited to Jeffrey A. Toretsky, Aykut Uren.
Application Number | 20170157089 15/365794 |
Document ID | / |
Family ID | 52993423 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170157089 |
Kind Code |
A1 |
Toretsky; Jeffrey A. ; et
al. |
June 8, 2017 |
METHODS AND COMPOSITIONS FOR TREATING CANCER
Abstract
Methods and compositions provided herein relate to the treatment
of cancer. In some embodiments, the compositions have utility in
the treatment of cancers including glioblastoma multiforme and lung
cancer.
Inventors: |
Toretsky; Jeffrey A.;
(Silver Spring, MD) ; Uren; Aykut; (Rockville,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GEORGETOWN UNIVERSITY |
WASHINGTON |
DC |
US |
|
|
Family ID: |
52993423 |
Appl. No.: |
15/365794 |
Filed: |
November 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15030713 |
Apr 20, 2016 |
9511050 |
|
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PCT/US2014/061418 |
Oct 20, 2014 |
|
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15365794 |
|
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61895308 |
Oct 24, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/404 20130101;
A61P 35/00 20180101; C07D 209/38 20130101; A61K 45/06 20130101;
A61K 47/40 20130101; A61P 11/00 20180101; A61P 25/00 20180101 |
International
Class: |
A61K 31/404 20060101
A61K031/404; A61K 47/40 20060101 A61K047/40; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0002] This invention was made with government support under NIH
Grant/Contract Numbers R01CA138212, R01CA133662, RC4CA156509
awarded by the National Institutes of Health of the United States
of America. The government has certain rights in the invention.
Claims
1. A method of treating a subject having a cancer selected from the
group consisting of lung cancer and glioblastoma multiforme, the
method comprising administering to a subject in need thereof an
effective amount of a compound having the structure: ##STR00085##
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein the lung cancer comprises a
non-small-cell lung carcinoma.
3. The method of claim 1, wherein the lung cancer comprises a
glioblastoma multiforme.
4. The method of claim 1, wherein the cancer comprises a
translocation comprising an ETS gene selected from the group
consisting of FLI1, ETV1, ETV4, ERG, ETS1, and ETS2.
5. The method of claim 1, wherein the compound is administered in
combination with an additional chemotherapeutic agent.
6. The method of claim 1, wherein the additional chemotherapeutic
agent is selected from the group consisting of cisplatin,
paclitaxel, gemcitabine, etoposide, and vinblastine.
7. The method of claim 1, wherein the compound is administered
parentally.
8. The method of claim 1, wherein the compound substantially
consists of the (S) enantiomer.
9. The method of claim 1, wherein the compound is administered in
combination with a .beta.-hydroxypropyl cyclodextrin excipient.
10. The method of claim 1, wherein the subject is mammalian.
11. The method of claim 1, wherein the subject is human.
12. A method of inhibiting the growth of a neoplastic cell selected
from the group consisting of a lung cell and a glial cell, the
method comprising contacting the cell with a compound having the
structure: ##STR00086##
13. The method of claim 12, wherein the cell is a glioblastoma
multiforme cell.
14. The method of claim 12, wherein the cell is a glioblastoma cell
selected from the group consisting of a DKMG cell, a DBTRG cell, a
42MGBA cell, a GAMG cell, a U87MG cell, a H4 cell, and a 8MGBA
cell.
15. The method of claim 12, wherein the cell is a non-small-cell
lung carcinoma cell.
16. The method of claim 12, wherein the cell is a lung cell
selected from the group consisting of a A549 cell, a H1944 cell, a
H358 cell, a H1395 cell, and a H596 cell.
17. The method of claim 12, wherein the cell is in vivo.
18. The method of claim 12, wherein the cell is in vitro.
19. The method of claim 12, wherein the subject is mammalian.
20. The method of claim 12, wherein the subject is human.
Description
INCORPORATION BY REFERENCE TO RELATED APPLICATIONS
[0001] Any and all priority claims identified in the Application
Data Sheet, or any correction thereto, are hereby incorporated by
reference under 37 CFR 1.57. This application is a continuation of
U.S. application Ser. No. 15/030,713, filed Apr. 20, 2016, which is
the national phase under 35 U.S.C. .sctn.371 of PCT International
Application No. PCT/US2014/061418, which has an International
filing date of Oct. 20, 2014, which claims the benefit of U.S.
Provisional Application No. 61/895,308 filed Oct. 24, 2013. Each of
the aforementioned applications is incorporated by reference herein
in its entirety, and each is hereby expressly made a part of this
specification.
FIELD OF THE INVENTION
[0003] Methods and compositions provided herein relate to the
treatment of cancer. In some embodiments, the compositions have
utility in the treatment of cancers including glioblastoma
multiforme and lung cancer.
BACKGROUND OF THE INVENTION
[0004] The Central Brain Tumor Registry of the United States
(CBTRUS) lists the total number of primary malignant brain tumor
deaths for all 50 states and the District of Columbia in 2012 is
estimated to be 13,700. Glioblastomas (GBMs) are the most common
brain malignancy with a median survival of only 14.6 months in
humans despite standard tri-modality treatment consisting of
surgical resection, post-operative radiation therapy and
temozolomide chemotherapy. Therapy is almost never curative because
the infiltrative nature of these tumors and their intrinsic
resistance to radiation and chemotherapy. Even with optimal
treatment, the median survival is less than 15 months, with only
10% of patients surviving 2 years without disease recurrence. New
therapeutic targets are clearly needed to improve patient survival
and quality of life for Glioblastomas and other cancers.
[0005] In addition, the Ewing's Sarcoma Family of Tumors (ESFT) are
highly aggressive tumors that occur in children, adolescents and
young adults in the bone and the soft tissues. They respond to
chemotherapy, yet 75% to 80% of the patients who have developed
metastatic ESFTs will die in five years despites high doses of
chemotherapy (Grier, H. E et al., N. Engl. J. Med. 348, 694-701
(2003)). ESFTs contain a well characterized chromosomal
translocation. This joins the Ewing's sarcoma gene (EWS), located
on chromosome 22, to an ets family gene, often friend leukemia
insertion (FLI)1 located on the chromosome 11, t(11:22) which lead
to the expression of various fusion proteins (Aykut Uren, Jeffrey A
Torestsky Ewing's sarcoma oncoproteins EWS-FLI1: the perfect target
without a therapeutic agent, Future Oncol. 1(4), 521-528
(2005)).
[0006] In vitro and in vivo studies have demonstrated that the
elimination of the oncoprotein, EWS-FLI1, leads to a decrease
proliferation of ESTF cell lines and a decrease of tumor volume.
EWS-FLI1 lacks enzymatic activity, however, the RHA helicase A
(RHA) increases EWS-FLI1-modulated oncogenesis, therefore the
protein-protein interactions between the two proteins is required
for the maintenance of the tumor growth (Hyariye N Erkizan et al. A
small molecule blocking oncogenic protein EWS-FlI1 interacting with
RHA helicase A inhibits growth of Ewing's sarcoma. Nature Medicine
15 (7) 750-756 (2009)). The paradigm of disrupting key protein
interactions may have utility in treatment of diseases including
sarcomas with similar translocations, and leukemias with MLL
translocations ((Heiman L J, Meltzer P. Mechanisms of sarcoma
development. Nat Rev Cancer 2003; 3(9):685-94); and Pui C H,
Relling M V, Downing J R. Acute lymphoblastic leukemia. N Engl J
Med 2004; 350(15):1535-48). Moreover, disordered proteins may be
excellent therapeutic targets based on their intrinsic biochemical
properties (Cheng Y, LeGall T, Oldfield C J, et al. Rational drug
design via intrinsically disordered protein. Trends Biotechnol
2006; 24(10):435-42).
[0007] Despite years of in vitro and xenograft studies with
antisense and siRNA directed towards EWS-FLI1, none of these is
heretofore practical as a human therapy based on inadequate
delivery and stability. Accordingly, there is a need for improved
therapies to treat disorders such as ESFTs.
SUMMARY OF THE INVENTION
[0008] In a generally applicable first aspect (i.e., independently
combinable with any of the aspects or embodiments identified
herein), a compound is provided of Formula I:
##STR00001## [0009] or a pharmaceutically acceptable salt thereof,
[0010] wherein R.sup.1 is selected from the group consisting of
hydrogen, C.sub.1-6 alkyl, one amino acid, two amino acids linked
together, three amino acids linked together,
[0010] ##STR00002## [0011] R.sup.3, R.sup.4, R.sup.5, R.sup.9,
R.sup.17 and R.sup.18 are each independently selected from the
group consisting of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6
alkoxy, --C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH,
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15; [0012]
R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are each independently
selected from the group consisting of hydrogen, halogen, C.sub.1-6
alkyl, C.sub.1-6 alkoxy, --C(.dbd.O)NH.sub.2, --NO.sub.2,
--NH.sub.2, --OH, --NH(R.sup.15), --N(R.sup.15).sub.2, and
--SR.sup.15; [0013] R.sup.6 is C.sub.1-6 dialkyl amine; [0014]
R.sup.7 is selected from the group consisting of hydrogen and
C.sub.1-6 alkyl; [0015] R.sup.8 and R.sup.15 are each independently
C.sub.1-6 alkyl; [0016] each R.sup.16 is independently hydrogen,
--OH, or C.sub.1-6 alkoxy; [0017] n is an integer from 0 to 4;
[0018] p is 1 or 3; and [0019] the dashed line represents an
optional double bond where said double bond has a configuration
selected from the group consisting of cis and trans, [0020] with
the proviso that at least one of R.sup.3, R.sup.4, R.sup.5,
R.sup.9, and R.sup.14 is selected from the group consisting of
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15.
[0021] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the compound of Formula I
is:
##STR00003## [0022] or a pharmaceutically acceptable salt
thereof.
[0023] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the compound of Formula I
is:
##STR00004## [0024] or a pharmaceutically acceptable salt
thereof.
[0025] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.1 is selected from the
group consisting of Leu, Leu-Asp, Leu-Asp-Ala,
--CH.sub.2--C(.dbd.O)--NHCH.sub.2COOH,
--CH.sub.2--C(.dbd.O)--(CH.sub.2)C(CH.sub.3).sub.2,
##STR00005##
[0026] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is selected from
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15.
[0027] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is
--N(CH.sub.3).sub.2.
[0028] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is --SCH.sub.3.
[0029] In an embodiment of the first aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the compound of Formula I
is:
##STR00006## [0030] or a pharmaceutically acceptable salt
thereof.
[0031] In a generally applicable second aspect (i.e. independently
combinable with any of the aspects or embodiments identified
herein), a method is provided of treating a cancer comprising
administering to a subject in need thereof an effective amount of
the compound of Formula I.
[0032] In an embodiment of the second aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the subject is mammalian.
[0033] In an embodiment of the second aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the subject is human.
[0034] In an embodiment of the second aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cancer is selected from the
group consisting of lung adenocarcinoma, and glioblastoma
multiforme.
[0035] In an embodiment of the second aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cancer comprises a
translocation comprising an ETS gene selected from the group
consisting of FLI1, ETV1, ETV4, ERG, ETS1, and ETS2.
[0036] In an embodiment of the second aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the compound is administered
parentally.
[0037] In a generally applicable third aspect (i.e. independently
combinable with any of the aspects or embodiments identified
herein), a method is provided of killing or inhibiting the growth
of a neoplastic cell comprising contacting the cell with an
effective amount of a compound of Formula I:
##STR00007## [0038] or a pharmaceutically acceptable salt thereof,
[0039] wherein R.sup.1 is selected from the group consisting of
hydrogen, C.sub.1-6 alkyl, one amino acid, two amino acids linked
together, three amino acids linked together,
[0039] ##STR00008## [0040] R.sup.3, R.sup.4, R.sup.5, R.sup.9, and
R.sup.14 are each independently selected from the group consisting
of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH, --NH(R.sup.15),
--N(R.sup.15).sub.2, and --SR.sup.15; [0041] R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 are each independently selected from the
group consisting of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6
alkoxy, --C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH,
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15; [0042]
R.sup.6 is C.sub.1-6 dialkyl amine; [0043] R.sup.7 is selected from
the group consisting of hydrogen and C.sub.1-6 alkyl; [0044]
R.sup.8 and R.sup.15 are each independently C.sub.1-6 alkyl; [0045]
each R.sup.16 is independently hydrogen, --OH, or C.sub.1-6 alkoxy;
[0046] R.sup.17 and R.sup.18 are independently H or F; [0047] n is
an integer from 0 to 4; [0048] p is 1 or 3; and [0049] the dashed
line represents an optional double bond where said double bond has
a configuration selected from the group consisting of cis and
trans, [0050] with the proviso that at least one of R.sup.3,
R.sup.4, R.sup.5, R.sup.9, and R.sup.14 is selected from the group
consisting of --NH(R.sup.15), --N(R.sup.15).sub.2, and
--SR.sup.15.
[0051] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the compound of Formula I
is:
##STR00009## [0052] or a pharmaceutically acceptable salt
thereof.
[0053] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), The compound of Formula I
is:
##STR00010## [0054] or a pharmaceutically acceptable salt
thereof.
[0055] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.1 is selected from the
group consisting of Leu, Leu-Asp, Leu-Asp-Ala,
--CH.sub.2--C(.dbd.O)--NHCH.sub.2COOH,
--CH.sub.2--C(.dbd.O)--(CH.sub.2)C(CH.sub.3).sub.2,
##STR00011##
[0056] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is selected from
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15.
[0057] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is
--N(CH.sub.3).sub.2.
[0058] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is --SCH.sub.3.
[0059] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), The compound of Formula I
is:
##STR00012## [0060] or a pharmaceutically acceptable salt
thereof.
[0061] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cell is mammalian.
[0062] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cell is human.
[0063] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cell is selected from the
group consisting of lung adenocarcinoma, and glioblastoma
multiforme.
[0064] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cell comprises a
translocation comprising an ETS gene selected from the group
consisting of FLI1, ETV1, ETV4, ERG, ETS1, and ETS2.
[0065] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cell is in vivo.
[0066] In an embodiment of the third aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), the cell is ex vivo.
[0067] In a generally applicable fourth aspect (i.e., independently
combinable with any of the aspects or embodiments identified
herein), a compound is provided of Formula I:
##STR00013## [0068] or a pharmaceutically acceptable salt thereof,
[0069] wherein R.sup.1 is selected from the group consisting of
hydrogen, C.sub.1-6 alkyl, one amino acid, two amino acids linked
together, three amino acids linked together,
[0069] ##STR00014## [0070] R.sup.3, R.sup.4, R.sup.5, R.sup.9, and
R.sup.14 are each independently selected from the group consisting
of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH, --NH(R.sup.15),
--N(R.sup.15).sub.2, and --SR.sup.15; [0071] R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 are each independently selected from the
group consisting of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6
alkoxy, --C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH,
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15; [0072]
R.sup.6 is C.sub.1-6 dialkyl amine; [0073] R.sup.7 is selected from
the group consisting of hydrogen and C.sub.1-6 alkyl; [0074]
R.sup.8 and R.sup.15 are each independently C.sub.1-6 alkyl; [0075]
each R.sup.16 is independently hydrogen, --OH, or C.sub.1-6 alkoxy;
[0076] R.sup.17 and R.sup.18 are independently H or F; [0077] n is
an integer from 0 to 4; [0078] p is 1 or 3; and [0079] the dashed
line represents an optional double bond where said double bond has
a configuration selected from the group consisting of cis and
trans, [0080] with the proviso that at least one of R.sup.3,
R.sup.4, R.sup.5, R.sup.9, and R.sup.14 is selected from the group
consisting of --NH(R.sup.15), --N(R.sup.15).sub.2, and
--SR.sup.15.
[0081] In an embodiment of the fourth aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R' is selected from the group
consisting of Leu, Leu-Asp, Leu-Asp-Ala,
--CH.sub.2--C(.dbd.O)--NHCH.sub.2COOH,
--CH.sub.2--C(.dbd.O)--(CH.sub.2)C(CH.sub.3).sub.2,
##STR00015##
[0082] In an embodiment of the fourth aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is selected from
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15.
[0083] In an embodiment of the fourth aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is
--N(CH.sub.3).sub.2.
[0084] In an embodiment of the fourth aspect, which is generally
applicable (i.e., independently combinable with any of the aspects
or embodiments identified herein), R.sup.3 is --SCH.sub.3.
[0085] In a generally applicable fifth aspect (i.e., independently
combinable with any of the aspects or embodiments identified
herein), pharmaceutical compositions are provided comprising
compounds of Formula I.
[0086] Any of the features of an embodiment of the first through
fifth aspects is applicable to all aspects and embodiments
identified herein. Moreover, any of the features of an embodiment
of the first through fifth aspects is independently combinable,
partly or wholly with other embodiments described herein in any
way, e.g., one, two, or three or more embodiments may be combinable
in whole or in part. Further, any of the features of an embodiment
of the first through fifth aspects may be made optional to other
aspects or embodiments. Any aspect or embodiment of a method can be
performed using a compound of another aspect or embodiment, and any
aspect or embodiment of a compound can be configured to be employed
in a method of another aspect or embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0087] FIG. 1 shows the structure of NSC635437 and a generic
structure for certain analogs.
[0088] FIG. 2 shows an example strategy to increase the potency of
YK-4-279.
[0089] FIG. 3A is a graph of the growth inhibition of TC71 and TC32
cells for various concentrations of YK-4-279 and PT-1-33. FIG. 3B
is a graph of the growth inhibition of TC71 cells for various
concentrations of YK-4-279, PT-1-33, and PT-1-55. FIG. 3C is a
graph of the growth inhibition of TC71 cells for various
concentrations of YK-4-279 and PT-1-123.
[0090] FIG. 4 is a photomicrograph of an immunoblot of protein
lysates from TC32 cells treated with YK-4-279 and co-precipitated
with RHA, EWS-FLI1 or total protein.
[0091] FIGS. 5A-5G are graphs of the relative optical density in
ELISA assays measuring inhibition of EWS-FLI1 binding to RHA by
various candidate agents.
[0092] FIG. 6A and FIG. 6B are graphs showing general trends for
relative luciferase activity for various concentrations of
candidate agents in luciferase assays measuring inhibition of
EWS-FLI1 binding to the NROB1 promoter.
[0093] FIG. 7A-FIG. 7I illustrate luciferase activity for various
concentrations of candidate agents in luciferase assays measuring
inhibition of EWS-FLI1 binding to the NROB1 promoter.
[0094] FIG. 8 depicts the cBioPortal for cancer genomics website
interface.
[0095] FIG. 9 depicts YK-4-279 binds to ERG and ETV1 with a KD of
11.7 .mu.M and 17.9 .mu.M respectively with steady state kinetics
measured on a Biacore T100 instrument.
[0096] FIG. 10 depicts GBM cell lines overexpress FLI1 which
correlates with YK-4-279 sensitivity. Top panel: immunoblot probed
with anti-FLI1. Ewing sarcoma TC32 included as positive control.
Expected size of FLI1, 50, EWS-FLI1 68 kDa. Bottom panel: graph of
IC50 and densiometry.
[0097] FIG. 11 depicts GEMM overexpression of FLI1 compared with
normal brain. RNA was extracted from normal and tumor tissues from
control and genetically modified mice. Hybridization followed by
normalization showed that FLI1 probes were significantly elevated
it the tumors but not normal brain tissues.
[0098] FIG. 12 depicts GBM expression of FLI1. Immunostaining
against FLI1 in human glioblastoma shows positive nuclear staining
in many of the tumor cells as well as in vessel endothelium and
inflammatory cells (40.times. objective).
[0099] FIGS. 13A and 13B illustrate that three days of treatment
with (S)-YK-4-279 or racemic shows significant tumor regression.
FIG. 13A: Mice with ES xenografts were treated with 400 mg/kg
compound or controls as indicated. Starting well-established tumors
(300 mm.sup.3), mice were treated with intraperitoneal compound for
three days, 6 total doses. FIG. 13B: H and E stained tumors from
same experiment.
[0100] FIG. 14 illustrates ERG induces expression of ZEB1 and ZEB2,
which activate EMT leading to lung cancer metastasis and drug
resistance.
