U.S. patent application number 17/751153 was filed with the patent office on 2022-09-08 for combination therapy for treating cancer.
The applicant listed for this patent is Epizyme, Inc.. Invention is credited to Heike KEILHACK, Sarah Kathleen KNUTSON, Kevin Wayne KUNTZ.
Application Number | 20220281854 17/751153 |
Document ID | / |
Family ID | 1000006351515 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220281854 |
Kind Code |
A1 |
KEILHACK; Heike ; et
al. |
September 8, 2022 |
COMBINATION THERAPY FOR TREATING CANCER
Abstract
The present invention relates to compositions comprising
inhibitors of human histone methyltransferase EZH2 and one or more
other therapeutic agents, particularly anticancer agents such as
prednisone, and methods of combination therapy for administering to
subjects in need thereof for the treatment of cancer.
Inventors: |
KEILHACK; Heike; (Belmont,
MA) ; KNUTSON; Sarah Kathleen; (Lincoln, MA) ;
KUNTZ; Kevin Wayne; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Epizyme, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
1000006351515 |
Appl. No.: |
17/751153 |
Filed: |
May 23, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16985404 |
Aug 5, 2020 |
11370781 |
|
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17751153 |
|
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|
|
16372657 |
Apr 2, 2019 |
10787440 |
|
|
16985404 |
|
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|
14394463 |
Oct 14, 2014 |
10301290 |
|
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PCT/US2013/036452 |
Apr 12, 2013 |
|
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16372657 |
|
|
|
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61785169 |
Mar 14, 2013 |
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61624194 |
Apr 13, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4745 20130101;
A61K 31/436 20130101; A61K 31/4412 20130101; A61K 31/573 20130101;
A61P 35/00 20180101; A61K 33/243 20190101; A61K 31/4439 20130101;
A61K 31/444 20130101; A61K 31/704 20130101; A61K 31/53 20130101;
A61K 31/675 20130101; A61K 31/5377 20130101; A61K 31/5355 20130101;
C07D 413/14 20130101; C07D 213/64 20130101; A61K 31/7068 20130101;
C07D 405/12 20130101; A61K 45/06 20130101 |
International
Class: |
C07D 405/12 20060101
C07D405/12; A61K 45/06 20060101 A61K045/06; A61K 31/436 20060101
A61K031/436; A61K 31/53 20060101 A61K031/53; A61K 31/573 20060101
A61K031/573; A61K 31/704 20060101 A61K031/704; A61K 31/7068
20060101 A61K031/7068; C07D 213/64 20060101 C07D213/64; A61K
31/4412 20060101 A61K031/4412; A61K 31/5377 20060101 A61K031/5377;
A61K 31/4439 20060101 A61K031/4439; C07D 413/14 20060101
C07D413/14; A61K 31/444 20060101 A61K031/444; A61P 35/00 20060101
A61P035/00; A61K 33/243 20060101 A61K033/243; A61K 31/5355 20060101
A61K031/5355; A61K 31/4745 20060101 A61K031/4745; A61K 31/675
20060101 A61K031/675 |
Claims
1. A method of treating cancer comprising administering to a
subject in need thereof a compound of Formula (IIa): ##STR00111##
or a pharmaceutically acceptable salt thereof, and prednisone,
wherein each of R.sub.a and R.sub.b, independently, is H or
C.sub.1-C.sub.6 alkyl; or R.sub.a and R.sub.b, together with the N
atom to which they are attached, form a 4 to 7-membered
heterocycloalkyl ring having 0 or 1 additional heteroatoms; wherein
the C.sub.1-C.sub.6 alkyl or the 4 to 7-membered heterocycloalkyl
ring are optionally substituted with one or more -Q.sub.3-T.sub.3,
in which Q.sub.3 is a bond or unsubstituted or substituted
C.sub.1-C.sub.3 alkyl linker, and T.sub.3 is H, halo, 4 to
7-membered heterocycloalkyl, C.sub.1-C.sub.3 alkyl, OR.sub.d,
COOR.sub.d, --S(O).sub.2R.sub.d, or --NR.sub.dR.sub.e, in which
each of R.sub.d and R.sub.e is independently H or C.sub.1-C.sub.6
alkyl, or -Q.sub.3T.sub.3 is oxo; R.sub.7 is C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.8 cycloalkyl, or 4 to 12-membered heterocycloalkyl,
each optionally substituted with one or more -Q.sub.5-T.sub.5, in
which Q.sub.5 is a bond, C(O), C(O)NR.sub.k, NR.sub.kC(O),
S(O).sub.2, or C.sub.1-C.sub.3 alkyl linker, R.sub.k being H or
C.sub.1-C.sub.6 alkyl, and T.sub.5 is H, halo, C.sub.1-C.sub.6
alkyl, hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O).sub.qR.sub.q
in which q is 0, 1, or 2 and R.sub.q is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and T.sub.5 is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
and R.sub.8 is H, methyl, or ethyl.
2. The method of claim 1, wherein the cancer is resistant or
refractory to at least one prior therapy.
3. The method of claim 1, wherein the compound of Formula (IIa) and
prednisone are administered simultaneously or sequentially.
4. The method of claim 1, wherein said subject has demonstrated
resistance to the compound of Formula (IIa) or a pharmaceutically
acceptable salt thereof when administered as a single agent.
5. The method of claim 1, wherein the cancer is resistant to at
least one prior monotherapy or at least one prior combination
therapy.
6. The method of claim 1, wherein the cancer is lymphoma, leukemia,
or melanoma.
7. The method of claim 6, wherein the lymphoma is follicular
lymphoma.
8. The method of claim 1, wherein the compound of Formula (IIa) is
Compound 44: ##STR00112## or a pharmaceutically acceptable salt
thereof.
9. A method of treating cancer comprising administering to a
subject in need thereof a compound of Formula (IIa): ##STR00113##
or a pharmaceutically acceptable salt thereof, and
cyclophosphamide, wherein each of R.sub.a and R.sub.b,
independently, is H or C.sub.1-C.sub.6 alkyl; or R.sub.a and
R.sub.b, together with the N atom to which they are attached, form
a 4 to 7-membered heterocycloalkyl ring having 0 or 1 additional
heteroatoms; wherein the C.sub.1-C.sub.6 alkyl or the 4 to
7-membered heterocycloalkyl ring are optionally substituted with
one or more -Q.sub.3-T.sub.3, in which Q.sub.3 is a bond or
unsubstituted or substituted C.sub.1-C.sub.3 alkyl linker, and
T.sub.3 is H, halo, 4 to 7-membered heterocycloalkyl,
C.sub.1-C.sub.3 alkyl, OR.sub.d, COOR.sub.d, --S(O).sub.2R.sub.a,
or --NR.sub.dR.sub.e, in which each of R.sub.a and R.sub.e is
independently H or C.sub.1-C.sub.6 alkyl, or -Q.sub.3T.sub.3 is
oxo; to R.sub.7 is C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8
cycloalkyl, or 4 to 12-membered heterocycloalkyl, each optionally
substituted with one or more -Q.sub.5-T.sub.5, in which Q.sub.5 is
a bond, C(O), C(O)NR.sub.k, NR.sub.kC(O), S(O).sub.2, or
C.sub.1-C.sub.3 alkyl linker, R.sub.k being H or C.sub.1-C.sub.6
alkyl, and T.sub.5 is H, halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O).sub.qR.sub.q
in which q is 0, 1, or 2 and R.sub.q is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and T.sub.5 is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
and R.sub.8 is H, methyl, or ethyl.
10. The method of claim 9, wherein the cancer is resistant or
refractory to at least one prior therapy.
11. The method of claim 9, wherein the compound of Formula (IIa)
and said cyclophosphamide are administered simultaneously or
sequentially.
12. The method of claim 9, wherein said subject has demonstrated
resistance to the compound of Formula (IIa) or a pharmaceutically
acceptable salt thereof when administered as a single agent.
13. The method of claim 9, wherein the cancer is resistant to at
least one prior monotherapy or at least one prior combination
therapy.
14. The method of claim 9, wherein the cancer is lymphoma,
leukemia, or melanoma.
15. The method of claim 14, wherein the lymphoma is follicular
lymphoma.
16. The method of claim 9, wherein the compound of Formula (IIa) is
Compound 44: ##STR00114## or a pharmaceutically acceptable salt
thereof.
17. A method of treating cancer comprising administering to a
subject in need thereof a compound of Formula (IIa): ##STR00115##
or a pharmaceutically acceptable salt thereof, and vincristine,
wherein each of R.sub.a and R.sub.b, independently, is H or
C.sub.1-C.sub.6 alkyl; or R.sub.a and R.sub.b, together with the N
atom to which they are attached, form a 4 to 7-membered
heterocycloalkyl ring having 0 or 1 additional heteroatoms; wherein
the C.sub.1-C.sub.6 alkyl or the 4 to 7-membered heterocycloalkyl
ring are optionally substituted with one or more -Q.sub.3-T.sub.3,
in which Q.sub.3 is a bond or unsubstituted or substituted
C.sub.1-C.sub.3 alkyl linker, and T.sub.3 is H, halo, 4 to
7-membered heterocycloalkyl, C.sub.1-C.sub.3 alkyl, OR.sub.d,
COOR.sub.d, --S(O).sub.2R.sub.d, or --NR.sub.dR.sub.e, in which
each of R.sub.d and R.sub.e is independently H or C.sub.1-C.sub.6
alkyl, or -Q.sub.3T.sub.3 is oxo; R.sub.7 is C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.8 cycloalkyl, or 4 to 12-membered heterocycloalkyl,
each optionally substituted with one or more -Q.sub.5-T.sub.5, in
which Q.sub.5 is a bond, C(O), C(O)NR.sub.k, NR.sub.kC(O),
S(O).sub.2, or C.sub.1-C.sub.3 alkyl linker, R.sub.k being H or
C.sub.1-C.sub.6 alkyl, and T.sub.5 is H, halo, C.sub.1-C.sub.6
alkyl, hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O).sub.qR.sub.q
in which q is 0, 1, or 2 and R.sub.q is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and T.sub.5 is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
and R.sub.8 is H, methyl, or ethyl.
18. The method of claim 17, wherein the cancer is resistant or
refractory to at least one prior therapy.
19. The method of claim 17, wherein the cancer is lymphoma,
leukemia, or melanoma.
20. The method of claim 17, wherein the compound of Formula (IIa)
is Compound 44: ##STR00116## or a pharmaceutically acceptable salt
thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/985,404, filed Aug. 5, 2020, which is a continuation of U.S.
application Ser. No. 16/372,657 filed Apr. 2, 2019 (now U.S. Pat.
No. 10,787,440), which is a continuation of U.S. application Ser.
No. 14/394,463 filed Oct. 14, 2014 (now U.S. Pat. No. 10,301,290),
which is a U.S. National Phase application of International
Application No. PCT/US2013/036452, filed Apr. 12, 2013, which
claims priority to, and the benefit of, the U.S. Provisional
Application No. 61/624,194 filed Apr. 13, 2012 and the U.S.
Provisional Application No. 61/785,169 filed Mar. 14, 2013, the
contents of each of which are incorporated by reference in their
entireties.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0002] The contents of the text file named "EPIZ009001WO_ST25.txt,"
which was created on Oct. 14, 2014 and is 45 KB in size, are hereby
incorporated by reference in their entireties.
PARTIES TO JOINT RESEARCH AGREEMENT
[0003] This invention was developed subject to a Joint Research
Agreement between Epizyme, Inc. and Eisai Co., Ltd.
FIELD OF THE INVENTION
[0004] This invention relates to compositions comprising inhibitors
of human histone methyltransferase EZH2, the catalytic subunit of
the PRC2 complex which catalyzes the mono-through tri-methylation
of lysine 27 on histone H3 (H3-K27), and one or more other
therapeutic agents, particularly anticancer agents, and methods of
combination therapy for treating cancer.
BACKGROUND OF THE INVENTION
[0005] Combination-therapy treatments for cancer have become more
common, in part due to the perceived advantage of attacking the
disease via multiple avenues. Although many effective
combination-therapy treatments have been identified over the past
few decades; in view of the continuing high number of deaths each
year resulting from cancer, a continuing need exists to identify
effective therapeutic regimens for use in anticancer treatment.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention features a composition
comprising a compound of Formula (IIa) below and one or more other
therapeutic agents or a pharmaceutically acceptable salt or ester
thereof.
##STR00001##
[0007] The compounds of Formula (IIa) can include one or more of
the following features:
[0008] Each of R.sub.a and R.sub.b, independently is H or
C.sub.1-C.sub.6 alkyl.
[0009] R.sub.a and R.sub.b, together with the N atom to which they
are attached, is a 4 to 7-membered heterocycloalkyl ring having 0
or 1 additional heteroatom, the C.sub.1-C.sub.6 alkyl and the 4 to
12-membered (e.g., 4 to 7-membered) heterocycloalkyl ring being
optionally substituted with one or more -Q.sub.3-T.sub.3.
[0010] Q.sub.3 is a bond or unsubstituted or substituted
C.sub.1-C.sub.3 alkyl linker.
[0011] T.sub.3 is H, halo, 4 to 7-membered heterocycloalkyl,
C.sub.1-C.sub.3 alkyl, OR.sub.d, COOR.sub.d, --S(O).sub.2R.sub.d,
or --NR.sub.dR.sub.e, each of R.sub.d and R.sub.e independently
being H or C.sub.1-C.sub.6 alkyl.
[0012] R.sub.7 is C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl
or 4 to 12-membered (e.g., 4 to 7-membered) heterocycloalkyl, each
optionally substituted with one or more -Q.sub.5-T.sub.5. For
example, R.sub.7 is not H.
[0013] R.sub.7 is 4 to 7-membered heterocycloalkyl optionally
substituted with one or more -Q.sub.5-T.sub.5.
[0014] R.sub.7 is piperidinyl, tetrahydropyran, cyclopentyl, or
cyclohexyl, each optionally substituted with one
-Q.sub.5-T.sub.5.
[0015] T.sub.5 is H, halo, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, or 4 to
12-membered (e.g., 4 to 7-membered) heterocycloalkyl.
[0016] Q.sub.5 is a bond and T.sub.5 is C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.8 cycloalkyl, or 4 to 12-membered (e.g., 4 to
7-membered) heterocycloalkyl.
[0017] Q.sub.5 is CO, S(O).sub.2, or NHC(O); and T.sub.5 is
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxyl, C.sub.3-C.sub.8
cycloalkyl, or 4 to 12-membered (e.g., 4 to 7-membered)
heterocycloalkyl.
[0018] Q.sub.5 is C.sub.1-C.sub.3 alkyl linker and T.sub.5 is H or
C.sub.6-C.sub.10 aryl.
[0019] Q.sub.5 is C.sub.1-C.sub.3 alkyl linker and T.sub.5 is
C.sub.3-C.sub.8 cycloalkyl, 4 to 7-membered heterocycloalkyl, or
S(O).sub.qR.sub.q.
[0020] R.sub.7 is cyclopentyl or cyclohexyl, each optionally
substituted with one -Q.sub.5-T.sub.5.
[0021] Q.sub.5 is NHC(O) and T.sub.5 is C.sub.1-C.sub.6 alkyl or
C.sub.1-C.sub.6 alkoxy.
[0022] R.sub.7 is isopropyl.
[0023] Each of R.sub.2 and R.sub.4, independently is H or
C.sub.1-C.sub.6 alkyl optionally substituted with amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, or
C.sub.6-C.sub.10 aryl.
[0024] R.sub.8 is H, methyl, or ethyl.
[0025] R.sub.8 is methyl.
[0026] R.sub.8 is ethyl.
[0027] R.sub.8 is 4 to 7-heterocycloalkyl, e.g.,
tetrahydropyran.
[0028] The present invention features a composition comprising a
compound selected from Table 1 or a pharmaceutically acceptable
salt or ester thereof and one or more other therapeutic agents.
[0029] The present invention features a composition comprising
Compound 44
##STR00002##
or a pharmaceutically acceptable salt or ester thereof and one or
more other therapeutic agents.
[0030] In this and other aspects of the invention, in one
embodiment the other therapeutic agents are anticancer agents.
[0031] In this and other aspects of the invention, in one
embodiment the other therapeutic agents are glucocorticoids.
[0032] In this and other aspects of the invention, in one
embodiment the other therapeutic agents are selected from
prednisone, prednisolone, cyclophosphamide, vincristine,
doxorubicin, mafosfamide, cisplatin, AraC, everolimus, decitabine,
dexamethasone, and analogs, derivatives, or combinations
thereof.
[0033] In this and other aspects of the invention, in one
embodiment the other therapeutic agent is prednisone, or an analog
or derivative thereof.
[0034] The present invention also provides pharmaceutical
compositions comprising a compound selected from those of Formula
(IIa) disclosed herein or pharmaceutical acceptable salts thereof
and one or more therapeutic agents, and a pharmaceutically
acceptable carrier.
[0035] The present invention also provides pharmaceutical
compositions comprising a compound selected from Table I, one or
more other therapeutic agents, or pharmaceutically acceptable salts
thereof, and a pharmaceutically acceptable carrier.
[0036] The present invention also provides pharmaceutical
compositions comprising a compound selected from those of Formula
(IIa) disclosed herein or pharmaceutically acceptable salts
thereof, one or more other therapeutic agents, and a
pharmaceutically acceptable carrier.
[0037] Another aspect of this invention is a method for treating or
preventing a disease by administering to a subject in need thereof
a therapeutically effective amount of a composition comprising a
compound of Formula (IIa), or a pharmaceutically acceptable salt
thereof, and one or more additional therapeutic agents. The disease
of the present invention can be influenced, treated, or prevented
by modulating the methylation status of histones or other proteins.
For example, the disease is cancer, a precancerous condition, or a
neurological disease. Preferably, the lymphoma is non-Hodgkin
lymphoma, follicular lymphoma or diffuse large B-cell lymphoma.
Alternatively, the leukemia is chronic myelogenous leukemia
(CIVIL). The precancerous condition is, e.g., myelodysplastic
syndromes (MDS, formerly known as preleukemia).
[0038] The subject of the present invention includes any human
subject who has been diagnosed with, has symptoms of, or is at risk
of developing a cancer or a precancerous condition. The subject of
the present invention includes any human subject expressing a
mutant EZH2. For example, a mutant EZH2 comprises one or more
mutations, wherein the mutation is a substitution, a point
mutation, a nonsense mutation, a missense mutation, a deletion, or
an insertion. A mutant EZH2 of the present invention may comprise a
mutation in the substrate pocket domain as defined in SEQ ID NO: 6.
A mutant EZH2 may have a substitution at amino acid Y641.
Preferably, the mutant EZH2 has one of the following mutations:
substitution of phenylalanine (F) for the wild type residue
tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F); a
substitution of histidine (H) for the wild type residue tyrosine
(Y) at amino acid position 641 of SEQ ID NO: 1 (Y641H); a
substitution of asparagine (N) for the wild type residue tyrosine
(Y) at amino acid position 641 of SEQ ID NO: 1 (Y641N); a
substitution of serine (S) for the wild type residue tyrosine (Y)
at amino acid position 641 of SEQ ID NO: 1 (Y641S); and a
substitution of cysteine (C) for the wild type residue tyrosine (Y)
at amino acid position 641 of SEQ ID NO: 1 (Y641C).
[0039] Other mutations of EZH2 may include, but are not limited to:
a substitution of glycine (G) for the wild type residue alanine (A)
at amino acid position 677 of SEQ ID NO: 1 (A677G); a substitution
of valine (V) for the wild type residue alanine (A) at amino acid
position 687 of SEQ ID NO: 1 (A687V); a substitution of methionine
(M) for the wild type residue valine (V) at amino acid position 674
of SEQ ID NO: 1 (V674M); a substitution of histidine (H) for the
wild type residue arginine (R) at amino acid position 685 of SEQ ID
NO: 1 (R685H); a substitution of cysteine (C) for the wild type
residue arginine (R) at amino acid position 685 of SEQ ID NO: 1
(R685C); a substitution of serine (S) for the wild type residue
asparagine (N) at amino acid position 322 of SEQ ID NO: 3 (N322S),
a substitution of glutamine (Q) for the wild type residue arginine
(R) at amino acid position 288 of SEQ ID NO: 3 (R288Q), a
substitution of isoleucine (I) for the wild type residue threonine
(T) at amino acid position 573 of SEQ ID NO: 3 (T573I), a
substitution of glutamic acid (E) for the wild type residue
aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3
(D664E), a substitution of glutamine (Q) for the wild type residue
arginine (R) at amino acid position 458 of SEQ ID NO: 5 (R458Q), a
substitution of lysine (K) for the wild type residue glutamic acid
(E) at amino acid position 249 of SEQ ID NO: 3 (E249K), a
substitution of cysteine (C) for the wild type residue arginine (R)
at amino acid position 684 of SEQ ID NO: 3 (R684C), a substitution
of histidine (H) for the wild type residue arginine (R) at amino
acid position 628 of SEQ ID NO: 11 (R628H), a substitution of
histidine (H) for the wild type residue glutamine (Q) at amino acid
position 501 of SEQ ID NO: 5 (Q501H), a substitution of asparagine
(N) for the wild type residue aspartic acid (D) at amino acid
position 192 of SEQ ID NO: 3 (D192N), a substitution of valine (V)
for the wild type residue aspartic acid (D) at amino acid position
664 of SEQ ID NO: 3 (D664V), a substitution of leucine (L) for the
wild type residue valine (V) at amino acid position 704 of SEQ ID
NO: 3 (V704L), a substitution of serine (S) for the wild type
residue proline (P) at amino acid position 132 of SEQ ID NO: 3
(P132S), a substitution of lysine (K) for the wild type residue
glutamic acid (E) at amino acid position 669 of SEQ ID NO: 11
(E669K), a substitution of threonine (T) for the wild type residue
alanine (A) at amino acid position 255 of SEQ ID NO: 3 (A255T), a
substitution of valine (V) for the wild type residue glutamic acid
(E) at amino acid position 726 of SEQ ID NO: 3 (E726V), a
substitution of tyrosine (Y) for the wild type residue cysteine (C)
at amino acid position 571 of SEQ ID NO: 3 (C571Y), a substitution
of cysteine (C) for the wild type residue phenylalanine (F) at
amino acid position 145 of SEQ ID NO: 3 (F145C), a substitution of
threonine (T) for the wild type residue asparagine (N) at amino
acid position 693 of SEQ ID NO: 3 (N693T), a substitution of serine
(S) for the wild type residue phenylalanine (F) at amino acid
position 145 of SEQ ID NO: 3 (F145S), a substitution of histidine
(H) for the wild type residue glutamine (Q) at amino acid position
109 of SEQ ID NO: 11 (Q109H), a substitution of cysteine (C) for
the wild type residue phenylalanine (F) at amino acid position 622
of SEQ ID NO: 11 (F622C), a substitution of arginine (R) for the
wild type residue glycine (G) at amino acid position 135 of SEQ ID
NO: 3 (G135R), a substitution of glutamine (Q) for the wild type
residue arginine (R) at amino acid position 168 of SEQ ID NO: 5
(R168Q), a substitution of arginine (R) for the wild type residue
glycine (G) at amino acid position 159 of SEQ ID NO: 3 (G159R), a
substitution of cysteine (C) for the wild type residue arginine (R)
at amino acid position 310 of SEQ ID NO: 5 (R310C), a substitution
of histidine (H) for the wild type residue arginine (R) at amino
acid position 561 of SEQ ID NO: 3 (R561H), a substitution of
histidine (H) for the wild type residue arginine (R) at amino acid
position 634 of SEQ ID NO: 11 (R634H), a substitution of arginine
(R) for the wild type residue glycine (G) at amino acid position
660 of SEQ ID NO: 3 (G660R), a substitution of cysteine (C) for the
wild type residue tyrosine (Y) at amino acid position 181 of SEQ ID
NO: 3 (Y181C), a substitution of arginine (R) for the wild type
residue histidine (H) at amino acid position 297 of SEQ ID NO: 3
(H297R), a substitution of serine (S) for the wild type residue
cysteine (C) at amino acid position 612 of SEQ ID NO: 11 (C612S), a
substitution of tyrosine (Y) for the wild type residue histidine
(H) at amino acid position 694 of SEQ ID NO: 3 (H694Y), a
substitution of alanine (A) for the wild type residue aspartic acid
(D) at amino acid position 664 of SEQ ID NO: 3 (D664A), a
substitution of threonine (T) for the wild type residue isoleucine
(I) at amino acid position 150 of SEQ ID NO: 3 (I150T), a
substitution of arginine (R) for the wild type residue isoleucine
(I) at amino acid position 264 of SEQ ID NO: 3 (I264R), a
substitution of leucine (L) for the wild type residue proline (P)
at amino acid position 636 of SEQ ID NO: 3 (P636L), a substitution
of threonine (T) for the wild type residue isoleucine (I) at amino
acid position 713 of SEQ ID NO: 3 (I713T), a substitution of
proline (P) for the wild type residue glutamine (Q) at amino acid
position 501 of SEQ ID NO: 5 (Q501P), a substitution of glutamine
(Q) for the wild type residue lysine (K) at amino acid position 243
of SEQ ID NO: 3 (K243Q), a substitution of aspartic acid (D) for
the wild type residue glutamic acid (E) at amino acid position 130
of SEQ ID NO: 5 (E130D), a substitution of glycine (G) for the wild
type residue arginine (R) at amino acid position 509 of SEQ ID NO:
3 (R509G), a substitution of histidine (H) for the wild type
residue arginine (R) at amino acid position 566 of SEQ ID NO: 3
(R566H), a substitution of histidine (H) for the wild type residue
aspartic acid (D) at amino acid position 677 of SEQ ID NO: 3
(D677H), a substitution of asparagine (N) for the wild type residue
lysine (K) at amino acid position 466 of SEQ ID NO: 5 (K466N), a
substitution of histidine (H) for the wild type residue arginine
(R) at amino acid position 78 of SEQ ID NO: 3 (R78H), a
substitution of methionine (M) for the wild type residue lysine (K)
at amino acid position 1 of SEQ ID NO: 6 (K6M), a substitution of
leucine (L) for the wild type residue serine (S) at amino acid
position 538 of SEQ ID NO: 3 (S538L), a substitution of glutamine
(Q) for the wild type residue leucine (L) at amino acid position
149 of SEQ ID NO: 3 (L149Q), a substitution of valine (V) for the
wild type residue leucine (L) at amino acid position 252 of SEQ ID
NO: 3 (L252V), a substitution of valine (V) for the wild type
residue leucine (L) at amino acid position 674 of SEQ ID NO: 3
(L674V), a substitution of valine (V) for the wild type residue
alanine (A) at amino acid position 656 of SEQ ID NO: 3 (A656V), a
substitution of aspartic acid (D) for the wild type residue alanine
(A) at amino acid position 731 of SEQ ID NO: 3 (Y731D), a
substitution of threonine (T) for the wild type residue alanine (A)
at amino acid position 345 of SEQ ID NO: 3 (A345T), a substitution
of aspartic acid (D) for the wild type residue alanine (A) at amino
acid position 244 of SEQ ID NO: 3 (Y244D), a substitution of
tryptophan (W) for the wild type residue cysteine (C) at amino acid
position 576 of SEQ ID NO: 3 (C576W), a substitution of lysine (K)
for the wild type residue asparagine (N) at amino acid position 640
of SEQ ID NO: 3 (N640K), a substitution of lysine (K) for the wild
type residue asparagine (N) at amino acid position 675 of SEQ ID
NO: 3 (N675K), a substitution of tyrosine (Y) for the wild type
residue aspartic acid (D) at amino acid position 579 of SEQ ID NO:
11 (D579Y), a substitution of isoleucine (I) for the wild type
residue asparagine (N) at amino acid position 693 of SEQ ID NO: 3
(N693I), and a substitution of lysine (K) for the wild type residue
asparagine (N) at amino acid position 693 of SEQ ID NO: 3
(N693K).