[0101] FIG. 15 illustrates YK-4-279 directly interacts with ERG
protein. Purified recombinant ERG was immobilized on Biacore CMS
microchips, and direct binding to eight different YK-4-279
concentrations (0.1-50 .mu.M) was determined by SPR. Steady state
KD was calculated using Biaevaluation software.
[0102] FIGS. 16A and 16B illustrates that YK-4-279 inhibits
transcriptional activity of ERG. FIG. 16A. Luciferase assays of
Cos-7 cells cotransfected with ERG and an Id-2 reporter luciferase
construct. YK-4-279 treatment decreased Id-2 promoter activity
without affecting ERG levels (*; p<0.001). FIG. 16B. VCaP cells
were treated with siERG or YK-4-279 for 48 hours and ERG target
mRNA and protein levels were determined. YK-4-279 treatment
resulted in decreased PLAU, ADAM19 and PLAT mRNA expression. PLAU
levels were also reduced.
[0103] FIG. 17 illustrates that NSCLC cell lines express ERG
protein. Total protein lysates from indicated NSCLC cell lines were
separated by PAGE. Expression of human ERG protein was confirmed by
western blotting using an anti-ERG antibody (upper panel).
Molecular weight markers are given on the left. Equal protein
loading was confirmed by stripping and re-blotting the same
membrane with an anti-beta-actin antibody (lower panel).
[0104] FIGS. 18A and 18B illustrate that ERG expression induces EMT
markers. H358 NSCLC cells were transfected with a cDNA coding for
human ERG protein. Increased ERG expression was detected by western
blotting (FIG. 18A). Real-time PCR analysis revealed higher
expression of ZEB1 and FOXC2 in ERG expressing cells (FIG. 18B).
Data is first normalized for 18S RNA and then expressed as fold
induction over empty vector transfected cells.
[0105] FIG. 19 illustrates that YK-4-279 inhibits EMT in NSCLC.
A549 cells expressed higher levels of ZEB1 and FOXC2 with increased
TGF-.beta. expression and reduced levels with ERG inhibition by
YK-4-279. Data was first normalized to 18S RNA and then fold
expression calculated by dividing to control for each group.
DETAILED DESCRIPTION
[0106] The following description and examples illustrate some
exemplary embodiments of the disclosed invention in detail. Those
of skill in the art will recognize that there are numerous
variations and modifications of this invention that are encompassed
by its scope. Accordingly, the description of a certain exemplary
embodiment should not be deemed to limit the scope of the present
invention.
[0107] A NCI/DTP library of three thousands small molecules was
screened for EWS-FLI1 binding using Surface Plasmon Resonance. The
compound, NSC635437, was selected as a suitable candidate for
further optimization and further study (FIG. 1). Of the first
series of analogs designed, YK-4-279, was the most active (FIG. 2).
YK-4-279 has been shown to functionally inhibit EWS-FLI1 and ESFT
cells and leads to caspase-3 activity increase (Hyariye N Erkizan
et al. A small molecule blocking oncogenic protein EWS-FlI1
interacting with RHA helicase A inhibits growth of Ewing's sarcoma.
Nature Medicine 15(7) 750-756 (2009)). The present application
relates to improved compounds and methods of using such compounds
to treat disorders such as lung adenocarcinoma, glioblastoma
multiforme, and cancers comprising a translocation comprising an
ETS gene selected from the group consisting of FLI1, ETV1, ETV4,
ERG, ETS1, and ETS2.
[0108] Other methods and compositions useful with those provided
herein are disclosed in Int. Pub. No. WO 2008/083326; U.S. Pub. No.
2010/0167994; U.S. Prov App. No. 61/623,349; and Int. Pub. No. WO
2013/155341, the disclosures of which are expressly incorporated
herein by reference in their entireties.
DEFINITIONS
[0109] As used herein, any "R" group(s) such as, without
limitation, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, R.sup.14, R.sup.15, R.sup.a, R.sup.b, represent
substituents that can be attached to the indicated atom. An R group
may be substituted or unsubstituted. If two "R" groups are
described as being "taken together" the R groups and the atoms they
are attached to can form a cycloalkyl, aryl, heteroaryl, or
heterocycle. For example, without limitation, if R.sup.1a and
R.sup.1b of an NR.sup.1aR.sup.1b group are indicated to be "taken
together," it means that they are covalently bonded to one another
to form a ring:
##STR00016##
[0110] Whenever a group is described as being "optionally
substituted" that group may be unsubstituted or substituted with
one or more of the indicated substituents. Likewise, when a group
is described as being "unsubstituted or substituted" if
substituted, the substituent(s) may be selected from one or more
the indicated substituents. If no substituents are indicated, it is
meant that the indicated "optionally substituted" or "substituted"
group may be substituted with one or more group(s) individually and
independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected
hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio,
cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,
N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy,
isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl,
sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl,
trihalomethanesulfonamido, an amino, a mono-substituted amino and a
di-substituted amino group, and protected derivatives thereof.
[0111] As used herein, "C.sub.a to C.sub.b" in which "a" and "b"
are integers refer to the number of carbon atoms in an alkyl,
alkenyl or alkynyl group, or the number of carbon atoms in the ring
of a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl or
heteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring
of the cycloalkyl, ring of the cycloalkenyl, ring of the
cycloalkynyl, ring of the aryl, ring of the heteroaryl or ring of
the heteroalicyclyl can contain from "a" to "b", inclusive, carbon
atoms. Thus, for example, a "C.sub.1 to C.sub.4 alkyl" group refers
to all alkyl groups having from 1 to 4 carbons, that is,
CH.sub.3--, CH.sub.3CH.sub.2--, CH.sub.3CH.sub.2CH.sub.2--,
(CH.sub.3).sub.2CH--, CH.sub.3CH.sub.2CH.sub.2CH.sub.2--,
CH.sub.3CH.sub.2CH(CH.sub.3)-- and (CH.sub.3).sub.3C--. If no "a"
and "b" are designated with regard to an alkyl, alkenyl, alkynyl,
cycloalkyl cycloalkenyl, cycloalkynyl, aryl, heteroaryl or
heteroalicyclyl group, the broadest range described in these
definitions is to be assumed.
[0112] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain that includes a fully saturated (no double or
triple bonds) hydrocarbon group. The alkyl group may have 1 to 20
carbon atoms (whenever it appears herein, a numerical range such as
"1 to 20" refers to each integer in the given range; e.g., "1 to 20
carbon atoms" means that the alkyl group may consist of 1 carbon
atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20
carbon atoms, although the present definition also covers the
occurrence of the term "alkyl" where no numerical range is
designated). The alkyl group may also be a medium size alkyl having
1 to 10 carbon atoms. The alkyl group could also be a lower alkyl
having 1 to 6 carbon atoms. The alkyl group of the compounds may be
designated as "C.sub.1-C.sub.4 alkyl" or similar designations. By
way of example only, "C.sub.1-C.sub.4 alkyl" indicates that there
are one to four carbon atoms in the alkyl chain, i.e., the alkyl
chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl,
iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include,
but are in no way limited to, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, tertiary butyl, pentyl and hexyl. The alkyl group
may be substituted or unsubstituted.
[0113] As used herein, "alkenyl" refers to an alkyl group that
contains in the straight or branched hydrocarbon chain one or more
double bonds. An alkenyl group may be unsubstituted or
substituted.
[0114] As used herein, "alkynyl" refers to an alkyl group that
contains in the straight or branched hydrocarbon chain one or more
triple bonds. An alkynyl group may be unsubstituted or
substituted.
[0115] As used herein, "cycloalkyl" refers to a completely
saturated (no double or triple bonds) mono- or multi-cyclic
hydrocarbon ring system. When composed of two or more rings, the
rings may be joined together in a fused fashion. Cycloalkyl groups
can contain 3 to 10 atoms in the ring(s) or 3 to 8 atoms in the
ring(s). A cycloalkyl group may be unsubstituted or substituted.
Typical cycloalkyl groups include, but are in no way limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
cyclooctyl.
[0116] As used herein, "cycloalkenyl" refers to a mono- or
multi-cyclic hydrocarbon ring system that contains one or more
double bonds in at least one ring; although, if there is more than
one, the double bonds cannot form a fully delocalized pi-electron
system throughout all the rings (otherwise the group would be
"aryl," as defined herein). When composed of two or more rings, the
rings may be connected together in a fused fashion. A cycloalkenyl
group may be unsubstituted or substituted.
[0117] As used herein, "cycloalkynyl" refers to a mono- or
multi-cyclic hydrocarbon ring system that contains one or more
triple bonds in at least one ring. If there is more than one triple
bond, the triple bonds cannot form a fully delocalized pi-electron
system throughout all the rings. When composed of two or more
rings, the rings may be joined together in a fused fashion. A
cycloalkynyl group may be unsubstituted or substituted.
[0118] As used herein, "aryl" refers to a carbocyclic (all carbon)
monocyclic or multicyclic aromatic ring system (including fused
ring systems where two carbocyclic rings share a chemical bond)
that has a fully delocalized pi-electron system throughout all the
rings. The number of carbon atoms in an aryl group can vary. For
example, the aryl group can be a C.sub.6-C.sub.14 aryl group, a
C.sub.6-C.sub.10 aryl group, or a C.sub.6 aryl group. Examples of
aryl groups include, but are not limited to, benzene, naphthalene
and azulene. An aryl group may be substituted or unsubstituted.
[0119] As used herein, "heteroaryl" refers to a monocyclic or
multicyclic aromatic ring system (a ring system with fully
delocalized pi-electron system) that contain(s) one or more
heteroatoms, that is, an element other than carbon, including but
not limited to, nitrogen, oxygen and sulfur. The number of atoms in
the ring(s) of a heteroaryl group can vary. For example, the
heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10
atoms in the ring(s) or 5 to 6 atoms in the ring(s). Furthermore,
the term "heteroaryl" includes fused ring systems where two rings,
such as at least one aryl ring and at least one heteroaryl ring, or
at least two heteroaryl rings, share at least one chemical bond.
Examples of heteroaryl rings include, but are not limited to,
furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole,
oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole,
1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole,
benzimidazole, indole, indazole, pyrazole, benzopyrazole,
isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole,
thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine,
purine, pteridine, quinoline, isoquinoline, quinazoline,
quinoxaline, cinnoline, and triazine. A heteroaryl group may be
substituted or unsubstituted.
[0120] As used herein, "heterocyclyl" or "heteroalicyclyl" refers
to three-, four-, five-, six-, seven-, eight-, nine-, ten-, up to
18-membered monocyclic, bicyclic, and tricyclic ring system wherein
carbon atoms together with from 1 to 5 heteroatoms constitute said
ring system. A heterocycle may optionally contain one or more
unsaturated bonds situated in such a way, however, that a fully
delocalized pi-electron system does not occur throughout all the
rings. The heteroatom(s) is an element other than carbon including,
but not limited to, oxygen, sulfur, and nitrogen. A heterocycle may
further contain one or more carbonyl or thiocarbonyl
functionalities, so as to make the definition include oxo-systems
and thio-systems such as lactams, lactones, cyclic imides, cyclic
thioimides and cyclic carbamates. When composed of two or more
rings, the rings may be joined together in a fused fashion.
Additionally, any nitrogens in a heteroalicyclic may be
quaternized. Heterocyclyl or heteroalicyclic groups may be
unsubstituted or substituted. Examples of such "heterocyclyl" or
"heteroalicyclyl" groups include but are not limited to,
1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane,
1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane,
1,3-dithiole, 1,3-dithiolane, 1,4-oxathiane,
tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide,
barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin,
dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline,
imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine,
oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane,
piperidine N-Oxide, piperidine, piperazine, pyrrolidine,
pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine,
2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran,
thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone,
and their benzo-fused analogs (e.g., benzimidazolidinone,
tetrahydroquinoline, 3,4-methylenedioxyphenyl).
[0121] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl
group connected, as a substituent, via a lower alkylene group. The
lower alkylene and aryl group of an aralkyl may be substituted or
unsubstituted. Examples include but are not limited to benzyl,
2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.
[0122] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)"
refer to a heteroaryl group connected, as a substituent, via a
lower alkylene group. The lower alkylene and heteroaryl group of
heteroaralkyl may be substituted or unsubstituted. Examples include
but are not limited to 2-thienylalkyl, 3-thienylalkyl, furylalkyl,
thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and
imidazolylalkyl, and their benzo-fused analogs.
[0123] A "(heteroalicyclyl)alkyl" and "(heterocyclyl)alkyl" refer
to a heterocyclic or a heteroalicyclylic group connected, as a
substituent, via a lower alkylene group. The lower alkylene and
heterocyclyl of a (heteroalicyclyl)alkyl may be substituted or
unsubstituted. Examples include but are not limited
tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl,
(piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and
(1,3-thiazinan-4-yl)methyl.
[0124] "Lower alkylene groups" are straight-chained --CH.sub.2--
tethering groups, forming bonds to connect molecular fragments via
their terminal carbon atoms. Examples include but are not limited
to methylene (--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--),
propylene (--CH.sub.2CH.sub.2CH.sub.2--), and butylene
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--). A lower alkylene group can
be substituted by replacing one or more hydrogen of the lower
alkylene group with a substituent(s) listed under the definition of
"substituted."
[0125] As used herein, "alkoxy" refers to the formula --OR wherein
R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl
or a cycloalkynyl is defined as above. A non-limiting list of
alkoxys is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy),
n-butoxy, iso-butoxy, sec-butoxy and tert-butoxy. An alkoxy may be
substituted or unsubstituted.
[0126] As used herein, "acyl" refers to a hydrogen, alkyl, alkenyl,
alkynyl, or aryl connected, as substituents, via a carbonyl group.
Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. An
acyl may be substituted or unsubstituted.
[0127] As used herein, "hydroxyalkyl" refers to an alkyl group in
which one or more of the hydrogen atoms are replaced by a hydroxy
group. Exemplary hydroxyalkyl groups include but are not limited
to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and
2,2-dihydroxyethyl. A hydroxyalkyl may be substituted or
unsubstituted.
[0128] As used herein, "haloalkyl" refers to an alkyl group in
which one or more of the hydrogen atoms are replaced by a halogen
(e.g., mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups
include but are not limited to, chloromethyl, fluoromethyl,
difluoromethyl, trifluoromethyl and 1-chloro-2-fluoromethyl,
2-fluoroisobutyl. A haloalkyl may be substituted or
unsubstituted.
[0129] As used herein, "haloalkoxy" refers to an alkoxy group in
which one or more of the hydrogen atoms are replaced by a halogen
(e.g., mono-haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such
groups include but are not limited to, chloromethoxy,
fluoromethoxy, difluoromethoxy, trifluoromethoxy,
1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. A haloalkoxy may
be substituted or unsubstituted.
[0130] As used herein, "aryloxy" and "arylthio" refers to RO-- and
RS--, in which R is an aryl, such as but not limited to phenyl.
Both an aryloxy and arylthio may be substituted or
unsubstituted.
[0131] A "sulfenyl" or "thio" group refers to an "--SR" group in
which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl. A sulfenyl may be substituted
or unsubstituted. The term "sulfenyl" or "thio" includes, but is
not limited to an --SH group (also referred to as a "thiol" group)
as well as an --SR.sub.A group (also referred to as a "thioether"
when R.sub.A is not hydrogen).
[0132] A "sulfinyl" group refers to an "--S(.dbd.O)--R" group in
which R can be the same as defined with respect to sulfenyl. A
sulfinyl may be substituted or unsubstituted.
[0133] A "sulfonyl" group refers to an "SO.sub.2R" group in which R
can be the same as defined with respect to sulfenyl. A sulfonyl may
be substituted or unsubstituted.
[0134] An "O-carboxy" group refers to a "RC(.dbd.O)O--" group in
which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl, as defined herein. An O-carboxy
may be substituted or unsubstituted.
[0135] The terms "ester" and "C-carboxy" refer to a "--C(.dbd.O)OR"
group in which R can be the same as defined with respect to
O-carboxy. An ester and C-carboxy may be substituted or
unsubstituted.
[0136] A "thiocarbonyl" group refers to a "--C(.dbd.S)R" group in
which R can be the same as defined with respect to O-carboxy. A
thiocarbonyl may be substituted or unsubstituted.
[0137] A "trihalomethanesulfonyl" group refers to an
"X.sub.3CSO.sub.2--" group wherein X is a halogen.
[0138] A "trihalomethanesulfonamido" group refers to an
"X.sub.3CS(O).sub.2N(R.sub.A)" group wherein X is a halogen and
R.sub.A is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl.
[0139] The term "amino" as used herein refers to a --N(R).sub.2
group, wherein R is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl. An amino may be substituted or
unsubstituted. The term "amino" includes, but is not limited to a
--NH.sub.2 group (also referred to as an "ammonium" group), a --NHR
group (also referred to as a "secondary amine" when R is not
hydrogen), or a --NR.sub.2 group (also referred to as a "tertiary
amine" when R is not hydrogen).
[0140] As used herein, the term "hydroxy" refers to a --OH
group.
[0141] A "cyano" group refers to a "--CN" group.
[0142] The term "azido" as used herein refers to a --N.sub.3
group.
[0143] An "isocyanato" group refers to a "--NCO" group.
[0144] A "thiocyanato" group refers to a "--CNS" group.
[0145] An "isothiocyanato" group refers to an "--NCS" group.
[0146] A "mercapto" group refers to an "--SH" group.
[0147] A "carbonyl" group refers to a C.dbd.O group.
[0148] An "S-sulfonamido" group refers to a
"--SO.sub.2N(R.sub.AR.sub.B)" group in which R.sub.A and R.sub.B
can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl. An S-sulfonamido may be
substituted or unsubstituted.
[0149] An "N-sulfonamido" group refers to a "RSO.sub.2N(R.sub.A)--"
group in which R and R.sub.A can be independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An
N-sulfonamido may be substituted or unsubstituted.
[0150] An "O-carbamyl" group refers to a
"--OC(.dbd.O)N(R.sub.AR.sub.B)" group in which R.sub.A and R.sub.B
can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl. An O-carbamyl may be
substituted or unsubstituted.
[0151] An "N-carbamyl" group refers to an "ROC(.dbd.O)N(R.sub.A)--"
group in which R and R.sub.A can be independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An
N-carbamyl may be substituted or unsubstituted.
[0152] An "O-thiocarbamyl" group refers to a
"--OC(.dbd.S)--N(R.sub.AR.sub.B)" group in which R.sub.A and
R.sub.B can be independently hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An
O-thiocarbamyl may be substituted or unsubstituted.
[0153] An "N-thiocarbamyl" group refers to an
"ROC(.dbd.S)N(R.sub.A)--" group in which R and R.sub.A can be
independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, or (heteroalicyclyl)alkyl. An N-thiocarbamyl may be
substituted or unsubstituted.
[0154] A "C-amido" group refers to a "--C(.dbd.O)N(R.sub.AR.sub.B)"
group in which R.sub.A and R.sub.B can be independently hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
aryl, heteroaryl, heteroalicyclyl, aralkyl, or
(heteroalicyclyl)alkyl. A C-amido may be substituted or
unsubstituted.
[0155] An "N-amido" group refers to a "RC(.dbd.O)N(R.sub.A)--"
group in which R and R.sub.A can be independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An
N-amido may be substituted or unsubstituted.
[0156] The term "halogen atom" or "halogen" as used herein, means
any one of the radio-stable atoms of column 7 of the Periodic Table
of the Elements, such as, fluorine, chlorine, bromine and
iodine.
[0157] Where the numbers of substituents is not specified (e.g.
haloalkyl), there may be one or more substituents present. For
example "haloalkyl" may include one or more of the same or
different halogens. As another example, "C.sub.1-C.sub.3
alkoxyphenyl" may include one or more of the same or different
alkoxy groups containing one, two or three atoms.
[0158] As used herein, the abbreviations for any protective groups,
amino acids and other compounds, are, unless indicated otherwise,
in accord with their common usage, recognized abbreviations, or the
IUPAC-IUB Commission on Biochemical Nomenclature (See, Biochem.
11:942-944 (1972)).
[0159] It is understood that the compounds described herein can be
labeled isotopically. Substitution with isotopes such as deuterium
may afford certain therapeutic advantages resulting from greater
metabolic stability, such as, for example, increased in vivo
half-life or reduced dosage requirements. Each chemical element as
represented in a compound structure may include any isotope of said
element. For example, in a compound structure a hydrogen atom may
be explicitly disclosed or understood to be present in the
compound. At any position of the compound that a hydrogen atom may
be present, the hydrogen atom can be any isotope of hydrogen,
including but not limited to hydrogen-1 (protium) and hydrogen-2
(deuterium). Thus, reference herein to a compound encompasses all
potential isotopic forms unless the context clearly dictates
otherwise.
[0160] It is understood that the methods and combinations described
herein include crystalline forms (also known as polymorphs, which
include the different crystal packing arrangements of the same
elemental composition of a compound), amorphous phases, salts,
solvates, and hydrates. In some embodiments, the compounds
described herein exist in solvated forms with pharmaceutically
acceptable solvents such as water, ethanol, or the like. In other
embodiments, the compounds described herein exist in unsolvated
form. Solvates contain either stoichiometric or non-stoichiometric
amounts of a solvent, and may be formed during the process of
crystallization with pharmaceutically acceptable solvents such as
water, ethanol, or the like. Hydrates are formed when the solvent
is water, or alcoholates are formed when the solvent is alcohol. In
addition, the compounds provided herein can exist in unsolvated as
well as solvated forms. In general, the solvated forms are
considered equivalent to the unsolvated forms for the purposes of
the compounds and methods provided herein.
[0161] Where a range of values is provided, it is understood that
the upper and lower limit, and each intervening value between the
upper and lower limit of the range is encompassed within the
embodiments.
Certain Synthetic Methods
[0162] In some embodiments, appropriate acetophenone (4.0 equiv.)
and catalytic amount of diethylamine (10 drops) were added to a
solution of 4,7-dichloroisatin (1.0 equiv.) in methanol (5 mL). The
mixture was stirred at room temperature until starting material
(4,7-dichloroisatin) disappeared completely. The resulted solution
was concentrated and applied to flash chromatography eluting with
Hexane/Ethyl acetate to afford pure product in quantitative yield.
Further purification was done by recrystallization with
Hexane/Ethyl acetate. NMR spectra were recorded using a Varian-400
spectrometer for .sup.1H (400 MHz), chemical shifts (.delta.) are
given in ppm downfield from tetramethylsilane as internal standard,
and coupling constants (J-values) are in hertz (Hz). Elemental
analyses were performed by Atlantic Microlabs.
[0163] Certain compounds provided herein can be prepared according
to the following synthesis schemes.
##STR00017##
[0164] In these schemes, ketone (4.0 equiv.) and a catalytic amount
of diethylamine (10 drops) are added to a solution of substituted
isatin (1.0 equiv.) in methanol (5 mL). The mixture is stirred at
room temperature until starting material (substituted isatin)
disappears completely. The resulting solution is concentrated and
applied to flash chromatography eluting with hexane/ethyl acetate
to afford pure product in quantitative yield. Further purification
is done by recrystallization with hexane/ethyl acetate.
[0165] The inhibitors incorporating a carbon-carbon double bond in
the group linking the two ring systems can be prepared from the
corresponding saturated inhibitor by reducing the compound using
synthetic techniques known in the art.
Certain Compounds
[0166] Certain compounds provided herein include compounds having a
Formula I:
##STR00018##
[0167] or a pharmaceutically acceptable salt thereof, wherein
R.sup.1 is selected from the group consisting of hydrogen,
C.sub.1-6 alkyl, one amino acid, two amino acids linked together,
three amino acids linked together,
##STR00019##
R.sup.3, R.sup.4, R.sup.5, R.sup.9, R.sup.14, R.sup.17 and R.sup.18
are each independently selected from the group consisting of
hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH--NH(R.sup.15),
--N(R.sup.15).sub.2, and --SR.sup.15; R.sup.10, R.sup.12, and
R.sup.13 are each independently selected from the group consisting
of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--C(.dbd.O)NH.sub.2, --NO.sub.2, --NH.sub.2, --OH, --NH(R.sup.15),
--N(R.sup.15).sub.2, and --SR.sup.15; R.sup.6 is C.sub.1-6 dialkyl
amine; R.sup.7 is selected from the group consisting of hydrogen
and C.sub.1-6 alkyl; R.sup.8 and R.sup.15 are each independently
C.sub.1-6 alkyl; each R.sup.16 is independently hydrogen, --OH, or
C.sub.1-6 alkoxy; n is an integer from 0 to 4; p is 1 or 3; and the
dashed line represents an optional double bond where said double
bond has a configuration selected from the group consisting of cis
and trans, with the proviso that at least one of R.sup.3, R.sup.4,
R.sup.5, R.sup.9, and R.sup.14 is selected from the group
consisting of --NH(R.sup.15), --N(R.sup.15).sub.2, and
--SR.sup.15.
[0168] In some embodiments, The compound of Formula I is:
##STR00020## [0169] or a pharmaceutically acceptable salt
thereof.
[0170] In some embodiments, The compound of Formula I is:
##STR00021## [0171] or a pharmaceutically acceptable salt
thereof.
[0172] In some embodiments, R' is selected from the group
consisting of Leu, Leu-Asp, Leu-Asp-Ala,
--CH.sub.2--C(.dbd.O)--NHCH.sub.2COOH,
--CH.sub.2--C(.dbd.O)--(CH.sub.2)C(CH.sub.3).sub.2,
##STR00022##
[0173] In some embodiments, R.sup.3 is selected from
--NH(R.sup.15), --N(R.sup.15).sub.2, and --SR.sup.15.
[0174] In some embodiments, R.sup.3 is --N(CH.sub.3).sub.2.
[0175] In some embodiments, R.sup.3 is --SCH.sub.3.
[0176] In some embodiments, The compound of Formula I is:
##STR00023## [0177] or a pharmaceutically acceptable salt
thereof.
[0178] Depending upon the substituents present, the small molecule
inhibitors can be in a form of a pharmaceutically acceptable salt.
The terms "pharmaceutically acceptable salt" as used herein are
broad terms, and is to be given its ordinary and customary meaning
to a person of ordinary skill in the art (and is not to be limited
to a special or customized meaning), and refers without limitation
to salts prepared from pharmaceutically acceptable, non-toxic acids
or bases. Suitable pharmaceutically acceptable salts include
metallic salts, e.g., salts of aluminum, zinc, alkali metal salts
such as lithium, sodium, and potassium salts, alkaline earth metal
salts such as calcium and magnesium salts; organic salts, e.g.,
salts of lysine, N,N'-dibenzylethylenediamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, meglumine
(N-methylglucamine), procaine, and tris; salts of free acids and
bases; inorganic salts, e.g., sulfate, hydrochloride, and
hydrobromide; and other salts which are currently in widespread
pharmaceutical use and are listed in sources well known to those of
skill in the art, such as, for example, The Merck Index. Any
suitable constituent can be selected to make a salt of the
therapeutic agents discussed herein, provided that it is non-toxic
and does not substantially interfere with the desired activity.
[0179] The compounds of preferred embodiments can include isomers,
racemates, optical isomers, enantiomers, diastereomers, tautomers,
and cis/trans conformers. All such isomeric forms are included
within preferred embodiments, including mixtures thereof. As
discussed above, the compounds of preferred embodiments may have
chiral centers, for example, they may contain asymmetric carbon
atoms and may thus exist in the form of enantiomers or
diastereoisomers and mixtures thereof, e.g., racemates. Asymmetric
carbon atom(s) can be present in the (R)-, (S)-, or
(R,S)-configuration, preferably in the (R)- or (S)-configuration,
or can be present as mixtures. Isomeric mixtures can be separated,
as desired, according to conventional methods to obtain pure
isomers.
[0180] The compounds can be in amorphous form, or in crystalline
forms. The crystalline forms of the compounds of preferred
embodiments can exist as polymorphs, which are included in
preferred embodiments. In addition, some of the compounds of
preferred embodiments may also form solvates with water or other
organic solvents. Such solvates are similarly included within the
scope of the preferred embodiments.
Certain Pharmaceutical Compositions
[0181] It is generally preferred to administer the inhibitors of
preferred embodiments in an intravenous or subcutaneous unit dosage
form; however, other routes of administration are also
contemplated. Contemplated routes of administration include but are
not limited to oral, parenteral, intravenous, and subcutaneous. The
inhibitors of preferred embodiments can be formulated into liquid
preparations for, e.g., oral administration. Suitable forms include
suspensions, syrups, elixirs, and the like. Particularly preferred
unit dosage forms for oral administration include tablets and
capsules. Unit dosage forms configured for administration once a
day are particularly preferred; however, in certain embodiments it
can be desirable to configure the unit dosage form for
administration twice a day, or more.
[0182] The pharmaceutical compositions of preferred embodiments are
preferably isotonic with the blood or other body fluid of the
recipient. The isotonicity of the compositions can be attained
using sodium tartrate, propylene glycol or other inorganic or
organic solutes. Sodium chloride is particularly preferred.
Buffering agents can be employed, such as acetic acid and salts,
citric acid and salts, boric acid and salts, and phosphoric acid
and salts. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like.
[0183] Viscosity of the pharmaceutical compositions can be
maintained at the selected level using a pharmaceutically
acceptable thickening agent. Methylcellulose is preferred because
it is readily and economically available and is easy to work with.
Other suitable thickening agents include, for example, xanthan gum,
carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the
like. The preferred concentration of the thickener will depend upon
the thickening agent selected. An amount is preferably used that
will achieve the selected viscosity. Viscous compositions are
normally prepared from solutions by the addition of such thickening
agents.
[0184] A pharmaceutically acceptable preservative can be employed
to increase the shelf life of the pharmaceutical compositions.
Benzyl alcohol can be suitable, although a variety of preservatives
including, for example, parabens, thimerosal, chlorobutanol, or
benzalkonium chloride can also be employed. A suitable
concentration of the preservative is typically from about 0.02% to
about 2% based on the total weight of the composition, although
larger or smaller amounts can be desirable depending upon the agent
selected. Reducing agents, as described above, can be
advantageously used to maintain good shelf life of the
formulation.
[0185] The inhibitors of preferred embodiments can be in admixture
with a suitable carrier, diluent, or excipient such as sterile
water, physiological saline, glucose, or the like, and can contain
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, gelling or viscosity enhancing additives,
preservatives, flavoring agents, colors, and the like, depending
upon the route of administration and the preparation desired. See,
e.g., "Remington: The Science and Practice of Pharmacy", Lippincott
Williams & Wilkins; 20th edition (Jun. 1, 2003) and
"Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18.sup.th and
19.sup.th editions (December 1985, and June 1990, respectively).
Such preparations can include complexing agents, metal ions,
polymeric compounds such as polyacetic acid, polyglycolic acid,
hydrogels, dextran, and the like, liposomes, microemulsions,
micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts
or spheroblasts. Suitable lipids for liposomal formulation include,
without limitation, monoglycerides, diglycerides, sulfatides,
lysolecithin, phospholipids, saponin, bile acids, and the like. The
presence of such additional components can influence the physical
state, solubility, stability, rate of in vivo release, and rate of
in vivo clearance, and are thus chosen according to the intended
application, such that the characteristics of the carrier are
tailored to the selected route of administration.
[0186] For oral administration, the pharmaceutical compositions can
be provided as a tablet, aqueous or oil suspension, dispersible
powder or granule, emulsion, hard or soft capsule, syrup or elixir.
Compositions intended for oral use can be prepared according to any
method known in the art for the manufacture of pharmaceutical
compositions and can include one or more of the following agents:
sweeteners, flavoring agents, coloring agents and preservatives.
Aqueous suspensions can contain the active ingredient in admixture
with excipients suitable for the manufacture of aqueous
suspensions.
[0187] Formulations for oral use can also be provided as hard
gelatin capsules, wherein the active ingredient(s) are mixed with
an inert solid diluent, such as calcium carbonate, calcium
phosphate, or kaolin, or as soft gelatin capsules. In soft
capsules, the inhibitors can be dissolved or suspended in suitable
liquids, such as water or an oil medium, such as peanut oil, olive
oil, fatty oils, liquid paraffin, or liquid polyethylene glycols.
Stabilizers and microspheres formulated for oral administration can
also be used. Capsules can include push-fit capsules made of
gelatin, as well as soft, sealed capsules made of gelatin and a
plasticizer, such as glycerol or sorbitol. The push-fit capsules
can contain the active ingredient in admixture with fillers such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers.
[0188] Tablets can be uncoated or coated by known methods to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period of time.
For example, a time delay material such as glyceryl monostearate
can be used. When administered in solid form, such as tablet form,
the solid form typically comprises from about 0.001 wt. % or less
to about 50 wt. % or more of active ingredient(s), preferably from
about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.
[0189] Tablets can contain the active ingredients in admixture with
non-toxic pharmaceutically acceptable excipients including inert
materials. For example, a tablet can be prepared by compression or
molding, optionally, with one or more additional ingredients.
Compressed tablets can be prepared by compressing in a suitable
machine the active ingredients in a free-flowing form such as
powder or granules, optionally mixed with a binder, lubricant,
inert diluent, surface active or dispersing agent. Molded tablets
can be made by molding, in a suitable machine, a mixture of the
powdered inhibitor moistened with an inert liquid diluent.
[0190] Preferably, each tablet or capsule contains from about 1 mg
or less to about 1,000 mg or more of an inhibitor of the preferred
embodiments, more preferably from about 10, 20, 30, 40, 50, 60, 70,
80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, or 900 mg. Most preferably, tablets
or capsules are provided in a range of dosages to permit divided
dosages to be administered. A dosage appropriate to the patient and
the number of doses to be administered daily can thus be
conveniently selected. In certain embodiments it can be preferred
to incorporate two or more of the therapeutic agents to be
administered into a single tablet or other dosage form (e.g., in a
combination therapy); however, in other embodiments it can be
preferred to provide the therapeutic agents in separate dosage
forms.
[0191] Suitable inert materials include diluents, such as
carbohydrates, mannitol, lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans, starch, and the like, or inorganic
salts such as calcium triphosphate, calcium phosphate, sodium
phosphate, calcium carbonate, sodium carbonate, magnesium
carbonate, and sodium chloride. Disintegrants or granulating agents
can be included in the formulation, for example, starches such as
corn starch, alginic acid, sodium starch glycolate, Amberlite,
sodium carboxymethylcellulose, ultramylopectin, sodium alginate,
gelatin, orange peel, acid carboxymethyl cellulose, natural sponge
and bentonite, insoluble cationic exchange resins, powdered gums
such as agar, karaya or tragacanth, or alginic acid or salts
thereof.
[0192] Binders can be used to form a hard tablet. Binders include
materials from natural products such as acacia, tragacanth, starch
and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl
cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose,
and the like.
[0193] Lubricants, such as stearic acid or magnesium or calcium
salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable
oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate,
polyethylene glycol, starch, talc, pyrogenic silica, hydrated
silicoaluminate, and the like, can be included in tablet
formulations.
[0194] Surfactants can also be employed, for example, anionic
detergents such as sodium lauryl sulfate, dioctyl sodium
sulfosuccinate and dioctyl sodium sulfonate, cationic such as
benzalkonium chloride or benzethonium chloride, or nonionic
detergents such as polyoxyethylene hydrogenated castor oil,
glycerol monostearate, polysorbates, sucrose fatty acid ester,
methyl cellulose, or carboxymethyl cellulose.
[0195] Controlled release formulations can be employed wherein the
amifostine or analog(s) thereof is incorporated into an inert
matrix that permits release by either diffusion or leaching
mechanisms. Slowly degenerating matrices can also be incorporated
into the formulation. Other delivery systems can include timed
release, delayed release, or sustained release delivery
systems.
[0196] Coatings can be used, for example, nonenteric materials such
as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols, or enteric materials such
as phthalic acid esters. Dyestuffs or pigments can be added for
identification or to characterize different combinations of
inhibitor doses
[0197] When administered orally in liquid form, a liquid carrier
such as water, petroleum, oils of animal or plant origin such as
peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic
oils can be added to the active ingredient(s). Physiological saline
solution, dextrose, or other saccharide solution, or glycols such
as ethylene glycol, propylene glycol, or polyethylene glycol are
also suitable liquid carriers. The pharmaceutical compositions can
also be in the form of oil-in-water emulsions. The oily phase can
be a vegetable oil, such as olive or arachis oil, a mineral oil
such as liquid paraffin, or a mixture thereof. Suitable emulsifying
agents include naturally-occurring gums such as gum acacia and gum
tragacanth, naturally occurring phosphatides, such as soybean
lecithin, esters or partial esters derived from fatty acids and
hexitol anhydrides, such as sorbitan mono-oleate, and condensation
products of these partial esters with ethylene oxide, such as
polyoxyethylene sorbitan mono-oleate. The emulsions can also
contain sweetening and flavoring agents.
[0198] Pulmonary delivery can also be employed. The compound is
delivered to the lungs while inhaling and traverses across the lung
epithelial lining to the blood stream. A wide range of mechanical
devices designed for pulmonary delivery of therapeutic products can
be employed, including but not limited to nebulizers, metered dose
inhalers, and powder inhalers, all of which are familiar to those
skilled in the art. These devices employ formulations suitable for
the dispensing of compound. Typically, each formulation is specific
to the type of device employed and can involve the use of an
appropriate propellant material, in addition to diluents,
adjuvants, and/or carriers useful in therapy.
[0199] The compound and/or other optional active ingredients are
advantageously prepared for pulmonary delivery in particulate form
with an average particle size of from 0.1 .mu.m or less to 10 .mu.m
or more, more preferably from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, or 0.9 .mu.m to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 .mu.m.
Pharmaceutically acceptable carriers for pulmonary delivery of
inhibitor include carbohydrates such as trehalose, mannitol,
xylitol, sucrose, lactose, and sorbitol. Other ingredients for use
in formulations can include DPPC, DOPE, DSPC, and DOPC. Natural or
synthetic surfactants can be used, including polyethylene glycol
and dextrans, such as cyclodextran. Bile salts and other related
enhancers, as well as cellulose and cellulose derivatives, and
amino acids can also be used. Liposomes, microcapsules,
microspheres, inclusion complexes, and other types of carriers can
also be employed.
[0200] Pharmaceutical formulations suitable for use with a
nebulizer, either jet or ultrasonic, typically comprise the
inhibitor dissolved or suspended in water at a concentration of
about 0.01 or less to 100 mg or more of inhibitor per mL of
solution, preferably from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, or 90 mg per mL of solution. The formulation can also
include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation can also contain a surfactant, to reduce or prevent
surface induced aggregation of the inhibitor caused by atomization
of the solution in forming the aerosol.
[0201] Formulations for use with a metered-dose inhaler device
generally comprise a finely divided powder containing the active
ingredients suspended in a propellant with the aid of a surfactant.
The propellant can include conventional propellants, such as
chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons,
and hydrocarbons. Preferred propellants include
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, and
combinations thereof. Suitable surfactants include sorbitan
trioleate, soya lecithin, and oleic acid.
[0202] Formulations for dispensing from a powder inhaler device
typically comprise a finely divided dry powder containing
inhibitor, optionally including a bulking agent, such as lactose,
sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount
that facilitates dispersal of the powder from the device, typically
from about 1 wt. % or less to 99 wt. % or more of the formulation,
preferably from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt.
% to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the
formulation.
[0203] When a compound of the preferred embodiments is administered
by intravenous, parenteral, or other injection, it is preferably in
the form of a pyrogen-free, parenterally acceptable aqueous
solution or oleaginous suspension. Suspensions can be formulated
according to methods well known in the art using suitable
dispersing or wetting agents and suspending agents. The preparation
of acceptable aqueous solutions with suitable pH, isotonicity,
stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for injection preferably
contains an isotonic vehicle such as 1,3-butanediol, water,
isotonic sodium chloride solution, Ringer's solution, dextrose
solution, dextrose and sodium chloride solution, lactated Ringer's
solution, or other vehicles as are known in the art. In addition,
sterile fixed oils can be employed conventionally as a solvent or
suspending medium. For this purpose, any bland fixed oil can be
employed including synthetic mono or diglycerides. In addition,
fatty acids such as oleic acid can likewise be used in the
formation of injectable preparations. The pharmaceutical
compositions can also contain stabilizers, preservatives, buffers,
antioxidants, or other additives known to those of skill in the
art.