[0040] Other mutations of EZH2 can include: a frameshift at amino
acid position 730, 391, 461, 441, 235, 254, 564, 662, 715, 405,
685, 64, 73, 656, 718, 374, 592, 505, 730, or 363 of SEQ ID NO: 3,
5 or 11, or the corresponding nucleotide position of the nucleic
acid sequence encoding SEQ ID NO: 3, 5, or 11; a deletion of
glutamic acid (E) and leucine (L) at amino acid positions 148 and
149 of SEQ ID NO: 3, 5 or 11, or a nonsense mutation at amino acid
position 733, 25, 317, 62, 553, 328, 58, 207, 123, 63, 137, or 60
of SEQ ID NO: 3, 5 or 11.
[0041] A subject of the present invention may have resistance to
any one or more other therapeutic agents or any of the compounds
described herein. For example, the subject may have resistance to
EZH inhibitors or prednisone.
[0042] The present invention features a method for inhibiting
cancer cell proliferation comprising contacting said cancer cells
with a composition comprising any compound of Formula (IIa) or
pharmaceutically acceptable salt thereof, and one or more
additional therapeutic agent. Inhibiting cancer cell proliferation
includes delaying cancer cell growth, inducing cell death, reducing
cancer cell viability, inhibiting or delaying tumor growth, or
reducing tumor size.
[0043] The present invention features methods of combination
therapy wherein any compound of Formula (IIa), or pharmaceutically
acceptable salt thereof, and one or more other therapeutic agents
are administered simultaneously or sequentially. For example, any
compound of Formula (IIa) or pharmaceutically acceptable salt
thereof may be administered prior to administration of one or more
other therapeutic agents. For example, any compound of Formula
(IIa) or pharmaceutically acceptable salt there or may be
administered prior to administration of a composition comprising
any compound of Formula (IIa) or pharmaceutically acceptable salt
thereof and one or more other therapeutic agents. Concurrent or
sequential administration of any compound of Formula (IIa) and/or
each therapeutic agent can be effected by any appropriate route
including, but not limited to, oral routes, intravenous routes,
intramuscular routes, and direct absorption through mucous membrane
tissues. The therapeutic agents can be administered by the same
route or by different routes.
[0044] The methods of combination therapy featured in the present
invention may result in a synergistic effect, wherein the effect of
a combination of compounds or other therapeutic agents is greater
than the sum of the effects resulting from administration of any of
the compounds or other therapeutic agents as single agents. A
synergistic effect may also be an effect that cannot be achieved by
administration of any of the compounds or other therapeutic agents
as single agents. The synergistic effect may include, but is not
limited to, an effect of treating cancer by reducing tumor size,
inhibiting tumor growth, or increasing survival of the subject. The
synergistic effect may also include reducing cancer cell viability,
inducing cancer cell death, and inhibiting or delaying cancer cell
growth.
[0045] Compositions of the present invention can be administered at
a dosage of 0.01 mg/kg per day to about 1000 mg/kg per day. Any
compound of Formula (IIa) or pharmaceutically acceptable salt
thereof may be administered at a dosage of 0.01 mg/kg per day to
about 1000 mg/kg per day. Any other therapeutic agent may be
administered at a dosage of 0.01 mg/kg per day to about 1000 mg/kg
per day.
[0046] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In the
specification, the singular forms also include the plural unless
the context clearly dictates otherwise. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents and other references mentioned herein are
incorporated by reference. The references cited herein are not
admitted to be prior art to the claimed invention. In the case of
conflict, the present specification, including definitions, will
control. In addition, the materials, methods and examples are
illustrative only and are not intended to be limiting.
[0047] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTIONS OF FIGURES
[0048] FIG. 1 is a schematic detailing in vitro treatment schedules
for determining the effect of combination therapy on cell
viability. (A) Administration of Compound 44 prior to
administration of Compound 44 and components of CHOP
(Cyclophosphamide, Doxorubicin, Vincristine, and Prednisolone). (B)
Administration of Compound 44 prior to CHOP components. (C)
Administration of components of CHOP prior to administration of
Compound 44 and CHOP components. After 4 days, cell viability was
determined.
[0049] FIG. 2 is four graphs measuring cancer cell viability in
vitro after treatment with Compound 44 and CHOP components alone or
in combination. WSU-DLCL2 cells were treated as shown in FIG. 1A.
Cells were first treated with varying concentrations of Compound
44. After four days, cells were treated with the combination of
varying concentrations of Compound 44 and (A) Mafofsamide
(Cyclophosphamide metabolite), (B) Doxorubicin, (C) Vincristine, or
(D) Prednisolone (Prednisone metabolite). Control cells were
treated with DMSO. Cell viability was normalized to the percentage
of cell viability in DMSO-treated for each Compound 44
concentration.
[0050] FIG. 3 is three graphs measuring cancer cell viability after
various treatment schedules. WSU-DCLC2 cells were treated with
increasing concentrations of Prednisolone and Compound 44. Cells
were treated with Compound 44 and Prednisolone according to the
treatment schedule in (A) FIG. 1A, (B) FIG. 1B, or (C) FIG. 1C.
Cell viability was normalized to the DMSO/DMSO-treated sample.
[0051] FIG. 4 is three graphs measuring cancer cell viability after
treatment with Prednisolone and Compound 44 in cell lines with
different EZH2 mutations. Cells were treated as depicted in FIG. 1A
and with increasing concentrations of Prednisolone and Compound 44.
Cell viability was normalized to the DMSO/DMSO-treated sample. (A)
WSU-DLCL2 cells express the Y641F mutation and are sensitive to
EZH2 inhibitors. (B) RL cells express the Y641N mutation are
resistant to EZH2 inhibitors and Prednisolone. (C) OCI-LY19 cells
are wild-type at Y641 and show sensitivity to Prednisolone, but do
not show sensitivity to Compound 44 or increased sensitivity to
combinations of Compound 44 plus Prednisolone.
[0052] FIG. 5 is five graphs showing a panel of pharmacokinetic
profiles for co-administration of Compound 44 and CHOP components
in vivo. BALB/c mice were administered a single oral administration
of Compound 44 and CHOP components. The concentration of Compound
44 (ng/mL) in the plasma was measured at various timepoints 0-24
hours after administration. Cyclophosphamide was administered by
intraperitoneal injection at 30 mg/kg. Vincristine was administered
by intravenous injection at 0.375 mg/kg. Doxorubicin was
administered by intravenous injection at 2.475 mg/kg. Prednisolone
was administered by oral administration at 0.15 mg/kg. Compound 44
was administered by oral administration at 225 mg/kg.
[0053] FIG. 6 is two graphs demonstrating the effect of Compound 44
and CHOP administration in SUDHL6 xenograft model on tumor growth
and survival. Athymic nude mice were subcutaneously injected with
1.times.10.sup.7 SUDHL6 human lymphoma cells. After tumors reached
a size of app. 120 mm.sup.3 Compound 44 was administered over 28
days at the indicated dosages either three times a day (TID) or
twice a day (BID). CHOP (Cyclophosphamide, Vincristine,
Doxorubicin, and Prednisone) was administered at day 1 and 8 to
mice receiving 225 mg/kg twice per day (225 mg/kg BID and CHOP).
Control mice did not receive any Compound 44 (vehicle) or CHOP only
(CHOP). Tumor volume was measured twice a week. Animals were
euthanized when tumor volume reached 2000 mm.sup.3 or 60 days after
the first dose. (A) For tumor growth delay analysis, median tumor
volume was calculated for each treatment group by measuring the
tumors twice a week for 60 days or until the tumor reached 2000
mm.sup.3. (B) Kaplan-meier curve depicts the survival rate of the
mice.
[0054] FIG. 7 is three graphs demonstrating the effect of Compound
44 and CHOP administration in WSU-DLCL2 xenograft model on tumor
growth and survival. SCID mice were subcutaneously injected with
1.times.10.sup.7 WSU-DLCL2 human lymphoma cells. After tumors
reached a size of app. 120 mm.sup.3 Compound 44 was administered
over 28 days at the indicated dosages at three times a day (TID),
twice a day (BID) or once a day (QD). CHOP (Cyclophosphamide,
Vincristine, Doxorubicin, and Prednisone) was administered at day 1
and 22 to mice receiving 225 mg/kg twice a day (225 mg/kg BID and
CHOP). Control mice either did not receive any Compound 44
(vehicle) or received CHOP only (CHOP). Tumor volume was measured
twice a week. Animals were euthanized 28 days after first dose. (A)
Treatment efficacy was determined by measuring mean tumor volume
over the course of treatment. (B) Concentration (ng/mL) of Compound
44 was measured in plasma from mice on day 28 at before
administration (trough) and three hours post-administration (post).
(C) Concentration (ng/g) of Compound 44 was measured from tumor
tissue from mice on day 28 three hours post-administration.
[0055] FIG. 8 is a western blot analysis and a graph showing the
histone methylation status from tumor samples from the WSU-DLCL2
xenograft model. Tumors were harvested 28 days after injection and
histones were extracted. (A) A Western Blot was probed with
antibodies specifically recognizing tri-methylated Lysine 27 of
histone H3 (H3K27me3) and total histone H3 proteins. (B)
Methylation status of tumors was determined by ELISA. H3K27
tri-methylation was detected and normalized to total histone H3
levels.
[0056] FIG. 9 is four graphs demonstrating the effect of Compound
44 and COP administration in a SUDHL10 xenograft model on tumor
growth and survival. SCID mice were subcutaneously injected with
1.times.10.sup.7 SUDHL10 human lymphoma cells. After tumors reached
a size of app. 120 mm.sup.3 Compound 44 was administered over 28
days at the indicated dosages twice a day (BID). COP
(Cyclophosphamide, Vincristine, and Prednisone) was administered at
day 1 and 22 to mice receiving 250 mg/kg twice a day (250 mg/kg BID
and COP). Control mice did not receive any Compound 44 (vehicle) or
COP only (COP). Tumor volume was measured twice a week. (A)
Treatment efficacy was determined by measuring mean tumor volume
over the course of treatment. (B) Mean tumor weight was determined
after mice were euthanized on day 28 after first dosing. (C) For
tumor growth delay analysis, mean tumor volume and SEM was
calculated for each treatment group by measuring the tumors twice a
week for 60 days or until the tumor reached 2000 mm.sup.3. (D)
Kaplan-Meier curve depicts the survival rate of the treated
mice.
[0057] FIG. 10 is four graphs showing the pharmacokinetic and
pharmacodynamic profiles after COP and Compound 44 administration
in the SUDHL10 xenograft model. Tumor-bearing mice were euthanized
after 28 days of treatment and tissues were harvested. (A)
Concentration (ng/mL) of Compound 44 was measured in plasma from
mice on day 28 before administration (trough) and
post-administration (post) by LC-MS/MS. Methylation status of
various tissues from tumor-bearing mice were determined by ELISA:
(B) tumor, (C) spleen, and (D) bone marrow. H3K27 tri-methylation
was detected and normalized to total histone H3 levels.
[0058] FIG. 11 is a structure model of partial EZH2 protein based
on the A chain of nuclear receptor binding SET domain protein 1
(NSD1) is provided below. This model corresponds to amino acid
residues 533-732 of EZH2 sequence of SEQ ID NO: 1.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention is based upon the discovery that EZH2
histone methyltransferase inhibitors and other anti-cancer agents
can be used in combination to treat certain tumors with superior
results than those achieved by treating tumors with EZH2 histone
methyltransferase inhibitors and the anti-cancer agents alone.
Accordingly, the present invention provides a composition
comprising an EZH2 histone methyltransferase inhibitor and one or
more other therapeutic agents, and methods for their use to treat
diseases the course of which can be influenced by modulating the
methylation status of histones or other proteins, e.g., cancer. In
a certain embodiment, the present invention features a composition
comprising a compound of Formula (IIa) and prednisone. The present
invention also includes methods for combination therapies
comprising EZH2 histone methyltransferase inhibitor and one or more
therapeutic agents, such as a compound of Formula (IIa) and
prednisone, to treat cancer, e.g., follicular lymphoma (FL) and
diffuse cell large B-cell lymphoma (DCLBL). Specifically, the
methods of the present invention are useful for treating or
preventing cancer or inhibiting cancer cell proliferation.
[0060] EZH2 is a histone methyltransferase that is the catalytic
subunit of the PRC2 complex which catalyzes the mono- through
tri-methylation of lysine 27 on histone H3 (H3-K27). Histone H3-K27
trimethylation is a mechanism for suppressing transcription of
specific genes that are proximal to the site of histone
modification. This trimethylation is known to be a cancer marker
with altered expression in cancer, such as prostate cancer (see,
e.g., U.S. Patent Application Publication No. 2003/0175736;
incorporated herein by reference in its entirety). Other studies
provided evidence for a functional link between dysregulated EZH2
expression, transcriptional repression, and neoplastic
transformation. Varambally et al. (2002) Nature 419(6907):624-9
Kleer et al. (2003) Proc Natl Acad Sci USA 100(20):11606-11.
[0061] An aspect of the present invention relates to methods for
treating or alleviating a symptom of cancer or precancerous
condition in a subject by administering to a subject expressing a
mutant EZH2 a therapeutically effective amount of an EZH2 inhibitor
and one or more other therapeutic agents. The mutant EZH2 of the
present invention refers to a mutant EZH2 polypeptide or a nucleic
acid sequence encoding a mutant EZH2 polypeptide. In certain
embodiments the mutant EZH2 comprises one or more mutations in its
substrate pocket domain as defined in SEQ ID NO: 6. For example,
the mutation may be a substitution, a point mutation, a nonsense
mutation, a misssense mutation, a deletion, or an insertion.
[0062] Human EZH2 nucleic acids and polypeptides have previously
been described. See, e.g., Chen et al. (1996) Genomics 38:30-7 [746
amino acids]; Swiss-Prot Accession No. Q15910 [746 amino acids];
GenBank Accession Nos. NM_004456 and NP_004447 (isoform a [751
amino acids]); and GenBank Accession Nos. NM_152998 and NP_694543
(isoform b [707 amino acids]), each of which is incorporated herein
by reference in its entirety.
TABLE-US-00001 Amino acid sequence of human EZH2 (Swiss-Prot
Accession No. Q15910) (SEQ ID NO: 1)
MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEW
KQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNF
MVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQ
YNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEEL
KEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFH
ATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPN
NSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKM
KPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPA
PAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQ
NFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVS
CKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDK
YMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGE
ELFFDYRYSQADALKYVGIEREMEIP Nucleotide sequence of human EZH2,
transcript variant 1 (GenBank Accession No. NM_004456) (SEQ ID NO:
2) ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgt
ttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccg
gcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcggg
acgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcg
gaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctga
tgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatctt
aaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctc
attgcgcgggactagggagtgttcggtgaccagtgacttggattttccaacacaagtcat
cccattaaagactctgaatgcagttgcttcagtacccataatgtattcttggtctcccct
acagcagaattttatggtggaagatgaaactgttttacataacattccttatatgggaga
tgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaa
agtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaa
tgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaag
agaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacctcg
gaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcac
agcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgcact
tcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagca
aagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcct
acatcgtaagtgcaattattcttttcatgcaacacccaacacttataagcggaagaacac
agaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagc
aaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccagg
aggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaa
tgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggaga
gaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaa
ttctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtgga
gtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaattt
ctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagt
caaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaa
aaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaagga
cggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagccttgtga
cagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttc
agagtgtcaaaaccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtg
cccgtgctacctggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgc
tgaccattgggacagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaa
aaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcc
tgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagc
tgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaa
tgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggt
aaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttt
tgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggc
tgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacc
tcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaa
tttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataattt
atagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttg
taccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaata
cttgaacttgtccttgttgaatc Full amino acid of EZH2, isoform a (GenBank
Accession No. NP_004447) (SEQ ID NO: 3)
MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEW
KQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNF
MVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQ
YNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEEL
KEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHRKC
NYSFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRR
GRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQ
TPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESS
IIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCP
CVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWD
SKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRG
KVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRA
IQTGEELFFDYRYSQADALKYVGIEREMEIP Nucleotide sequence of human EZH2,
transcript variant 2 (GenBank Accession No. NM_152998) (SEQ ID NO:
4) ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgt
ttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccg
gcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcggg
acgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcg
gaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctga
tgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatctt
aaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctc
attgcgcgggactagggaggtggaagatgaaactgttttacataacattccttatatggg
agatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgg
gaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggt
gaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaaga
aagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacc
tcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataaggg
cacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgc
acttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagaga
gcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgctt
cctacatccttttcatgcaacacccaacacttataagcggaagaacacagaaacagctct
agacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgc
tgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaag
aggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatc
aaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataa
agaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtca
aacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgc
tgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgc
taggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctag
catcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaa
acaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaa
ccatgtttacaactatcaaccctgtgatcatccacggcagccttgtgacagttcgtgccc
ttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaa
ccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtgcccgtgctacct
ggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgctgaccattggga
cagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaaaaagcatctatt
gctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaa
tgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagg
gaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggt
ggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactg
ctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagc
catccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaa
gtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctc
ctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaaga
acatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagt
ttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatt
tttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtc
cttgttgaatc Full amino acid of EZH2, isoform b (GenBank Accession
No. NP_694543) (SEQ ID NO: 5)
MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKIL
ERTEILNQEWKQRRIQPVHILTSVSSLRGTREVEDETVLHNIPYMGDEVL
DQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDD
GDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEATSSMFPDKGTAEE
LKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRC
FKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALT
AERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETG
GENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASM
FRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPP
RKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIA
QNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCG
AADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFIS
EYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANH
SVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGI EREMEIP Full
amino acid of EZH2, isoform e (GenBank Accession No.
NP_001190178.1) (SEQ ID NO: 11)
MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHI
LTSCSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEEL
IKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFP
SDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRC
FKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLP
NNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPEN
VEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLW
AAHCRKIQLKKGQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSK
KHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKG
NKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP
Homo sapiens enhancer of zeste homolog 2 (Drosophila)(EZH2),
transcript variant 5, nucleotide sequence (GenBank Accession No.
NM_001203249.1) (SEQ ID NO: 12)
GACGACGTTCGCGGCGGGGAACTCGGAGTAGCTTCGCCTCTGACGTTTCCCCACGACGCACCCCGAAATC
CCCCTGAGCTCCGGCGGTCGCGGGCTGCCCTCGCCGCCTGGTCTGGCTTTATGCTAAGTTTGAGGGAAGA
GTCGAGCTGCTCTGCTCTCTATTGATTGTGTTTCTGGAGGGCGTCCTGTTGAATTCCCACTTCATTGTGT
ACATCCCCTTCCGTTCCCCCCAAAAATCTGTGCCACAGGGTTACTTTTTGAAAGCGGGAGGAATCGAGAA
GCACGATCTTTTGGAAAACTTGGTGAACGCCTAAATAATCATGGGCCAGACTGGGAAGAAATCTGAGAAG
GGACCAGTTTGTTGGCGGAAGCGTGTAAAATCAGAGTACATGCGACTGAGACAGCTCAAGAGGTTCAGAC
GAGCTGATGAAGTAAAGAGTATGTTTAGTTCCAATCGTCAGAAAATTTTGGAAAGAACGGAAATCTTAAA
CCAAGAATGGAAACAGCGAAGGATACAGCCTGTGCACATCCTGACTTCTTGTTCGGTGACCAGTGACTTG
GATTTTCCAACACAAGTCATCCCATTAAAGACTCTGAATGCAGTTGCTTCAGTACCCATAATGTATTCTT
GGTCTCCCCTACAGCAGAATTTTATGGTGGAAGATGAAACTGTTTTACATAACATTCCTTATATGGGAGA
TGAAGTTTTAGATCAGGATGGTACTTTCATTGAAGAACTAATAAAAAATTATGATGGGAAAGTACACGGG
GATAGAGAATGTGGGTTTATAAATGATGAAATTTTTGTGGAGTTGGTGAATGCCCTTGGTCAATATAATG
ATGATGACGATGATGATGATGGAGACGATCCTGAAGAAAGAGAAGAAAAGCAGAAAGATCTGGAGGATCA
CCGAGATGATAAAGAAAGCCGCCCACCTCGGAAATTTCCTTCTGATAAAATTTTTGAAGCCATTTCCTCA
ATGTTTCCAGATAAGGGCACAGCAGAAGAACTAAAGGAAAAATATAAAGAACTCACCGAACAGCAGCTCC
CAGGCGCACTTCCTCCTGAATGTACCCCCAACATAGATGGACCAAATGCTAAATCTGTTCAGAGAGAGCA
AAGCTTACACTCCTTTCATACGCTTTTCTGTAGGCGATGTTTTAAATATGACTGCTTCCTACATCCTTTT
CATGCAACACCCAACACTTATAAGCGGAAGAACACAGAAACAGCTCTAGACAACAAACCTTGTGGACCAC
AGTGTTACCAGCATTTGGAGGGAGCAAAGGAGTTTGCTGCTGCTCTCACCGCTGAGCGGATAAAGACCCC
ACCAAAACGTCCAGGAGGCCGCAGAAGAGGACGGCTTCCCAATAACAGTAGCAGGCCCAGCACCCCCACC
ATTAATGTGCTGGAATCAAAGGATACAGACAGTGATAGGGAAGCAGGGACTGAAACGGGGGGAGAGAACA
ATGATAAAGAAGAAGAAGAGAAGAAAGATGAAACTTCGAGCTCCTCTGAAGCAAATTCTCGGTGTCAAAC
ACCAATAAAGATGAAGCCAAATATTGAACCTCCTGAGAATGTGGAGTGGAGTGGTGCTGAAGCCTCAATG
TTTAGAGTCCTCATTGGCACTTACTATGACAATTTCTGTGCCATTGCTAGGTTAATTGGGACCAAAACAT
GTAGACAGGTGTATGAGTTTAGAGTCAAAGAATCTAGCATCATAGCTCCAGCTCCCGCTGAGGATGTGGA
TACTCCTCCAAGGAAAAAGAAGAGGAAACACCGGTTGTGGGCTGCACACTGCAGAAAGATACAGCTGAAA
AAGGGTCAAAACCGCTTTCCGGGATGCCGCTGCAAAGCACAGTGCAACACCAAGCAGTGCCCGTGCTACC
TGGCTGTCCGAGAGTGTGACCCTGACCTCTGTCTTACTTGTGGAGCCGCTGACCATTGGGACAGTAAAAA
TGTGTCCTGCAAGAACTGCAGTATTCAGCGGGGCTCCAAAAAGCATCTATTGCTGGCACCATCTGACGTG
GCAGGCTGGGGGATTTTTATCAAAGATCCTGTGCAGAAAAATGAATTCATCTCAGAATACTGTGGAGAGA
TTATTTCTCAAGATGAAGCTGACAGAAGAGGGAAAGTGTATGATAAATACATGTGCAGCTTTCTGTTCAA
CTTGAACAATGATTTTGTGGTGGATGCAACCCGCAAGGGTAACAAAATTCGTTTTGCAAATCATTCGGTA
AATCCAAACTGCTATGCAAAAGTTATGATGGTTAACGGTGATCACAGGATAGGTATTTTTGCCAAGAGAG
CCATCCAGACTGGCGAAGAGCTGTTTTTTGATTACAGATACAGCCAGGCTGATGCCCTGAAGTATGTCGG
CATCGAAAGAGAAATGGAAATCCCTTGACATCTGCTACCTCCTCCCCCCTCCTCTGAAACAGCTGCCTTA
GCTTCAGGAACCTCGAGTACTGTGGGCAATTTAGAAAAAGAACATGCAGTTTGAAATTCTGAATTTGCAA
AGTACTGTAAGAATAATTTATAGTAATGAGTTTAAAAATCAACTTTTTATTGCCTTCTCACCAGCTGCAA
AGTGTTTTGTACCAGTGAATTTTTGCAATAATGCAGTATGGTACATTTTTCAACTTTGAATAAAGAATAC
TTGAACTTGTCCTTGTTGAATC
[0063] A structure model of partial EZH2 protein based on the A
chain of nuclear receptor binding SET domain protein 1 (NSD1) is
provided in FIG. 11. This model corresponds to amino acid residues
533-732 of EZH2 sequence of SEQ ID NO: 1.
[0064] The corresponding amino acid sequence of the structure model
seen in FIG. 11 is provided below. The residues in the substrate
pocket domain are underlined. The residues in the SET domain are
shown italic.
TABLE-US-00002 (SEQ ID NO: 6)
SCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDP
DLCLTCGAADHWDSKNVSCKNCSIQRGSKKH RYS AD
[0065] The catalytic site of EZH2 is believed to reside in a
conserved domain of the protein known as the SET domain. The amino
acid sequence of the SET domain of EZH2 is provided by the
following partial sequence spanning amino acid residues 613-726 of
Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1):
TABLE-US-00003 (SEQ ID NO: 7)
HLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYM
CSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFA
KRAIQTGEELFFDY.