[0204] The duration of the injection can be adjusted depending upon
various factors, and can comprise a single injection administered
over the course of a few seconds or less, to 0.5, 0.1, 0.25, 0.5,
0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, or 24 hours or more of continuous
intravenous administration.
[0205] The compounds of the preferred embodiments can additionally
employ adjunct components conventionally found in pharmaceutical
compositions in their art-established fashion and at their
art-established levels. Thus, for example, the compositions can
contain additional compatible pharmaceutically active materials for
combination therapy (such as supplementary antimicrobials,
antipruritics, astringents, local anesthetics, anti-inflammatory
agents, reducing agents, chemotherapeutics and the like), or can
contain materials useful in physically formulating various dosage
forms of the preferred embodiments, such as excipients, dyes,
thickening agents, stabilizers, preservatives or antioxidants.
Anti-cancer agents that can be used in combination with the
compounds of preferred embodiments include, but are not limited to,
vinca alkaloids such as vinblastine and vincristine; anthracyclines
such as doxorubicin, daunorubicin, epirubicin; anthracenes such as
bisantrene and mitoxantrone; epipodophyllo-toxins such as etoposide
and teniposide; and other anticancer drugs such as actinomyocin D,
mithomycin C, mitramycin, methotrexate, docetaxel, etoposide
(VP-16), paclitaxel, docetaxel, and adriamycin); and
immunosuppressants (e.g., cyclosporine A, tacrolimus). In some
embodiments, the compounds, compositions and methods provided
herein may be in combination with histone deacetylase inhibitors
(HDAC), aurora kinase inhibitors, demethylating agents (such as
5-AZA cytidine), immunotherapy with natural killer cells, IGF-IR
antibodies, Ewing antigen antibodies, immunosuppressive drugs, and
hydroxyurea. Examples of histone deacetylase inhibitors include
vorinostat, romidepsin, panobinostat, valproic acid, belinostat,
mocetinostat, givinostat, and trichostatin A. Examples of aurora
kinase inhibitors include ZM447439, hesperadin, and VX-680.
Examples of demethylating agents include 5-azacytidine,
5-azadeoxycytidine, and procaine. Examples of immunosuppressive
drugs include 6-mercaptopurine, and azathioprine.
Certain Kits
[0206] The compounds of the preferred embodiments can be provided
to an administering physician or other health care professional in
the form of a kit. The kit is a package which houses a container
which contains the compounds in a suitable pharmaceutical
composition, and instructions for administering the pharmaceutical
composition to a subject. The kit can optionally also contain one
or more additional therapeutic agents, e.g., chemotherapeutics
currently employed for treating the sarcomas described herein. For
example, a kit containing one or more compositions comprising
compounds of the preferred embodiments in combination with one or
more additional chemotherapeutic agents can be provided, or
separate pharmaceutical compositions containing an inhibitor of the
preferred embodiments and additional therapeutic agents can be
provided. The kit can also contain separate doses of a compound of
the preferred embodiments for serial or sequential administration.
The kit can optionally contain one or more diagnostic tools and
instructions for use. The kit can contain suitable delivery
devices, e.g., syringes, and the like, along with instructions for
administering the inhibitor(s) and any other therapeutic agent. The
kit can optionally contain instructions for storage, reconstitution
(if applicable), and administration of any or all therapeutic
agents included. The kits can include a plurality of containers
reflecting the number of administrations to be given to a
subject.
Certain Therapeutic Methods
[0207] Some embodiments provided herein relate to methods of
treating the Ewing's sarcoma family of tumors (ESFT). ESFT contains
the unique fusion protein EWS-FLI1. ESFT affects patients between
the ages of 3 and 40 years, with most cases occurring in the second
decade. Although the embryologic cell type from which ESFT are
derived is unknown, the tumor often grows in close proximity to
bone, but can occur as a soft-tissue mass. Over 40% of patients who
present with localized tumors will develop recurrent disease and
the majority of these will die from ESFT, while 75-80% of patients
who present with metastatic ESFT will die within 5 years despite
high-dose chemotherapy (Grier H E, Krailo M D, Tarbell N J, et al.
Addition of ifosfamide and etoposide to standard chemotherapy for
Ewing's sarcoma and primitive neuroectodermal tumor of bone. N Engl
J Med 2003; 348(8):694-701). These survival rates have not improved
for the past 20 years, even after dose-intensifying chemotherapy.
To improve survival and reduce therapy-related morbidity, novel
targeted strategies for treating ESFT patients, as provided in the
preferred embodiments, can be employed.
[0208] ESFT are characterized by a translocation, occurring in 95%
of tumors, between the central exons of the EWS gene (Ewing
Sarcoma) located on chromosome 22 to the central exons of an ets
family gene; either FLI1 (Friend Leukemia Insertion) located on
chromosome 11, t(11; 22), or ERG located on chromosome 21, t(21;
22). The EWS-FLI1 fusion transcript encodes a 55 kDa protein
(electrophoretic motility of approximately 68 kD) with two primary
domains. The EWS domain is a potent transcriptional activator,
while the FLI1 domain contains a highly conserved ets DNA binding
domain (May W A, Lessnick S L, Braun B S, et al. The Ewing's
sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional
activator and is a more powerful transforming gene than FLI-1. Mol
Cell Biol 1993; 13(12):7393-8); the resulting EWS-FLI1 fusion
protein acts as an aberrant transcription factor. EWS-FLI1
transformation of mouse fibroblasts requires both the EWS and FLI1
functional domains to be intact (May W A, Gishizky M L, Lessnick S
L, et al. Ewing sarcoma 11; 22 translocation produces a chimeric
transcription factor that requires the DNA-binding domain encoded
by FLI1 for transformation. Proc Natl Acad Sci USA 1993;
90(12):5752-6).
[0209] EWS-FLI1 is an outstanding therapeutic target, in that it is
expressed only in tumor cells and is required to maintain the
growth of ESFT cell lines. Reduced expression levels of EWS-FLI1
using either antisense oligodeoxynucleotides (ODN) (Toretsky J A,
Connell Y, Neckers L, Bhat N K. Inhibition of EWS-FLI-1 fusion
protein with antisense oligodeoxynucleotides. J Neurooncol 1997;
31(1-2): 9-16; Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y.
EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of
human Ewing's sarcoma and primitive neuroectodermal tumor cells. J
Clin Invest 1997; 99(2):239-47) or small interfering RNAs (siRNA)
(Ouchida M, Ohno T, Fujimura Y, Rao V N, Reddy E S. Loss of
tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to
EWS-fusion transcripts. Oncogene 1995; 11(6):1049-54; Maksimenko A,
Malvy C, Lambert G, et al. Oligonucleotides targeted against a
junction oncogene are made efficient by nanotechnologies. Pharm Res
2003; 20(10):1565-7; Kovar H, Aryee D N, Jug G, et al. EWS/FLI-1
antagonists induce growth inhibition of Ewing tumor cells in vitro.
Cell Growth Differ 1996; 7(4):429-37) cause decreased proliferation
of ESFT cell lines and regression of tumors in nude mice. Recent
advances in nanotechnology have improved the delivery and
controlled release of siRNA, yet neither antisense ODN nor siRNA
reduction of EWS-FLI1 in humans is possible with current
technologies (Maksimenko A, Malvy C, Lambert G, et al.
Oligonucleotides targeted against a junction oncogene are made
efficient by nanotechnologies. Pharm Res 2003; 20(10):1565-7;
Lambert G, Bertrand J R, Fattal E, et al. EWS fli-1 antisense
nanocapsules inhibits Ewing sarcoma-related tumor in mice. Biochem
Biophys Res Commun 2000; 279(2):401-6). One interesting approach to
EWS-FLI1 targeting used comparative expression between siRNA
reduced EWS-FLI1 and a library of small molecules, which led to a
current clinical trial with Ara-C (Stegmaier K, Wong J S, Ross K N,
et al. Signature-based small molecule screening identifies cytosine
arabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS medicine
2007; 4(4):e122). This method of identifying Ara-C also indicated
doxorubicin and puromycin would reduce EWS-FLI1 levels. Doxorubicin
is currently used as standard therapy for ESFT patients and yet,
survival is far from acceptable (Grier H E, Krailo M D, Tarbell N
J, et al. Addition of ifosfamide and etoposide to standard
chemotherapy for Ewing's sarcoma and primitive neuroectodermal
tumor of bone. N Engl J Med 2003; 348(8):694-701). The use of Ara-C
in ESFT patients is currently being evaluated in a Phase II trial.
While it is hoped that this represents a needed clinical
breakthrough, it certainly demonstrates the importance of small
molecule targeting of EWS-FLI1. The preferred embodiments provide
small molecule protein-protein interaction inhibitors (SMPPII) that
disrupt EWS-FLI1 from critical protein partners, thereby achieving
tumor specificity and more precise targeting of EWS-FLI1.
[0210] There is sufficient evidence to conclude that EWS-FLI1
fusion protein functions differently than either untranslocated EWS
or FLI1 (May W A, Gishizky M L, Lessnick S L, et al. Ewing sarcoma
11; 22 translocation produces a chimeric transcription factor that
requires the DNA-binding domain encoded by FLI1 for transformation.
Proc Natl Acad Sci USA 1993; 90(12):5752-6). Changes in gene
expression profiles of EWS-FLI1-expressing cell lines (Braun B S,
Frieden R, Lessnick S L, May W A, Denny C T. Identification of
target genes for the Ewing's sarcoma EWS/FLI fusion protein by
representational difference analysis. Mol Cell Biol 1995;
15(8):4623-30) or tumor cells taken from ESFT patients, compared to
tumors lacking EWS-FLI1 expression, indicate that EWS-FLI1 may play
a role in transcriptional regulation (Khan J, Wei J S, Ringner M,
et al. Classification and diagnostic prediction of cancers using
gene expression profiling and artificial neural networks. Nat Med
2001; 7(6):673-9; Baer C, Nees M, Breit S, et al. Profiling and
functional annotation of mRNA gene expression in pediatric
rhabdomyosarcoma and Ewing's sarcoma. Int J Cancer 2004;
110(5):687-94). While a clear picture of the mechanism of
EWS-FLI1-regulated gene expression has yet to emerge, this activity
is likely the result of direct or secondary interactions between
EWS-FLI1 and regulators of RNA synthesis and splicing (Uren A,
Toretsky J A. Ewing's Sarcoma Oncoprotein EWS-FLI1: the Perfect
Target without a Therapeutic Agent. Future Onc 2005;
1(4):521-8).
[0211] EWS-FLI1 is a great therapeutic target since it is only
expressed in tumor cells; however, the ability to target this
tumor-specific oncogene has previously not been successful. One of
the challenges towards small molecule development is that EWS-FLI1
lacks any know enzymatic domains, and enzyme domains have been
thought to be critical for targeted therapeutics. In addition,
EWS-FLI1 is a disordered protein, indicating that it does not
exhibit a rigid structure that can be used for structure based drug
design (Uren A, Tcherkasskaya O, Toretsky J A. Recombinant EWS-FLI1
oncoprotein activates transcription. Biochemistry 2004;
43(42):13579-89). In fact, the disordered nature of EWS-FLI1 is
critical for its transcriptional regulation (Ng K P, Potikyan G,
Savene R O, Denny C T, Uversky V N, Lee K A. Multiple aromatic side
chains within a disordered structure are critical for transcription
and transforming activity of EWS family oncoproteins. Proc Natl
Acad Sci USA 2007; 104(2):479-84). Disordered proteins are
considered as more attractive targets for small molecule
protein-protein interaction inhibitors specifically because of
their biochemical disordered properties (Cheng Y, LeGall T,
Oldfield C J, et al. Rational drug design via intrinsically
disordered protein. Trends Biotechnol 2006; 24(10):435-42).
[0212] EWS-FLI1 binds RNA helicase A in vitro and in vivo. It is
believed that protein-protein interactions of EWS-FLI1 may
contribute to its oncogenic potential; therefore, novel proteins
have been sought that directly interact with and functionally
modulate EWS-FLI1. Recombinant EWS-FLI1 that is transcriptionally
active (Uren A, Tcherkasskaya O, Toretsky J A. Recombinant EWS-FLI1
oncoprotein activates transcription. Biochemistry 2004;
43(42):13579-89) was used as a target for screening a commercial
peptide phage display library. Twenty-eight novel peptides that
differentially bind to EWS-FLI1 were identified from phage
sequencing. A National Center for Biotechnology Information
database search for human proteins homologous to these peptides
identified a peptide that was homologous to aa 823-832 of the human
RNA helicase A, (RHA, gene bank accession number A47363) (Toretsky
J A, Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is
enhanced by RNA helicase A. Cancer Res 2006; 66(11):5574-81).
[0213] RHA, a member of the highly conserved DEXD/H box helicase
family of proteins, is an integral, multifunctional member of the
human transcriptome (Zhang S, Grosse F. Multiple functions of
nuclear DNA helicase II (RNA helicase A) in nucleic acid
metabolism. Acta Biochim Biophys Sin (Shanghai) 2004; 36(3):177-83;
von Hippel P H, Delagoutte E. A general model for nucleic acid
helicases and their "coupling" within macromolecular machines. Cell
2001; 104(2):177-90). These proteins are involved in diverse
functions in a variety of organisms, from archaea, eubacteria,
lower and higher eukaryotes and a number of viruses, including the
positive-sense RNA viruses of the Flavivirus family. RHA is a
transcriptional coactivator for NF-.kappa.B, and has been shown to
form complexes with Creb-binding protein (CBP) (Nakajima T, Uchida
C, Anderson S F, et al. RNA helicase A mediates association of CBP
with RNA polymerase II. Cell 1997; 90(6):1107-12), RNA Polymerase
II (Nakajima T, Uchida C, Anderson S F, et al. RNA helicase A
mediates association of CBP with RNA polymerase II. Cell 1997;
90(6):1107-12), the breast cancer tumor suppressor BRCA1 (Anderson
S F, Schlegel B P, Nakajima T, Wolpin E S, Parvin J D. BRCA1
protein is linked to the RNA polymerase II holoenzyme complex via
RNA helicase A. Nat Genet 1998; 19(3):254-6), and, most recently,
EWS-FLI1 (Toretsky J A, Erkizan V, Levenson A, et al. Oncoprotein
EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 2006;
66(11):5574-81). EWS-FLI1 binds to a region of RHA that is unique
and not known as a binding site for any of the other RHA binding
partners (Toretsky J A, Erkizan V, Levenson A, et al. Oncoprotein
EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 2006;
66(11):5574-81). RHA expression enhanced EWS-FLI1 mediated
anchorage-independent colony formation, while an inactivating
mutation of RHA prevented colony formation (Toretsky J A, Erkizan
V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is enhanced by
RNA helicase A. Cancer Res 2006; 66(11):5574-81). This structural
and function interaction is the basis for the therapeutic agents of
preferred embodiments.
[0214] Despite the importance of transcription in tumorigenesis,
the role of helicases in this process has not been well-studied.
RHA is an integral member of the human transcriptome with diverse
functions (Zhang S, Grosse F. Multiple functions of nuclear DNA
helicase II (RNA helicase A) in nucleic acid metabolism. Acta
Biochim Biophys Sin (Shanghai) 2004; 36(3):177-83; von Hippel P H,
Delagoutte E. A general model for nucleic acid helicases and their
"coupling" within macromolecular machines. Cell 2001;
104(2):177-90). Our recently published data show that RHA interacts
with the multifunctional EWS-FLI1 oncoprotein (Toretsky J A,
Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is
enhanced by RNA helicase A. Cancer Res 2006; 66(11):5574-81). This
interaction could account for the observed ability of EWS-FLI1 to
function in both transcription initiation and post-transcriptional
RNA modification. RNA helicases are also known to bind and act as a
bridge for some of the same factors that have been identified as
binding partners for EWS-FLI1, including the splicing factor U1C
(Chen J Y, Stands L, Staley J P, Jackups R R, Jr., Latus L J, Chang
T H. Specific alterations of U1-C protein or U1 small nuclear RNA
can eliminate the requirement of Prp28p, an essential DEAD box
splicing factor. Mol Cell 2001; 7(1):227-32; Knoop L L, Baker S J.
The splicing factor U1C represses EWS/FLI-mediated transactivation.
J Biol Chem 2000; 275(32):24865-71), Creb-binding protein (CBP)
(Nakajima T, Uchida C, Anderson S F, et al. RNA helicase A mediates
association of CBP with RNA polymerase II. Cell 1997;
90(6):1107-12) and RNA Polymerase II (Nakajima T, Uchida C,
Anderson S F, et al. RNA helicase A mediates association of CBP
with RNA polymerase II. Cell 1997; 90(6):1107-12). RHA may perform
a similar function for EWS-FLI1 and RNA Pol II, acting in the
recruitment of key processing proteins. RHA may also contribute to
ESFT oncogenesis by maintaining EWS-FLI1 as part of a large
transcriptional complex whose function relies on the ATPase
activity of RHA as an energy source. Finally, helicases, like RHA,
can stabilize mRNA species (Iost I, Dreyfus M. mRNAs can be
stabilized by DEAD-box proteins. Nature 1994; 372(6502):193-6). The
stabilization and metabolism of EWS-FLI1 transcribed mRNA by RHA
may augment the oncogenic nature of EWS-FLI1.
[0215] While EWS-FLI1 is quite specific to ESFT cells, EWS and RHA
are ubiquitously expressed. The region between EWS-FLI1 and RHA are
targeted by molecular therapeutics that may have specificity; since
EWS-FLI1 is expressed only in tumors and the interaction points
with RHA may be unique. Therapeutic agents, namely, small molecule
protein-protein interaction inhibitors, are provided herein to
inhibit EWS-FLI1 function.
[0216] Most translocation-fusion protein sarcomas portend a poor
prognosis, including ESFT. The chromosomal translocation t(11; 22),
leading to the unique and critical fusion protein EWS-FLI1, is a
perfect cancer target. Many other sarcomas share similar
translocation variants (Table 2. from Helman L J, Meltzer P.
Mechanisms of sarcoma development. Nat Rev Cancer 2003;
3(9):685-94).
[0217] EWS-FLI1 translocations have been reported in solid
pseudopapillaryneoplasms of the pancreas (Maitra A., et al.,
Detection of t(11; 22)(q24; q12) translocation and EWS-FLI-1 fusion
transcript in a case of solid pseudopapillary tumor of the
pancreas. Pediatr Dev Pathol 2000; 3:603-605), however the role of
EWS-FLI1 in all solid pseudopaillary neoplasms remains to be
resolved (Katharina Tiemann et al., Solid pseudopapillary neoplasms
of the pancreas are associated with FLI-1 expression, but not with
EWS/FLI-1 translocation).
[0218] EWS or FLI1 homologues are partners in translocations that
occur in a wide range of sarcomas and leukemias. EWS, or its
homologue TLS or FUS, is involved in chromosomal translocations of
clear cell sarcoma, myxoid liposarcoma, desmoplastic small round
cell tumor, chondrosarcoma and acute myeloid leukemia. FLI1 belongs
to the ets family of genes. The FLI1 homologue ERG is translocated
in approximately 10% of Ewing's sarcomas and 20% of acute myeloid
leukemias. This suggests that EWS-FLI1 can serve as model system
that might impact upon a family of diseases (related by
translocation partners) that affect a large number of patients
(Uren A., Tcherkasskaya O. and Toretsky J. A. Recombinant EWS-FLI1
oncoprotein activates transcription. Biochemistry 43(42) 13579-89
(2004)).
[0219] ERG is also translocated in prostate cancer, where the
TMPRSS2:ERG fusion suggests a distinct molecular subtype that may
define risk for disease progression (F. Demichelis et al.,
TMPRSS2:ERG gene fusion associated with lethal cancer in a watchful
waiting cohort. Oncogene (2007)26, 4596-4599). Other diseases where
translocations of EWS or FLI1 family members have been observed
include congenital fibrosarcoma and cellular mesoblastic nephroma
where the ets family member ETV6 is juxtaposed with NTRK3. Other
translocation gene fusions include chronic myeloid leukemia that
leads to expression of the BCR-ABL fusion protein, and synovial
sarcoma where the SYT gene from chromosome 18 is juxtaposed with
either SSX1 or SSX2 from the X chromosome (Aykut Uren and Jeffrey
A. Toretsky, Pediatric malignancies provide unique cancer therapy
targets. Curr Opin Pediatr 17:14-19 (2005)).
[0220] Therefore, the therapeutic agents of the preferred
embodiments have potential for application in many other tumors.
More broadly, some of the most difficult leukemias also have
translocation-generated fusion proteins involving the mixed-lineage
leukemia gene (MLL, 11q23), and our work could serve as a paradigm
for a very treatment-resistant group of cancers (Pui C H, Chessells
J M, Camitta B, et al. Clinical heterogeneity in childhood acute
lymphoblastic leukemia with 11q23 rearrangements. Leukemia 2003;
17(4):700-6.). Thus embodiments include cancers where
translocations have occurred. Translocation fusion genes are listed
in Table 1.
TABLE-US-00001 TABLE 1 Translocation Genes Type of fusion gene
Ewing's sarcoma t(11; 22)(q24; q12) EWSR1-FLI1 Transcription factor
t(21; 22)(q22; q12) EWSR1-ERG Transcription factor t(7; 22)(p22;
q12) EWSR1-ETV1 Transcription factor t(17; 22)(q21; q12) EWSR1-ETV4
Transcription factor t(2; 22)(q33; q12) EWSR1-FEV Transcription
factor Clear-cell sarcoma t(12; 22)(q13; q12) EWSR1-ATF1
Transcription factor Desmoplastic small round-cell tumor t(11;
22)(p13: q12) EWSR1-WT1 Transcription factor Myxoid chondrosarcoma
t(9; 22)(q22-31; q11-12) EWSR1-NR4A3 Transcription factor Myxoid
liposarcoma t(12; 16)(q13; p11) FUS-DDIT3 Transcription factor
t(12; 22)(q13; q12) EWSR1-DDIT3 Transcription factor Alveolar
rhabdomyosarcoma t(2; 13)(q35; q14) PAX3-FOXO1A Transcription
factor t(1; 13)(p36; q14) PAX7-FOXO1A Transcription factor Synovial
sarcoma t(X; 18)(p11; q11) SYT-SSX Transcription factor
Dermatofibrosarcoma protuberans t(17; 22)(q22; q13) COL1A1-PDGFB
Growth factor Congenital fibrosarcoma t(12; 15)(p13; q25)
ETV6-NTRK3 Transcription-factor receptor Inflammatory
myofibroblastic tumor 2p23 rearrangements TMP3-ALK; TMP4-
Growth-factor ALK receptor Alveolar soft-part sarcoma t(X;
17)(p11.2; q25) ASPL-TFE3 Transcription factor
Certain Indications
[0221] Certain compounds, compositions and methods provided herein
can be used to treat a number of disorders such as a tumor
comprising a translocation gene fusion, Ewing's sarcoma, clear cell
sarcoma, myxoid liposarcoma, desmoplastic small round-cell tumor,
myxoid chondrosarcoma, acute myeloid leukemia, congenital
fibrosarcoma, prostate cancer, breast cancer, and pancreatic
cancer. In some embodiments, the cancer is lung adenocarcinoma, or
glioblastoma multiforme. In some embodiments, the cancer comprises
a translocation comprising an ETS gene selected from the group
consisting of FLI1, ETV1, ETV4, ERG, ETS1, and ETS2.
EXAMPLES
[0222] The following examples, including experiments and results
achieved, are provided for illustrative purposes only and are not
to be construed as limiting the present invention. Where chemical
structures depict atoms having an unfilled valency, it is to be
understood that the valency is satisfied with one or more hydrogen
atoms.
Example 1--Synthesis of 4,7 Dichloroisatin Analogs
##STR00024##
[0224] An appropriate acetophenone and 4, 7-dichloroisatin were
condensed in the presence of a catalytic amount of diethylamine to
prepare the desired compound in quantitative yield. Example
compounds: R.sup.1=4'-CN (PT-1-11); 2'-OCH.sub.3 (PT-1-12);
3'-OCH.sub.3 (PT-1-18); 2',4'-OCH.sub.3 (PT-1-19); 2',3'-OCH.sub.3
(PT-1-20); 3',4'OCH.sub.3 (PT-1-21); 3',5'OCH.sub.3 (PT-1-22);
2',3',4',-OCH.sub.3 (PT-1-23); 3',4',5'-OCH.sub.3 (PT-1-13);
4'-OC.sub.2H.sub.5 (PT-1-14); 4'-CF.sub.3 (PT-1-15); 4'-OCF.sub.3
(PT-1-16); 4'-N(CH.sub.3).sub.2 (PT-1-17); 4'-OPh (PT-1-60);
4'-SCH.sub.3 (PT-1-67); and 4'-C(CH.sub.3).sub.2 (PT-1-67).
Example 2--Synthesis of Dehydrated 4,7 Dichloroisatin Analogs
##STR00025##
[0226] A solution of 4,7-dichloroisatin in 96% H.sub.2SO.sub.4 was
stirred at room temperature to yield the reduced analogs. Example
compounds: R2=4'-OCH3 (PT-1-33); 2',4'-OCH3 (PT-1-39);
2',3',4',-OCH3 (PT-1-41); 4'-OC2H5 (PT-1-43); and 4'-N(CH3)2
(PT-1-38).
Example 3--Synthesis of Reduced 4,7 Dichloroisatin Analogs
##STR00026##
[0227] Example 4--Synthesis of Reduced 4,7 Dichloroisatin Pyridine
Derivatives
##STR00027##
[0228] Example 5--Biological Activity of Certain Compounds
[0229] Compounds provided in Table 2 were prepared using methods
similar to those described herein. The structures and IC.sub.50
activities of particular compounds in PANC1 (a human pancreatic
carcinoma), TC32 (human ESFT cell line), and TC71 (human ESFT cell
line) cells are summarized in Table 2.
TABLE-US-00002 TABLE 2 IC.sub.50 (.mu.M) Example Structure PANC 1
TC32 TC71 YK-4-275 ##STR00028## 11 40 23.95 YK-4-279 ##STR00029##
19.98; 33.96 0.9395; 0.7657 0.9178; 1.426 YK-4-280 ##STR00030## 40
12.11 30.08 YK-4-281 ##STR00031## 40 7.218 29.61 YK-4-283
##STR00032## 12.66 8.911 25.96 YK-4-284 ##STR00033## 40 40 40
YK-4-285 ##STR00034## 40 40 40 YK-4-286 ##STR00035## 40 4.631 9.149
YK-4-287 ##STR00036## 12.6 6.32 15.82 YK-4-288 ##STR00037## 40
3.002 9.345 YK-4-289 ##STR00038## 40 40 40 PT-1-11 ##STR00039## 40
10.34 12.28 PT-1-14 ##STR00040## 11.11 2.698 3.568 PT-1-15
##STR00041## 10.91 2.952 6.941 PT-1-17 ##STR00042## 40; 40 0.2589;
0.2836 0.4008; 0.2945 PT-1-18 ##STR00043## 40 40 40 PT-1-19
##STR00044## 22.94 2.609 2.819 PT-1-22 ##STR00045## 40 8.988 40
PT-1-23 ##STR00046## 40 2.698 4.422 PT-1-38 ##STR00047## 15.5; 40
0.2908; 0.3833 40; 0.5682 PT-1-39 ##STR00048## 5.413; 6.763 1.052;
1.664 1.806; 2.318 PT-1-41 ##STR00049## 2.855; 5.158 1.194; 1.611
2.142; 1.599 PT-1-43 ##STR00050## 10.98 1.409 5.655 PT-1-53
##STR00051## 2.202 40 4.08 PT-1-54 ##STR00052## 2.127; 40 1.498;
2.57 1.362; 2.202 PT-1-60 ##STR00053## 40 40 40 PT-1-64 40 32.8 40
PT-1-67 ##STR00054## 28.1; 40 0.9822; 1.203 0.9086; 1.409 PT-1-69
##STR00055## 40 40 40 PT-1-267 ##STR00056## 40 40 40 PT-1-271
##STR00057## 40 40 40 PT-1-275 ##STR00058## 40 40 40 PT-2-39
##STR00059## 40 40 40 PT-2-52 ##STR00060## 40 40 40 PT-2-56
##STR00061## 40 12.36 40 PT-2-59 ##STR00062## 40 40 40 PT-2-64
##STR00063## 40 40 40 PT-2-69 ##STR00064## 40; 40 2.178; 2.305
0.7145; 2.341 PT-2-71 ##STR00065## 40 40 40 YK-4-276 ##STR00066##
40 40 40 YK-4-277 ##STR00067## 40 40 40 YK-4-278 ##STR00068## 40 40
40 YK-4-282 ##STR00069## 40 40 40 PT-1-12 ##STR00070## 40 40 40
PT-1-13 ##STR00071## 40 40 40 PT-1-16 ##STR00072## 40 40 40 PT-1-20
##STR00073## 40 40 40 PT-1-21 ##STR00074## 40 40 40 PT-1-33
##STR00075## 40 1.035 1.636 PT-2-37 ##STR00076## 40 40 40 PT-2-78
##STR00077## 40 40 40 PT-2-79 ##STR00078## 11.19 12.13 16.98
PT-2-47 ##STR00079## PT-2-39 ##STR00080## PT-2-99 ##STR00081##
PT-2-94 ##STR00082## PT-2-84 ##STR00083## PT-2-89 ##STR00084##
Example 6--Growth Inhibition of EWS-FLI1 Cells with Substituted
Analogs
[0230] The effects of the YK-4-279 analogs on the ESFT cells were
tested by determining their growth inhibition. The IC50 of the lead
compound was 900 nM for cells growing in monolayer. Growth
inhibition of ESFT cells was measured for various concentrations of
particular compounds. Growth inhibition of TC71 and TC32 cells was
measured for various concentrations of YK-4-279 and PT-1-33 (FIG.
3A). Growth inhibition of TC71 cells was measured for various
concentrations of YK-4-279, PT-1-33, and PT-1-55 (FIG. 3B). Growth
inhibition of TC71 cells was measured for various concentrations of
YK-4-279 and PT-1-123 (FIG. 3C). Some of the analogs had similar
activity to YK-4-279. The dehydrated analogs and the alcohol
analogs showed a similar activity against ESFT cells (FIG. 3A).
Modifications of the ketone did not improve the activity of
compounds (FIG. 3B and FIG. 3C).
Example 7--Apoptosis of EWS-FLI1 Cells
[0231] Immunoblots were prepared from protein lysates from TC32
cells treated with YK-4-279 and co-precipitated with RHA, EWS-FLI1
or total protein (FIG. 4). YK-4-279 did not directly affect the
level of EWS-FLI1 or RHA but did disrupt their interactions. The
disruption of the interaction of RHA with EWS-FLI1 presents an
avenue for the development of a class of small molecules as
potential therapeutics against the Ewing's family sarcoma tumors.
While YK-4-279 disrupted the protein-protein interaction, PT-1-17
appeared to be more potent in the TC71 cells. Dehydrated analogs of
YK-4-279 did not significantly increase the potency of the
compounds.
Example 8--Disruption of EWS-FLI1/RHA Binding
[0232] The activity of candidate small molecules to disrupt binding
between EWS-FLI1 and the His-tagged RHA protein, His-Tag RHA
(647-1075), was screened in an ELISA assay. Briefly, candidate
agents were incubated with RHA on plates coated with EWS-FLI1.
After washing the plates, the amount of RHA that remained bound to
the plates was determined using a primary anti-RHA antibody, and a
secondary signal antibody.
[0233] Wells in a 96-well plate were incubated with 100 .mu.l/well
20 nM EWS-FLI1 protein solution (1M imidazole, 20 mM Tris, 500 mM
NaCl) overnight at 4.degree. C. Plates were washed with PBS,
blocked with 150 .mu.l/well 4% BSA for at least 2 h at room
temperature, and then washed again with ELISA wash solution
(PBS+0.1% T20, 200 .mu.l/well). Plates were incubated for 1 hour at
room temperature with 100 .mu.l/well candidate agent in PBS (10
.mu.M or 50 .mu.M final), or DMSO control. Plates were incubated
overnight at 4.degree. C. with 100 .mu.l/well 20 nM His-RHA protein
solution (0.5 M imidazole, 125 mM NaCl, 20 mM Tris), and then
washed with ELISA wash solution (PBS+0.1% T20, 200 .mu.l/well). RHA
bound to the plates was detected by incubating plates for 1 hour at
room temperature with 100 .mu.l/well primary anti-RHA antibody
(1:1000 goat Anti-DHX9/EB09297, Everest), and then washing with
ELISA wash solution (PBS+0.1% T20, 200 .mu.l/well). Primary
antibody was detected by incubating plates for 1 hour at room
temperature with 100 .mu.l/well secondary anti-goat antibody (1:500
donkey anti-goat IgG-HRP: sc-2020), and then washing with ELISA
wash solution (PBS+0.1% T20, 200 .mu.l/well). A horseradish
peroxidase assay kit was used to determine the amount of secondary
anti-goat antibody in each well (Bio-Rad--TMB Peroxidase EIA
Substrate Kit #172-1066), with plates read at 450 nm. A relatively
lower optical density indicating lower amounts of HRP indicate a
candidate agent with increased inhibitory activity for EWS-FLI1-RHA
binding. The results are summarized in FIGS. 5A-5G. FIG. 5A
summarizes results for the following candidate molecules: YK-4-275,
YK-4-285, PT-1-12, PT-1-18, PT-1-19, PT-1-20, PT-1-21, PT-1-22,
PT-1-23, PT-1-175. FIG. 5B summarizes results for the following
candidate molecules: PT-2-84, PT-2-59, PT-1-17, PT-2-71, PT-2-89,
PT-1-123, PT-1-15, PT-1-60, PT-1-67, PT-1-69. FIG. 5C summarizes
results for the following candidate molecules: YK-4-285, YK-4-286,
PT-1-33, PT-1-38, PT-1-271, PT-1-52, PT-1-56, PT-1-64, PT-2-94,
PT-1-267). FIG. 5D summarizes results for the following candidate
molecules: YK-4-282, YK-4-287, YK-4-2 80, YK-4-289, YK-4-288,
YK-4-278, YK-4-276, YK-4-283, YK-4-277, YK-4-281 FIG. 5E summarizes
results for the following candidate molecules: PT-1-54, YK-4-279
(S), YK-4-279 (R), PT-1-55, PT-2-75, PT-2-39, PT-2-79, PT-1-16,
PT-1-13, PT-2-64. FIG. 5F summarizes results for the following
candidate molecules: YK-4-284, PT-1-14, PT-1-39, PT-1-41, PT-1-43,
PT-1-53, PT-2-56, PT-2-52, PT-1-61, PT-1-183. FIG. 5G summarizes
results for the following candidate molecules: PT-1-275, PT-2-69,
PT-2-99, YK-4-288, PT-1-19, PT-1-20, PT-1-69, PT-2-89, PT-1-17,
PT-2-94.
Example 9-Disruption of EWS-FLI1 Transcription Factor Activity
[0234] The activity of candidate small molecules to disrupt
EWS-FLI1 transcription factor activity was screened using a
luciferase assay in which EWS-FLI1 binding to the NROB1 promoter
increases luciferase expression. Briefly, cells were transfected
with a vector containing the NROB1 promoter driving luciferase
expression, and an EWS-FLI1 expression vector. Transfected cells
were treated with various concentrations of a candidate agent, and
any change in the relative level of luciferase expression was
determined. COST cells were plated in 96-well plates and
transfected with pciNEO/EF vector and pGL3-NROB1. Controls included
transfections with each vector only. Transfected cells were treated
with various concentrations of a candidate agent, and treated cells
were assays for luciferase activity. Decreased luciferase activity
indicates a candidate agent with inhibitory activity in EWS-FLI1
acting as a transcription factor, promoting transcription of
luciferase. FIG. 6A and FIG. 6B show general trends for relative
luciferase activity for various concentrations of candidate agents.
FIGS. 7A-7I show inhibitory activity for various concentrations of
candidate agents.
Example 10-Treating Glioblastoma Multiforme
[0235] Glioblastoma multiforme (GBM) is a very well annotated tumor
from the perspective of its genetics that have led to molecular
segregation into classic, proneural, neural, and mesenchymal
categories (Purow B W, Schiff D. Glioblastoma genetics: in rapid
flux. Discov Med. 2010 February; 9(45):125-31. PubMed PMID:
20193638. Pubmed Central PMCID: 3365574). The genetic alterations
that categorize GBM include constitutive activation of signaling
pathways, loss of tumor suppressors, mutations in metabolic
pathways, abnormal DNA repair, and loss of mitotic regulators
(Suzuki E, Williams S, Sato S, Gilkeson G, Watson D K, Zhang X K.
The transcription factor Fli-1 regulates monocyte, macrophage and
dendritic cell development in mice. Immunology. 2013 July;
139(3):318-27. PubMed PMID: 23320737. Pubmed Central PMCID:
3701178; Chow L M, Endersby R, Zhu X, Rankin S, Qu C, Zhang J, et
al. Cooperativity within and among Pten, p53, and Rb pathways
induces high-grade astrocytoma in adult brain. Cancer Cell. 2011
Mar. 8; 19(3):305-16. PubMed PMID: 21397855. Pubmed Central PMCID:
3060664; Solomon D A, Kim T, Diaz-Martinez L A, Fair J, Elkahloun A
G, Harris B T, et al. Mutational inactivation of STAG2 causes
aneuploidy in human cancer. Science. 2011 Aug. 19;
333(6045):1039-43. PubMed PMID: 21852505. Epub 2011/08/20. eng).
Even within these categories, GBM is recognized as a tumor with
significant intratumoral heterogeneity (Garraway L A, Lander E S.
Lessons from the cancer genome. Cell. 2013 Mar. 28; 153(1):17-37.
PubMed PMID: 23540688; Nabilsi N H, Deleyrolle L P, Darst R P, Riva
A, Reynolds B A, Kladde M P. Multiplex mapping of chromatin
accessibility and DNA methylation within targeted single molecules
identifies epigenetic heterogeneity in neural stem cells and
glioblastoma. Genome Res. 2013 Oct. 8. PubMed PMID: 24105770).
Despite this extraordinary variability in genetics, little
attention has been focused on transcriptional regulators. One
reason transcription factors have been less well studied in GBM may
be the absence of effective small molecule inhibitors. The
exception to this is p53, whose wild-type function can be sustained
with the small molecule protein interaction inhibitor Nutlin-3
(Vassilev L T. p53 Activation by small molecules: application in
oncology. J Med Chem. 2005 Jul. 14; 48(14):4491-9. PubMed PMID:
15999986).
[0236] Despite the genetic diversity, many clinical trials have
been completed that evaluate targeted and non-targeted therapeutics
for GBM. Despite all of these trials, including radiation therapy
and molecularly guided surgical resection, progress towards
effective, long-term GBM therapy has been unsuccessful for the vast
majority of patients (Yin A A, Cheng J X, Zhang X, Liu B L. The
treatment of glioblastomas: A systematic update on clinical Phase
III trials. Crit Rev Oncol Hematol. 2013 September; 87(3):265-82.
PubMed PMID: 23453191). A most recent VEGFR small molecule
inhibitor plus temozolomide phase III trial also showed no
improvement over standard of care temozolomide plus radiation
therapy (Batchelor T T, Mulholland P, Neyns B, Nabors L B, Campone
M, Wick A, et al. Phase III Randomized Trial Comparing the Efficacy
of Cediranib As Monotherapy, and in Combination With Lomustine,
Versus Lomustine Alone in Patients With Recurrent Glioblastoma. J
Clin Oncol. 2013 Sep. 10; 31(26):3212-8. PubMed PMID: 23940216). A
significant challenge to any GBM therapy is overcoming the
blood-brain barrier (BBB), and this has impacted many potential
targeted therapies (Juratli T A, Schackert G, Krex D. Current
status of local therapy in malignant gliomas--a clinical review of
three selected approaches. Pharmacol Ther. 2013 September;
139(3):341-58. PubMed PMID: 23694764).