The tyrosine (Y) residue shown underlined in SEQ ID NO: 7 is Tyr641
(Y641) in Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1).
[0066] The SET domain of GenBank Accession No. NP_004447 (SEQ ID
NO: 3) spans amino acid residues 618-731 and is identical to SEQ ID
NO:6. The tyrosine residue corresponding to Y641 in Swiss-Prot
Accession No. Q15910 shown underlined in SEQ ID NO: 7 is Tyr646
(Y646) in GenBank Accession No. NP_004447 (SEQ ID NO: 3).
[0067] The SET domain of GenBank Accession No. NP_694543 (SEQ ID
NO: 5) spans amino acid residues 574-687 and is identical to SEQ ID
NO: 7. The tyrosine residue corresponding to Y641 in Swiss-Prot
Accession No. Q15910 shown underlined in SEQ ID NO: 7 is Tyr602
(Y602) in GenBank Accession No. NP_694543 (SEQ ID NO: 5).
[0068] The nucleotide sequence encoding the SET domain of GenBank
Accession No. NP_004447 is
TABLE-US-00004 (SEQ ID NO: 8)
catctattgctggcaccatctgacgtggcaggctgggggatttttatcaa
agatcctgtgcagaaaaatgaattcatctcagaatactgtggagagatta
tttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatg
tgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccg
caagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgct
atgcaaaagttatgatggttaacggtgatcacaggataggtatttttgcc
aagagagccatccagactggcgaagagctgttttttgattac,
where the codon encoding Y641 is shown underlined.
[0069] For purposes of this application, amino acid residue Y641 of
human EZH2 is to be understood to refer to the tyrosine residue
that is or corresponds to Y641 in Swiss-Prot Accession No.
Q15910.
TABLE-US-00005 Full amino acid sequence of Y641 mutant EZH2 (SEQ ID
NO: 9) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKIL
ERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKT
LNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELI
KNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQ
KDLEDHRDDKESRPPRKFPSDKIFEATSSMFPDKGTAEELKEKYKELTEQ
QLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFH
ATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRP
GGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEK
KDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDN
FCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWA
AHCRKTQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCS
SECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVS
CKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEXCGEIISQDE
ADRRGKVYDKYMCSFLFNLMNDFVVDATRKGNKIRFANHSVNPNCYAKVM
MVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP Wherein x can be any
amino acid residue other than tyrosine (Y)
[0070] Also for purposes of this application, a Y641 mutant of
human EZH2, and, equivalently, a Y641 mutant of EZH2, is to be
understood to refer to a human EZH2 in which the amino acid residue
corresponding to Y641 of wild-type human EZH2 is substituted by an
amino acid residue other than tyrosine.
[0071] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a single amino acid residue
corresponding to Y641 of wild-type human EZH2 by an amino acid
residue other than tyrosine.
[0072] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of phenylalanine (F) for the single amino
acid residue corresponding to Y641 of wild-type human EZH2. The
Y641 mutant of EZH2 according to this embodiment is referred to
herein as a Y641F mutant or, equivalently, Y641F.
[0073] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of histidine (H) for the single amino
acid residue corresponding to Y641 of wild-type human EZH2. The
Y641 mutant of EZH2 according to this embodiment is referred to
herein as a Y641H mutant or, equivalently, Y641H.
[0074] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of asparagine (N) for the single amino
acid residue corresponding to Y641 of wild-type human EZH2. The
Y641 mutant of EZH2 according to this embodiment is referred to
herein as a Y641N mutant or, equivalently, Y641N.
[0075] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of serine (S) for the single amino acid
residue corresponding to Y641 of wild-type human EZH2. The Y641
mutant of EZH2 according to this embodiment is referred to herein
as a Y641S mutant or, equivalently, Y641S.
[0076] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of cysteine (C) for the single amino acid
residue corresponding to Y641 of wild-type human EZH2. The Y641
mutant of EZH2 according to this embodiment is referred to herein
as a Y641C mutant or, equivalently, Y641C.
[0077] In one embodiment the amino acid sequence of a A677 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a non-alanine amino acid, preferably
glycine (G) for the single amino acid residue corresponding to A677
of wild-type human EZH2. The A677 mutant of EZH2 according to this
embodiment is referred to herein as an A677 mutant, and preferably
an A677G mutant or, equivalently, A677G.
[0078] In one embodiment the amino acid sequence of a A687 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a non-alanine amino acid, preferably
valine (V) for the single amino acid residue corresponding to A687
of wild-type human EZH2. The A687 mutant of EZH2 according to this
embodiment is referred to herein as an A687 mutant and preferably
an A687V mutant or, equivalently, A687V.
[0079] In one embodiment the amino acid sequence of a R685 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a non-arginine amino acid, preferably
histidine (H) or cysteine (C) for the single amino acid residue
corresponding to R685 of wild-type human EZH2. The R685 mutant of
EZH2 according to this embodiment is referred to herein as an R685
mutant and preferably an R685C mutant or an R685H mutant or,
equivalently, R685H or R685C.
[0080] In one embodiment the amino acid sequence of a mutant of
EZH2 differs from the amino acid sequence of wild-type human EZH2
in one or more amino acid residues in its substrate pocket domain
as defined in SEQ ID NO: 6. The mutant of EZH2 according to this
embodiment is referred to herein as an EZH2 mutant.
[0081] Other exemplary substitution amino acid mutation includes a
substitution at amino acid position 677, 687, 674, 685, or 641 of
SEQ ID NO: 1, such as, but is not limited to a substitution of
glycine (G) for the wild type residue alanine (A) at amino acid
position 677 of SEQ ID NO: 1 (A677G); a substitution of valine (V)
for the wild type residue alanine (A) at amino acid position 687 of
SEQ ID NO: 1 (A687V); a substitution of methionine (M) for the wild
type residue valine (V) at amino acid position 674 of SEQ ID NO: 1
(V674M); a substitution of histidine (H) for the wild type residue
arginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685H); a
substitution of cysteine (C) for the wild type residue arginine (R)
at amino acid position 685 of SEQ ID NO: 1 (R685C); a substitution
of phenylalanine (F) for the wild type residue tyrosine (Y) at
amino acid position 641 of SEQ ID NO: 1 (Y641F); a substitution of
histidine (H) for the wild type residue tyrosine (Y) at amino acid
position 641 of SEQ ID NO: 1 (Y641H); a substitution of asparagine
(N) for the wild type residue tyrosine (Y) at amino acid position
641 of SEQ ID NO: 1 (Y641N); a substitution of serine (S) for the
wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID
NO: 1 (Y641S); or a substitution of cysteine (C) for the wild type
residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1
(Y641C).
[0082] The mutation of the present invention may also include a
substitution of serine (S) for the wild type residue asparagine (N)
at amino acid position 322 of SEQ ID NO: 3 (N322S), a substitution
of glutamine (Q) for the wild type residue arginine (R) at amino
acid position 288 of SEQ ID NO: 3 (R288Q), a substitution of
isoleucine (I) for the wild type residue threonine (T) at amino
acid position 573 of SEQ ID NO: 3 (T573I), a substitution of
glutamic acid (E) for the wild type residue aspartic acid (D) at
amino acid position 664 of SEQ ID NO: 3 (D664E), a substitution of
glutamine (Q) for the wild type residue arginine (R) at amino acid
position 458 of SEQ ID NO: 5 (R458Q), a substitution of lysine (K)
for the wild type residue glutamic acid (E) at amino acid position
249 of SEQ ID NO: 3 (E249K), a substitution of cysteine (C) for the
wild type residue arginine (R) at amino acid position 684 of SEQ ID
NO: 3 (R684C), a substitution of histidine (H) for the wild type
residue arginine (R) at amino acid position 628 of SEQ ID NO: 11
(R628H), a substitution of histidine (H) for the wild type residue
glutamine (Q) at amino acid position 501 of SEQ ID NO: 5 (Q501H), a
substitution of asparagine (N) for the wild type residue aspartic
acid (D) at amino acid position 192 of SEQ ID NO: 3 (D192N), a
substitution of valine (V) for the wild type residue aspartic acid
(D) at amino acid position 664 of SEQ ID NO: 3 (D664V), a
substitution of leucine (L) for the wild type residue valine (V) at
amino acid position 704 of SEQ ID NO: 3 (V704L), a substitution of
serine (S) for the wild type residue proline (P) at amino acid
position 132 of SEQ ID NO: 3 (P132S), a substitution of lysine (K)
for the wild type residue glutamic acid (E) at amino acid position
669 of SEQ ID NO: 11 (E669K), a substitution of threonine (T) for
the wild type residue alanine (A) at amino acid position 255 of SEQ
ID NO: 3 (A255T), a substitution of valine (V) for the wild type
residue glutamic acid (E) at amino acid position 726 of SEQ ID NO:
3 (E726V), a substitution of tyrosine (Y) for the wild type residue
cysteine (C) at amino acid position 571 of SEQ ID NO: 3 (C571Y), a
substitution of cysteine (C) for the wild type residue
phenylalanine (F) at amino acid position 145 of SEQ ID NO: 3
(F145C), a substitution of threonine (T) for the wild type residue
asparagine (N) at amino acid position 693 of SEQ ID NO: 3 (N693T),
a substitution of serine (S) for the wild type residue
phenylalanine (F) at amino acid position 145 of SEQ ID NO: 3
(F145S), a substitution of histidine (H) for the wild type residue
glutamine (Q) at amino acid position 109 of SEQ ID NO: 11 (Q109H),
a substitution of cysteine (C) for the wild type residue
phenylalanine (F) at amino acid position 622 of SEQ ID NO: 11
(F622C), a substitution of arginine (R) for the wild type residue
glycine (G) at amino acid position 135 of SEQ ID NO: 3 (G135R), a
substitution of glutamine (Q) for the wild type residue arginine
(R) at amino acid position 168 of SEQ ID NO: 5 (R168Q), a
substitution of arginine (R) for the wild type residue glycine (G)
at amino acid position 159 of SEQ ID NO: 3 (G159R), a substitution
of cysteine (C) for the wild type residue arginine (R) at amino
acid position 310 of SEQ ID NO: 5 (R310C), a substitution of
histidine (H) for the wild type residue arginine (R) at amino acid
position 561 of SEQ ID NO: 3 (R561H), a substitution of histidine
(H) for the wild type residue arginine (R) at amino acid position
634 of SEQ ID NO: 11 (R634H), a substitution of arginine (R) for
the wild type residue glycine (G) at amino acid position 660 of SEQ
ID NO: 3 (G660R), a substitution of cysteine (C) for the wild type
residue tyrosine (Y) at amino acid position 181 of SEQ ID NO: 3
(Y181C), a substitution of arginine (R) for the wild type residue
histidine (H) at amino acid position 297 of SEQ ID NO: 3 (H297R), a
substitution of serine (S) for the wild type residue cysteine (C)
at amino acid position 612 of SEQ ID NO: 11 (C612S), a substitution
of tyrosine (Y) for the wild type residue histidine (H) at amino
acid position 694 of SEQ ID NO: 3 (H694Y), a substitution of
alanine (A) for the wild type residue aspartic acid (D) at amino
acid position 664 of SEQ ID NO: 3 (D664A), a substitution of
threonine (T) for the wild type residue isoleucine (I) at amino
acid position 150 of SEQ ID NO: 3 (I150T), a substitution of
arginine (R) for the wild type residue isoleucine (I) at amino acid
position 264 of SEQ ID NO: 3 (I264R), a substitution of leucine (L)
for the wild type residue proline (P) at amino acid position 636 of
SEQ ID NO: 3 (P636L), a substitution of threonine (T) for the wild
type residue isoleucine (I) at amino acid position 713 of SEQ ID
NO: 3 (I713T), a substitution of proline (P) for the wild type
residue glutamine (Q) at amino acid position 501 of SEQ ID NO: 5
(Q501P), a substitution of glutamine (Q) for the wild type residue
lysine (K) at amino acid position 243 of SEQ ID NO: 3 (K243Q), a
substitution of aspartic acid (D) for the wild type residue
glutamic acid (E) at amino acid position 130 of SEQ ID NO: 5
(E130D), a substitution of glycine (G) for the wild type residue
arginine (R) at amino acid position 509 of SEQ ID NO: 3 (R509G), a
substitution of histidine (H) for the wild type residue arginine
(R) at amino acid position 566 of SEQ ID NO: 3 (R566H), a
substitution of histidine (H) for the wild type residue aspartic
acid (D) at amino acid position 677 of SEQ ID NO: 3 (D677H), a
substitution of asparagine (N) for the wild type residue lysine (K)
at amino acid position 466 of SEQ ID NO: 5 (K466N), a substitution
of histidine (H) for the wild type residue arginine (R) at amino
acid position 78 of SEQ ID NO: 3 (R78H), a substitution of
methionine (M) for the wild type residue lysine (K) at amino acid
position 1 of SEQ ID NO: 6 (K6M), a substitution of leucine (L) for
the wild type residue serine (S) at amino acid position 538 of SEQ
ID NO: 3 (S538L), a substitution of glutamine (Q) for the wild type
residue leucine (L) at amino acid position 149 of SEQ ID NO: 3
(L149Q), a substitution of valine (V) for the wild type residue
leucine (L) at amino acid position 252 of SEQ ID NO: 3 (L252V), a
substitution of valine (V) for the wild type residue leucine (L) at
amino acid position 674 of SEQ ID NO: 3 (L674V), a substitution of
valine (V) for the wild type residue alanine (A) at amino acid
position 656 of SEQ ID NO: 3 (A656V), a substitution of aspartic
acid (D) for the wild type residue alanine (A) at amino acid
position 731 of SEQ ID NO: 3 (Y731D), a substitution of threonine
(T) for the wild type residue alanine (A) at amino acid position
345 of SEQ ID NO: 3 (A345T), a substitution of aspartic acid (D)
for the wild type residue alanine (A) at amino acid position 244 of
SEQ ID NO: 3 (Y244D), a substitution of tryptophan (W) for the wild
type residue cysteine (C) at amino acid position 576 of SEQ ID NO:
3 (C576W), a substitution of lysine (K) for the wild type residue
asparagine (N) at amino acid position 640 of SEQ ID NO: 3 (N640K),
a substitution of lysine (K) for the wild type residue asparagine
(N) at amino acid position 675 of SEQ ID NO: 3 (N675K), a
substitution of tyrosine (Y) for the wild type residue aspartic
acid (D) at amino acid position 579 of SEQ ID NO: 11 (D579Y), a
substitution of isoleucine (I) for the wild type residue asparagine
(N) at amino acid position 693 of SEQ ID NO: 3 (N693I), and a
substitution of lysine (K) for the wild type residue asparagine (N)
at amino acid position 693 of SEQ ID NO: 3 (N693K).
[0083] The mutation of the present invention may be a frameshift at
amino acid position 730, 391, 461, 441, 235, 254, 564, 662, 715,
405, 685, 64, 73, 656, 718, 374, 592, 505, 730, or 363 of SEQ ID
NO: 3, 5 or 11 or the corresponding nucleotide position of the
nucleic acid sequence encoding SEQ ID NO: 3, 5, or 11. The mutation
of the EZH2 may also be an insertion of a glutamic acid (E) between
amino acid positions 148 and 149 of SEQ ID NO: 3, 5 or 21. Another
example of EZH2 mutation is a deletion of glutamic acid (E) and
leucine (L) at amino acid positions 148 and 149 of SEQ ID NO: 3, 5
or 11. The mutant EZH2 may further comprise a nonsense mutation at
amino acid position 733, 25, 317, 62, 553, 328, 58, 207, 123, 63,
137, or 60 of SEQ ID NO: 3, 5 or 11.
[0084] Cells heterozygous for EZH2 would be expected to display a
malignant phenotype due to the efficient formation of H3-K27me1 by
the WT enzyme and the efficient, subsequent transition of this
progenitor species to H3-K27me2, and, especially, H3-K27me3, by the
mutant enzyme form(s).
[0085] Previous results point to dependency on enzymatic coupling
between enzymes that perform H3-K27 mono-methylation and certain
mutant forms of EZH2 for pathogenesis in follicular lymphoma and
diffuse large B-cell lymphoma. For example, cells expressing Y641
mutant EZH2 may be more sensitive to small molecule EZH2 inhibitors
than cells expressing WT EZH2. Specifically, cells expressing Y641
mutant EZH2 show reduced growing, dividing or proliferation, or
even undergo apoptosis or necrosis after the treatment of EZH2
inhibitors. In contrast, cells expressing WT EZH2 are not
responsive to the anti-proliferative effect of the EZH2 inhibitors
(U.S. Patent Application No. 61/381,684; incorporated herein by
reference in its entirety.)
[0086] An aspect of the present invention is a method for treating
or alleviating a symptom of cancer or precancerous condition in a
subject by administering to a subject expressing a mutant EZH2
comprising a mutation in the substrate pocket domain as defined in
SEQ ID NO: 6 a therapeutically effective amount of an EZH2
inhibitor as described herein, e.g., a compound of Formula (IIa) in
combination with another agent suitable to be administered together
simultaneously, sequentially, or in alternation.
[0087] Another aspect of the invention is a method for inhibiting
in a subject conversion of H3-K27 to trimethylated H3-K27. The
inhibition can involve inhibiting in a subject conversion of
unmethylated H3-K27 to monomethylated H3-K27, conversion of
monomethylated H3-K27 to dimethylated H3-K27, conversion of
dimethylated H3-K27 to trimethylated H3-K27, or any combination
thereof, including, for example, conversion of monomethylated
H3-K27 to dimethylated H3-K27 and conversion of dimethylated H3-K27
to trimethylated H3-K27. As used herein, unmethylated H3-K27 refers
to histone H3 with no methyl group covalently linked to the amino
group of lysine 27. As used herein, monomethylated H3-K27 refers to
histone H3 with a single methyl group covalently linked to the
amino group of lysine 27. Monomethylated H3-K27 is also referred to
herein as H3-K27me1. As used herein, dimethylated H3-K27 refers to
histone H3 with two methyl groups covalently linked to the amino
group of lysine 27. Dimethylated H3-K27 is also referred to herein
as H3-K27me2. As used herein, trimethylated H3-K27 refers to
histone H3 with three methyl groups covalently linked to the amino
group of lysine 27. Trimethylated H3-K27 is also referred to herein
as H3-K27me3.
[0088] Histone H3 is a 136 amino acid long protein, the sequence of
which is known. See, for example, GenBank Accession No. CAB02546,
the content of which is incorporated herein by reference. As
disclosed further herein, in addition to full-length histone H3,
peptide fragments of histone H3 comprising the lysine residue
corresponding to K27 of full-length histone H3 can be used as
substrate for EZH2 (and likewise for mutant forms of EZH2) to
assess conversion of H3-K27 ml to H3-K27m2 and conversion of
H3-K27m2 to H3-K27m3. In one embodiment, such peptide fragment
corresponds to amino acid residues 21-44 of histone H3. Such
peptide fragment has the amino acid sequence
LATKAARKSAPATGGVKKPHRYRP (SEQ ID NO: 10).
[0089] A composition of the present invention comprises a compound
of Formula (IIa) and one or more other therapeutic agents, or a
pharmaceutically acceptable salt thereof. The compounds of Formula
(IIa) are suitable for administration as part of a combination
therapy with one or more other therapeutic agents or treatment
modality, suitable to be administered together, sequentially, or in
alternation. Other compounds of Formula (IIa) suitable for the
methods of the invention are described in U.S. Publication
20120264734, the contents of which are hereby incorporated by
reference in their entireties.
[0090] The present invention provides the compounds of Formula
(IIa):
##STR00003##
or a pharmaceutically acceptable salts or esters thereof, wherein
R.sub.7, R.sub.8, R.sub.a, and R.sub.b are defined herein.
[0091] The compounds of Formula (IIa) can include one or more of
the following features:
[0092] For example, each of R.sub.a and R.sub.b independently is H
or C.sub.1-C.sub.6 alkyl optionally substituted with one or more
-Q.sub.3-T.sub.3.
[0093] For example, one of R.sub.a and R.sub.b is H.
[0094] For example, R.sub.a and R.sub.b, together with the N atom
to which they are attached, form a 4 to 7-membered heterocycloalkyl
ring having 0 or 1 additional heteroatoms to the N atom (e.g.,
azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,
oxazolidinyl, isoxazolidinyl, triazolidinyl, piperidinyl,
1,2,3,6-tetrahydropyridinyl, piperazinyl, morpholinyl,
1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,
2,5-diazabicyclo[2.2.1]heptanyl, and the like) and the ring is
optionally substituted with one or more -Q.sub.3-T.sub.3.
[0095] For example, R.sub.a and R.sub.b, together with the N atom
to which they are attached, form azetidinyl, pyrrolidinyl,
imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,
triazolidinyl, tetrahyrofuranyl, piperidinyl,
1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl, and the
ring is optionally substituted with one or more
-Q.sub.3-T.sub.3.
[0096] For example, one or more -Q.sub.3-T.sub.3 are oxo.
[0097] For example, Q.sub.3 is a bond or unsubstituted or
substituted C.sub.1-C.sub.3 alkyl linker.
[0098] For example, T.sub.3 is H, halo, 4 to 7-membered
heterocycloalkyl, C.sub.1-C.sub.3 alkyl, OR.sub.d, COOR.sub.d,
--S(O).sub.2R.sub.d, or --NR.sub.dR.sub.e.
[0099] For example, each of R.sub.d and R.sub.e independently being
H or C.sub.1-C.sub.6 alkyl.
[0100] For example, R.sub.7 is C.sub.3-C.sub.8 cycloalkyl or 4 to
7-membered heterocycloalkyl, each optionally substituted with one
or more -Q.sub.5-T.sub.5.
[0101] For example, R.sub.7 is piperidinyl, tetrahydropyran,
tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl,
or cycloheptyl, each optionally substituted with one or more
-Q.sub.5-T.sub.5.
[0102] For example, R.sub.7 is cyclopentyl cyclohexyl or
tetrahydro-2H-thiopyranyl, each of which is optionally substituted
with one or more -Q.sub.5-T.sub.5.
[0103] For example, Q.sub.5 is NHC(O) and T.sub.5 is
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy, each
[0104] For example, one or more -Q.sub.5-T.sub.5 are oxo.
[0105] For example, R.sub.7 is 1-oxide-tetrahydro-2H-thiopyranyl or
1,1-dioxide-tetrahydro-2H-thiopyranyl.
[0106] For example, Q.sub.5 is a bond and T.sub.5 is amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino.
[0107] For example, Q.sub.5 is CO, S(O).sub.2, or NHC(O); and
T.sub.5 is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxyl,
C.sub.3-C.sub.8 cycloalkyl, or 4 to 7-membered
heterocycloalkyl.
[0108] For example, R.sub.8 is H or C.sub.1-C.sub.6 alkyl which is
optionally substituted with one or more substituents selected from
the group consisting of halo, hydroxyl, COOH,
C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6
alkylamino.
[0109] For example, R.sub.8 is H, methyl, or ethyl.
[0110] In one embodiment, the compound of the invention is Compound
44
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0111] In one embodiment, the compound of the invention is the
compound itself, i.e., the free base or "naked" molecule. In
another embodiment, the compound is a salt thereof, e.g., a
mono-HCl or tri-HCl salt, mono-HBr or tri-HBr salt of the naked
molecule.
[0112] Representative compounds of the present invention include
compounds listed in Table 1.
[0113] In the table below, each occurrence of
##STR00005##
should be construed as
##STR00006##
TABLE-US-00006 TABLE 1 Compound Number Structure MS (M + 1).sup.+ 1
##STR00007## 501.39 2 ##STR00008## 543.22 3 ##STR00009## 486.21 4
##STR00010## 529.30 11 ##STR00011## 558.45 12 ##STR00012## 559.35
13 ##STR00013## 517.3 14 ##STR00014## 557.4 16 ##STR00015## 515.4
20 ##STR00016## 614.4 21 ##STR00017## 614.4 27 ##STR00018## 516.35
36 ##STR00019## 557.35 39 ##STR00020## 572.35 40 ##STR00021##
572.35 42 ##STR00022## 572.4 43 ##STR00023## 572.6 44 ##STR00024##
573.40 47 ##STR00025## 530.35 59 ##STR00026## 587.40 60
##STR00027## 601.30 61 ##STR00028## 599.35 62 ##STR00029## 601.35
63 ##STR00030## 613.35 65 ##STR00031## 531.30 66 ##STR00032##
586.40 67 ##STR00033## 585.25 68 ##STR00034## 585.35 69
##STR00035## 557.25 70 ##STR00036## 573.40 71 ##STR00037## 573.40
72 ##STR00038## 575.35 73 ##STR00039## 572.10 74 ##STR00040##
575.35 75 ##STR00041## 571.25 76 ##STR00042## 587.40 77
##STR00043## 587.45 78 ##STR00044## 587.20 79 ##STR00045## 589.35
80 ##STR00046## 589.30 81 ##STR00047## 607.35 82 ##STR00048##
543.40 83 ##STR00049## 559.80 84 ##STR00050## 561.25 85
##STR00051## 86 ##STR00052## 585.37 87 ##STR00053## 600.30 88
##STR00054## 587.40 89 ##STR00055## 503.40 90 ##STR00056## 517.30
91 ##STR00057## 531.35 92 ##STR00058## 545.40 93 ##STR00059##
557.35 94 ##STR00060## 559.20 95 ##STR00061## 599.35 (M + Na) 96
##STR00062## 577.25 97 ##STR00063## 571.40 98 ##STR00064## 547.35
99 ##STR00065## 561.30 100 ##STR00066## 591.25 101 ##STR00067##
546.35 102 ##STR00068## 560.20 103 ##STR00069## 567.30 104
##STR00070## 585.25 105 ##STR00071## 585.40 107 ##STR00072## 108
##STR00073## 530.35 114 ##STR00074## 573.25 115 ##STR00075## 642.45
116 ##STR00076## 545.15 117 ##STR00077## 489.20 119 ##STR00078##
609.35 122 ##STR00079## 587.55 124 ##STR00080## 650.85 125
##STR00081## 614.75 126 ##STR00082## 572.35 127 ##STR00083## 656.65
128 ##STR00084## 586.45 129 ##STR00085## 628.35 130 ##STR00086##
591.2 131 ##STR00087## 587.35 132 ##STR00088## 589.25 133
##STR00089## 605.25 135 ##STR00090## 621.40 136 ##STR00091## 621.45
137 ##STR00092## 589.35 138 ##STR00093## 627.5 141 ##STR00094##
614.65 142 ##STR00095## 603.45 143 ##STR00096## 578.35 144
##STR00097## 609.15 146 ##STR00098## 641.50 178 ##STR00099##
593.60
[0114] As used herein, "alkyl", "C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5 or C.sub.6 alkyl" or "C.sub.1-C.sub.6 alkyl" is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or
C.sub.6 straight chain (linear) saturated aliphatic hydrocarbon
groups and C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated
aliphatic hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and
C.sub.6 alkyl groups. Examples of alkyl include, moieties having
from one to six carbon atoms, such as, but not limited to, methyl,
ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl,
s-pentyl or n-hexyl.
[0115] In certain embodiments, a straight chain or branched alkyl
has six or fewer carbon atoms (e.g., C.sub.1-C.sub.6 for straight
chain, C.sub.3-C.sub.6 for branched chain), and in another
embodiment, a straight chain or branched alkyl has four or fewer
carbon atoms.
[0116] As used herein, the term "cycloalkyl" refers to a saturated
or unsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g.,
fused, bridged, or spiro rings) system having 3 to 30 carbon atoms
(e.g., C.sub.3-C.sub.10). Examples of cycloalkyl include, but are
not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl,
cycloheptenyl, and adamantyl. The term "heterocycloalkyl" refers to
a saturated or unsaturated nonaromatic 3-8 membered monocyclic,
7-12 membered bicyclic (fused, bridged, or spiro rings), or 11-14
membered tricyclic ring system (fused, bridged, or spiro rings)
having one or more heteroatoms (such as O, N, S, or Se), unless
specified otherwise. Examples of heterocycloalkyl groups include,
but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl,
dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl,
imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,
triazolidinyl, tetrahyrofuranyl, oxiranyl, azetidinyl, oxetanyl,
thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl,
dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl,
1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,
2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl,
2,6-diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl and
the like.
[0117] The term "optionally substituted alkyl" refers to
unsubstituted alkyl or alkyl having designated substituents
replacing one or more hydrogen atoms on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0118] An "arylalkyl" or an "aralkyl" moiety is an alkyl
substituted with an aryl (e.g., phenylmethyl (benzyl)). An
"alkylaryl" moiety is an aryl substituted with an alkyl (e.g.,
methylphenyl).
[0119] As used herein, "alkyl linker" is intended to include
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or C.sub.6 straight
chain (linear) saturated divalent aliphatic hydrocarbon groups and
C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated aliphatic
hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl linker is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and
C.sub.6 alkyl linker groups. Examples of alkyl linker include,
moieties having from one to six carbon atoms, such as, but not
limited to, methyl (--CH.sub.2--), ethyl (--CH.sub.2CH.sub.2--),
n-propyl (--CH.sub.2CH.sub.2CH.sub.2--), i-propyl
(--CHCH.sub.3CH.sub.2--), n-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), s-butyl
(--CHCH.sub.3CH.sub.2CH.sub.2--), i-butyl
(--C(CH.sub.3).sub.2CH.sub.2--), n-pentyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), s-pentyl
(--CHCH.sub.3CH.sub.2CH.sub.2CH.sub.2--) or n-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--).
[0120] "Alkenyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
that contain at least one double bond. For example, the term
"alkenyl" includes straight chain alkenyl groups (e.g., ethenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl), and branched alkenyl groups. In certain embodiments, a
straight chain or branched alkenyl group has six or fewer carbon
atoms in its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). The term "C.sub.2-C.sub.6"
includes alkenyl groups containing two to six carbon atoms. The
term "C.sub.3-C.sub.6" includes alkenyl groups containing three to
six carbon atoms.
[0121] The term "optionally substituted alkenyl" refers to
unsubstituted alkenyl or alkenyl having designated substituents
replacing one or more hydrogen atoms on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or
heteroaromatic moiety.
[0122] "Alkynyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
which contain at least one triple bond. For example, "alkynyl"
includes straight chain alkynyl groups (e.g., ethynyl, propynyl,
butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl),
and branched alkynyl groups. In certain embodiments, a straight
chain or branched alkynyl group has six or fewer carbon atoms in
its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). The term "C.sub.2-C.sub.6"
includes alkynyl groups containing two to six carbon atoms. The
term "C.sub.3-C.sub.6" includes alkynyl groups containing three to
six carbon atoms.
[0123] The term "optionally substituted alkynyl" refers to
unsubstituted alkynyl or alkynyl having designated substituents
replacing one or more hydrogen atoms on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0124] Other optionally substituted moieties (such as optionally
substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)
include both the unsubstituted moieties and the moieties having one
or more of the designated substituents. For example, substituted
heterocycloalkyl includes those substituted with one or more alkyl
groups, such as 2,2,6,6-tetramethyl-piperidinyl and
2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.
[0125] "Aryl" includes groups with aromaticity, including
"conjugated," or multicyclic systems with at least one aromatic
ring and do not contain any heteroatom in the ring structure.
Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl,
etc.
[0126] "Heteroaryl" groups are aryl groups, as defined above,
except having from one to four heteroatoms in the ring structure,
and may also be referred to as "aryl heterocycles" or
"heteroaromatics." As used herein, the term "heteroaryl" is
intended to include a stable 5-, 6-, or 7-membered monocyclic or
7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic
ring which consists of carbon atoms and one or more heteroatoms,
e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1,
2, 3, 4, 5, or 6 heteroatoms, independently selected from the group
consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be
substituted or unsubstituted (i.e., N or NR wherein R is H or other
substituents, as defined). The nitrogen and sulfur heteroatoms may
optionally be oxidized (i.e., N.fwdarw.O and S(O).sub.p, where p=1
or 2). It is to be noted that total number of S and O atoms in the
aromatic heterocycle is not more than 1.
[0127] Examples of heteroaryl groups include pyrrole, furan,
thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine,
pyrimidine, and the like.
[0128] Furthermore, the terms "aryl" and "heteroaryl" include
multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic,
e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,
isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran,
deazapurine, indolizine.
[0129] In the case of multicyclic aromatic rings, only one of the
rings needs to be aromatic (e.g., 2,3-dihydroindole), although all
of the rings may be aromatic (e.g., quinoline). The second ring can
also be fused or bridged.
[0130] The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring
can be substituted at one or more ring positions (e.g., the
ring-forming carbon or heteroatom such as N) with such substituents
as described above, for example, alkyl, alkenyl, alkynyl, halogen,
hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and
heteroaryl groups can also be fused or bridged with alicyclic or
heterocyclic rings, which are not aromatic so as to form a
multicyclic system (e.g., tetralin, methylenedioxyphenyl).
[0131] As used herein, "carbocycle" or "carbocyclic ring" is
intended to include any stable monocyclic, bicyclic or tricyclic
ring having the specified number of carbons, any of which may be
saturated, unsaturated, or aromatic. Carbocycle includes cycloalkyl
and aryl. For example, a C.sub.3-C.sub.14 carbocycle is intended to
include a monocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms. Examples of carbocycles
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl,
cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,
indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are also
included in the definition of carbocycle, including, for example,
[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane
and [2.2.2]bicyclooctane. A bridged ring occurs when one or more
carbon atoms link two non-adjacent carbon atoms. In one embodiment,
bridge rings are one or two carbon atoms. It is noted that a bridge
always converts a monocyclic ring into a tricyclic ring. When a
ring is bridged, the substituents recited for the ring may also be
present on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl)
and spiro rings are also included.
[0132] As used herein, "heterocycle" or "heterocyclic group"
includes any ring structure (saturated, unsaturated, or aromatic)
which contains at least one ring heteroatom (e.g., N, O or S).
Heterocycle includes heterocycloalkyl and heteroaryl. Examples of
heterocycles include, but are not limited to, morpholine,
pyrrolidine, tetrahydrothiophene, piperidine, piperazine, oxetane,
pyran, tetrahydropyran, azetidine, and tetrahydrofuran.
[0133] Examples of heterocyclic groups include, but are not limited
to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,
pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,
pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and
xanthenyl.
[0134] The term "substituted," as used herein, means that any one
or more hydrogen atoms on the designated atom is replaced with a
selection from the indicated groups, provided that the designated
atom's normal valency is not exceeded, and that the substitution
results in a stable compound. When a substituent is oxo or keto
(i.e., .dbd.O), then 2 hydrogen atoms on the atom are replaced.
Keto substituents are not present on aromatic moieties. Ring double
bonds, as used herein, are double bonds that are formed between two
adjacent ring atoms (e.g., C.dbd.C, C.dbd.N or N.dbd.N). "Stable
compound" and "stable structure" are meant to indicate a compound
that is sufficiently robust to survive isolation to a useful degree
of purity from a reaction mixture, and formulation into an
efficacious therapeutic agent.
[0135] When a bond to a substituent is shown to cross a bond
connecting two atoms in a ring, then such substituent may be bonded
to any atom in the ring. When a substituent is listed without
indicating the atom via which such substituent is bonded to the
rest of the compound of a given formula, then such substituent may
be bonded via any atom in such formula. Combinations of
substituents and/or variables are permissible, but only if such
combinations result in stable compounds.
[0136] When any variable (e.g., R.sub.1) occurs more than one time
in any constituent or formula for a compound, its definition at
each occurrence is independent of its definition at every other
occurrence. Thus, for example, if a group is shown to be
substituted with 0-2 R.sub.1 moieties, then the group may
optionally be substituted with up to two R.sub.1 moieties and
R.sub.1 at each occurrence is selected independently from the
definition of R.sub.1. Also, combinations of substituents and/or
variables are permissible, but only if such combinations result in
stable compounds.
[0137] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.
[0138] As used herein, "halo" or "halogen" refers to fluoro,
chloro, bromo and iodo. The term "perhalogenated" generally refers
to a moiety wherein all hydrogen atoms are replaced by halogen
atoms. The term "haloalkyl" or "haloalkoxyl" refers to an alkyl or
alkoxyl substituted with one or more halogen atoms.
[0139] The term "carbonyl" includes compounds and moieties which
contain a carbon connected with a double bond to an oxygen atom.
Examples of moieties containing a carbonyl include, but are not
limited to, aldehydes, ketones, carboxylic acids, amides, esters,
anhydrides, etc.
[0140] The term "carboxyl" refers to --COOH or its C.sub.1-C.sub.6
alkyl ester.
[0141] "Acyl" includes moieties that contain the acyl radical
(R--C(O)--) or a carbonyl group. "Substituted acyl" includes acyl
groups where one or more of the hydrogen atoms are replaced by, for
example, alkyl groups, alkynyl groups, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety.
[0142] "Aroyl" includes moieties with an aryl or heteroaromatic
moiety bound to a carbonyl group. Examples of aroyl groups include
phenylcarboxy, naphthyl carboxy, etc.
[0143] "Alkoxyalkyl," "alkylaminoalkyl," and "thioalkoxyalkyl"
include alkyl groups, as described above, wherein oxygen, nitrogen,
or sulfur atoms replace one or more hydrocarbon backbone carbon
atoms.
[0144] The term "alkoxy" or "alkoxyl" includes substituted and
unsubstituted alkyl, alkenyl and alkynyl groups covalently linked
to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals
include, but are not limited to, methoxy, ethoxy, isopropyloxy,
propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy
groups include halogenated alkoxy groups. The alkoxy groups can be
substituted with groups such as alkenyl, alkynyl, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy and trichloromethoxy.
[0145] The term "ether" or "alkoxy" includes compounds or moieties
which contain an oxygen bonded to two carbon atoms or heteroatoms.
For example, the term includes "alkoxyalkyl," which refers to an
alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen
atom which is covalently bonded to an alkyl group.
[0146] The term "ester" includes compounds or moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
[0147] The term "thioalkyl" includes compounds or moieties which
contain an alkyl group connected with a sulfur atom. The thioalkyl
groups can be substituted with groups such as alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxyacid,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl,
alkoxyl, amino (including alkylamino, dialkylamino, arylamino,
diarylamino and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moieties.
[0148] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0149] The term "thioether" includes moieties which contain a
sulfur atom bonded to two carbon atoms or heteroatoms. Examples of
thioethers include, but are not limited to alkthioalkyls,
alkthioalkenyls, and alkthioalkynyls. The term "alkthioalkyls"
include moieties with an alkyl, alkenyl, or alkynyl group bonded to
a sulfur atom which is bonded to an alkyl group. Similarly, the
term "alkthioalkenyls" refers to moieties wherein an alkyl, alkenyl
or alkynyl group is bonded to a sulfur atom which is covalently
bonded to an alkenyl group; and alkthioalkynyls" refers to moieties
wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur
atom which is covalently bonded to an alkynyl group.
[0150] As used herein, "amine" or "amino" refers to unsubstituted
or substituted --NH.sub.2. "Alkylamino" includes groups of
compounds wherein nitrogen of --NH.sub.2 is bound to at least one
alkyl group. Examples of alkylamino groups include benzylamino,
methylamino, ethylamino, phenethylamino, etc. "Dialkylamino"
includes groups wherein the nitrogen of --NH.sub.2 is bound to at
least two additional alkyl groups. Examples of dialkylamino groups
include, but are not limited to, dimethylamino and diethylamino.
"Arylamino" and "diarylamino" include groups wherein the nitrogen
is bound to at least one or two aryl groups, respectively.
"Aminoaryl" and "aminoaryloxy" refer to aryl and aryloxy
substituted with amino. "Alkylarylamino," "alkylaminoaryl" or
"arylaminoalkyl" refers to an amino group which is bound to at
least one alkyl group and at least one aryl group. "Alkaminoalkyl"
refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen
atom which is also bound to an alkyl group. "Acylamino" includes
groups wherein nitrogen is bound to an acyl group. Examples of
acylamino include, but are not limited to, alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido groups.
[0151] The term "amide" or "aminocarboxy" includes compounds or
moieties that contain a nitrogen atom that is bound to the carbon
of a carbonyl or a thiocarbonyl group. The term includes
"alkaminocarboxy" groups that include alkyl, alkenyl or alkynyl
groups bound to an amino group which is bound to the carbon of a
carbonyl or thiocarbonyl group. It also includes "arylaminocarboxy"
groups that include aryl or heteroaryl moieties bound to an amino
group that is bound to the carbon of a carbonyl or thiocarbonyl
group. The terms "alkylaminocarboxy", "alkenylaminocarboxy",
"alkynylaminocarboxy" and "arylaminocarboxy" include moieties
wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively,
are bound to a nitrogen atom which is in turn bound to the carbon
of a carbonyl group. Amides can be substituted with substituents
such as straight chain alkyl, branched alkyl, cycloalkyl, aryl,
heteroaryl or heterocycle. Substituents on amide groups may be
further substituted.
[0152] Compounds of the present invention that contain nitrogens
can be converted to N-oxides by treatment with an oxidizing agent
(e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen
peroxides) to afford other compounds of the present invention.
Thus, all shown and claimed nitrogen-containing compounds are
considered, when allowed by valency and structure, to include both
the compound as shown and its N-oxide derivative (which can be
designated as NO or N.fwdarw.O or N.sup.+--O.sup.-). Furthermore,
in other instances, the nitrogens in the compounds of the present
invention can be converted to N-hydroxy or N-alkoxy compounds. For
example, N-hydroxy compounds can be prepared by oxidation of the
parent amine by an oxidizing agent such as m-CPBA. All shown and
claimed nitrogen-containing compounds are also considered, when
allowed by valency and structure, to cover both the compound as
shown and its N-hydroxy (i.e., N--OH) and N-alkoxy (i.e., N--OR,
wherein R is substituted or unsubstituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkynyl, 3-14-membered
carbocycle or 3-14-membered heterocycle) derivatives.
[0153] In the present specification, the structural formula of the
compound represents a certain isomer for convenience in some cases,
but the present invention includes all isomers, such as geometrical
isomers, optical isomers based on an asymmetrical carbon,
stereoisomers, tautomers, and the like. In addition, a crystal
polymorphism may be present for the compounds represented by the
formula. It is noted that any crystal form, crystal form mixture,
or anhydride or hydrate thereof is included in the scope of the
present invention. Furthermore, so-called metabolite which is
produced by degradation of the present compound in vivo is included
in the scope of the present invention.
[0154] "Isomerism" means compounds that have identical molecular
formulae but differ in the sequence of bonding of their atoms or in
the arrangement of their atoms in space. Isomers that differ in the
arrangement of their atoms in space are termed "stereoisomers."
Stereoisomers that are not mirror images of one another are termed
"diastereoisomers," and stereoisomers that are non-superimposable
mirror images of each other are termed "enantiomers" or sometimes
optical isomers. A mixture containing equal amounts of individual
enantiomeric forms of opposite chirality is termed a "racemic
mixture."
[0155] A carbon atom bonded to four nonidentical substituents is
termed a "chiral center."
[0156] "Chiral isomer" means a compound with at least one chiral
center. Compounds with more than one chiral center may exist either
as an individual diastereomer or as a mixture of diastereomers,
termed "diastereomeric mixture." When one chiral center is present,
a stereoisomer may be characterized by the absolute configuration
(R or S) of that chiral center. Absolute configuration refers to
the arrangement in space of the substituents attached to the chiral
center. The substituents attached to the chiral center under
consideration are ranked in accordance with the Sequence Rule of
Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit.
1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413;
Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al.,
Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).
[0157] "Geometric isomer" means the diastereomers that owe their
existence to hindered rotation about double bonds or a cycloalkyl
linker (e.g., 1,3-cylcobutyl). These configurations are
differentiated in their names by the prefixes cis and trans, or Z
and E, which indicate that the groups are on the same or opposite
side of the double bond in the molecule according to the
Cahn-Ingold-Prelog rules.
[0158] It is to be understood that the compounds of the present
invention may be depicted as different chiral isomers or geometric
isomers. It should also be understood that when compounds have
chiral isomeric or geometric isomeric forms, all isomeric forms are
intended to be included in the scope of the present invention, and
the naming of the compounds does not exclude any isomeric
forms.
[0159] Furthermore, the structures and other compounds discussed in
this invention include all atropic isomers thereof "Atropic
isomers" are a type of stereoisomer in which the atoms of two
isomers are arranged differently in space. Atropic isomers owe
their existence to a restricted rotation caused by hindrance of
rotation of large groups about a central bond. Such atropic isomers
typically exist as a mixture, however as a result of recent
advances in chromatography techniques, it has been possible to
separate mixtures of two atropic isomers in select cases.
[0160] "Tautomer" is one of two or more structural isomers that
exist in equilibrium and is readily converted from one isomeric
form to another. This conversion results in the formal migration of
a hydrogen atom accompanied by a switch of adjacent conjugated
double bonds. Tautomers exist as a mixture of a tautomeric set in
solution. In solutions where tautomerization is possible, a
chemical equilibrium of the tautomers will be reached. The exact
ratio of the tautomers depends on several factors, including
temperature, solvent and pH. The concept of tautomers that are
interconvertable by tautomerizations is called tautomerism.
[0161] Of the various types of tautomerism that are possible, two
are commonly observed. In keto-enol tautomerism a simultaneous
shift of electrons and a hydrogen atom occurs. Ring-chain
tautomerism arises as a result of the aldehyde group (--CHO) in a
sugar chain molecule reacting with one of the hydroxy groups (--OH)
in the same molecule to give it a cyclic (ring-shaped) form as
exhibited by glucose.
[0162] Common tautomeric pairs are: ketone-enol, amide-nitrile,
lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings
(e.g., in nucleobases such as guanine, thymine and cytosine),
imine-enamine and enamine-enamine. An example of keto-enol
equilibria is between pyridin-2(1H)-ones and the corresponding
pyridin-2-ols, as shown below.
##STR00100##
[0163] It is to be understood that the compounds of the present
invention may be depicted as different tautomers. It should also be
understood that when compounds have tautomeric forms, all
tautomeric forms are intended to be included in the scope of the
present invention, and the naming of the compounds does not exclude
any tautomer form.
[0164] The term "crystal polymorphs", "polymorphs" or "crystal
forms" means crystal structures in which a compound (or a salt or
solvate thereof) can crystallize in different crystal packing
arrangements, all of which have the same elemental composition.
Different crystal forms usually have different X-ray diffraction
patterns, infrared spectral, melting points, density hardness,
crystal shape, optical and electrical properties, stability and
solubility. Recrystallization solvent, rate of crystallization,
storage temperature, and other factors may cause one crystal form
to dominate. Crystal polymorphs of the compounds can be prepared by
crystallization under different conditions.
[0165] The compounds of Formula (IIa) disclosed herein include the
compounds themselves, as well as their salts, their esters, their
solvates, and their prodrugs, if applicable. A salt, for example,
can be formed between an anion and a positively charged group
(e.g., amino) on an aryl- or heteroaryl-substituted benzene
compound. Suitable anions include chloride, bromide, iodide,
sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate,
methanesulfonate, trifluoroacetate, glutamate, glucuronate,
glutarate, malate, maleate, succinate, fumarate, tartrate,
tosylate, salicylate, lactate, naphthalenesulfonate, and acetate
(e.g., trifluoroacetate). The term "pharmaceutically acceptable
anion" refers to an anion suitable for forming a pharmaceutically
acceptable salt. Likewise, a salt can also be formed between a
cation and a negatively charged group (e.g., carboxylate) on an
aryl- or heteroaryl-substituted benzene compound. Suitable cations
include sodium ion, potassium ion, magnesium ion, calcium ion, and
an ammonium cation such as tetramethylammonium ion. The aryl- or
heteroaryl-substituted benzene compounds also include those salts
containing quaternary nitrogen atoms. In the salt form, it is
understood that the ratio of the compound to the cation or anion of
the salt can be 1:1, or any ration other than 1:1, e.g., 3:1, 2:1,
1:2, or 1:3.
[0166] Examples of prodrugs include esters and other
pharmaceutically acceptable derivatives, which, upon administration
to a subject, are capable of providing active aryl- or
heteroaryl-substituted benzene compounds.
[0167] Additionally, the compounds of the present invention, for
example, the salts of the compounds, can exist in either hydrated
or unhydrated (the anhydrous) form or as solvates with other
solvent molecules. Nonlimiting examples of hydrates include
monohydrates, dihydrates, etc. Nonlimiting examples of solvates
include ethanol solvates, acetone solvates, etc.
[0168] "Solvate" means solvent addition forms that contain either
stoichiometric or non stoichiometric amounts of solvent. Some
compounds have a tendency to trap a fixed molar ratio of solvent
molecules in the crystalline solid state, thus forming a solvate.
If the solvent is water the solvate formed is a hydrate; and if the
solvent is alcohol, the solvate formed is an alcoholate. Hydrates
are formed by the combination of one or more molecules of water
with one molecule of the substance in which the water retains its
molecular state as H2O.
[0169] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another but differs
slightly in composition (as in the replacement of one atom by an
atom of a different element or in the presence of a particular
functional group, or the replacement of one functional group by
another functional group). Thus, an analog is a compound that is
similar or comparable in function and appearance, but not in
structure or origin to the reference compound.
[0170] As defined herein, the term "derivative" refers to compounds
that have a common core structure, and are substituted with various
groups as described herein. For example, all of the compounds
represented by Formula (I) are aryl- or heteroaryl-substituted
benzene compounds, and have Formula (I) as a common core.
[0171] The term "bioisostere" refers to a compound resulting from
the exchange of an atom or of a group of atoms with another,
broadly similar, atom or group of atoms. The objective of a
bioisosteric replacement is to create a new compound with similar
biological properties to the parent compound. The bioisosteric
replacement may be physicochemically or topologically based.
Examples of carboxylic acid bioisosteres include, but are not
limited to, acyl sulfonimides, tetrazoles, sulfonates and
phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96,
3147-3176, 1996.
[0172] The present invention is intended to include all isotopes of
atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example and without limitation, isotopes of hydrogen
include tritium and deuterium, and isotopes of carbon include C-13
and C-14.
[0173] Any compound of Formula (IIa) of the present invention, as
described herein, may be an EZH2 inhibitor.
[0174] In certain aspects of the invention an inhibitor of EZH2
"selectively inhibits" histone methyltransferase activity of the
mutant EZH2 when it inhibits histone methyltransferase activity of
the mutant EZH2 more effectively than it inhibits histone
methyltransferase activity of wild-type EZH2. For example, in one
embodiment the selective inhibitor has an IC50 for the mutant EZH2
that is at least 40 percent lower than the IC50 for wild-type EZH2.
In one embodiment the selective inhibitor has an IC50 for the
mutant EZH2 that is at least 50 percent lower than the IC50 for
wild-type EZH2. In one embodiment the selective inhibitor has an
IC50 for the mutant EZH2 that is at least 60 percent lower than the
IC50 for wild-type EZH2. In one embodiment the selective inhibitor
has an IC50 for the mutant EZH2 that is at least 70 percent lower
than the IC50 for wild-type EZH2. In one embodiment the selective
inhibitor has an IC50 for the mutant EZH2 that is at least 80
percent lower than the IC50 for wild-type EZH2. In one embodiment
the selective inhibitor has an IC50 for the mutant EZH2 that is at
least 90 percent lower than the IC50 for wild-type EZH2.
[0175] In one embodiment, the selective inhibitor of a mutant EZH2
exerts essentially no inhibitory effect on wild-type EZH2.
[0176] In certain aspects of the invention the inhibitor inhibits
conversion of H3-K27me2 to H3-K27me3. In one embodiment the
inhibitor is said to inhibit trimethylation of H3-K27. Since
conversion of H3-K27me1 to H3-K27me2 precedes conversion of
H3-K27me2 to H3-K27me3, an inhibitor of conversion of H3-K27me1 to
H3-K27me2 naturally also inhibits conversion of H3-K27me2 to
H3-K27me3, i.e., it inhibits trimethylation of H3-K27. It is also
possible to inhibit conversion of H3-K27me2 to H3-K27me3 without
inhibition of conversion of H3-K27me1 to H3-K27me2. Inhibition of
this type would also result in inhibition of trimethylation of
H3-K27, albeit without inhibition of dimethylation of H3-K27.