[0237] The use of microRNA (miRNA) both to understand the biology
of GBM and develop novel therapies led to a novel discovery that
diacylglycerol kinase alpha may be a potential target, and small
molecule optimizations are currently underway for these inhibitors
(Dominguez C L, Floyd D H, Xiao A, Mullins G R, Kefas B A, Xin W,
et al. Diacylglycerol kinase alpha is a critical signaling node and
novel therapeutic target in glioblastoma and other cancers. Cancer
Discov. 2013 July; 3(7):782-97. PubMed PMID: 23558954. Pubmed
Central PMCID: 3710531). In addition, the miRNA have been used to
create bioinformatics models that do suggest a network of
transcriptional regulation is clearly important for GBM oncogenesis
(Sun J, Gong X, Purow B, Zhao Z. Uncovering MicroRNA and
Transcription Factor Mediated Regulatory Networks in Glioblastoma.
PLoS computational biology. 2012; 8(7):e1002488. PubMed PMID:
22829753. Pubmed Central PMCID: 3400583).
Friend Leukemia Insertion-1 (FLI1) is a Putative Novel GBM
Target.
[0238] Transcription factors are the focal driving oncogene in many
cancers, yet have been considered `undruggable` since they lack
enzymatic activity. To date, despite the TCGA database for GBM, the
therapeutic targeting of critical transcriptional nodes has not
occurred. The ets family transcription factor FLI1 is expressed in
GBM based upon querying the TCGA database (FIG. 8). Early ETS-1
studies correlated ETS-1 expression with malignant potential in
human astrocytic tumors (Kitange G, Kishikawa M, Nakayama T, Naito
S, Iseki M, Shibata S. Expression of the Ets-1 proto-oncogene
correlates with malignant potential in human astrocytic tumors. Mod
Pathol. 1999 June; 12(6):618-26. PubMed PMID: 10392639). In
addition, studies showed that ETS-1 may drive angiogenesis in
astrocytic tumors (Valter M M, Hugel A, Huang H J, Cavenee W K,
Wiestler O D, Pietsch T, et al. Expression of the Ets-1
transcription factor in human astrocytomas is associated with
Fms-like tyrosine kinase-1 (Flt-1)/vascular endothelial growth
factor receptor-1 synthesis and neoangiogenesis. Cancer Res. 1999
Nov. 1; 59(21):5608-14. PubMed PMID: 10554042). Many studies
suggest a significant role for ets family ELK members in GBM
transcription and overall biology (Day B W, Stringer B W,
Spanevello M D, Charmsaz S, Jamieson P R, Ensbey K S, et al. ELK4
neutralization sensitizes glioblastoma to apoptosis through
downregulation of the anti-apoptotic protein Mcl-1. Neuro Oncol.
2011 November; 13(11):1202-12. PubMed PMID: 21846680. Pubmed
Central PMCID: 3199151; Shukla A A, Jain M, Chauhan S S.
Ets-1/Elk-1 is a critical mediator of dipeptidyl-peptidase III
transcription in human glioblastoma cells. Febs J. 2010 April;
277(8):1861-75. PubMed PMID: 20236318; Uht R M, Amos S, Martin P M,
Riggan A E, Hussaini I M. The protein kinase C-eta isoform induces
proliferation in glioblastoma cell lines through an ERK/Elk-1
pathway. Oncogene. 2007 May 3; 26(20):2885-93. PubMed PMID:
17146445). While one immunohistochemical study did not find FLI1
expression in GBM, however, there are significant challenges in
antibody selection and antigen retrieval that may have impacted on
these negative results (Mhawech-Fauceglia P, Herrmann F R, Bshara
W, Odunsi K, Terracciano L, Sauter G, et al. Friend leukaemia
integration-1 expression in malignant and benign tumours: a
multiple tumour tissue microarray analysis using polyclonal
antibody. J Clin Pathol. 2007 June; 60(6):694-700. PubMed PMID:
16917000. Pubmed Central PMCID: 195505).
[0239] Using the cBioPortal for cancer genomics website interface,
a subset of ets family members involved in cancer was evaluated.
These alterations include amplifications (solid red bar), mutations
(small green square), and mRNA upregulation (open red bars). See
FIG. 8.
[0240] FLI1 targeting in GBM is further supported based upon its
transcriptional activation of MDM2 (Truong A H, Cervi D, Lee J,
Ben-David Y. Direct transcriptional regulation of MDM2 by Fli-1.
Oncogene. 2005 Feb. 3; 24(6):962-9. PubMed PMID: 15592502). In this
case, high MDM2 would cause degradation of p53, leading to loss of
a key tumor suppressor protein. Of note, there is a loose
correlation among the seven GBM cell lines between those with high
FLI1 and high MDM2 (Table 3 and FIG. 10). In hematopoietic
development, FLI1 is clearly an important protein, as noted by
multiple immune defects when protein is eliminated by homologous
recombination (Suzuki E, Williams S, Sato S, Gilkeson G, Watson D
K, Zhang X K. The transcription factor Fli-1 regulates monocyte,
macrophage and dendritic cell development in mice. Immunology. 2013
July; 139(3):318-27. PubMed PMID: 23320737. Pubmed Central PMCID:
3701178; Kruse E A, Loughran S J, Baldwin T M, Josefsson E C, Ellis
S, Watson D K, et al. Dual requirement for the ETS transcription
factors Fli-1 and Erg in hematopoietic stem cells and the
megakaryocyte lineage. Proc Natl Acad Sci USA. 2009 Aug. 18;
106(33):13814-9. PubMed PMID: 19666492. Pubmed Central PMCID:
2728977; Liu F, Walmsley M, Rodaway A, Patient R. Fli1 acts at the
top of the transcriptional network driving blood and endothelial
development. Curr Biol. 2008 Aug. 26; 18(16):1234-40. PubMed PMID:
18718762). While FLI1 is critical from embryogenesis, it is not
likely to be critical in mature tissues since its expression is
limited to subsets of immune cells and endothelium (Watson D K,
Smyth F E, Thompson D M, Cheng J Q, Testa J R, Papas T S, et al.
The ERGB/Fli-1 gene: isolation and characterization of a new member
of the family of human ETS transcription factors. Cell Growth
Differ. 1992 October; 3(10):705-13. PubMed PMID: 1445800; Truong A
H, Ben-David Y. The role of Fli-1 in normal cell function and
malignant transformation. Oncogene. 2000 Dec. 18; 19(55):6482-9.
PubMed PMID: 11175364; Prasad D D, Rao V N, Reddy E S. Structure
and expression of human Fli-1 gene. Cancer Res. 1992;
52(20):5833-7). In addition, an approach to targeting FLI1 is to
disrupt it from protein interactions with YK-4-279 rather than
eliminating its expression.
YK-4-279 Inhibits the Function of Ets Family Members ERG, ETV1, and
EWS-FLI1
[0241] In the childhood/young adult cancer, Ewing sarcoma, the EWS
transcription activation domain is fused to an ets family member
leading to the novel fusion protein, EWS-FLI1. We identified and
validated small molecule YK-4-279 that prevents the binding of
EWS-FLI1 to RHA leading to cellular apoptosis in a panel of Ewing
sarcoma cell lines (Erkizan H V, Kong Y, Merchant M, Schlottmann S,
Barber-Rotenberg J S, Yuan L, et al. A small molecule blocking
oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits
growth of Ewing's sarcoma. Nat Med. 2009 July; 15(7):750-6. PubMed
PMID: 19584866. eng). We also demonstrated reduced tumor growth in
Ewing Sarcoma xenograft models while not affecting the growth of
non-EWS-FLI1 containing tumors at similar dosages. As an important
proof of specificity, only the (S) enantiomer of YK-4-279 is able
to inhibit the functional activities of EWS-FLI1 including binding
to RHA, transcript activation, and alternative splicing
(Barber-Rotenberg J S, Selvanathan S P, Kong Y, Erkizan H V, Snyder
T M, Hong P S, et al. Single Enantiomer of YK-4-279 Demonstrates
Specificity in Targeting the Oncogene EWS-FLI1. Oncotarget. 2012
February; 3(2):172-82. PubMed PMID: 22383402. Epub 2012/03/03.
eng). Advanced prostate cancers over-express ERG, ETV1 or ETV4 by
either chromosomal translocation or gene amplification. ERG, ETV1,
or ETV4 activity have been directly implicated to increase invasion
and metastasis. All three are ets family proteins that share
significant homology to FLI1, and have essentially identical DNA
binding domains. Prostate cells driven by ERG or ETV1 showed
significantly decreased invasion when treated with YK-4-279 (Rahim
S, Beauchamp E M, Kong Y, Brown M L, Toretsky J A, Uren A. YK-4-279
Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion. PLoS
ONE. 2011; 6(4):e19343. PubMed PMID: 21559405. Pubmed Central
PMCID: 3084826. Epub 2011/05/12. eng). This cross-tumoral activity
based upon the homology of ets transcription factors led us to
explore additional tumors that might be in part driven by an ets
transcription factor.
[0242] The ets family member FLI1 may therefore be a novel
molecular target and YK-4-279 a potential targeted therapeutic in
GBM. Orthotopic xenograft and genetically engineered mouse models
of GBM are helpful for proof-of-principle studies to support a
rationale for advancement to human clinical trials. FLI1 may be a
novel vital target for GBM and that YK-4-279 may be useful as a
future therapeutic.
[0243] GBM is one of the targeted tumor types of The Cancer Genome
Atlas (TCGA), with multiple dataset available for analysis.
Alterations in FLI1 as well as highly homologous proteins have been
searched and it was found that 23% of GBM specimens had alterations
that support FLI1 as a novel target (FIG. 8).
[0244] Considering the close homology between FLI1, ERG and ETV1,
the binding of YK-4-279 to ERG and ETV1 was evaluated (Rahim S,
Beauchamp E M, Kong Y, Brown M L, Toretsky J A, Uren A. YK-4-279
Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion. PLoS
ONE. 2011; 6(4):e19343. PubMed PMID: 21559405. Pubmed Central
PMCID: 3084826. Epub 2011/05/12. eng). The affinity (KD) of
YK-4-279 for EWS-FLI1 was measured to be 9.5 .mu.M (Erkizan H V,
Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L, et
al. A small molecule blocking oncogenic protein EWS-FLI1
interaction with RNA helicase A inhibits growth of Ewing's sarcoma.
Nat Med. 2009 July; 15(7):750-6. PubMed PMID: 19584866. Eng;
Barber-Rotenberg J S, Selvanath). The steady state kinetics of
YK-4-279 binding to recombinant ERG and ETV1 using surface plasmon
resonance had a binding affinity (KD) of 11.7 .mu.M for ERG and
17.4 .mu.M for ETV1, whereas it bound the non-specific protein BSA
with a weak affinity of 122.4 .mu.M. FIG. 9 shows that YK-4-279
binds to ERG and ETV1 with a KD of 11.7 .mu.M and 17.9 .mu.M
respectively. Steady state kinetics were measured on a Biacore T100
instrument, as previously described (Erkizan H V, Kong Y, Merchant
M, Schlottmann S, Barber-Rotenberg J S, Yuan L, et al. A small
molecule blocking oncogenic protein EWS-FLI1 interaction with RNA
helicase A inhibits growth of Ewing's sarcoma. Nat Med. 2009 July;
15(7):750-6. PubMed PMID: 19584866. Eng; Barber-Rotenberg J S,
Selvanath), SPR sensograms are not shown.
[0245] A large sequencing project of GBM that included analysis of
XX cell lines has been completed (Solomon D A, Kim T, Diaz-Martinez
L A, Fair J, Elkahloun A G, Harris B T, et al. Mutational
inactivation of STAG2 causes aneuploidy in human cancer. Science.
2011 Aug. 19; 333(6045):1039-43. PubMed PMID: 21852505. Epub
2011/08/20. eng). Seven cell lines were selected with a spectrum of
genetic abnormalities that occur in GBM. Table 3 shows the
heterogeneity of GBM cell lines. A panel of GBM cell lines were
acquired that represent the heterogeneity of the disease. The (+)
indicates the expression of the listed protein, either wild-type or
mutant. The (-) indicates the absence of expression on an
immunoblot. These cell lines were used to evaluate the expression
of FLI1 as well as sensitivity to the inhibitor YK-4-279 (FIG.
3).
TABLE-US-00003 TABLE 3 DKMG DBTRG 42MGBA GAMG U87MG H4 8MGBA EGFR +
- + + + - + Myc + - + + + + + PTEN + - - + + - + MDM2 + + - + - - -
p53 + + + + - - + p14ARF - - - - - - + 21WAF1/CIP + + + + + + +
CDK4 + + + + + + + CDK6 + + + + - + + p16INK4a - - - - - - +
p18INK4c + + + + - + + RB + + - + + + -
[0246] Six of the 7 GBM cell lines (85%) demonstrated FLI1
expression by immunoblot (FIG. 10, top panel). Growth of each of
these cell lines was reduced by YK-4-279 with IC50 ranging from 0.5
up to 9.9 .mu.M and an inverse correlation was observed between the
level of FLI1 and the sensitivity to YK-4-279 (r2=0.8, FIG.
10).
[0247] To evaluate the potential of FLI1 as a GBM target, two
transgenic models were analyzed (Chow L M, Endersby R, Zhu X,
Rankin S, Qu C, Zhang J, et al. Cooperativity within and among
Pten, p53, and Rb pathways induces high-grade astrocytoma in adult
brain. Cancer Cell. 2011 Mar. 8; 19(3):305-16. PubMed PMID:
21397855. Pubmed Central PMCID: 3060664). Very low expression for
two FLI1 probe sets comparing normal brainstem, brainstem
astrocytes, and cortical astrocytes was observed (FIG. 11).
However, 20 of 22 double knock-out (PTEN/p53) and 13 of 14 triple
knockout (PTEN/p53/Rb) tumors had significant expression of the
FLI1 based upon the two probesets analyzed (FIG. 11).
[0248] In order to determine the FLI1 expression level in human
GBM, a panel of GBM was stained with FLI1 antibody. The panel
consisted of six randomly selected grade 4 tumors; four of six
(66%) showed convincing IHC staining for FLI1 after an antibody was
optimized to eliminate cross-reacting proteins and non-specific
signal (FIG. 12). Four additional tumors were considered
unevaluable since the internal positive control, endothelial cells,
were not positive.
[0249] One of the significant challenges in getting novel targeted
therapy into GBM is overcoming the blood-brain barrier (BBB). As
part of a pharmacokinetic evaluation of YK-4-279, tissue levels
were measured and compared these with plasma in 12 mice that
received 75 mg/kg IV racemic YK-4-279. Levels of YK-4-279 in brain
tissue were 74% that of the Ewing sarcoma pretibial xenograft
tumor, which would be adequate for inhibiting FLI1. In addition,
when rat pharmacokinetics were performed using IV injection of
compound, rats became somnolent after rapid injection, which did
not occur with slower infusion, thus supporting an ability to pass
into the central nervous system across the BBB.
[0250] The data provided herein identifies FLI1 as a putative
target for GBM. The combination of TCGA data, a panel of cell
lines, GEMM model, and panel of IHC from human tumors support
further validation of FLU.
Validation of FLI1 as a Novel Target in GBM
[0251] FLI1 as putative target is validated by evaluating whether
it is necessary for GBM cell growth. Whether FLI1 is a potential
oncogene in glial stem cells is determined. GBM cells are compared
with normal human astrocytes and glial stem cells for the
importance of FLI1 in order to address the therapeutic index of
targeting FLU. The comparison with normal brain cells is useful to
establish FLI1 as a valid target with a preferable therapeutic
index.
GBM Cell Lines Require FLI1 Expression for Survival, Growth, and
Invasion are Identified
[0252] Using an shRNA vector that is tagged with EGFP which targets
the 3'UTR of FLI1 GBM cell lines require FLI1 expression for
survival, growth, and invasion are identified. The shRNA are
infected into cells using a lentiviral system. Thus, the relative
importance of FLI1 in seven GBM cell lines which span much of the
genotypic heterogeneity is evaluated (Table 3). After FLI1 is
reduced with shRNA, changes are measure between scrambled shRNA
control and FLI1 reduction in monolayer growth, invasion,
anchorage-independent growth, and tumorigenesis assays. Cell
culture experiments are performed as previously reported (Erkizan H
V, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L,
et al. A small molecule blocking oncogenic protein EWS-FLI1
interaction with RNA helicase A inhibits growth of Ewing's sarcoma.
Nat Med. 2009 July; 15(7):750-6. PubMed PMID: 19584866. Eng; Rahim
S, Beauchamp E M, Kong Y, Brown M L, Toretsky J A, Uren A. YK-4-279
Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion. PLoS
ONE. 2011; 6(4):e19343. PubMed PMID: 21559405. Pubmed Central
PMCID: 3084826. Epub 2011/05/12. eng). Invasion assays are
performed using tumor cells and their invasion through umbilical
endothelial cells using an electrical-impedance based technique
that monitors and quantifies in real-time the invasion of
endothelial cells by malignant tumor cells. The xCELLigence
instrument, manufactured by Roche, which measures changes in
electrical impedance as cells attach and then as tumor cells
disrupt this attachment is used (Rahim S, Beauchamp E M, Kong Y,
Brown M L, Toretsky J A, Uren A. YK-4-279 Inhibits ERG and ETV1
Mediated Prostate Cancer Cell Invasion. PLoS ONE. 2011;
6(4):e19343. PubMed PMID: 21559405. Pubmed Central PMCID: 3084826.
Epub 2011/05/12. Eng; Rahim S, Uren A. A real-time electrical
impedance based technique to measure invasion of endothelial cell
monolayer by cancer cells. Journal of visualized experiments: JoVE.
2011 (50). PubMed PMID: 21490581. Pubmed Central PMCID: 3169283).
Xenograft experiments use polyclonal shRNA reduced FLI1 in all
seven cell lines. Each shRNA FLI1 and scrambled cell lines are
stereotactically injected into 5 athymic mice (assisted by
Fiandanca). (7 cell lines, 5 animals per cell line, +/-FLI1=70).
Growth is monitored by MRI in the GU Animal Imaging shared resource
at 7-10 day intervals. Calculations of tumor growth kinetics are
performed by region of interest analysis as described, using Bruker
Paravision 5.0 software or ImageJ (NIH) (Truong A H, Cervi D, Lee
J, Ben-David Y. Direct transcriptional regulation of MDM2 by Fli-1.
Oncogene. 2005 Feb. 3; 24(6):962-9. PubMed PMID: 15592502; Pimanda
J E, Chan W Y, Donaldson U, Bowen M, Green A R, Gottgens B.
Endoglin expression in the endothelium is regulated by Fli-1, Erg,
and Elf-1 acting on the promoter and a -8-kb enhancer. Blood. 2006
Jun. 15; 107(12):4737-45. PubMed PMID: 16484587). Growth statistics
of tumors are calculated to build a time-dependent profile of
progression and treatment.
Transfection of Normal Human Astrocytes with a Full-Length FLI1
cDNA
[0253] To determine the transformative effect of FLI1 upon
astrocytes, normal human astrocytes are transfected with a
full-length FLI1 cDNA using lentiviral system and evaluate for
transformation in soft agar and in vivo orthotopic injection
assays. Control (empty vector) and FLI1 transfected polycolonal
cells are placed in soft agar for anchorage-independent growth
assays (Erkizan H V, Kong Y, Merchant M, Schlottmann S,
Barber-Rotenberg J S, Yuan L, et al. A small molecule blocking
oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits
growth of Ewing's sarcoma. Nat Med. 2009 July; 15(7):750-6. PubMed
PMID: 19584866. eng). Xenograft studies are performed as described
above. (2 cell lines+/-FLI1, five animals per cell line=20
animals). Animals are imaged by MRI to assess tumor growth, as
described above, every 10 days.