[0177] In one embodiment the inhibitor inhibits conversion of
H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to
H3-K27me3. Such inhibitor may directly inhibit the conversion of
H3-K27me1 to H3-K27me2 alone. Alternatively, such inhibitor may
directly inhibit both the conversion of H3-K27me1 to H3-K27me2 and
the conversion of H3-K27me2 to H3-K27me3.
[0178] In certain aspects of the invention, the inhibitor compound
inhibits histone methyltransferase activity. Inhibition of histone
methyltransferase activity can be detected using any suitable
method. The inhibition can be measured, for example, either in
terms of rate of histone methyltransferase activity or as product
of histone methyltransferase activity.
[0179] The inhibition is a measurable inhibition compared to a
suitable control. In one embodiment, inhibition is at least 10
percent inhibition compared to a suitable control. That is, the
rate of enzymatic activity or the amount of product with the
inhibitor is less than or equal to 90 percent of the corresponding
rate or amount made without the inhibitor. In various other
embodiments, inhibition is at least 20, 25, 30, 40, 50, 60, 70, 75,
80, 90, or 95 percent inhibition compared to a suitable control. In
one embodiment, inhibition is at least 99 percent inhibition
compared to a suitable control. That is, the rate of enzymatic
activity or the amount of product with the inhibitor is less than
or equal to 1 percent of the corresponding rate or amount made
without the inhibitor.
[0180] A composition of the present invention comprises a compound
of Formula (IIa), or a pharmaceutically acceptable salt thereof,
and one or more other therapeutic agents, or a pharmaceutically
acceptable salt thereof. The present invention provides for the
administration of a compound of Formula (IIa) or a pharmaceutically
acceptable salt thereof, and one or more therapeutic agents or a
pharmaceutically acceptable salt thereof, as a co-formulation or
separate formulations, wherein the administration of formulations
is simultaneous, sequential, or in alternation. In certain
embodiments, the other therapeutic agents can be an agent that is
recognized in the art as being useful to treat the disease or
condition being treated by the composition of the present
invention. In other embodiment, the other therapeutic agent can be
an agent that is not recognized in the art as being useful to treat
the disease or condition being treated by the composition of the
present invention. In one aspect, the other therapeutic agents can
be an agent that imparts a beneficial attribute to the composition
of the present invention (e.g., an agent that affects the viscosity
of the composition). The beneficial attribute to the composition of
the present invention includes, but is not limited to,
pharmacokinetic or pharmacodynamic co-action resulting from the
combination of a compound of Formula (IIa) and one or more other
therapeutic agents. For example, the one or more other therapeutic
agents can be anticancer agents or chemotherapeutic agents. For
example, the one or more other therapeutic agents can be
glucocorticoids. For example, the one or more other therapeutic
agents can be selected from prednisone, prednisolone,
cyclophosphamide, vincristine, doxorubicin, mafosfamide, cisplatin,
AraC, everolimus, decitabine, dexamethasone, or functional analogs,
derivatives, prodrugs, and metabolites thereof. In another aspect,
the other therapeutic agent can be Prednisone or its active
metabolite, Prednisolone.
[0181] The therapeutic agents set forth below are for illustrative
purposes and not intended to be limiting. The present invention
includes at least one other therapeutic agent selected from the
lists below. The present invention can include more than one other
therapeutic agent, e.g., two, three, four, or five other
therapeutic agents such that the composition of the present
invention can perform its intended function.
[0182] In one embodiment, the other therapeutic agent is an
anticancer agent. In one embodiment, the anticancer agent is a
compound that affects histone modifications, such as an HDAC
inhibitor. In certain embodiments, an anticancer agent is selected
from the group consisting of chemotherapeutics (such as 2CdA, 5-FU,
6-Mercaptopurine, 6-TG, Abraxane.TM. Accutane.RTM., Actinomycin-D,
Adriamycin.RTM., Alimta.RTM., all-trans retinoic acid,
amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane.RTM.,
Camptosar.RTM., CeeNU.RTM., Clofarabine, Clolar.TM. Cytoxan.RTM.,
daunorubicin hydrochloride, DaunoXome.RTM., Dacogen.RTM., DIC,
Doxil.RTM., Ellence.RTM., Eloxatin.RTM., Emcyt.RTM., etoposide
phosphate, Fludara.RTM., FUDR.RTM., Gemzar.RTM., Gleevec.RTM.,
hexamethylmelamine, Hycamtin.RTM., Hydrea.RTM., Idamycin.RTM.,
Ifex.RTM., ixabepilone, Ixempra.RTM., L-asparaginase,
Leukeran.RTM., liposomal Ara-C, L-PAM, Lysodren, Matulane.RTM.,
mithracin, Mitomycin-C, Myleran.RTM., Navelbine.RTM.,
Neutrexin.RTM., nilotinib, Nipent.RTM., Nitrogen Mustard,
Novantrone.RTM., Oncaspar.RTM., Panretin.RTM., Paraplatin.RTM.,
Platinol.RTM., prolifeprospan 20 with carmustine implant,
Sandostatin.RTM., Targretin.RTM., Tasigna.RTM., Taxotere.RTM.,
Temodar.RTM., TESPA, Trisenox.RTM., Valstar.RTM., Velban.RTM.,
Vidaza.TM., vincristine sulfate, VM 26, Xeloda.RTM. and
Zanosar.RTM.); biologics (such as Alpha Interferon, Bacillus
Calmette-Guerin, Bexxar.RTM., Campath.RTM., Ergamisol.RTM.,
Erlotinib, Herceptin.RTM., Interleukin-2, Iressa.RTM.,
lenalidomide, Mylotarg.RTM., Ontak.RTM., Pegasys.RTM.,
Revlimid.RTM., Rituxan.RTM., Tarceva.TM., Thalomid.RTM.,
Tykerb.RTM., Velcade.RTM. and Zevalin.TM.); corticosteroids, (such
as dexamethasone sodium phosphate, DeltaSone.RTM. and
Delta-Cortef.RTM.); hormonal therapies (such as Arimidex.RTM.,
Aromasin.RTM., Casodex.RTM., Cytadren.RTM., Eligard.RTM.,
Eulexin.RTM., Evista.RTM., Faslodex.RTM., Femara.RTM.,
Halotestin.RTM., Megace.RTM., Nilandron.RTM., Nolvadex.RTM.,
Plenaxis.TM. and Zoladex.RTM.); and radiopharmaceuticals (such as
Iodotope.RTM., Metastron.RTM., Phosphocol.RTM. and Samarium
SM-153).
[0183] In another embodiment, the other therapeutic agent is a
chemotherapeutic agent (also referred to as an anti-neoplastic
agent or anti-proliferative agent), selected from the group
including an alkylating agent; an antibiotic; an anti-metabolite; a
detoxifying agent; an interferon; a polyclonal or monoclonal
antibody; an EGFR inhibitor; a HER2 inhibitor; a histone
deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTOR
inhibitor; a multi-kinase inhibitor; a serine/threonine kinase
inhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; a
taxane or taxane derivative, an aromatase inhibitor, an
anthracycline, a microtubule targeting drug, a topoisomerase poison
drug, an inhibitor of a molecular target or enzyme (e.g., a kinase
or a protein methyltransferase), a cytidine analogue drug or any
chemotherapeutic, anti-neoplastic or anti-proliferative agent
listed in www.cancer.org/docroot/cdg/cdg_0.asp.
[0184] Exemplary alkylating agents include, but are not limited to,
cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran);
melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex);
lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin);
carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine
(Mustargen); busulfan (Myleran); carboplatin (Paraplatin);
cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa
(Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).
[0185] Exemplary antibiotics include, but are not limited to,
doxorubicin (Adriamycin); doxorubicin liposomal (Doxil);
mitoxantrone (Novantrone); bleomycin (Blenoxane); daunorubicin
(Cerubidine); daunorubicin liposomal (DaunoXome); dactinomycin
(Cosmegen); epirubicin (Ellence); idarubicin (Idamycin); plicamycin
(Mithracin); mitomycin (Mutamycin); pentostatin (Nipent); or
valrubicin (Valstar).
[0186] Exemplary anti-metabolites include, but are not limited to,
fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea
(Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta);
fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine
Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine
(Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia);
pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar);
cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX;
Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or
cytarabine (Tarabine PFS).
[0187] Exemplary detoxifying agents include, but are not limited
to, amifostine (Ethyol) or mesna (Mesnex).
[0188] Exemplary interferons include, but are not limited to,
interferon alfa-2b (Intron A) or interferon alfa-2a
(Roferon-A).
[0189] Exemplary polyclonal or monoclonal antibodies include, but
are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra);
bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux);
panitumumab (Vectibix); tositumomab/iodine131 tositumomab (Bexxar);
alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin);
gemtuzumab (Mylotarg); eculizumab (Soliris) ordenosumab.
[0190] Exemplary EGFR inhibitors include, but are not limited to,
gefitinib (Iressa); lapat.sub.inib (Tykerb); .sub.cetuximab
(Erbitux); .sub.erlolinib (Tarceva); .sub.panitumumab (Vectibix);
PKI-166; canertinib (CI-1033); matuzumab (Emd7200) or EKB-569.
[0191] Exemplary HER2 inhibitors include, but are not limited to,
trastuzumab (Herceptin); la.sub.patinib (Tykerb) or AC-480.
[0192] Histone Deacetylase Inhibitors include, but are not limited
to, vorinostat (Zolinza).
[0193] Exemplary hormones include, but are not limited to,
tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol
(Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur);
fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar
La.; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex);
bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone
(Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera);
estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston);
degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis);
or testolactone (Teslac).
[0194] Exemplary mitotic inhibitors include, but are not limited
to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere);
vincristine (Oncovin; Vincasar PFS); vinblastine (Velban);
etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon);
ixabepiione (Ixempra); nocodazole; epothilone; vinorelbine
(Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan
(Hycamtin); amsacrine or lamellarin D (LAM-D).
[0195] Exemplary MTOR inhibitors include, but are not limited to,
everolimus (Afinitor) or temsirolimus (Torisel); rapamune,
ridaforolimus; or AP23573.
[0196] Exemplary VEGF/VEGFR inhibitors include, but are not limited
to, bevacizumab (Avastin); .sub.sorafenib (Nexavar); .sub.sunitinib
(Sutent); ranibizumab; pegaptanib; or vandetinib.
[0197] Exemplary microtubule targeting drugs include, but are not
limited to, paclitaxel, docetaxel, vincristine, vinblastin,
nocodazole, epothilones and navelbine.
[0198] Exemplary topoisomerase poison drugs include, but are not
limited to, teniposide, etoposide, adriamycin, camptothecin,
daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin and
idarubicin.
[0199] Exemplary taxanes or taxane derivatives include, but are not
limited to, paclitaxel and docetaxol.
[0200] Exemplary general chemotherapeutic, anti-neoplastic,
anti-proliferative agents include, but are not limited to,
altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis;
Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib
(Velcade) asparaginase (Elspar); levamisole (Ergamisol); mitotane
(Lysodren); procarbazine (Matulane); pegaspargase (Oncaspar);
denileukin diftitox (Ontak); porfimer (Photofrin); aldesleukin
(Proleukin); lenalidomide (Revlimid); bexarotene (Targretin);
thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide
(Trisenox); verteporfin (Visudyne); mimosine (Leucenol); (1M
tegafur--0.4 M 5-chloro-2,4-dihydroxypyrimidine--1 M potassium
oxonate), or lovastatin.
[0201] In another aspect, the other therapeutic agent is a
chemotherapeutic agent or a cytokine such as G-CSF (granulocyte
colony stimulating factor).
[0202] In yet another aspect, the other therapeutic agents can be
standard chemotherapy combinations such as, but not restricted to,
CMF (cyclophosphamide, methotrexate and 5-fluorouracil), CAF
(cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin
and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and
cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and
paclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP),
Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a
molar ratio of 1:0.4:1), Camptothecin-11 (CPT-11, Irinotecan or
Camptosar.TM.), CHOP (cyclophosphamide, hydroxydaunorubicin,
oncovin, and prednisone or prednisolone), R-CHOP (rituximab,
cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone or
prednisolone), or CMFP (cyclophosphamide, methotrexate,
5-fluorouracil and prednisone).
[0203] In another aspect, the other therapeutic agents can be an
inhibitor of an enzyme, such as a receptor or non-receptor kinase.
Receptor and non-receptor kinases are, for example, tyrosine
kinases or serine/threonine kinases. Kinase inhibitors described
herein are small molecules, polynucleic acids, polypeptides, or
antibodies.
[0204] Exemplary kinase inhibitors include, but are not limited to,
Bevacizumab (targets VEGF), BMW 2992 (targets EGFR and Erb2),
Cetuximab/Erbitux (targets Erb1), Imatinib/Gleevic (targets
Bcr-Abl), Trastuzumab (targets Erb2), Gefitinib/Iressa (targets
EGFR), Ranibizumab (targets VEGF), Pegaptanib (targets VEGF),
Erlotinib/Tarceva (targets Erb1), Nilotinib (targets Bcr-Abl),
Lapatinib (targets Erb1 and Erb2/Her2), GW-572016/lapatinib
ditosylate (targets HER2/Erb2), Panitumumab/Vectibix (targets
EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targets
including RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166
(targets EGFR), Canertinib/CI-1033 (targets EGFR),
Sunitinib/SU-11464/Sutent (targets EGFR and FLT3),
Matuzumab/Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474
(targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3),
Vatalanib/Ptk787/ZK222584 (targets VEGR), CEP-701 (targets FLT3),
SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3),
VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets
SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets
JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK),
sorafenib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3,
PDGFR-B, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src),
AC-220 (targets Flt3), AC-480 (targets all HER proteins, "panHER"),
Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit),
Denosumab (targets RANKL, inhibits SRC), AMG888 (targets HER3), and
AP24534 (multiple targets including Flt3).
[0205] Exemplary serine/threonine kinase inhibitors include, but
are not limited to, Rapamune (targets mTOR/FRAP1), Deforolimus
(targets mTOR), Certican/Everolimus (targets mTOR/FRAP1), AP23573
(targets mTOR/FRAP1), Eril/Fasudil hydrochloride (targets RHO),
Flavopiridol (targets CDK), Seliciclib/CYC202/Roscovitrine (targets
CDK), SNS-032/BMS-387032 (targets CDK), Ruboxistaurin (targets
PKC), Pkc412 (targets PKC), Bryostatin (targets PKC), KAI-9803
(targets PKC), SF1126 (targets PI3K), VX-680 (targets Aurora
kinase), Azd1152 (targets Aurora kinase), Arry-142886/AZD-6244
(targets MAP/MEK), SCIO-469 (targets MAP/MEK), GW681323 (targets
MAP/MEK), CC-401 (targets JNK), CEP-1347 (targets JNK), and PD
332991 (targets CDK).
[0206] Exemplary tyrosine kinase inhibitors include, but are not
limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib
(Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab
(Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib
(Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus
(Afinitor); alemtuzumab (Campath); .sub.gemtuzumab (Mylotarg);
.sub.temsirolimus (Torisel); .sub.pazopanib (Votrient);
.sub.dasatinib (Sprycel); .sub.nilotinib (Tasigna); vatalanib
(Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322;
Azd0530; BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131;
AC-220; or AMG888.
[0207] The present invention provides methods for combination
therapy in which a composition comprising a compound of Formula
(IIa) or a pharmaceutically acceptable salt thereof, and one or
more other therapeutic agents are administered to a subject in need
for treatment of a disease or cancer. The combination therapy can
also be administered to cancer cells to inhibit proliferation or
induce cell death. In one aspect, a compound of Formula (IIa) or a
pharmaceutically acceptable salt thereof is administered prior to
administration of the composition of the present invention
comprising a compound of Formula (IIa) or a pharmaceutically
acceptable salt thereof, and one or more other therapeutic agents.
In one aspect, a compound of Formula (IIa) or a pharmaceutically
acceptable salt thereof is administered prior to administration of
one or more therapeutic agents, such that the other therapeutic
agents are administered either in a single composition or in two or
more compositions, e.g. administered simultaneously, sequentially,
or in alternation.
[0208] In one embodiment, a composition of the present invention
includes a compound of Formula (IIa) or a pharmaceutically
acceptable salt thereof, and one or more anticancer agents, e.g.,
CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and
prednisone or prednisolone) or R-CHOP (rituximab, cyclophosphamide,
hydroxydaunorubicin, oncovin, prednisone or prednisolone). In one
embodiment, a composition of the present invention includes a
compound of Formula (IIa) or a pharmaceutically acceptable salt
thereof, and prednisone or prednisolone. Methods of the present
invention include the combination therapy of administering a
compound of Formula (IIa) or a pharmaceutically acceptable salt
thereof, and anticancer agents, wherein the anticancer agents are
CHOP, R-CHOP, prednisone, or prednisolone.
[0209] In certain embodiments, "combination therapy" is intended to
embrace administration of these therapeutic agents in a sequential
manner, wherein each therapeutic agent is administered at a
different time, as well as administration of these therapeutic
agents, or at least two of the therapeutic agents concurrently, or
in a substantially simultaneous manner. Simultaneous administration
can be accomplished, for example, by administering to the subject a
single capsule having a fixed ratio of each therapeutic agent or in
multiple, single capsules for each of the therapeutic agents.
Sequential or substantially simultaneous administration of each
therapeutic agent can be effected by any appropriate route
including, but not limited to, oral routes, intravenous routes,
intramuscular routes, and direct absorption through mucous membrane
tissues. The therapeutic agents can be administered by the same
route or by different routes. For example, a first therapeutic
agent of the combination selected may be administered by
intravenous injection while the other therapeutic agents of the
combination may be administered orally. Alternatively, for example,
all therapeutic agents may be administered orally or all
therapeutic agents may be administered by intravenous injection.
Therapeutic agents may also be administered in alternation.
[0210] In certain aspects of the invention, the combination
therapies featured in the present invention can result in a
synergistic effect in the treatment of a disease or cancer. A
"synergistic effect" is defined as where the efficacy of a
combination of therapeutic agents is greater than the sum of the
effects of any of the agents given alone. A synergistic effect may
also be an effect that cannot be achieved by administration of any
of the compounds or other therapeutic agents as single agents. The
synergistic effect may include, but is not limited to, an effect of
treating cancer by reducing tumor size, inhibiting tumor growth, or
increasing survival of the subject. The synergistic effect may also
include reducing cancer cell viability, inducing cancer cell death,
and inhibiting or delaying cancer cell growth.
[0211] In certain aspects of the invention "combination therapy"
also embraces the administration of the therapeutic agents as
described above in further combination with other biologically
active ingredients and non-drug therapies (e.g., surgery or
radiation treatment). Where the combination therapy further
comprises a non-drug treatment, the non-drug treatment may be
conducted at any suitable time so long as a beneficial effect from
the co-action of the combination of the therapeutic agents and
non-drug treatment is achieved. For example, in appropriate cases,
the beneficial effect is still achieved when the non-drug treatment
is temporally removed from the administration of the therapeutic
agents, perhaps by days or even weeks.
[0212] In another aspect, a composition of the present invention,
or a pharmaceutically acceptable salt, prodrug, metabolite, analog
or derivative thereof, may be administered in combination with
radiation therapy. Radiation therapy can also be administered in
combination with a composition of the present invention and another
chemotherapeutic agent described herein as part of a multiple agent
therapy.
[0213] The present invention also provides pharmaceutical
compositions comprising a compound of Formula (IIa) or
pharmaceutically acceptable salts thereof, and one or more other
therapeutic agent disclosed herein, mixed with pharmaceutically
suitable carriers or excipient(s) at doses to treat or prevent a
disease or condition as described herein. In one aspect, the
present invention also provides pharmaceutical compositions
comprising any compound of Table I or pharmaceutically acceptable
salts thereof, and one or more therapeutic agents, mixed with
pharmaceutically suitable carriers or excipient (s) at doses to
treat or prevent a disease or condition as described herein. In
another aspect, the present invention also provides pharmaceutical
compositions comprising Compound 44
##STR00101##
or pharmaceutically acceptable salts thereof, and one or more
therapeutic agents, mixed with pharmaceutically suitable carriers
or excipient(s) at doses to treat or prevent a disease or condition
as described herein. The pharmaceutical compositions of the present
invention can also be administered in combination with other
therapeutic agents or therapeutic modalities simultaneously,
sequentially, or in alternation.
[0214] Mixtures of compositions of the present invention can also
be administered to the patient as a simple mixture or in suitable
formulated pharmaceutical compositions. For example, one aspect of
the invention relates to a pharmaceutical composition comprising a
therapeutically effective dose of an EZH2 inhibitor of Formula
(IIa), or a pharmaceutically acceptable salt, hydrate, enantiomer
or stereoisomer thereof; one or more other therapeutic agent, and a
pharmaceutically acceptable diluent or carrier.
[0215] A "pharmaceutical composition" is a formulation containing
the compounds of the present invention in a form suitable for
administration to a subject. In one embodiment, the pharmaceutical
composition is in bulk or in unit dosage form. The unit dosage form
is any of a variety of forms, including, for example, a capsule, an
IV bag, a tablet, a single pump on an aerosol inhaler or a vial.
The quantity of active ingredient (e.g., a formulation of the
disclosed compound or salt, hydrate, solvate or isomer thereof) in
a unit dose of composition is an effective amount and is varied
according to the particular treatment involved. One skilled in the
art will appreciate that it is sometimes necessary to make routine
variations to the dosage depending on the age and condition of the
patient. The dosage will also depend on the route of
administration. A variety of routes are contemplated, including
oral, pulmonary, rectal, parenteral, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, inhalational, buccal,
sublingual, intrapleural, intrathecal, intranasal, and the like.
Dosage forms for the topical or transdermal administration of a
compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants. In
one embodiment, the active compound is mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants that are required.
[0216] As used herein, the phrase "pharmaceutically acceptable"
refers to those compounds, anions, cations, materials,
compositions, carriers, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0217] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the
specification and claims includes both one and more than one such
excipient.
[0218] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), and transmucosal administration. Solutions
or suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0219] A composition of the invention can be administered to a
subject in many of the well-known methods currently used for
chemotherapeutic treatment. For example, for treatment of cancers,
a compound of the invention may be injected directly into tumors,
injected into the blood stream or body cavities or taken orally or
applied through the skin with patches. The dose chosen should be
sufficient to constitute effective treatment but not so high as to
cause unacceptable side effects. The state of the disease condition
(e.g., cancer, precancer, and the like) and the health of the
patient should preferably be closely monitored during and for a
reasonable period after treatment.
[0220] The term "therapeutically effective amount", as used herein,
refers to an amount of a pharmaceutical agent to treat, ameliorate,
or prevent an identified disease or condition, or to exhibit a
detectable therapeutic or inhibitory effect. The effect can be
detected by any assay method known in the art. The precise
effective amount for a subject will depend upon the subject's body
weight, size, and health; the nature and extent of the condition;
and the therapeutic or combination of therapeutics selected for
administration. Therapeutically effective amounts for a given
situation can be determined by routine experimentation that is
within the skill and judgment of the clinician. In a preferred
aspect, the disease or condition to be treated is cancer. In
another aspect, the disease or condition to be treated is a cell
proliferative disorder.
[0221] In certain embodiments the therapeutically effective amount
of each pharmaceutical agent used in combination will be lower when
used in combination in comparison to monotherapy with each agent
alone. Such lower therapeutically effective amount could afford for
lower toxicity of the therapeutic regimen.
[0222] For any compound, the therapeutically effective amount can
be estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. Therapeutic/prophylactic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio between
toxic and therapeutic effects is the therapeutic index, and it can
be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions that exhibit large therapeutic indices are preferred.
The dosage may vary within this range depending upon the dosage
form employed, sensitivity of the patient, and the route of
administration.
[0223] Dosage and administration are adjusted to provide sufficient
levels of the active agent(s) or to maintain the desired effect.
Factors which may be taken into account include the severity of the
disease state, general health of the subject, age, weight, and
gender of the subject, diet, time and frequency of administration,
drug combination(s), reaction sensitivities, and tolerance/response
to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0224] The pharmaceutical compositions containing active compounds
of the present invention may be manufactured in a manner that is
generally known, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Pharmaceutical compositions
may be formulated in a conventional manner using one or more
pharmaceutically acceptable carriers comprising excipients and/or
auxiliaries that facilitate processing of the active compounds into
preparations that can be used pharmaceutically. Of course, the
appropriate formulation is dependent upon the route of
administration chosen.
[0225] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol and sorbitol, and sodium chloride in
the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0226] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0227] Oral compositions generally include an inert diluent or an
edible pharmaceutically acceptable carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic administration, the active compound can be
incorporated with excipients and used in the form of tablets,
troches, or capsules. Oral compositions can also be prepared using
a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is applied orally and swished and expectorated or
swallowed. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, troches and the like can contain any of
the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0228] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0229] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0230] The active compounds can be prepared with pharmaceutically
acceptable carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0231] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.
[0232] In therapeutic applications, the dosages of the EZH2
inhibitor compounds described herein, other therapeutic agents
described herein, compositions comprising a compound of Formula
(IIa) and one or more other therapeutic agents, or the
pharmaceutical compositions used in accordance with the invention
vary depending on the agent, the age, weight, and clinical
condition of the recipient patient, and the experience and judgment
of the clinician or practitioner administering the therapy, among
other factors affecting the selected dosage. Generally, the dose
should be sufficient to result in slowing, and preferably
regressing, the growth of the tumors and also preferably causing
complete regression of the cancer. Dosages can range from about
0.01 mg/kg per day to about 5000 mg/kg per day. In preferred
aspects, dosages can range from about 1 mg/kg per day to about 1000
mg/kg per day. In an aspect, the dose will be in the range of about
0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day;
about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day;
or about 0.1 mg to about 1 g/day, in single, divided, or continuous
doses (which dose may be adjusted for the patient's weight in kg,
body surface area in m.sup.2, and age in years). An effective
amount of a pharmaceutical agent is that which provides an
objectively identifiable improvement as noted by the clinician or
other qualified observer. For example, regression of a tumor in a
patient may be measured with reference to the diameter of a tumor.
Decrease in the diameter of a tumor indicates regression.
Regression is also indicated by failure of tumors to reoccur after
treatment has stopped. As used herein, the term "dosage effective
manner" refers to amount of an active compound to produce the
desired biological effect in a subject or cell.
[0233] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0234] The composition of the present invention is capable of
further forming salts. The composition of the present invention is
capable of forming more than one salt per molecule, e.g., mono-,
di-, tri-. All of these forms are also contemplated within the
scope of the claimed invention.