[0254] Glial stem cells are evaluated for both for their innate
expression of FLI1 and the evaluation of oncogenic effects when
FLI1 is exogenously expressed. Published (Lelievre E, Lionneton F,
Mattot V, Spruyt N, Soncin F. Ets-1 regulates fli-1 expression in
endothelial cells. Identification of ETS binding sites in the fli-1
gene promoter. J Biol Chem. 2002 Jul. 12; 277(28):25143-51. PubMed
PMID: 11991951) and novel cell lines are used. To evaluate
anchorage-independent growth and invasion assays, for both control
and FLU expression are performed. In addition, these cell lines are
used in xenograft experiments, both transfected with control and
FLI1 expression (always proven by immunoblots prior to evaluation).
These cells are all be carefully grown in minimal growth media with
exogenous growth factors rather than serum to maintain their
pristine neural qualities (Rossi S, Orvieto E, Furlanetto A,
Laurino L, Ninfo V, Dei Tos A P. Utility of the immunohistochemical
detection of FLI-1 expression in round cell and vascular neoplasm
using a monoclonal antibody. Mod Pathol. 2004 May; 17(5):547-52.
PubMed PMID: 15001993). (3 cell lines+/-FLI1, five animals per cell
line=15 animals).
[0255] The correlation between YK-4-279 toxicity and FLI1 levels is
measured. YK-4-279 targets the FLI1 component of EWS-FLI1
(Barber-Rotenberg J S, Selvanathan S P, Kong Y, Erkizan H V, Snyder
T M, Hong P S, et al. Single Enantiomer of YK-4-279 Demonstrates
Specificity in Targeting the Oncogene EWS-FLI1. Oncotarget. 2012
February; 3(2):172-82. PubMed PMID: 22383402. Epub 2012 Mar. 3.
Eng; Rahim S, Beauchamp E M, Kong Y, Brown M L, Toretsky J A, Uren
A. YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell
Invasion. PLoS ONE. 2011; 6(4):e19343. PubMed PMID: 21559405.
Pubmed Central PMCID: 3084826. Epub 2011 May 12. eng). Following an
evaluation of the effect of FLI1 inhibitor YK-4-279 upon a panel of
cell lines and correlation of inhibition with FLI1 expression,
these results are compared to the shRNA data. Preliminary data for
7 GBM cell lines with correlation to FLI1 expression. This is
expanded to more cell lines with methodology used to generate FIG.
9. Non-tumor glial cell lines are included for these
comparisons.
[0256] Whether FLI1 expression correlates with other known GBM
genotypes and phenotypes is determined. A series of on-line
informatics tools from TCGA as well as correlation with our cell
line data is used. The analysis includes FLI1 cDNA or protein
expression and evaluates whether FLI1 correlates with other known
GBM genetic events, such as loss of PTEN, p16, p53 mutation, IDH1
point mutation or EGFR mutation. This is expanded to include
patient tumors that are resected at diagnosis and inclusion of FLI1
into the panel of genetic markers evaluated.
Utilizing Animal Models of GBM to Evaluate YK-4-279 as a
Therapy
[0257] To establish whether YK-4-279 is effective in either slowing
growth or causing regression of GBM, YK-4-279 is administered to
mice with established GBM. This evaluates both xenograft and
transgenic models of GBM.
[0258] To establish a method for evaluating intracranial lesions in
groups of mice, rather than one at a time with MM. While the MRI
provides a highly detailed graphic and metabolic spectrum of GBM
tumors, the time to screen mice limits its use for larger studies.
Two GBM cell lines are created, selected from the GBM orthotopic
screening of seven cell lines that are used for Xenogen
intracranial imaging. The two GBM cell lines are selected based
upon their requirements for FLI1, as determined above and growth
kinetics. These two GBM cell lines are stably transfected with
luciferase so that groups of animals can be screened/monitored
using the Xenogen system. For more detailed studies and volumetric
comparison, selected animals are evaluated using MM. Cell lines are
screened in vitro followed by an orthotopic xenograft pilot study
(2 cell lines.times.5 animals=10 animals). Prior to Xenogen
imaging, animals are injected with luciferase substrate
intraperitoneally.
[0259] YK-4-279 upon xenograft GBM tumors is evaluated by treating
animals seven days after tumor injection and those with symptomatic
tumors. Early tumors are screened with Xenogen, and size correlated
with MRI, and treatment started when animals 5 mm.sup.3 lesions.
YK-4-279 passes the blood-brain barrier. Animals are treated with
BID injections of YK-4-279 similar to the dose that led to
regression of Ewing sarcoma tumors (FIG. 13). The study will
measure brain tumor volumes, animal symptoms, and overall survival.
At necropsy, tumors and normal adjacent brain will be evaluated by
immunohistochemistry for GBM markers, apoptosis, and FLI1 regulated
target genes. FIG. 13 illustrates that three days of treatment with
(S)-YK-4-279 or racemic shows significant tumor regression. FIG.
13A: Mice with ES xenografts were treated with 400 mg/kg compound
or controls as indicated. Starting well-established tumors (300
mm.sup.3), mice were treated with intraperitoneal compound for
three days, 6 total doses. FIG. 13B: H and E stained tumors from
same experiment.
[0260] Since the vast majority of patients with GBM present with
symptoms and relatively large tumors, YK-4-279 is tested against
larger, symptomatic tumors. Thus, YK-4-279 is evaluated on upon
well-established GBM, 20 mm.sup.3, by MM, by treating animals after
tumors are established and at first signs of symptoms. Tumors at
this time are detectable with Xenogen. Animals undergo Xenogen
evaluation twice a week while on treatment. The study compares
tumor volumes, animal symptoms, and overall survival. At necropsy,
tumors and normal adjacent brain are evaluated by
immunohistochemistry for GBM markers, apoptosis, and FLI1 regulated
target genes.
[0261] The GEMM model of GBM is used. Animals are bred as published
(Chow L M, Endersby R, Zhu X, Rankin S, Qu C, Zhang J, et al.
Cooperativity within and among Pten, p53, and Rb pathways induces
high-grade astrocytoma in adult brain. Cancer Cell. 2011 Mar. 8;
19(3):305-16. PubMed PMID: 21397855. Pubmed Central PMCID:
3060664). At approximately 90 days of life, animals have MRI
evaluation every 10-14 days to look for onset of GBM. In Experiment
1 animals are treated with YK-4-279 or control starting at day 90
(10 animals in control and treated=20 animals). In Experiment 2
animals are treated at the onset of symptoms or MRI measured tumor
of greater than 2 mm in any dimension (10 animals in control and
treated=20 animals). Following treatment, animals are evaluated as
described above. Administration of YK-4-279 is using the
intraperitoneal route.
[0262] Data is provided to support further exploration of FLI1 and
potentially other ets family members as drivers of GBM.
[0263] References pertaining to selected cancers include the
following: CBTRUS Statistical Report: Primary Brain and Central
Nervous System Tumors Diagnosed in the United States in 2004-2008
(Mar. 23, 2012 Revision). Central Brain Tumor Registry of the
United States [Internet]. 2012; http://www.cbtrus.org. Available
from: http://www.cbtrus.org.
Example 11--Use of YK-4-279 for Treating Lung Cancer
[0264] Epithelial-to-mesenchymal transition (EMT) is a key
component of the pathogenesis of carcinomas. EMT induces
significant changes in cell morphology and behavior that impart
metastatic and drug-resistant phenotypes. Moreover, there is
evidence suggesting that EMT participates in the generation of
cancer stem cells. Lung cancer is the leading cause of
cancer-related mortality, mainly because it is typically diagnosed
at advanced stages that are difficult to treat. Advances in our
understanding of the molecular genetics of cancers have identified
individual molecules required for tumorigenesis. This has led to
the development of targeted therapies that are successful for
treating certain cancers. Examples of these molecular targets
include cell surface growth factor receptors and intracellular
protein tyrosine kinases. Unfortunately, such treatments have not
significantly improved overall survival or quality of life for
patients with lung cancer.
[0265] Recent discoveries described here have led to the hypothesis
that the product of the E-26 Transforming Sequence (ETS)-related
gene ERG, a member of the ETS family of transcription factors,
plays an important role in EMT. Moreover, it induces EMT and the
malignant progression of epithelial cells through direct
up-regulation of the expression of zinc finger E-box binding
homeobox 1 and 2 genes (ZEB1, ZEB2). ZEB1 is linked to EMT in lung
cancer cells, and inhibiting its expression using siRNA not only
reverses EMT but also inhibits tumor growth in vitro and in vivo.
Because lung cancer cells express high levels of ERG, ERG may
induce EMT through ZEB. Experiments are conducted that determine
whether ERG participates in EMT of lung cancer cells mediated by
ZEB1/2. New formulations of YK-4-279 are produced and evaluated for
treating lung cancer.
[0266] ERG as a transcription factor modulates expression of many
genes that are important for carcinogenesis. Earlier observations
suggested that oncogenic properties of ERG include its ability to
induce epithelial-to-mesenchymal transition (EMT). In different
experimental systems, ERG has been shown to induce expression of
zinc finger E-box binding homeobox 1 and 2 genes (ZEB1, ZEB2),
which are positive regulators of EMT in cancer cells. Since EMT
results in metastasis and drug resistance in NSCLC, inhibition of
molecular pathways leading to EMT may have significant clinical
utility.
[0267] The role of ERG in mediating EMT in lung cancer cells is
determined. EMT and drug resistance phenotypes of NSCLC cell lines
in response to changes in ERG expression is determined. If ERG
induces EMT like it does in other epithelial tumors is determined.
Further, if the ERG mediated EMT in NSCLC is through ZEB1/2 genes
is determined. These experiments involve inhibition of ERG and ZEB
expression in NSCLC cells by RNAi technologies. EMT phenotype is
evaluated by real-time PCR and western blotting for established EMT
markers.
[0268] Formulations of YK-4-279 that can be administered
parenterally are produced and the effects of YK-4-279 on the
proliferation and malignant properties of lung cancer cells are
determined. An examples excipient is .beta.-hydroxypropyl
cyclodextrin ((3-HPCD). NSCLC cells are treated with YK-4-279 and
their response is measured in multiple in vitro and in vivo models.
Cell viability, chemotaxis, endothelial cell invasion and xenograft
growth in immunocompromised mice is measured. The potential synergy
between YK-4-279 and most common chemotherapeutic agents for NSCLC
is determined. The properties of drug resistance and high
metastatic potential of NSCLC cells, mediated by EMT, contribute
significantly to the poor prognosis of patients with NSCLC.
[0269] The properties of drug resistance and high metastatic
potential of NSCLC cells, mediated by EMT, contribute significantly
to the poor prognosis of patients with NSCLC. A specific protein to
reverse the EMT phenotype in lung cancer is targeted using a small
molecule. FIG. 14 illustrates ERG induces expression of ZEB1 and
ZEB2, which activate EMT leading to lung cancer metastasis and drug
resistance.
[0270] Targeting drugs to specific molecules required for the
growth of cancer cells remains a difficult challenge despite recent
advances in molecular and cellular biology. Although proteins that
drive the unregulated reproduction of cancer cells are known, only
a few have served as targets of effective therapies. Examples
include an intracellular protein tyrosine kinase whose activity is
inhibited by a small molecule called imatinib mesylate used to
treat chronic myelogenous leukemia; and a monoclonal antibody
(trastuzumab) used to treat breast cancers, is targeted to a cell
surface growth factor receptor. These limited but significant and
highly encouraging successes have stimulated continuing and robust
research by the pharmaceutical industry. Inhibiting the activity of
an enzyme or the activation of receptor are well-established goals
of drug development for numerous diseases in addition to cancer,
because the biochemistry of these proteins is so well understood.
In contrast, the biochemistry involved in the binding of proteins
to one another is much more complex and poorly understood, and
inhibiting these interactions has therefore received relatively
little attention.
[0271] ERG is a further challenge for designing a targeted therapy,
because it localizes to the nucleus and lacks enzyme activity.
YK-4-279 is a small molecule that inhibits ERG's transcriptional
activity, and is investigated for its role in NSCLC. Whether
YK-4-279, by binding to and interfering with ERG function required
for EMT is determined (FIG. 14), can be used to treat NSCLC.
[0272] ERG is an oncogenic protein. The E-26 Transforming Sequence
(ETS)-related gene ERG encodes a member of the ETS family of
transcription factors that is essential for endothelial
homeostasis, differentiation, and angiogenesis in many tissues (Liu
F, Patient R. Genome-wide analysis of the zebrafish ETS family
identifies three genes required for hemangioblast differentiation
or angiogenesis. Circulation research. 2008; 103:1147-54; Sashida
G, Bazzoli E, Menendez S, Liu Y, Nimer S D. The oncogenic role of
the ETS transcription factors MEF and ERG. Cell cycle. 2010;
9:3457-9). Evidence suggests that the activities of certain genes
that are regulated by ERG are required for angiogenesis. For
example, VE-cadherin, which requires ERG for its expression, is
essential for endothelial junctional stability and endothelial
survival, both critical processes in angiogenesis (Yuan L,
Sacharidou A, Stratman A N, Le Bras A, Zwiers P J, Spokes K, et al.
RhoJ is an endothelial cell-restricted Rho GTPase that mediates
vascular morphogenesis and is regulated by the transcription factor
ERG. Blood. 2011; 118:1145-53). In carcinogenesis, ETS
transcription factors are involved in the regulation of numerous
genes that participate in processes required for metastasis,
including degradation of the extracellular matrix, and formation of
cell-to-cell and cell-to-matrix junctions (Lelievre E, Lionneton F,
Soncin F, Vandenbunder B. The Ets family contains transcriptional
activators and repressors involved in angiogenesis. The
international journal of biochemistry & cell biology. 2001;
33:391-407).
[0273] Specific examples include the receptor for vascular
endothelial growth factor, endoglin, matrix metalloproteinases,
collagenase 1, and heme oxygenase 1. ERG is overexpressed in
hematopoietic and epithelial cell cancers and acts as a potent
oncogene in human prostate cancers (Chen Y, Chi P, Rockowitz S,
Iaquinta P J, Shamu T, Shukla S, et al. ETS factors reprogram the
androgen receptor cistrome and prime prostate tumorigenesis in
response to PTEN loss. Nature medicine. 2013; Rahim S, Uren A.
Emergence of ETS transcription factors as diagnostic tools and
therapeutic targets in prostate cancer. American journal of
translational research. 2013; 5:254-68; Turner D P, Watson D K. ETS
transcription factors: oncogenes and tumor suppressor genes as
therapeutic targets for prostate cancer. Expert review of
anticancer therapy. 2008; 8:33-42).
[0274] ERG and EMT signifies poor clinical outcome in NSCLC.
Notable findings leading to our interest in the role of ERG in lung
cancers include the detection of relatively high levels of ERG
expression in NSCLCs and the presence of alternatively spliced
versions of ERG in 100% of lung tumor samples compared with normal
tissue (Xi L, Feber A, Gupta V, Wu M, Bergemann A D, Landreneau R
J, et al. Whole genome exon arrays identify differential expression
of alternatively spliced, cancer-related genes in lung cancer.
Nucleic acids research. 2008; 36:6535-47). Analysis of mRNA
expression by micro array: ERG mRNA expression ranking is in top 8%
(Ramaswamy S, Ross K N, Lander E S, Golub T R. A molecular
signature of metastasis in primary solid tumors. Nature genetics.
2003; 33:49-54), and in top 11% (Ding L, Getz G, Wheeler D A,
Mardis E R, McLellan M D, Cibulskis K, et al. Somatic mutations
affect key pathways in lung adenocarcinoma. Nature. 2008;
455:1069-75) in NSCLC tissue samples. Since ERG target genes are
involved in EMT phenotype we hypothesized that ERG mediated EMT may
contribute to malignant phenotype of NSCLC.
[0275] EMT was first described in early embryonic development when
cells lose their epithelial characteristics and acquire mesenchymal
phenotypes (Sato M, Shames D S, Hasegawa Y. Emerging evidence of
epithelial-to-mesenchymal transition in lung carcinogenesis.
Respirology. 2012; 17:1048-59). As EMT progresses, cells acquire a
more motile and invasive phenotype. Therefore, EMT emerged as an
important component of carcinogenesis. The associations between EMT
and NSCLC local invasion, angiogenesis, distant metastasis as well
as drug resistance and anti-apoptotic phenotypes have been
demonstrated by numerous studies conducted in vivo and in vitro
(Table 4). For example, the expression of molecules involved in EMT
correlate with the clinico-pathological features of NSCLC,
including increased metastasis and shortened overall survival of
patients (Dauphin M, Barbe C, Lemaire S, Nawrocki-Raby B, Lagonotte
E, Delepine G, et al. Vimentin expression predicts the occurrence
of metastases in non small cell lung carcinomas. Lung cancer. 2013;
81:117-22). Moreover, the important role of EMT in carcinogenesis
is indicated by cellular phenotypes characteristic of stem cells
(Mani S A, Guo W, Liao M J, Eaton E N, Ayyanan A, Zhou A Y, et al.
The epithelial-mesenchymal transition generates cells with
properties of stem cells. Cell. 2008; 133:704-15). Taken together,
these studies provide strong justification for inhibiting EMT that
occurs in the development of lung cancer. Table 4 lists molecules
involved in EMT that correlate with clinical features in NSCLC.
TABLE-US-00004 TABLE 4 EMT Genes Clinical features References
Epithelial Longer overall Nakata S, Sugio K, Uramoto H, Oyama T,
Cadherin survival Hanagiri T, Morita M, et al. The methylation
status and protein expression of CDH1, p16(INK4A), and fragile
histidine triad in nonsmall cell lung carcinoma: epigenetic
silencing, clinical features, and prognostic significance. Cancer.
2006; 106: 2190-9. Negative for lymph Kase S, Sugio K, Yamazaki K,
Okamoto T, node metastasis Yano T, Sugimachi K. Expression of E-
cadherin and beta-catenin in human non-small cell lung cancer and
the clinical significance. Clinical cancer research: an official
journal of the American Association for Cancer Research. 2000; 6:
4789-96. SLUG Postoperative relapse Shih JY, Tsai MF, Chang TH,
Chang YL, Yuan A, Yu CJ, et al. Transcription repressor slug
promotes carcinoma invasion and predicts outcome of patients with
lung adenocarcinoma. Clinical cancer research: an official journal
of the American Association for Cancer Research. 2005; 11: 8070-8.
Shorter overall Chiou SH, Wang ML, Chou YT, Chen CJ, survival Hong
CF, Hsieh WJ, et al. Coexpression of Oct4 and Nanog enhances
malignancy in lung adenocarcinoma by inducing cancer stem cell-
like properties and epithelial-mesenchymal transdifferentiation.
Cancer research. 2010; 70: 10433-44. SNAIL Shorter overall Yanagawa
J, Walser TC, Zhu LX, Hong L, survival Fishbein MC, Mah V, et al.
Snail promotes CXCR2 ligand-dependent tumor progression in
non-small cell lung carcinoma. Clinical cancer research: an
official journal of the American Association for Cancer Research.
2009; 15: 6820-9. TWIST Shorter overall Hung JJ, Yang MH, Hsu HS,
Hsu WH, Liu JS, survival Wu KJ. Prognostic significance of hypoxia-
HIF-1 alpha Shorter overall inducible factor-1alpha, TWIST1 and
Snail survival expression in resectable non-small cell lung Shorter
recurrence cancer. Thorax. 2009; 64: 1082-9. free survival
[0276] ERG target gene ZEB1 mediates EMT. EMT is a complex cellular
response that involves multiple signaling pathways. The zinc finger
E-box-binding homeobox (ZEB) proteins are key regulators of EMT
(Takeyama Y, Sato M, Horio M, Hase T, Yoshida K, Yokoyama T, et al.
Knockdown of ZEB1, a master epithelial-to-mesenchymal transition
(EMT) gene, suppresses anchorage-independent cell growth of lung
cancer cells. Cancer letters. 2010; 296:216-24). In particular,
ZEB1 plays a predominant role in the EMT-associated carcinogenic
phenotypes of lung cancer by regulating the expression of genes
that encode proteins that participate in EMT. For example,
inhibition of ZEB1 expression by siRNA in lung cancer cell lines
results in the reversal of EMT, increased sensitivity to docetaxel,
and reduced growth of lung cancer cells in vitro and in vivo (Ren
J, Chen Y, Song H, Chen L, Wang R. Inhibition of ZEB1 reverses EMT
and chemoresistance in docetaxel-resistant human lung
adenocarcinoma cell line. Journal of cellular biochemistry. 2013;
114:1395-403). Most important, ERG mediates EMT in prostate cancer
cells through the ZEB axis (Leshem O, Madar S, Kogan-Sakin I, Kamer
I, Goldstein I, Brosh R, et al. TMPRSS2/ERG promotes epithelial to
mesenchymal transition through the ZEB1/ZEB2 axis in a prostate
cancer model. PloS one. 2011; 6:e21650). Moreover, miR-30
suppresses EMT in prostate cancer cells by directly targeting ERG
expression (Kao C J, Martiniez A, Shi X B, Yang J, Evans C P, Dobi
A, et al. miR-30 as a tumor suppressor connects EGF/Src signal to
ERG and EMT. Oncogene. 2013). These and other studies indicate that
it is reasonable to conclude that efforts to target ZEB1 and ZEB2
to reverse EMT in lung cancer may be successful. Because RNAi
technologies are not advanced enough for clinical applications, it
will be necessary to identify alternative mechanisms to inhibit
ZEB1 expression in lung cancers. It is known that ERG binding sites
are present in the ZEB1 and ZEB2 promoter regions (Leshem O, Madar
S, Kogan-Sakin I, Kamer I, Goldstein I, Brosh R, et al. TMPRSS2/ERG
promotes epithelial to mesenchymal transition through the ZEB1/ZEB2
axis in a prostate cancer model. PloS one. 2011; 6:e21650). In 2009
we discovered YK-4-279 as an inhibitor of EWS-FLI1, a fusion
protein encoded by a tumor-specific rearranged gene in Ewing
Sarcoma (Erkizan H V, Kong Y, Merchant M, Schlottmann S,
Barber-Rotenberg J S, Yuan L, et al. A small molecule blocking
oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits
growth of Ewing's sarcoma. Nature medicine. 2009; 15:750-6). More
recently, based on the homology between two ETS members, FLI1 and
ERG, YK-4-279 binds directly to ERG and inhibits its
transcriptional activity (Rahim S, Beauchamp E M, Kong Y, Brown M
L, Toretsky J A, Uren A. YK-4-279 inhibits ERG and ETV1 mediated
prostate cancer cell invasion. PloS one. 2011; 6:e19343).
Inhibition of ERG function in lung cancer cells may reverse EMT
mediated by ZEB proteins and lead to inhibition of metastatic
growth and increased sensitivity to chemotherapeutic drugs.
YK-4-279 Binds ERG
[0277] YK-4-279, a small molecule that binds directly to EWS-FLI1
and inhibits the growth of Ewing Sarcoma cells (Erkizan H V, Kong
Y, Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L, et al.
A small molecule blocking oncogenic protein EWS-FLI1 interaction
with RNA helicase A inhibits growth of Ewing's sarcoma. Nature
medicine. 2009; 15:750-6). EWS-FLI1 is the product of a chromosomal
translocation. FLI1 is an ETS family transcription factor with a
conserved DNA binding domain. Alignment of FLI1 with another ETS
family member, ERG, amino acid sequence shows significant
similarities (63.5% identity, 80.2% homology). Commercially
available recombinant ERG protein (Origene, Rockville, Md.) was
obtained and measured direct binding affinity for YK-4-279 on a
Biacore T-100 (FIG. 15), which detects molecular interactions in
real time without a reporter moiety. Recombinant protein was
immobilized on Biacore microchips and binding was measured in the
presence of varying YK-4-279 concentrations. We detected YK-4-279
binding to ERG with a steady state KD of 11.7 .mu.M. FIG. 15
illustrates YK-4-279 directly interacts with ERG protein. Purified
recombinant ERG was immobilized on Biacore CMS microchips, and
direct binding to eight different YK-4-279 concentrations (0.1-50
.mu.M) was determined by SPR. Steady state KD was calculated using
Biaevaluation software.
[0278] YK-4279 inhibits transcriptional activity of ERG. ETS
proteins control expression of target genes encoding proteins that
participate in diverse biochemical processes, many of which
contribute to oncogenic growth (Hollenhorst P C, McIntosh L P,
Graves B J. Genomic and biochemical insights into the specificity
of ETS transcription factors. Annual review of biochemistry. 2011;
80:437-71). The effect of YK-4-279 on ERG's transcriptional
activity was tested utilizing promoter reporter assays and
endogenous gene expression profiling. YK-4-279 inhibited ERG
activation of an ETS target gene promoter (Id2) controlling
luciferase expression in COST cells (FIG. 16A). VCaP prostate
cancer cells possess the TMPRSS2/ERG fusion gene, where ERG induces
expression of specific endogenous target genes such as PLAU, ADAM19
and PLAT. Real time (RT)-PCR analysis revealed that YK-4-279
significantly inhibited their expression but not that of ERG
expression (FIG. 16B). These findings were extended to the protein
level for ERG and PLAU. Inhibition by YK-4-279 was comparable to
that observed when cells were treated with ERG siRNA (FIG. 16B)
(Rahim S, Beauchamp E M, Kong Y, Brown M L, Toretsky J A, Uren A.
YK-4-279 inhibits ERG and ETV1 mediated prostate cancer cell
invasion. PloS one. 2011; 6:e19343). Since epithelial cells do not
express FLI1, YK-4-279's effects were most likely due to inhibiting
ERG. This provides further evidence that YK-4-279 is not simply a
general inhibitor of transcription and translation (ERG mRNA and
protein levels did not change), but it specifically inhibits the
transcriptional activities of ETS family proteins.
ERG is Expressed in NSCLC Cell Lines and Induces EMT Markers
[0279] Five NSCLC cell lines were examined to confirm that
significant ERG expression is present similar to what was observed
in human tumor samples. A western blot analysis of A549, H1944,
H358, H1395, and H596 cell lysates were performed (FIG. 17). Four
out of 5 cell lines expressed high levels of ERG protein. H358 cell
line that expressed very little ERG will be used as a negative
control in our studies.
[0280] Earlier work in other tumor types suggested that ERG may
induce EMT. In order to test, if the same effect exists in NSCLC
cells, an ERG expression vector was transfected to H358 cells,
which has relatively very low levels of endogenous ERG protein
(FIG. 18A). When the H358 cells expressed high level of ERG
protein, we observed a significant increase in expression of two
EMT markers, ZEB1 and Foxc2 (FIG. 18B), suggesting that ERG can
induce EMT in NSCLC cells.
[0281] FIGS. 18A and 18B illustrate that ERG expression induces EMT
markers. H358 NSCLC cells were transfected with a cDNA coding for
human ERG protein. Increased ERG expression was detected by western
blotting (FIG. 18A). Real-time PCR analysis revealed higher
expression of ZEB1 and FOXC2 in ERG expressing cells (FIG. 18B).
Data is first normalized for 18S RNA and then expressed as fold
induction over empty vector transfected cells.
YK-4-279 Inhibits Expression of ERG Dependent EMT Markers
[0282] A549 cells express relatively high levels of ERG protein
(FIG. 17). We evaluated the EMT marker gene expression in these
cells by real-time quantitative PCR (FIG. 19). TGF-.beta., a known
EMT inducer, treatment of A549 cells resulted in increased ZEB1 and
FOXC2 expression. When the untransfected A549 cells were treated
with the ERG inhibitor, YK-4-279, we observed the complete
opposite, both ZEB and FOXC2 expression was inhibited.
[0283] The preliminary data presented here confirms that YK-4-279
directly binds to ERG protein and inhibit its function as a
transcription factor. Furthermore, we demonstrated that ERG can
induce EMT markers in NSCLC cells and this effect can be reversed
by YK-4-279.
[0284] Experiments summarized in this section test the hypothesis
that ERG contributes to the pathogenesis of NSCLC by inducing
(EMT), and that ERG can serve as a target for lung cancer therapy.
We establish that ERG may be successfully inhibited by YK-4-279 as
a novel therapeutic approach.
The Role of ERG in Mediating EMT in Lung Cancer Cells
[0285] We assess the effects of modulating the levels of ERG
expression on lung cancer cell lines, particularly regarding EMT.
The H358 cell line, derived from a non-small cell lung cancer,
expresses relatively low levels of endogenous ERG protein (FIG.
17). We introduce an ERG-expression vector to elevate ERG levels in
these cells. Other lung cancer cell lines (A549, H1944, H1395, and
H596) express very high levels of endogenous ERG. We use RNA
interference techniques (shRNA or siRNA) targeted to ERG to reduce
its expression levels in these cells. In each case, the effect on
EMT is assessed by determining the expression levels of mRNAs and
their cognate proteins that serve as specific markers for EMT as
follows: E-cadherin, vimentin, Snail, Slug, and ZEB1. A heightened
EMT expression profile in H358 cells overexpressing ERG or a
reduced EMT expression profile in cells (A549, H1944, H1395, and
H596) in which ERG expression has been inhibited by RNA
interference is observed, we extend these studies by determining
the effects of ZEB1 and ZEB 2 siRNAs. The hypothesis that ERG
mediates EMT functions through ZEB1 and ZEB2 is supported if the
effect of ERG on EMT is diminished when ZEB1 and ZEB2 expression is
inhibited.
[0286] We determine IC50 values for common chemotherapeutic agents,
cisplatin, paclitaxel, gemcitabine, etoposide, and vinblastine on
five NSCLC cell lines. Cell viability is determined by electric
impedance and WST assays. Once the baseline IC50 values are
established, we repeat the experiment with altered EGR expression.
ERG expression is inhibited in A549, H1944, H1395, and H596 cells
with shRNA. If stable shRNA expression and reduced ERG protein
expression cannot be achieved, we will perform these experiments
with transient transfection of siRNA targeting ERG. Reducing ERG
expression in NSCLC cell lines is expected to shift the IC50 curves
significantly to the left such that the cells become more sensitive
these chemotherapeutic agents. To complement these experiments we
establish a stable H358 cell line that express high levels of ERG
protein from a mammalian expression vector. In this cell line we
see a significant shift to right in the IC50 curve such that the
cells become more resistant to chemotherapy.
[0287] New formulations of YK-4-279 that can be administered
parenterally are produced and the effects of YK-4-279 on the
proliferation and malignant properties of lung cancer cells are
determined.
[0288] The lead excipient is .beta.-hydroxypropyl cyclodextrin
(.beta.-HPCD), while .beta.-HPCD is a clinically viable vehicle.
The top seven formulations are compared to kinetics for HP.beta.CD.
CD-1 are injected IP followed by time-points at 0, 5, 10, 15, 30,
60, 120, 180, 240 and 480 minutes. A 24 hour point also checks for
delayed clearance. A series of CD-1 mice with IV injection followed
by time-points at 0, 5, 10, 15, 30, 60, 120, 180, 240 are used to
measure absorption levels. Plasma are analyzed and pharmacokinetic
parameters calculated. The goal of these studies is to determine if
there is a superior preparation to HP.beta.CD by comparing
absorption and half-life. If a formulation can achieve IP
absorption and sustain plasma levels of greater than 3 .mu.M for 24
hours, we consider this a significant improvement. This allows us
to evaluate daily dosing in comparison with continuous infusion
therapy. The use of a daily dose rather than continuous IV is
preferred for future animal and clinical studies.
[0289] We inhibit ERG function by treating cells with YK-4-279.
Changes in EMT markers are determined as described above to confirm
that YK-4-279 treatment results in the same EMT marker expression
profile as the lack of ERG (siRNA or shRNA). The goal of following
experiments is to assess the effects on functional outcomes when
EMT is altered. For this purpose, we evaluate surrogate markers of
the malignant phenotype as follows: cell motility, chemotaxis,
invasion of an endothelial cell monolayer, growth on plastic,
growth in soft agar, and in vivo growth as xenografts. In parallel
experiments, cells are treated with YK-4-279 and varying
concentrations of different chemotherapeutic agents for NSCLC,
including cisplatin, paclitaxel, gemcitabine, etoposide, and
vinblastine. We observe a synergistic inhibitory effect induced by
YK-4-279 in combination with these drugs. To support the hypotheses
that inhibiting ERG expression diminishes EMT through ZEB1/2, we
determine whether enforced overexpression of ZEB1/2 reverses the
effects of YK-4-279 on cell phenotype.
[0290] We test the effects of YK-4-279 on NSCLC cell motility and
invasion. We test YK-4-279 for its ability to inhibit NSCLC cell
invasion using the xCELLigence system. This new method allows
real-time measurement of cell motility in a classical Boyden
chamber format with a layer of gold electrodes on the underneath
surface of the porous membrane (xCELLigence sim-plates). As the
cells move from the upper chamber through the membrane towards a
chemoattractant in the lower chamber, they increase electric
impedance on the under surface of the membrane, which is recorded
in real-time. The same instrument is also used for measuring
invasion through an endothelial monolayer (Rahim S, Uren A. A
real-time electrical impedance based technique to measure invasion
of endothelial cell monolayer by cancer cells. Journal of
visualized experiments: JoVE. 2011). In this experimental format,
human umblical vein endothelial cells (HUVEC) grow on regular cell
culture plates with gold electrodes on the surface (xCELLigence
E-plates). Once the endothelial cells form a stable monolayer,
NSCLC cells are added on top. As the cancer cells break tight
junctions between endothelial cells and penetrate through the
endothelial monolayer, they alter the electric impedance. These
experiments allow us to evaluate if YK-4-279 alters motility,
chemotaxis and invasive phenotype of NSCLC cells
[0291] We perform synergy studies in cell culture by titrating
YK-4-279 and chemotherapeutic agents (cisplatin, paclitaxel,
gemcitabine, etoposide, and vinblastine). Cell death is used as the
end point and any potential synergy is calculated by combination
index (CI) isobologram equation method (Chou T C. Theoretical
basis, experimental design, and computerized simulation of
synergism and antagonism in drug combination studies.
Pharmacological reviews. 2006; 58:621-81).
[0292] We test the effect of ERG inhibition on growth of 3
different human NSCLC xenografts (two with high ERG expression and
one with low ERG expression). Cell suspension prepared in matrigel
is subcutaneously implanted into four- to six-week-old male SCID
mice. Adjusting for take rate, two groups of animals (10
animals/xenograft line) are administered the inhibitor YK-4-279,
and the carrier only placebo based on the formulation studies for
each xenograft line. Drug treatment start when the tumors reach to
200 mm3 size. Tumor growth and body weight is measured twice
weekly. All experimental groups have a power of 83% with p<0.05
to detect a 35% difference in total tumor volume. The animals are
harvested at eight weeks or earlier if animals become compromised
(primary tumor reaching to 2000 mm.sup.3, primary tumor ulcerating,
or mice showing signs of pain and distress). Half of the tumor
tissue is embedded in paraffin for immunohistochemical analysis and
the other half flash frozen for molecular analysis. We hypothesize
that blocking ERG activity in NSCLC xenografts may result in a
reduction in primary tumor size.
[0293] While the disclosure has been illustrated and described in
detail in the drawings and foregoing description, such illustration
and description are to be considered illustrative or exemplary and
not restrictive. The disclosure is not limited to the disclosed
embodiments. Variations to the disclosed embodiments can be
understood and effected by those skilled in the art in practicing
the claimed disclosure, from a study of the drawings, the
disclosure and the appended claims.
[0294] All references cited herein are incorporated herein by
reference in their entirety. To the extent publications and patents
or patent applications incorporated by reference contradict the
disclosure contained in the specification, the specification is
intended to supersede and/or take precedence over any such
contradictory material.
[0295] Unless otherwise defined, all terms (including technical and
scientific terms) are to be given their ordinary and customary
meaning to a person of ordinary skill in the art, and are not to be
limited to a special or customized meaning unless expressly so
defined herein. It should be noted that the use of particular
terminology when describing certain features or aspects of the
disclosure should not be taken to imply that the terminology is
being re-defined herein to be restricted to include any specific
characteristics of the features or aspects of the disclosure with
which that terminology is associated. Terms and phrases used in
this application, and variations thereof, especially in the
appended claims, unless otherwise expressly stated, should be
construed as open ended as opposed to limiting. As examples of the
foregoing, the term `including` should be read to mean `including,
without limitation,` `including but not limited to,` or the like;
the term `comprising` as used herein is synonymous with
`including,` `containing,` or `characterized by,` and is inclusive
or open-ended and does not exclude additional, unrecited elements
or method steps; the term `having` should be interpreted as `having
at least;` the term `includes` should be interpreted as `includes
but is not limited to;` the term `example` is used to provide
exemplary instances of the item in discussion, not an exhaustive or
limiting list thereof; adjectives such as `known`, `normal`,
`standard`, and terms of similar meaning should not be construed as
limiting the item described to a given time period or to an item
available as of a given time, but instead should be read to
encompass known, normal, or standard technologies that may be
available or known now or at any time in the future; and use of
terms like `preferably,` `preferred,` desired,' or `desirable,` and
words of similar meaning should not be understood as implying that
certain features are critical, essential, or even important to the
structure or function of the invention, but instead as merely
intended to highlight alternative or additional features that may
or may not be utilized in a particular embodiment of the invention.
Likewise, a group of items linked with the conjunction `and` should
not be read as requiring that each and every one of those items be
present in the grouping, but rather should be read as `and/or`
unless expressly stated otherwise. Similarly, a group of items
linked with the conjunction `or` should not be read as requiring
mutual exclusivity among that group, but rather should be read as
`and/or` unless expressly stated otherwise.
[0296] Where a range of values is provided, it is understood that
the upper and lower limit, and each intervening value between the
upper and lower limit of the range is encompassed within the
embodiments.
[0297] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity. The indefinite article "a" or "an" does
not exclude a plurality. A single processor or other unit may
fulfill the functions of several items recited in the claims. The
mere fact that certain measures are recited in mutually different
dependent claims does not indicate that a combination of these
measures cannot be used to advantage. Any reference signs in the
claims should not be construed as limiting the scope.
[0298] It will be further understood by those within the art that
if a specific number of an introduced claim recitation is intended,
such an intent will be explicitly recited in the claim, and in the
absence of such recitation no such intent is present. For example,
as an aid to understanding, the following appended claims may
contain usage of the introductory phrases "at least one" and "one
or more" to introduce claim recitations. However, the use of such
phrases should not be construed to imply that the introduction of a
claim recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
embodiments containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should typically be interpreted to mean "at least one" or "one
or more"); the same holds true for the use of definite articles
used to introduce claim recitations. In addition, even if a
specific number of an introduced claim recitation is explicitly
recited, those skilled in the art will recognize that such
recitation should typically be interpreted to mean at least the
recited number (e.g., the bare recitation of "two recitations,"
without other modifiers, typically means at least two recitations,
or two or more recitations). Furthermore, in those instances where
a convention analogous to "at least one of A, B, and C, etc." is
used, in general such a construction is intended in the sense one
having skill in the art would understand the convention (e.g., "a
system having at least one of A, B, and C" would include but not be
limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, and/or A, B, and C
together, etc.). In those instances where a convention analogous to
"at least one of A, B, or C, etc." is used, in general such a
construction is intended in the sense one having skill in the art
would understand the convention (e.g., "a system having at least
one of A, B, or C" would include but not be limited to systems that
have A alone, B alone, C alone, A and B together, A and C together,
B and C together, and/or A, B, and C together, etc.). It will be
further understood by those within the art that virtually any
disjunctive word and/or phrase presenting two or more alternative
terms, whether in the description, claims, or drawings, should be
understood to contemplate the possibilities of including one of the
terms, either of the terms, or both terms. For example, the phrase
"A or B" will be understood to include the possibilities of "A" or
"B" or "A and B."
[0299] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification are to be
understood as being modified in all instances by the term `about.`
Accordingly, unless indicated to the contrary, the numerical
parameters set forth herein are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of any claims in any
application claiming priority to the present application, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0300] Furthermore, although the foregoing has been described in
some detail by way of illustrations and examples for purposes of
clarity and understanding, it is apparent to those skilled in the
art that certain changes and modifications may be practiced.
Therefore, the description and examples should not be construed as
limiting the scope of the invention to the specific embodiments and
examples described herein, but rather to also cover all
modification and alternatives coming with the true scope and spirit
of the invention.
* * * * *
References