[0235] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the compounds of the present invention wherein the
parent compound is modified by making acid or base salts thereof.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines, alkali or organic salts of acidic residues such as
carboxylic acids, and the like. The pharmaceutically acceptable
salts include the conventional non-toxic salts or the quaternary
ammonium salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. For example, such
conventional non-toxic salts include, but are not limited to, those
derived from inorganic and organic acids selected from
2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic,
benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic,
ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic,
gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic,
hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic,
hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic,
maleic, malic, mandelic, methane sulfonic, napsylic, nitric,
oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
polygalacturonic, propionic, salicyclic, stearic, subacetic,
succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene
sulfonic, and the commonly occurring amine acids, e.g., glycine,
alanine, phenylalanine, arginine, etc.
[0236] Other examples of pharmaceutically acceptable salts include
hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic
acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid,
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, muconic acid, and the like. The present invention also
encompasses salts formed when an acidic proton present in the
parent compound either is replaced by a metal ion, e.g., an alkali
metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like.
[0237] It should be understood that all references to
pharmaceutically acceptable salts include solvent addition forms
(solvates) or crystal forms (polymorphs) as defined herein, of the
same salt.
[0238] The composition of the present invention may also be
prepared as esters, for example, pharmaceutically acceptable
esters. For example, a carboxylic acid function group in a compound
can be converted to its corresponding ester, e.g., a methyl, ethyl
or other ester. Also, an alcohol group in a compound can be
converted to its corresponding ester, e.g., acetate, propionate or
other ester.
[0239] The composition of the present invention can also be
prepared as prodrugs, for example, pharmaceutically acceptable
prodrugs. The terms "pro-drug" and "prodrug" are used
interchangeably herein and refer to any compound which releases an
active parent drug in vivo. Since prodrugs are known to enhance
numerous desirable qualities of pharmaceuticals (e.g., solubility,
bioavailability, manufacturing, etc.), the compounds of the present
invention can be delivered in prodrug form. Thus, the present
invention is intended to cover prodrugs of the presently claimed
compounds, methods of delivering the same and compositions
containing the same. "Prodrugs" are intended to include any
covalently bonded carriers that release an active parent drug of
the present invention in vivo when such prodrug is administered to
a subject. Prodrugs in the present invention are prepared by
modifying functional groups present in the compound in such a way
that the modifications are cleaved, either in routine manipulation
or in vivo, to the parent compound. Prodrugs include compounds of
the present invention wherein a hydroxy, amino, sulfhydryl, carboxy
or carbonyl group is bonded to any group that may be cleaved in
vivo to form a free hydroxyl, free amino, free sulfhydryl, free
carboxy or free carbonyl group, respectively.
[0240] Examples of prodrugs include, but are not limited to, esters
(e.g., acetate, dialkylaminoacetates, formates, phosphates,
sulfates and benzoate derivatives) and carbamates (e.g.,
N,N-dimethylaminocarbonyl) of hydroxy functional groups, esters
(e.g., ethyl esters, morpholinoethanol esters) of carboxyl
functional groups, N-acyl derivatives (e.g., N-acetyl)N-Mannich
bases, Schiff bases and enaminones of amino functional groups,
oximes, acetals, ketals and enol esters of ketone and aldehyde
functional groups in compounds of the invention, and the like, See
Bundegaard, H., Design of Prodrugs, p1-92, Elesevier, N.Y.-Oxford
(1985).
[0241] The composition, or pharmaceutically acceptable salts,
esters or prodrugs thereof, are administered orally, nasally,
transdermally, pulmonary, inhalationally, buccally, sublingually,
intraperintoneally, subcutaneously, intramuscularly, intravenously,
rectally, intrapleurally, intrathecally and parenterally. In one
embodiment, the compound is administered orally. One skilled in the
art will recognize the advantages of certain routes of
administration.
[0242] The dosage regimen utilizing the compounds is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the severity of
the condition to be treated; the route of administration; the renal
and hepatic function of the patient; and the particular compound or
salt thereof employed. An ordinarily skilled physician or
veterinarian can readily determine and prescribe the effective
amount of the drug required to prevent, counter, or arrest the
progress of the condition.
[0243] Techniques for formulation and administration of the
disclosed compounds of the invention can be found in Remington: the
Science and Practice of Pharmacy, 19.sup.th edition, Mack
Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds
described herein, and the pharmaceutically acceptable salts
thereof, are used in pharmaceutical preparations in combination
with a pharmaceutically acceptable carrier or diluent. Suitable
pharmaceutically acceptable carriers include inert solid fillers or
diluents and sterile aqueous or organic solutions. The compounds
will be present in such pharmaceutical compositions in amounts
sufficient to provide the desired dosage amount in the range
described herein.
[0244] All percentages and ratios used herein, unless otherwise
indicated, are by weight. Other features and advantages of the
present invention are apparent from the different examples. The
provided examples illustrate different components and methodology
useful in practicing the present invention. The examples do not
limit the claimed invention. Based on the present disclosure the
skilled artisan can identify and employ other components and
methodology useful for practicing the present invention.
[0245] The present invention provides compositions and methods for
treating conditions and diseases the course of which can be
influenced by modulating the methylation status of histones or
other proteins, wherein said methylation status is mediated at
least in part by the activity of EZH2. Modulation of the
methylation status of histones can in turn influence the level of
expression of target genes activated by methylation, and/or target
genes suppressed by methylation. The method includes administering
to a subject in need of such treatment, a therapeutically effective
amount of a composition of the present invention or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, to a subject in need of such treatment.
[0246] Based at least on the fact that abnormal histone methylation
has been found to be associated with certain cancers and
precancerous conditions, a method for treating cancer or a
precancerous condition with a mutant EZH2 in a subject comprises
administering to the subject in need thereof a therapeutically
effective amount of a compound that inhibits methylation. In one
embodiment a method for treating cancer or a precancerous condition
in a subject comprises administering to the subject in need thereof
a therapeutically effective amount of a compound that inhibits
conversion of unmethylated H3-K27 to monomethylated H3-K27
(H3-K27me1). In one embodiment a method for treating cancer or a
precancerous condition in a subject comprises administering to the
subject in need thereof a therapeutically effective amount of a
compound that inhibits conversion of monomethylated H3-K27
(H3-K27me1) to dimethylated H3-K27 (H3-K27me2). In one embodiment a
method for treating cancer or a precancerous condition in a subject
comprises administering to the subject in need thereof a
therapeutically effective amount of a compound that inhibits
conversion of H3-K27me2 to trimethylated H3-K27 (H3-K27me3). In one
embodiment a method for treating cancer or a precancerous condition
in a subject comprises administering to the subject in need thereof
a therapeutically effective amount of a compound that inhibits both
conversion of H3-K27me1 to H3-K27me2 and conversion of H3-K27me2 to
H3-K27me3. It is important to note that disease-specific increase
in methylation can occur at chromatin in key genomic loci in the
absence of a global increase in cellular levels of histone or
protein methylation. For example, it is possible for aberrant
hypermethylation at key disease-relevant genes to occur against a
backdrop of global histone or protein hypomethylation.
[0247] Modulators of methylation can be used for modulating cell
proliferation, generally. For example, in some cases excessive
proliferation may be reduced with agents that decrease methylation,
whereas insufficient proliferation may be stimulated with agents
that increase methylation. Accordingly, diseases that may be
treated include hyperproliferative diseases, such as benign cell
growth and malignant cell growth (cancer).
[0248] The disorder in which EZH2-mediated protein methylation
plays a part can be cancer, a cell proliferative disorder, or a
precancerous condition. The present invention further provides the
use of a composition of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, to a subject in need of such treatment, for the
preparation of a medicament useful for the treatment of cancer.
Exemplary cancers that may be treated include lymphomas, including
non-Hodgkin lymphoma, follicular lymphoma (FL) and diffuse large
B-cell lymphoma (DLBCL); melanoma; and leukemia, including CML.
Exemplary precancerous condition includes myelodisplastic syndrome
(MDS; formerly known as preleukemia).
[0249] In general, compounds that are methylation modulators can be
used for modulating cell proliferation, generally. For example, in
some cases excessive proliferation may be reduced with agents that
decrease methylation, whereas insufficient proliferation may be
stimulated with agents that increase methylation. Accordingly,
diseases that may be treated by the compounds of the invention
include hyperproliferative diseases, such as benign cell growth and
malignant cell growth.
[0250] As used herein, a "subject in need thereof" is a subject
having a disorder in which EZH2-mediated protein methylation plays
a part, or a subject having an increased risk of developing such
disorder relative to the population at large. A subject in need
thereof can have a precancerous condition. Preferably, a subject in
need thereof has cancer. A "subject" includes a mammal. The mammal
can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat,
fowl, dog, cat, cow, horse, goat, camel, sheep or a pig.
Preferably, the mammal is a human.
[0251] The subject of the present invention includes any human
subject who has been diagnosed with, has symptoms of, or is at risk
of developing a cancer or a precancerous condition. The subject of
the present invention includes any human subject expressing a
mutant EZH2. For example, a mutant EZH2 comprises one or more
mutations, wherein the mutation is a substitution, a point
mutation, a nonsense mutation, a missense mutation, a deletion, or
an insertion or any other EZH2 mutation described herein.
[0252] A subject in need thereof may have refractory or resistant
cancer. "Refractory or resistant cancer" means cancer that does not
respond to treatment. The cancer may be resistant at the beginning
of treatment or it may become resistant during treatment. In some
embodiments, the subject in need thereof has cancer recurrence
following remission on most recent therapy. In some embodiments,
the subject in need thereof received and failed all known effective
therapies for cancer treatment. In some embodiments, the subject in
need thereof received at least one prior therapy. In certain
embodiments the prior therapy is monotherapy. In certain
embodiments the prior therapy is combination therapy.
[0253] In some embodiments, a subject in need thereof may have a
secondary cancer as a result of a previous therapy. "Secondary
cancer" means cancer that arises due to or as a result from
previous carcinogenic therapies, such as chemotherapy.
[0254] The subject may also exhibit resistance to EZH2 histone
methyltransferase inhibitors or any other therapeutic agent.
[0255] The invention also features a method of selecting a
combination therapy for a subject having cancer. The method
includes the steps of: detecting one or more EZH2 mutations
described herein in a sample from the subject; and selecting, based
on the presence of the one or more EZH2 mutations, a combination
therapy for treating cancer. In one embodiment, the therapy
includes administering to the subject a composition of the
invention. In one embodiment, the method further includes
administrating to the subject a therapeutically effective amount of
a composition of the invention. An EZH2 mutation can be detected
using any suitable method known in the art. More methods are
described in U.S. patent publication US 20130040906, which is
incorporated herein by reference in their entireties.
[0256] The methods and uses described herein may include steps of
detecting one or more EZH2 mutations described herein in a sample
from a subject in need thereof prior to and/or after the
administration of a composition of the invention (e.g., a
composition comprising a compound of Formula (IIa) or
pharmaceutically acceptable salts thereof, and one or more
therapeutic agents) to the subject. The presence of the one or more
EZH2 mutations described herein in the tested sample indicates the
subject is responsive to the combination therapy of the
invention.
[0257] The present invention provides personalized medicine,
treatment and/or cancer management for a subject by genetic
screening of one or more EZH2 mutations described herein in the
subject. For example, the present invention provides methods for
treating or alleviating a symptom of cancer or a precancerous
condition in a subject in need thereof by determining
responsiveness of the subject to a combination therapy and when the
subject is responsive to the combination therapy, administering to
the subject a composition of the invention. The responsiveness is
determined by obtaining a sample from the subject and detecting one
or more EZH2 mutations described herein, and the presence of such
one or more EZH2 mutations described herein indicates that the
subject is responsive to the composition of the invention. Once the
responsiveness of a subject is determined, a therapeutically
effective amount of a composition, for example, a composition
comprising a compound of Formula (IIa) or pharmaceutically
acceptable salts thereof, and one or more therapeutic agents, can
be administered. The therapeutically effective amount of a
composition can be determined by one of ordinary skill in the
art.
[0258] As used herein, the term "responsiveness" is interchangeable
with terms "responsive", "sensitive", and "sensitivity", and it is
meant that a subject is showing therapeutic responses when
administered a composition of the invention, e.g., tumor cells or
tumor tissues of the subject undergo apoptosis and/or necrosis,
and/or display reduced growing, dividing, or proliferation. This
term is also meant that a subject will or has a higher probability,
relative to the population at large, of showing therapeutic
responses when administered a composition of the invention, e.g.,
tumor cells or tumor tissues of the subject undergo apoptosis
and/or necrosis, and/or display reduced growing, dividing, or
proliferation.
[0259] By "sample" it means any biological sample derived from the
subject, includes but is not limited to, cells, tissues samples,
body fluids (including, but not limited to, mucus, blood, plasma,
serum, urine, saliva, and semen), tumor cells, and tumor tissues.
Preferably, the sample is selected from bone marrow, peripheral
blood cells, blood, plasma and serum. Samples can be provided by
the subject under treatment or testing. Alternatively samples can
be obtained by the physician according to routine practice in the
art.
[0260] As used herein, the term "cell proliferative disorder"
refers to conditions in which unregulated or abnormal growth, or
both, of cells can lead to the development of an unwanted condition
or disease, which may or may not be cancerous. Exemplary cell
proliferative disorders of the invention encompass a variety of
conditions wherein cell division is deregulated. Exemplary cell
proliferative disorder include, but are not limited to, neoplasms,
benign tumors, malignant tumors, pre-cancerous conditions, in situ
tumors, encapsulated tumors, metastatic tumors, liquid tumors,
solid tumors, immunological tumors, hematological tumors, cancers,
carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing
cells. The term "rapidly dividing cell" as used herein is defined
as any cell that divides at a rate that exceeds or is greater than
what is expected or observed among neighboring or juxtaposed cells
within the same tissue. A cell proliferative disorder includes a
precancer or a precancerous condition. A cell proliferative
disorder includes cancer. Preferably, the methods provided herein
are used to treat or alleviate a symptom of cancer. The term
"cancer" includes solid tumors, as well as, hematologic tumors
and/or malignancies. A "precancer cell" or "precancerous cell" is a
cell manifesting a cell proliferative disorder that is a precancer
or a precancerous condition. A "cancer cell" or "cancerous cell" is
a cell manifesting a cell proliferative disorder that is a cancer.
Any reproducible means of measurement may be used to identify
cancer cells or precancerous cells. Cancer cells or precancerous
cells can be identified by histological typing or grading of a
tissue sample (e.g., a biopsy sample). Cancer cells or precancerous
cells can be identified through the use of appropriate molecular
markers.
[0261] Exemplary non-cancerous conditions or disorders include, but
are not limited to, rheumatoid arthritis; inflammation; autoimmune
disease; lymphoproliferative conditions; acromegaly; rheumatoid
spondylitis; osteoarthritis; gout, other arthritic conditions;
sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic
shock syndrome; asthma; adult respiratory distress syndrome;
chronic obstructive pulmonary disease; chronic pulmonary
inflammation; inflammatory bowel disease; Crohn's disease;
psoriasis; eczema; ulcerative colitis; pancreatic fibrosis; hepatic
fibrosis; acute and chronic renal disease; irritable bowel
syndrome; pyresis; restenosis; cerebral malaria; stroke and
ischemic injury; neural trauma; Alzheimer's disease; Huntington's
disease; Parkinson's disease; acute and chronic pain; allergic
rhinitis; allergic conjunctivitis; chronic heart failure; acute
coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme
disease; Reiter's syndrome; acute synovitis; muscle degeneration,
bursitis; tendonitis; tenosynovitis; herniated, ruptures, or
prolapsed intervertebral disk syndrome; osteopetrosis; thrombosis;
restenosis; silicosis; pulmonary sarcosis; bone resorption
diseases, such as osteoporosis; graft-versus-host reaction;
Multiple Sclerosis; lupus; fibromyalgia; AIDS and other viral
diseases such as Herpes Zoster, Herpes Simplex I or II, influenza
virus and cytomegalovirus; and diabetes mellitus.
[0262] Exemplary cancers include, but are not limited to,
adrenocortical carcinoma, AIDS-related cancers, AIDS-related
lymphoma, anal cancer, anorectal cancer, cancer of the anal canal,
appendix cancer, childhood cerebellar astrocytoma, childhood
cerebral astrocytoma, basal cell carcinoma, skin cancer
(non-melanoma), biliary cancer, extrahepatic bile duct cancer,
intrahepatic bile duct cancer, bladder cancer, uringary bladder
cancer, bone and joint cancer, osteosarcoma and malignant fibrous
histiocytoma, brain cancer, brain tumor, brain stem glioma,
cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,
ependymoma, medulloblastoma, supratentorial primitive
neuroectodeimal tumors, visual pathway and hypothalamic glioma,
breast cancer, bronchial adenomas/carcinoids, carcinoid tumor,
gastrointestinal, nervous system cancer, nervous system lymphoma,
central nervous system cancer, central nervous system lymphoma,
cervical cancer, childhood cancers, chronic lymphocytic leukemia,
chronic myelogenous leukemia, chronic myeloproliferative disorders,
colon cancer, colorectal cancer, cutaneous T-cell lymphoma,
lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial
cancer, esophageal cancer, extracranial germ cell tumor,
extragonadal germ cell tumor, extrahepatic bile duct cancer, eye
cancer, intraocular melanoma, retinoblastoma, gallbladder cancer,
gastric (stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian
germ cell tumor, gestational trophoblastic tumor glioma, head and
neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma,
hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet
cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer,
renal cancer, kidney cancer, laryngeal cancer, acute lymphoblastic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, hairy cell leukemia, lip and oral
cavity cancer, liver cancer, lung cancer, non-small cell lung
cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin
lymphoma, primary central nervous system lymphoma, Waldenstram
macroglobulinemia, medulloblastoma, melanoma, intraocular (eye)
melanoma, merkel cell carcinoma, mesothelioma malignant,
mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer
of the tongue, multiple endocrine neoplasia syndrome, mycosis
fungoides, myelodysplastic syndromes,
myelodysplastic/myeloproliferative diseases, chronic myelogenous
leukemia, acute myeloid leukemia, multiple myeloma, chronic
myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma,
oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian
cancer, ovarian epithelial cancer, ovarian low malignant potential
tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal
sinus and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytoma, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
prostate cancer, rectal cancer, renal pelvis and ureter,
transitional cell cancer, retinoblastoma, rhabdomyosarcoma,
salivary gland cancer, ewing family of sarcoma tumors, Kaposi
Sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin
cancer (non-melanoma), skin cancer (melanoma), merkel cell skin
carcinoma, small intestine cancer, soft tissue sarcoma, squamous
cell carcinoma, stomach (gastric) cancer, supratentorial primitive
neuroectodermal tumors, testicular cancer, throat cancer, thymoma,
thymoma and thymic carcinoma, thyroid cancer, transitional cell
cancer of the renal pelvis and ureter and other urinary organs,
gestational trophoblastic tumor, urethral cancer, endometrial
uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal
cancer, vulvar cancer, and Wilm's Tumor.
[0263] A "cell proliferative disorder of the hematologic system" is
a cell proliferative disorder involving cells of the hematologic
system. A cell proliferative disorder of the hematologic system can
include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms,
myelodysplasia, benign monoclonal gammopathy, lymphomatoid
granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic
myelocytic leukemia, agnogenic myeloid metaplasia, and essential
thrombocythemia. A cell proliferative disorder of the hematologic
system can include hyperplasia, dysplasia, and metaplasia of cells
of the hematologic system. Preferably, compositions of the present
invention may be used to treat a cancer selected from the group
consisting of a hematologic cancer of the present invention or a
hematologic cell proliferative disorder of the present invention. A
hematologic cancer of the present invention can include multiple
myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's
lymphoma, childhood lymphomas, and lymphomas of lymphocytic and
cutaneous origin), leukemia (including childhood leukemia,
hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic
leukemia, chronic lymphocytic leukemia, chronic myelocytic
leukemia, chronic myelogenous leukemia, and mast cell leukemia),
myeloid neoplasms and mast cell neoplasms.
[0264] A "cell proliferative disorder of the lung" is a cell
proliferative disorder involving cells of the lung. Cell
proliferative disorders of the lung can include all forms of cell
proliferative disorders affecting lung cells. Cell proliferative
disorders of the lung can include lung cancer, a precancer or
precancerous condition of the lung, benign growths or lesions of
the lung, and malignant growths or lesions of the lung, and
metastatic lesions in tissue and organs in the body other than the
lung. Preferably, compositions of the present invention may be used
to treat lung cancer or cell proliferative disorders of the lung.
Lung cancer can include all forms of cancer of the lung. Lung
cancer can include malignant lung neoplasms, carcinoma in situ,
typical carcinoid tumors, and atypical carcinoid tumors. Lung
cancer can include small cell lung cancer ("SCLC"), non-small cell
lung cancer ("NSCLC"), squamous cell carcinoma, adenocarcinoma,
small cell carcinoma, large cell carcinoma, adenosquamous cell
carcinoma, and mesothelioma. Lung cancer can include "scar
carcinoma," bronchioalveolar carcinoma, giant cell carcinoma,
spindle cell carcinoma, and large cell neuroendocrine carcinoma.
Lung cancer can include lung neoplasms having histologic and
ultrastructural heterogeneity (e.g., mixed cell types).
[0265] Cell proliferative disorders of the lung can include all
forms of cell proliferative disorders affecting lung cells. Cell
proliferative disorders of the lung can include lung cancer,
precancerous conditions of the lung. Cell proliferative disorders
of the lung can include hyperplasia, metaplasia, and dysplasia of
the lung. Cell proliferative disorders of the lung can include
asbestos-induced hyperplasia, squamous metaplasia, and benign
reactive mesothelial metaplasia. Cell proliferative disorders of
the lung can include replacement of columnar epithelium with
stratified squamous epithelium, and mucosal dysplasia. Individuals
exposed to inhaled injurious environmental agents such as cigarette
smoke and asbestos may be at increased risk for developing cell
proliferative disorders of the lung. Prior lung diseases that may
predispose individuals to development of cell proliferative
disorders of the lung can include chronic interstitial lung
disease, necrotizing pulmonary disease, scleroderma, rheumatoid
disease, sarcoidosis, interstitial pneumonitis, tuberculosis,
repeated pneumonias, idiopathic pulmonary fibrosis, granulomata,
asbestosis, fibrosing alveolitis, and Hodgkin's disease.
[0266] A "cell proliferative disorder of the colon" is a cell
proliferative disorder involving cells of the colon. Preferably,
the cell proliferative disorder of the colon is colon cancer.
Preferably, compositions of the present invention may be used to
treat colon cancer or cell proliferative disorders of the colon.
Colon cancer can include all forms of cancer of the colon. Colon
cancer can include sporadic and hereditary colon cancers. Colon
cancer can include malignant colon neoplasms, carcinoma in situ,
typical carcinoid tumors, and atypical carcinoid tumors. Colon
cancer can include adenocarcinoma, squamous cell carcinoma, and
adenosquamous cell carcinoma. Colon cancer can be associated with a
hereditary syndrome selected from the group consisting of
hereditary nonpolyposis colorectal cancer, familial adenomatous
polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's
syndrome and juvenile polyposis. Colon cancer can be caused by a
hereditary syndrome selected from the group consisting of
hereditary nonpolyposis colorectal cancer, familial adenomatous
polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's
syndrome and juvenile polyposis.
[0267] Cell proliferative disorders of the colon can include all
forms of cell proliferative disorders affecting colon cells. Cell
proliferative disorders of the colon can include colon cancer,
precancerous conditions of the colon, adenomatous polyps of the
colon and metachronous lesions of the colon. A cell proliferative
disorder of the colon can include adenoma. Cell proliferative
disorders of the colon can be characterized by hyperplasia,
metaplasia, and dysplasia of the colon. Prior colon diseases that
may predispose individuals to development of cell proliferative
disorders of the colon can include prior colon cancer. Current
disease that may predispose individuals to development of cell
proliferative disorders of the colon can include Crohn's disease
and ulcerative colitis. A cell proliferative disorder of the colon
can be associated with a mutation in a gene selected from the group
consisting of p53, ras, FAP and DCC. An individual can have an
elevated risk of developing a cell proliferative disorder of the
colon due to the presence of a mutation in a gene selected from the
group consisting of p53, ras, FAP and DCC.
[0268] A "cell proliferative disorder of the pancreas" is a cell
proliferative disorder involving cells of the pancreas. Cell
proliferative disorders of the pancreas can include all forms of
cell proliferative disorders affecting pancreatic cells. Cell
proliferative disorders of the pancreas can include pancreas
cancer, a precancer or precancerous condition of the pancreas,
hyperplasia of the pancreas, and dysaplasia of the pancreas, benign
growths or lesions of the pancreas, and malignant growths or
lesions of the pancreas, and metastatic lesions in tissue and
organs in the body other than the pancreas. Pancreatic cancer
includes all forms of cancer of the pancreas. Pancreatic cancer can
include ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic
giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like
giant cell carcinoma, mucinous cystadenocarcinoma, acinar
carcinoma, unclassified large cell carcinoma, small cell carcinoma,
pancreatoblastoma, papillary neoplasm, mucinous cystadenoma,
papillary cystic neoplasm, and serous cystadenoma. Pancreatic
cancer can also include pancreatic neoplasms having histologic and
ultrastructural heterogeneity (e.g., mixed cell types).
[0269] A "cell proliferative disorder of the prostate" is a cell
proliferative disorder involving cells of the prostate. Cell
proliferative disorders of the prostate can include all forms of
cell proliferative disorders affecting prostate cells. Cell
proliferative disorders of the prostate can include prostate
cancer, a precancer or precancerous condition of the prostate,
benign growths or lesions of the prostate, and malignant growths or
lesions of the prostate, and metastatic lesions in tissue and
organs in the body other than the prostate. Cell proliferative
disorders of the prostate can include hyperplasia, metaplasia, and
dysplasia of the prostate.
[0270] A "cell proliferative disorder of the skin" is a cell
proliferative disorder involving cells of the skin. Cell
proliferative disorders of the skin can include all forms of cell
proliferative disorders affecting skin cells. Cell proliferative
disorders of the skin can include a precancer or precancerous
condition of the skin, benign growths or lesions of the skin,
melanoma, malignant melanoma and other malignant growths or lesions
of the skin, and metastatic lesions in tissue and organs in the
body other than the skin. Cell proliferative disorders of the skin
can include hyperplasia, metaplasia, and dysplasia of the skin.
[0271] A "cell proliferative disorder of the ovary" is a cell
proliferative disorder involving cells of the ovary. Cell
proliferative disorders of the ovary can include all forms of cell
proliferative disorders affecting cells of the ovary. Cell
proliferative disorders of the ovary can include a precancer or
precancerous condition of the ovary, benign growths or lesions of
the ovary, ovarian cancer, malignant growths or lesions of the
ovary, and metastatic lesions in tissue and organs in the body
other than the ovary. Cell proliferative disorders of the skin can
include hyperplasia, metaplasia, and dysplasia of cells of the
ovary.
[0272] A "cell proliferative disorder of the breast" is a cell
proliferative disorder involving cells of the breast. Cell
proliferative disorders of the breast can include all forms of cell
proliferative disorders affecting breast cells. Cell proliferative
disorders of the breast can include breast cancer, a precancer or
precancerous condition of the breast, benign growths or lesions of
the breast, and malignant growths or lesions of the breast, and
metastatic lesions in tissue and organs in the body other than the
breast. Cell proliferative disorders of the breast can include
hyperplasia, metaplasia, and dysplasia of the breast.
[0273] A cell proliferative disorder of the breast can be a
precancerous condition of the breast. Compositions of the present
invention may be used to treat a precancerous condition of the
breast. A precancerous condition of the breast can include atypical
hyperplasia of the breast, ductal carcinoma in situ (DCIS),
intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular
neoplasia, and stage 0 or grade 0 growth or lesion of the breast
(e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ). A
precancerous condition of the breast can be staged according to the
TNM classification scheme as accepted by the American Joint
Committee on Cancer (AJCC), where the primary tumor (T) has been
assigned a stage of TO or Tis; and where the regional lymph nodes
(N) have been assigned a stage of NO; and where distant metastasis
(M) has been assigned a stage of M0.
[0274] The cell proliferative disorder of the breast can be breast
cancer. Preferably, compositions of the present invention may be
used to treat breast cancer. Breast cancer includes all forms of
cancer of the breast. Breast cancer can include primary epithelial
breast cancers. Breast cancer can include cancers in which the
breast is involved by other tumors such as lymphoma, sarcoma or
melanoma. Breast cancer can include carcinoma of the breast, ductal
carcinoma of the breast, lobular carcinoma of the breast,
undifferentiated carcinoma of the breast, cystosarcoma phyllodes of
the breast, angiosarcoma of the breast, and primary lymphoma of the
breast. Breast cancer can include Stage I, II, IIIA, IIIB, IIIC and
IV breast cancer. Ductal carcinoma of the breast can include
invasive carcinoma, invasive carcinoma in situ with predominant
intraductal component, inflammatory breast cancer, and a ductal
carcinoma of the breast with a histologic type selected from the
group consisting of comedo, mucinous (colloid), medullary,
medullary with lymphocytic infiltrate, papillary, scirrhous, and
tubular. Lobular carcinoma of the breast can include invasive
lobular carcinoma with predominant in situ component, invasive
lobular carcinoma, and infiltrating lobular carcinoma. Breast
cancer can include Paget's disease, Paget's disease with
intraductal carcinoma, and Paget's disease with invasive ductal
carcinoma. Breast cancer can include breast neoplasms having
histologic and ultrastructural heterogeneity (e.g., mixed cell
types).
[0275] Preferably, compound of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph,
or solvate thereof, may be used to treat breast cancer. A breast
cancer that is to be treated can include familial breast cancer. A
breast cancer that is to be treated can include sporadic breast
cancer. A breast cancer that is to be treated can arise in a male
subject. A breast cancer that is to be treated can arise in a
female subject. A breast cancer that is to be treated can arise in
a premenopausal female subject or a postmenopausal female subject.
A breast cancer that is to be treated can arise in a subject equal
to or older than 30 years old, or a subject younger than 30 years
old. A breast cancer that is to be treated has arisen in a subject
equal to or older than 50 years old, or a subject younger than 50
years old. A breast cancer that is to be treated can arise in a
subject equal to or older than 70 years old, or a subject younger
than 70 years old.
[0276] A breast cancer that is to be treated can be typed to
identify a familial or spontaneous mutation in BRCA1, BRCA2, or
p53. A breast cancer that is to be treated can be typed as having a
HER2/neu gene amplification, as overexpressing HER2/neu, or as
having a low, intermediate or high level of HER2/neu expression. A
breast cancer that is to be treated can be typed for a marker
selected from the group consisting of estrogen receptor (ER),
progesterone receptor (PR), human epidermal growth factor
receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met. A breast cancer
that is to be treated can be typed as ER-unknown, ER-rich or
ER-poor. A breast cancer that is to be treated can be typed as
ER-negative or ER-positive. ER-typing of a breast cancer may be
performed by any reproducible means. ER-typing of a breast cancer
may be performed as set forth in Onkologie 27: 175-179 (2004). A
breast cancer that is to be treated can be typed as PR-unknown,
PR-rich, or PR-poor. A breast cancer that is to be treated can be
typed as PR-negative or PR-positive. A breast cancer that is to be
treated can be typed as receptor positive or receptor negative. A
breast cancer that is to be treated can be typed as being
associated with elevated blood levels of CA 15-3, or CA 27-29, or
both.
[0277] A breast cancer that is to be treated can include a
localized tumor of the breast. A breast cancer that is to be
treated can include a tumor of the breast that is associated with a
negative sentinel lymph node (SLN) biopsy. A breast cancer that is
to be treated can include a tumor of the breast that is associated
with a positive sentinel lymph node (SLN) biopsy. A breast cancer
that is to be treated can include a tumor of the breast that is
associated with one or more positive axillary lymph nodes, where
the axillary lymph nodes have been staged by any applicable method.
A breast cancer that is to be treated can include a tumor of the
breast that has been typed as having nodal negative status (e.g.,
node-negative) or nodal positive status (e.g., node-positive). A
breast cancer that is to be treated can include a tumor of the
breast that has metastasized to other locations in the body. A
breast cancer that is to be treated can be classified as having
metastasized to a location selected from the group consisting of
bone, lung, liver, or brain. A breast cancer that is to be treated
can be classified according to a characteristic selected from the
group consisting of metastatic, localized, regional,
local-regional, locally advanced, distant, multicentric, bilateral,
ipsilateral, contralateral, newly diagnosed, recurrent, and
inoperable.
[0278] A compound of the present invention, or a pharmaceutically
acceptable salt, ester, prodrug, metabolite, polymorph or solvate
thereof, may be used to treat or prevent a cell proliferative
disorder of the breast, or to treat or prevent breast cancer, in a
subject having an increased risk of developing breast cancer
relative to the population at large. A subject with an increased
risk of developing breast cancer relative to the population at
large is a female subject with a family history or personal history
of breast cancer. A subject with an increased risk of developing
breast cancer relative to the population at large is a female
subject having a germ-line or spontaneous mutation in BRCA1 or
BRCA2, or both. A subject with an increased risk of developing
breast cancer relative to the population at large is a female
subject with a family history of breast cancer and a germ-line or
spontaneous mutation in BRCA1 or BRCA2, or both. A subject with an
increased risk of developing breast cancer relative to the
population at large is a female who is greater than 30 years old,
greater than 40 years old, greater than 50 years old, greater than
60 years old, greater than 70 years old, greater than 80 years old,
or greater than 90 years old. A subject with an increased risk of
developing breast cancer relative to the population at large is a
subject with atypical hyperplasia of the breast, ductal carcinoma
in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ
(LCIS), lobular neoplasia, or a stage 0 growth or lesion of the
breast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in
situ).
[0279] A breast cancer that is to be treated can histologically
graded according to the Scarff-Bloom-Richardson system, wherein a
breast tumor has been assigned a mitosis count score of 1, 2, or 3;
a nuclear pleiomorphism score of 1, 2, or 3; a tubule formation
score of 1, 2, or 3; and a total Scarff-Bloom-Richardson score of
between 3 and 9. A breast cancer that is to be treated can be
assigned a tumor grade according to the International Consensus
Panel on the Treatment of Breast Cancer selected from the group
consisting of grade 1, grade 1-2, grade 2, grade 2-3, or grade
3.
[0280] A cancer that is to be treated can be staged according to
the American Joint Committee on Cancer (AJCC) TNM classification
system, where the tumor (T) has been assigned a stage of TX, T1,
T1mic, T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and where
the regional lymph nodes (N) have been assigned a stage of NX, N0,
N1, N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant
metastasis (M) can be assigned a stage of MX, M0, or M1. A cancer
that is to be treated can be staged according to an American Joint
Committee on Cancer (AJCC) classification as Stage I, Stage IIA,
Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. A
cancer that is to be treated can be assigned a grade according to
an AJCC classification as Grade GX (e.g., grade cannot be
assessed), Grade 1, Grade 2, Grade 3 or Grade 4. A cancer that is
to be treated can be staged according to an AJCC pathologic
classification (pN) of pNX, pN0, PN0 (I-), PN0 (I+), PN0 (mol-),
PN0 (mol+), PN1, PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3,
pN3a, pN3b, or pN3c.
[0281] A cancer that is to be treated can include a tumor that has
been determined to be less than or equal to about 2 centimeters in
diameter. A cancer that is to be treated can include a tumor that
has been determined to be from about 2 to about 5 centimeters in
diameter. A cancer that is to be treated can include a tumor that
has been determined to be greater than or equal to about 3
centimeters in diameter. A cancer that is to be treated can include
a tumor that has been determined to be greater than 5 centimeters
in diameter. A cancer that is to be treated can be classified by
microscopic appearance as well differentiated, moderately
differentiated, poorly differentiated, or undifferentiated. A
cancer that is to be treated can be classified by microscopic
appearance with respect to mitosis count (e.g., amount of cell
division) or nuclear pleiomorphism (e.g., change in cells). A
cancer that is to be treated can be classified by microscopic
appearance as being associated with areas of necrosis (e.g., areas
of dying or degenerating cells). A cancer that is to be treated can
be classified as having an abnormal karyotype, having an abnormal
number of chromosomes, or having one or more chromosomes that are
abnormal in appearance. A cancer that is to be treated can be
classified as being aneuploid, triploid, tetraploid, or as having
an altered ploidy. A cancer that is to be treated can be classified
as having a chromosomal translocation, or a deletion or duplication
of an entire chromosome, or a region of deletion, duplication or
amplification of a portion of a chromosome.
[0282] A cancer that is to be treated can be evaluated by DNA
cytometry, flow cytometry, or image cytometry. A cancer that is to
be treated can be typed as having 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90% of cells in the synthesis stage of cell division
(e.g., in S phase of cell division). A cancer that is to be treated
can be typed as having a low S-phase fraction or a high S-phase
fraction.
[0283] As used herein, a "normal cell" is a cell that cannot be
classified as part of a "cell proliferative disorder". A normal
cell lacks unregulated or abnormal growth, or both, that can lead
to the development of an unwanted condition or disease. Preferably,
a normal cell possesses normally functioning cell cycle checkpoint
control mechanisms.
[0284] As used herein, "contacting a cell" refers to a condition in
which a compound or other composition of matter is in direct
contact with a cell, or is close enough to induce a desired
biological effect in a cell.
[0285] As used herein, "candidate compound" refers to a compound of
the present invention, or a pharmaceutically acceptable salt,
ester, prodrug, metabolite, polymorph or solvate thereof, that has
been or will be tested in one or more in vitro or in vivo
biological assays, in order to determine if that compound is likely
to elicit a desired biological or medical response in a cell,
tissue, system, animal or human that is being sought by a
researcher or clinician. A candidate compound is a compound of the
present invention, or a pharmaceutically acceptable salt, ester,
prodrug, metabolite, polymorph or solvate thereof. The biological
or medical response can be the treatment of cancer. The biological
or medical response can be treatment or prevention of a cell
proliferative disorder. In vitro or in vivo biological assays can
include, but are not limited to, enzymatic activity assays,
electrophoretic mobility shift assays, reporter gene assays, in
vitro cell viability assays, and the assays described herein.
[0286] As used herein, "treating" or "treat" describes the
management and care of a patient for the purpose of combating a
disease, condition, or disorder and includes the administration of
a compound of the present invention, or a pharmaceutically
acceptable salt, prodrug, metabolite, polymorph or solvate thereof,
to alleviate the symptoms or complications of a disease, condition
or disorder, or to eliminate the disease, condition or
disorder.
[0287] A composition of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, can also be used to prevent a disease, condition
or disorder. As used herein, "preventing" or "prevent" describes
reducing or eliminating the onset of the symptoms or complications
of the disease, condition or disorder.
[0288] As used herein, the term "alleviate" is meant to describe a
process by which the severity of a sign or symptom of a disorder is
decreased. Importantly, a sign or symptom can be alleviated without
being eliminated. In a preferred embodiment, the administration of
pharmaceutical compositions of the invention leads to the
elimination of a sign or symptom, however, elimination is not
required. Effective dosages are expected to decrease the severity
of a sign or symptom. For instance, a sign or symptom of a disorder
such as cancer, which can occur in multiple locations, is
alleviated if the severity of the cancer is decreased within at
least one of multiple locations.
[0289] As used herein, the term "severity" is meant to describe the
potential of cancer to transform from a precancerous, or benign,
state into a malignant state. Alternatively, or in addition,
severity is meant to describe a cancer stage, for example,
according to the TNM system (accepted by the International Union
Against Cancer (UICC) and the American Joint Committee on Cancer
(AJCC)) or by other art-recognized methods. Cancer stage refers to
the extent or severity of the cancer, based on factors such as the
location of the primary tumor, tumor size, number of tumors, and
lymph node involvement (spread of cancer into lymph nodes).
Alternatively, or in addition, severity is meant to describe the
tumor grade by art-recognized methods (see, National Cancer
Institute, www.cancer.gov). Tumor grade is a system used to
classify cancer cells in terms of how abnormal they look under a
microscope and how quickly the tumor is likely to grow and spread.
Many factors are considered when determining tumor grade, including
the structure and growth pattern of the cells. The specific factors
used to determine tumor grade vary with each type of cancer.
Severity also describes a histologic grade, also called
differentiation, which refers to how much the tumor cells resemble
normal cells of the same tissue type (see, National Cancer
Institute, www.cancer.gov). Furthermore, severity describes a
nuclear grade, which refers to the size and shape of the nucleus in
tumor cells and the percentage of tumor cells that are dividing
(see, National Cancer Institute, www.cancer.gov).
[0290] In another aspect of the invention, severity describes the
degree to which a tumor has secreted growth factors, degraded the
extracellular matrix, become vascularized, lost adhesion to
juxtaposed tissues, or metastasized. Moreover, severity describes
the number of locations to which a primary tumor has metastasized.
Finally, severity includes the difficulty of treating tumors of
varying types and locations. For example, inoperable tumors, those
cancers which have greater access to multiple body systems
(hematological and immunological tumors), and those which are the
most resistant to traditional treatments are considered most
severe. In these situations, prolonging the life expectancy of the
subject and/or reducing pain, decreasing the proportion of
cancerous cells or restricting cells to one system, and improving
cancer stage/tumor grade/histological grade/nuclear grade are
considered alleviating a sign or symptom of the cancer.
[0291] As used herein the term "symptom" is defined as an
indication of disease, illness, injury, or that something is not
right in the body. Symptoms are felt or noticed by the individual
experiencing the symptom, but may not easily be noticed by others.
Others are defined as non-health-care professionals.
[0292] As used herein the term "sign" is also defined as an
indication that something is not right in the body. But signs are
defined as things that can be seen by a doctor, nurse, or other
health care professional.
[0293] Cancer is a group of diseases that may cause almost any sign
or symptom. The signs and symptoms will depend on where the cancer
is, the size of the cancer, and how much it affects the nearby
organs or structures. If a cancer spreads (metastasizes), then
symptoms may appear in different parts of the body.
[0294] The disorder in which EZH2-mediated protein methylation
plays a part can be a neurological disease. The compound of this
invention can thus also be used for treating neurologic diseases
such as epilepsy, schizophrenia, bipolar disorder or other
psychological and/or psychiatric disorders, neuropathies, skeletal
muscle atrophy, and neurodegenerative diseases, e.g., a
neurodegenerative disease. Exemplary neurodegenerative diseases
include: Alzheimer's, Amyotrophic Lateral Sclerosis (ALS), and
Parkinson's disease. Another class of neurodegenerative diseases
includes diseases caused at least in part by aggregation of
poly-glutamine. Diseases of this class include: Huntington's
Diseases, Spinalbulbar Muscular Atrophy (SBMA or Kennedy's Disease)
Dentatorubropallidoluysian Atrophy (DRPLA), Spinocerebellar Ataxia
1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease
(MJD; SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar
Ataxia 7 (SCAT), and Spinocerebellar Ataxia 12 (SCA12).
[0295] Any other disease in which epigenetic methylation, which is
mediated by EZH2, plays a role may be treatable or preventable
using compositions and methods described herein.
[0296] Treating cancer can result in a reduction in size of a
tumor. A reduction in size of a tumor may also be referred to as
"tumor regression". Preferably, after treatment, tumor size is
reduced by 5% or greater relative to its size prior to treatment;
more preferably, tumor size is reduced by 10% or greater; more
preferably, reduced by 20% or greater; more preferably, reduced by
30% or greater; more preferably, reduced by 40% or greater; even
more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75% or greater. Size of a tumor may be
measured by any reproducible means of measurement. The size of a
tumor may be measured as a diameter of the tumor.
[0297] Treating cancer can result in a reduction in tumor volume.
Preferably, after treatment, tumor volume is reduced by 5% or
greater relative to its size prior to treatment; more preferably,
tumor volume is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75% or greater. Tumor volume may be measured by any
reproducible means of measurement.
[0298] Treating cancer results in a decrease in number of tumors.
Preferably, after treatment, tumor number is reduced by 5% or
greater relative to number prior to treatment; more preferably,
tumor number is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. Number of tumors may be measured by any reproducible
means of measurement. The number of tumors may be measured by
counting tumors visible to the naked eye or at a specified
magnification. Preferably, the specified magnification is 2.times.,
3.times., 4.times., 5.times., 10.times., or 50.times..
[0299] Treating cancer can result in a decrease in number of
metastatic lesions in other tissues or organs distant from the
primary tumor site. Preferably, after treatment, the number of
metastatic lesions is reduced by 5% or greater relative to number
prior to treatment; more preferably, the number of metastatic
lesions is reduced by 10% or greater; more preferably, reduced by
20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. The number of metastatic lesions may be measured by any
reproducible means of measurement. The number of metastatic lesions
may be measured by counting metastatic lesions visible to the naked
eye or at a specified magnification. Preferably, the specified
magnification is 2.times., 3.times., 4.times., 5.times., 10.times.,
or 50.times..
[0300] Treating cancer can result in an increase in average
survival time of a population of treated subjects in comparison to
a population receiving carrier alone. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. An increase in average survival time of a population may be
measured, for example, by calculating for a population the average
length of survival following initiation of treatment with an active
compound. An increase in average survival time of a population may
also be measured, for example, by calculating for a population the
average length of survival following completion of a first round of
treatment with an active compound.
[0301] Treating cancer can result in an increase in average
survival time of a population of treated subjects in comparison to
a population of untreated subjects. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. An increase in average survival time of a population may be
measured, for example, by calculating for a population the average
length of survival following initiation of treatment with an active
compound. An increase in average survival time of a population may
also be measured, for example, by calculating for a population the
average length of survival following completion of a first round of
treatment with an active compound.
[0302] Treating cancer can result in increase in average survival
time of a population of treated subjects in comparison to a
population receiving monotherapy with a drug that is not a compound
of the present invention, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof. Preferably, the
average survival time is increased by more than 30 days; more
preferably, by more than 60 days; more preferably, by more than 90
days; and most preferably, by more than 120 days. An increase in
average survival time of a population may be measured by any
reproducible means. An increase in average survival time of a
population may be measured, for example, by calculating for a
population the average length of survival following initiation of
treatment with an active compound. An increase in average survival
time of a population may also be measured, for example, by
calculating for a population the average length of survival
following completion of a first round of treatment with an active
compound.
[0303] Treating cancer can result in a decrease in the mortality
rate of a population of treated subjects in comparison to a
population receiving carrier alone. Treating cancer can result in a
decrease in the mortality rate of a population of treated subjects
in comparison to an untreated population. Treating cancer can
result in a decrease in the mortality rate of a population of
treated subjects in comparison to a population receiving
monotherapy with a drug that is not a compound of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, analog or derivative thereof. Preferably, the mortality
rate is decreased by more than 2%; more preferably, by more than
5%; more preferably, by more than 10%; and most preferably, by more
than 25%. A decrease in the mortality rate of a population of
treated subjects may be measured by any reproducible means. A
decrease in the mortality rate of a population may be measured, for
example, by calculating for a population the average number of
disease-related deaths per unit time following initiation of
treatment with an active compound. A decrease in the mortality rate
of a population may also be measured, for example, by calculating
for a population the average number of disease-related deaths per
unit time following completion of a first round of treatment with
an active compound.
[0304] Treating cancer can result in a decrease in tumor growth
rate. Preferably, after treatment, tumor growth rate is reduced by
at least 5% relative to number prior to treatment; more preferably,
tumor growth rate is reduced by at least 10%; more preferably,
reduced by at least 20%; more preferably, reduced by at least 30%;
more preferably, reduced by at least 40%; more preferably, reduced
by at least 50%; even more preferably, reduced by at least 50%; and
most preferably, reduced by at least 75%. Tumor growth rate may be
measured by any reproducible means of measurement. Tumor growth
rate can be measured according to a change in tumor diameter per
unit time.
[0305] Treating cancer can result in a decrease in tumor regrowth.
Preferably, after treatment, tumor regrowth is less than 5%; more
preferably, tumor regrowth is less than 10%; more preferably, less
than 20%; more preferably, less than 30%; more preferably, less
than 40%; more preferably, less than 50%; even more preferably,
less than 50%; and most preferably, less than 75%. Tumor regrowth
may be measured by any reproducible means of measurement. Tumor
regrowth is measured, for example, by measuring an increase in the
diameter of a tumor after a prior tumor shrinkage that followed
treatment. A decrease in tumor regrowth is indicated by failure of
tumors to reoccur after treatment has stopped.
[0306] Treating or preventing a cell proliferative disorder can
result in a reduction in the rate of cellular proliferation.
Preferably, after treatment, the rate of cellular proliferation is
reduced by at least 5%; more preferably, by at least 10%; more
preferably, by at least 20%; more preferably, by at least 30%; more
preferably, by at least 40%; more preferably, by at least 50%; even
more preferably, by at least 50%; and most preferably, by at least
75%. The rate of cellular proliferation may be measured by any
reproducible means of measurement. The rate of cellular
proliferation is measured, for example, by measuring the number of
dividing cells in a tissue sample per unit time.
[0307] Treating or preventing a cell proliferative disorder can
result in a reduction in the proportion of proliferating cells.
Preferably, after treatment, the proportion of proliferating cells
is reduced by at least 5%; more preferably, by at least 10%; more
preferably, by at least 20%; more preferably, by at least 30%; more
preferably, by at least 40%; more preferably, by at least 50%; even
more preferably, by at least 50%; and most preferably, by at least
75%. The proportion of proliferating cells may be measured by any
reproducible means of measurement. Preferably, the proportion of
proliferating cells is measured, for example, by quantifying the
number of dividing cells relative to the number of nondividing
cells in a tissue sample. The proportion of proliferating cells can
be equivalent to the mitotic index.
[0308] Treating or preventing a cell proliferative disorder can
result in a decrease in size of an area or zone of cellular
proliferation. Preferably, after treatment, size of an area or zone
of cellular proliferation is reduced by at least 5% relative to its
size prior to treatment; more preferably, reduced by at least 10%;
more preferably, reduced by at least 20%; more preferably, reduced
by at least 30%; more preferably, reduced by at least 40%; more
preferably, reduced by at least 50%; even more preferably, reduced
by at least 50%; and most preferably, reduced by at least 75%. Size
of an area or zone of cellular proliferation may be measured by any
reproducible means of measurement. The size of an area or zone of
cellular proliferation may be measured as a diameter or width of an
area or zone of cellular proliferation.
[0309] Treating or preventing a cell proliferative disorder can
result in a decrease in the number or proportion of cells having an
abnormal appearance or morphology. Preferably, after treatment, the
number of cells having an abnormal morphology is reduced by at
least 5% relative to its size prior to treatment; more preferably,
reduced by at least 10%; more preferably, reduced by at least 20%;
more preferably, reduced by at least 30%; more preferably, reduced
by at least 40%; more preferably, reduced by at least 50%; even
more preferably, reduced by at least 50%; and most preferably,
reduced by at least 75%. An abnormal cellular appearance or
morphology may be measured by any reproducible means of
measurement. An abnormal cellular morphology can be measured by
microscopy, e.g., using an inverted tissue culture microscope. An
abnormal cellular morphology can take the form of nuclear
pleiomorphism.
[0310] As used herein, the term "selectively" means tending to
occur at a higher frequency in one population than in another
population. The compared populations can be cell populations.
Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, acts selectively on a cancer or precancerous cell
but not on a normal cell. Preferably, a compound of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, acts selectively to
modulate one molecular target (e.g., a target protein
methyltransferase) but does not significantly modulate another
molecular target (e.g., a non-target protein methyltransferase).
The invention also provides a method for selectively inhibiting the
activity of an enzyme, such as a protein methyltransferase.
Preferably, an event occurs selectively in population A relative to
population B if it occurs greater than two times more frequently in
population A as compared to population B. An event occurs
selectively if it occurs greater than five times more frequently in
population A. An event occurs selectively if it occurs greater than
ten times more frequently in population A; more preferably, greater
than fifty times; even more preferably, greater than 100 times; and
most preferably, greater than 1000 times more frequently in
population A as compared to population B. For example, cell death
would be said to occur selectively in cancer cells if it occurred
greater than twice as frequently in cancer cells as compared to
normal cells.
[0311] A composition of the present invention, e.g., a composition
comprising any compound of Formula (IIa) or pharmaceutically
acceptable salt thereof, and one or more other therapeutic agents,
such as prednisone, can modulate the activity of a molecular target
(e.g., a target protein methyltransferase). Modulating refers to
stimulating or inhibiting an activity of a molecular target.
Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, modulates the activity of a molecular target if it
stimulates or inhibits the activity of the molecular target by at
least 2-fold relative to the activity of the molecular target under
the same conditions but lacking only the presence of said compound.
More preferably, a compound of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, modulates the activity of a molecular target if it
stimulates or inhibits the activity of the molecular target by at
least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold,
at least 100-fold relative to the activity of the molecular target
under the same conditions but lacking only the presence of said
compound. The activity of a molecular target may be measured by any
reproducible means. The activity of a molecular target may be
measured in vitro or in vivo. For example, the activity of a
molecular target may be measured in vitro by an enzymatic activity
assay or a DNA binding assay, or the activity of a molecular target
may be measured in vivo by assaying for expression of a reporter
gene.
[0312] A composition of the present invention does not
significantly modulate the activity of a molecular target if the
addition of the compound does not stimulate or inhibit the activity
of the molecular target by greater than 10% relative to the
activity of the molecular target under the same conditions but
lacking only the presence of said compound.
[0313] As used herein, the term "isozyme selective" means
preferential inhibition or stimulation of a first isoform of an
enzyme in comparison to a second isoform of an enzyme (e.g.,
preferential inhibition or stimulation of a protein
methyltransferase isozyme alpha in comparison to a protein
methyltransferase isozyme beta). Preferably, a compound of the
present invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, demonstrates a minimum of
a fourfold differential, preferably a tenfold differential, more
preferably a fifty fold differential, in the dosage required to
achieve a biological effect. Preferably, a compound of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, demonstrates this
differential across the range of inhibition, and the differential
is exemplified at the IC50, i.e., a 50% inhibition, for a molecular
target of interest.
[0314] Administering a composition of the present invention to a
cell or a subject in need thereof can result in modulation (i.e.,
stimulation or inhibition) of an activity of a protein
methyltransferase of interest.
[0315] Administering a compound of the present invention, e.g., a
composition comprising any compound of Formula (IIa) or
pharmaceutically acceptable salt thereof, and one or more other
therapeutic agents, such as prednisone, to a cell or a subject in
need thereof results in modulation (i.e., stimulation or
inhibition) of an activity of an intracellular target (e.g.,
substrate). Several intracellular targets can be modulated with the
compounds of the present invention, including, but not limited to,
protein methyltrasferase.
[0316] Activating refers to placing a composition of matter (e.g.,
protein or nucleic acid) in a state suitable for carrying out a
desired biological function. A composition of matter capable of
being activated also has an unactivated state. An activated
composition of matter may have an inhibitory or stimulatory
biological function, or both.
[0317] Elevation refers to an increase in a desired biological
activity of a composition of matter (e.g., a protein or a nucleic
acid). Elevation may occur through an increase in concentration of
a composition of matter.
[0318] As used herein, "a cell cycle checkpoint pathway" refers to
a biochemical pathway that is involved in modulation of a cell
cycle checkpoint. A cell cycle checkpoint pathway may have
stimulatory or inhibitory effects, or both, on one or more
functions comprising a cell cycle checkpoint. A cell cycle
checkpoint pathway is comprised of at least two compositions of
matter, preferably proteins, both of which contribute to modulation
of a cell cycle checkpoint. A cell cycle checkpoint pathway may be
activated through an activation of one or more members of the cell
cycle checkpoint pathway. Preferably, a cell cycle checkpoint
pathway is a biochemical signaling pathway.
[0319] As used herein, "cell cycle checkpoint regulator" refers to
a composition of matter that can function, at least in part, in
modulation of a cell cycle checkpoint. A cell cycle checkpoint
regulator may have stimulatory or inhibitory effects, or both, on
one or more functions comprising a cell cycle checkpoint. A cell
cycle checkpoint regulator can be a protein or not a protein.
[0320] Treating cancer or a cell proliferative disorder can result
in cell death, and preferably, cell death results in a decrease of
at least 10% in number of cells in a population. More preferably,
cell death means a decrease of at least 20%; more preferably, a
decrease of at least 30%; more preferably, a decrease of at least
40%; more preferably, a decrease of at least 50%;
[0321] most preferably, a decrease of at least 75%. Number of cells
in a population may be measured by any reproducible means. A number
of cells in a population can be measured by fluorescence activated
cell sorting (FACS), immunofluorescence microscopy and light
microscopy. Methods of measuring cell death are as shown in Li et
al., Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect,
cell death occurs by apoptosis.
[0322] Preferably, an effective amount of a composition of the
present invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, is not significantly
cytotoxic to normal cells. A therapeutically effective amount of a
compound is not significantly cytotoxic to normal cells if
administration of the compound in a therapeutically effective
amount does not induce cell death in greater than 10% of normal
cells.
[0323] A therapeutically effective amount of a compound does not
significantly affect the viability of normal cells if
administration of the compound in a therapeutically effective
amount does not induce cell death in greater than 10% of normal
cells. In an aspect, cell death occurs by apoptosis.
[0324] Contacting a cell with a composition of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, can induce or activate
cell death selectively in cancer cells. Administering to a subject
in need thereof a compound of the present invention, or a
pharmaceutically acceptable salt, prodrug, metabolite, polymorph or
solvate thereof, can induce or activate cell death selectively in
cancer cells. Contacting a cell with a composition of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, can induce cell death
selectively in one or more cells affected by a cell proliferative
disorder. Preferably, administering to a subject in need thereof a
composition of the present invention, or a pharmaceutically
acceptable salt, prodrug, metabolite, polymorph or solvate thereof,
induces cell death selectively in one or more cells affected by a
cell proliferative disorder.
[0325] The present invention relates to a method of treating or
preventing cancer by administering a composition of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, to a subject in need
thereof, where administration of the composition of the present
invention, or a pharmaceutically acceptable salt, prodrug,
metabolite, polymorph or solvate thereof, results in one or more of
the following: prevention of cancer cell proliferation by
accumulation of cells in one or more phases of the cell cycle (e.g.
G1, G1/S, G2/M), or induction of cell senescence, or promotion of
tumor cell differentiation; promotion of cell death in cancer cells
via cytotoxicity, necrosis or apoptosis, without a significant
amount of cell death in normal cells, antitumor activity in animals
with a therapeutic index of at least 2. As used herein,
"therapeutic index" is the maximum tolerated dose divided by the
efficacious dose.
[0326] One skilled in the art may refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et al., Molecular Cloning, A Laboratory Manual (3.sup.rd
edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(2000); Coligan et al., Current Protocols in Immunology, John Wiley
& Sons, N.Y.; Enna et al., Current Protocols in Pharmacology,
John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological
Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa., 18.sup.th edition (1990). These
texts can, of course, also be referred to in making or using an
aspect of the invention.
Example 1: Synthesis of
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl
(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-bip-
henyl]-3-carboxamide
##STR00102##
[0327] Step 1: Synthesis of 5-bromo-2-methyl-3-nitrobenzoic
acid
##STR00103##
[0329] To stirred solution of 2-methyl-3-nitrobenzoic acid (100 g,
552 mmol) in conc. H.sub.2SO.sub.4 (400 mL),
1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol)
was added in a portion wise manner at room temperature and the
reaction mixture was then stirred at room temperature for 5 h. The
reaction mixture was poured onto ice cold water, the precipitated
solid was filtered off, washed with water and dried under vacuum to
afford the desired compound as a solid (140 g, 98%). The isolated
compound was taken directly into the next step. .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 8.31 (s, 1H), 8.17 (s, 1H), 2.43
(s, 3H).
Step 2: Synthesis of methyl 5-bromo-2-methyl-3-nitrobenzoate
##STR00104##
[0331] To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic
acid (285 g, 1105 mmol) in DMF (2.8 L) at room temperature was
added sodium carbonate (468 g, 4415 mmol) followed by addition of
methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture
was heated at 60.degree. C. for 8 h. After completion (monitored by
TLC), the reaction mixture was filtered (to remove sodium
carbonate) and washed with ethyl acetate (1 L.times.3). The
combined filtrate was washed with water (3L.times.5) and the
aqueous phase was back extracted with ethyl acetate (1 L.times.3).
The combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure to afford
the title compound as a solid (290 g, 97% yield). The isolated
compound was taken directly into the next step. .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s,
3H), 2.59 (s, 3H).
Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate
##STR00105##
[0333] To a stirred solution of methyl
5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol (1.5
L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved
in 1.5 L water). The resulting mixture was stirred at 80.degree. C.
to which iron powder (472 g, 8451 mmol) was added in a portion wise
manner. The resulting reaction mixture was heated at 80.degree. C.
for 12 h. Upon completion as determined by TLC, the reaction
mixture was hot filtered over Celite.RTM. and the celite bed was
washed with methanol (5 L) followed by washing with 30% MeOH in DCM
(5 L). The combined filtrate was concentrated in-vacuo, the residue
obtained was diluted with aqueous sodium bicarbonate solution (2 L)
and extracted with ethyl acetate (5L.times.3). The combined organic
layers were dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure to afford the title compound as
a solid (220 g, 85%). The compound was taken directly into the next
step. .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.37 (s, 1H), 6.92
(s, 1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).
Step 4: Synthesis of methyl
5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate
##STR00106##
[0335] To a stirred solution of methyl
3-amino-5-bromo-2-methylbenzoate (15 g, 61.5 mmol) and
dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) in dichloroethane (300
mL) was added acetic acid (22 g, 369 mmol) and the reaction mixture
stirred at room temperature for 15 minutes, then the reaction
mixture was cooled to 0.degree. C. and sodium triacetoxyborohydride
(39 g, 184 mmol) was added. The reaction mixture was stirred
overnight at room temperature. Upon completion of the reaction as
determined by TLC, aqueous sodium bicarbonate solution was added to
the reaction mixture until a pH of 7-8 was obtained. The organic
phase was separated and the aqueous phase was extracted with ethyl
acetate. The combined organic layers were dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure.
The crude compound was purified by column chromatography (100-200
mesh silica gel) eluting with ethyl acetate:hexane to afford the
desired compound as a solid (14 g, 69%). .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 7.01 (s, 1H), 6.98 (s, 1H), 5.00 (d, 1H, J=7.6
Hz), 3.84-3.87 (m, 2H), 3.79 (s, 3H), 3.54-3.56 (m, 1H), 3.43 (t,
2H, J=12 Hz), 2.14 (s, 3H), 1.81-1.84 (m, 2H), 1.47-1.55 (m,
2H).
Step 5: Synthesis of methyl 5-bromo-3-(ethyl
(tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate
##STR00107##
[0337] To a stirred solution of methyl
5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14
g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde
(3.75 g, 85.2 mmol) and acetic acid (15.3 g, 256 mmol). The
resulting reaction mixture was stirred at room temperature for 15
minutes. The mixture was cooled to 0.degree. C. and sodium
triacetoxyborohydride (27 g, 128 mmol) was added. The reaction
mixture was stirred at room temperature for 3 hours. Upon
completion of the reaction as determined by TLC, aqueous sodium
bicarbonate solution was added to the reaction mixture until a pH
7-8 was obtained, the organic phase was separated and the aqueous
phase was extracted with ethyl acetate. The combined organic layers
were dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure. The crude compound was purified by column
chromatography (100-200 mesh silica gel) eluting with ethyl
acetate:hexane to afford the desired compound as a viscous liquid
(14 g, 93%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 7.62 (s,
1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H),
2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1.37-1.50 (m,
2H), 0.87 (t, 3H, J=6.8 Hz).
Step 6: Synthesis of 5-bromo-N-((4, 6-dimethyl-2-oxo-1,
2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-2-methylbenzamide
##STR00108##
[0339] To a stirred solution of 5-bromo-3-(ethyl
(tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate (14 g, 39.4
mmol) in ethanol (100 mL) was added aqueous NaOH (2.36 g, 59.2 mmol
in 25 mL water) and the resulting mixture was stirred at 60.degree.
C. for 1 h. Upon completion of the reaction as determined by TLC,
the solvent was removed under reduced pressure and the residue
obtained was acidified with 1N HCl until a pH 7 was obtained and
then aqueous citric acid solution was added until a pH 5-6 was
obtained. The aqueous layer was extracted with 10% MeOH in DCM (200
mL.times.3), the combined organic layers were dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure to
give the respective acid (14 g, 100%).
[0340] The above acid (14 g, 40.9 mmol) was then dissolved in DMSO
(70 mL) and 3-(amino methyl)-4, 6-dimethylpyridin-2(1H)-one (12.4
g, 81.9 mmol) was added to it. The reaction mixture was stirred at
room temperature for 15 minutes, then PYBOP (31.9 g, 61.4 mmol) was
added and stirring was continued for overnight at room temperature.
Upon completion of the reaction as determined by TLC, the reaction
mixture was poured onto ice-cold water (700 mL), stirred for 30
minutes and the precipitated solid was collected by filtration,
washed with water (500 mL) and air dried. The solid obtained was
stirred with acetonitrile (75 mL.times.2), filtered and air dried.
The solid obtained was again stirred with 5% MeOH in DCM (100 mL),
filtered and dried completely under vacuum to afford the title
compound as a solid (14 g, 74%). .sup.1H NMR (DMSO-d.sub.6, 400
MHz) .delta. 11.47 (s, 1H), 8.23 (t, 1H), 7.30 (s, 1H), 7.08 (s,
1H), 5.85 (s, 1H), 4.23 (d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=10.4 Hz),
3.20-3.26 (m, 2H), 3.00-3.07 (m, 1H), 2.91-2.96 (m, 2H), 2.18 (s,
3H), 2.14 (s, 3H), 2.10 (s, 3H), 1.58-1.60 (m, 2H), 1.45-1.50 (m,
2H), 0.78 (t, 3H, J=6.8 Hz).
Step 7: Synthesis of N-((4, 6-dimethyl-2-oxo-1,
2-dihydropyridin-3-yl) methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-4-methyl-4'-(morpholinomethyl)-[1,
1'-biphenyl]-3-carboxamide
##STR00109##
[0342] To a stirred solution of 5-bromo-N-((4, 6-dimethyl-2-oxo-1,
2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-2-methylbenzamide (14 g, 29.5 mmol) in dioxane/water mixture
(70 mL/14 mL) was added 4-(4-(4, 4, 5, 5-tetramethyl-1, 3,
2-dioxaborolan-2-yl) benzyl) morpholine (13.4 g, 44.2 mmol)
followed by addition of Na.sub.2CO.sub.3 (11.2 g, 106.1 mmol). The
solution was purged with argon for 15 minutes and then Pd
(PPh.sub.3).sub.4 (3.40 g, 2.94 mmol) was added and the solution
was again purged with argon for a further 10 min. The reaction
mixture was heated at 100.degree. C. for 4 h. After completion
(monitored by TLC), the reaction mixture was diluted with water and
extracted with 10% MeOH/DCM. The combined organic layers were dried
over anhydrous sodium sulphate, filtered and concentrated under
reduced pressure. The crude compound was purified by column
chromatography (100-200 mesh silica gel) eluting with methanol:DCM
to the title compound as a solid (12 g, 71%). Analytical Data:
LCMS: 573.35 (M+1).sup.+; HPLC: 99.5% (@ 254 nm) (R.sub.t; 3.999;
Method: Column: YMC ODS-A 150 mm.times.4.6 mm.times.5; Mobile
Phase: A; 0.05% TFA in water/B; 0.05% TFA in acetonitrile; Inj.
Vol: 10 .mu.L, Col. Temp.: 30.degree. C.; Flow rate: 1.4 mL/min.;
Gradient: 5% B to 95% B in 8 min, Hold for 1.5 min, 9.51-12 min 5%
B); .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.46 (s, 1H), 8.19
(t, 1H), 7.57 (d, 2H, J=7.2 Hz), 7.36-7.39 (m, 3H), 7.21 (s, 1H),
5.85 (s, 1H), 4.28 (d, 2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57
(bs, 4H), 3.48 (s, 2H), 3.24 (t, 2H, J=10.8 Hz), 3.07-3.09 (m, 2H),
3.01 (m, 1H), 2.36 (m, 4H), 2.24 (s, 3H), 2.20 (s, 3H), 2.10 (s,
3H), 1.64-1.67 (m, 2H), 1.51-1.53 (m, 2H), 0.83 (t, 3H, J=6.4
Hz).
Step 8: Synthesis of
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl
(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-bip-
henyl]-3-carboxamide trihydrochloride
##STR00110##
[0344] N-((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl)
methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-4-methyl-4'-(morpholinomethyl)-[1,
1'-biphenyl]-3-carboxamide (12 g, 21.0 mmol) was dissolved in
methanolic HCl (200 mL) and stirred at room temperature for 3 h.
After three hours of stirring, the reaction mixture was
concentrated under reduced pressure. The solid obtained was stirred
with ether (100 mL.times.2) to afford the desired salt as a solid
(11 g, 77%). Analytical Data of the tri-HCl salt: LCMS: 573.40
(M+1).sup.+; HPLC: 99.1% (@ 254 nm) (R.sub.t; 3.961; Method:
Column: YMC ODS-A 150 mm.times.4.6 mm.times.5; Mobile Phase: A;
0.05% TFA in water/B; 0.05% TFA in acetonitrile; Inj. Vol: 10
.mu.L, Col. Temp.: 30.degree. C.; Flow rate: 1.4 mL/min.; Gradient:
5% B to 95% B in 8 min, Hold for 1.5 min, 9.51-12 min 5% B); H NMR
(D.sub.2O 400 MHz) .delta. 7.92 (bs, 1H) 7.80 (s, 1H), 7.77 (d, 2H,
J=8 Hz), 7.63 (s, 1H), 7.61 (s, 1H), 6.30 (s, 1H), 4.48 (s, 2H),
4.42 (s, 2H), 4.09-4.11 (m, 4H), 3.95-3.97 (m, 2H), 3.77 (t, 3H,
J=10.4 Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H),
2.35 (s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1.76 (m, 2H), 1.04 (t,
3H, J=6.8 Hz).
Example 2: Combination Therapy of Compound 44 and CHOP
Components
[0345] The human lymphoma cell lines WSU-DLCL2 (DSMZ; ACC 575),
OCI-Ly19 (DSMZ; ACC 528), RL (ATCC; CRL-2261) were obtained from
the indicated sources and maintained in RPMI-1640 medium
supplemented with 10% fetal bovine serum and 2 mM glutamine. The
cells were cultured in tissue culture flasks in a humidified
incubator at 37.degree. C., in an atmosphere of 5% CO.sub.2 and 95%
air.
[0346] The effect of combination therapy of Compound 44 with each
individual CHOP (Cyclophosphamide, Vincristine, Doxorubicin, and
Prednisolone) component on cancer cell viability was investigated
in vitro. The dosage schedule is depicted in FIG. 1A. WSU-DLCL2
human lymphoma cells were treated with increasing concentrations of
Compound 44. After 4 days, a combination of increasing
concentrations of Compound 44 and each CHOP component were
administered to the cells. After 4 days, cell viability was
determined using Millipore Guava ViaCount Reagent and flow
cytometry analysis. The percentage of viable cells for each sample
was normalized to the percentage of viable cells for the
DMSO-treated samples within each Compound 44 concentration
group.
[0347] Cells treated with Compound 44 and CHOP components alone
showed a decrease in cell viability. Cells treated with Compound 44
with Mafofsamide (Cyclophosphamide metabolite) (FIG. 2A) and
Doxorubicin (FIG. 2B) did not exhibit reduced cell viability at
increasing concentrations of Mafofsamide or Doxorubicin.
Combination therapy of Compound 44 with Vincristine (FIG. 2C)
showed reduced cell viability at the highest concentration Compound
44. Importantly, combination therapy utilizing Compound 44 and
Prednisolone (Prednisone metabolite) (FIG. 2D) showed synergistic
reduction in cell viability at the 2 highest doses of Compound 44
and all doses of Prednisolone.
Example 3: Synergistic Effects of Compound 44 and Prednisolone
Combination Therapy is Dependent on Dosage Schedule
[0348] To investigate the role of the timing of administration of
Compound 44 and Prednisolone on cell viability, WSU-DLCL2 cells
were treated with different dosing schedules, as depicted in FIG.
1. Cell viability was determined by staining with Millipore Guava
ViaCount Reagent and then analyzed by flow cytometry.
[0349] Administration of Compound 44 prior to co-administration of
Compound 44 and Prednisolone resulted in reduced cell viability
(FIG. 3A). Cells treated as depicted in FIG. 1A. Cell viability was
normalized to the DMSO/DMSO sample, thereby revealing the effect of
treatment with Compound 44 alone. Increasing concentrations of
Compound 44 resulted in a reduction of cell growth. Importantly,
increasing concentrations of Prednisolone in combination with
Compound 44 resulted in additional reduction of cell growth
compared to treating the cells with Compound 44 or Prednisolone
independently as single agents. Therefore, combination therapy of
Compound 44 and Prednisolone, wherein Compound 44 is administered
first causes a synergistic effect of reducing cancer cell
viability.
[0350] Administration of Compound 44 prior to administration of
Prednisolone resulted in reduced cell viability (FIG. 3B). Cells
were treated as depicted in FIG. 1B. Cell viability was normalized
to the DMSO-treated sample for each Compound 44 concentration
group. Treatment with increasing concentrations of Prednisolone as
a single agent after a prior administration of Compound 44 also
resulted in additional reduction of cell viability when compared to
cells treated with Prednisolone alone, thereby demonstrating the
synergistic effect of combination therapy with Compound 44 and
Prednisolone.
[0351] Administration of Prednisone prior to co-administration of
Compound 44 and Prednisolone did not reduce cell viability (FIG.
3C). Cells were treated as depicted in FIG. 1C. Cell viability was
normalized to the DMSO-treated sample for each Compound 44
concentration group. These results demonstrated that administration
of Prednisolone prior to treatment with a composition comprising
Compound 44 and Prednisolone did not cause a synergistic effect on
cell viability.
[0352] These data clearly show that the combination therapy of
Compound 44 and Prednisolone decreases cancer cell viability or
induces cancer cell death. Specifically, combination therapy
wherein the cells are treated with Compound 44 prior to treatment
with Prednisolone or a combination of Prenisolone and Compound 44
results in a synergistic effect on cell viability, wherein the
reduction of cell viability is greater than that induced by
treatment of either Prednisolone or Compound 44 as single
agents.
Example 4: Compound 44 and Prednisolone Syngery is Dependent on
EZH2 Mutation
[0353] Different human lymphoma cancer cell lines harboring
different EZH2 mutations were analyzed for their responsiveness to
Compound 44 and Prednisolone. Cells were treated with dosing
schedules as in FIG. 1A, where cells are first treated with
Compound 44, and then after 4 days, are treated with a combination
of Compound 44 and Prednisolone. Cell viability was determined 4
days later using Millipore Guava ViaCount reagent and flow
cytometry analysis. Percentage of cell viability was normalized to
percentage of the DMSO-treated sample.
[0354] Lymphoma cells expressing wild-type (WT) EZH2, OCI-LY19 cell
line, are resistant to treatment with EZH2 inhibitors. Accordingly,
treatment with increasing concentrations of Compound 44 does not
affect cell viability. Increasing concentrations of Prednisolone
does not have an additional or synergistic effect on cell viability
(FIG. 4A).
[0355] WSU-DLCL2 cells harboring an Y641F mutation are sensitive to
treatment with EZH2 inhibitors, as evidenced by a decrease in cell
viability at increasing concentrations of Compound 44 when
administered as a single agent. Moreover, when treated in
combination with Prednisolone, the cells exhibit reduced cell
viability (FIG. 4B).
[0356] RL cells harbor an Y641N mutation and are resistant to EZH2
inhibitor treatment. Administration of increasing concentrations of
Compound 44 alone does not result in a reduction in cell viability.
However, co-administration of Compound 44 with Prednisolone
resulted in a synergistic effect, wherein the cell viability was
decreased greater than that observed when Compound 44 and
Prednisolone are administered as single agents (FIG. 4C).
[0357] Taken together, these results suggest that combination
therapy with Compound 44 and Prednisolone may result in a
synergistic effect in reducing cancer cell viability and increasing
cancer cell death in cells that express a mutant EZH2. Moreover,
cells that are resistant to either drug, Compound 44 or
Prednisolone, when administered as a single agent, become sensitive
to the combination treatment and cell viability is reduced.
Example 5: Pharmacokinetic Analysis of Compound 44 and CHOP
Components Co-Administration In Vivo
[0358] Pharmacokinetic analysis of Compound 44 in combination with
each of the CHOP components (Cyclophosphamide, Vincristine,
Doxorubicin, and Prednisolone) was performed to determine the
absorption or distribution of Compound 44 in vivo. Male BALB/c
between 8-12 weeks old and weighing 20-40 g were obtained from In
vivo, Bengaluru, India. Animals were administered one of the CHOP
components as a single agent, or in combination with Compound 44.
Cyclophosphamide was administered by intraperitoneal injection at
30 mg/kg. Vincristine was administered by intravenous injection at
0.375 mg/kg. Doxorubicin was administered by intravenous injection
at 2.475 mg/kg. Prednisolone was administered by oral
administration at 0.15 mg/kg. Compound 44 was administered at 225
mg/kg by oral administration. Plasma samples were taken at various
timepoints over a course of 24 hours after administration.
[0359] The extraction procedure for plasma study samples or the
spiked plasma calibration standards were identical: A 25 .mu.L
sample of either study sample or spiked calibration standard was
added to individual pre-labeled micro-centrifuge tubes. A volume of
100 .mu.L of IS (Glipizide, 500 ng/mL) prepared in ACN was then
added to the micro-centrifuge tubes except in blank sample where
acetonitrile was added and vortexed for 5 minutes. Samples were
centrifuged for 10 minutes at the speed of 15000 rpm (20600 g) at
4.degree. C. Following centrifugation, 100 .mu.L of the supernatant
was sampled from each centrifuge tube and transferred into insert
vials. These vials were loaded in auto-sampler for the LC/MS/NIS
analysis. Calibration standards were prepared by spiking 10 .mu.L
of analyte (Compound 44, Cyclophosphamide, Doxorubicin, Vincristine
and Prednisolone) in 190 .mu.L of blank mouse plasma.
[0360] NonCompartmental-Analysis module in WinNonlin.RTM. (Version
5.2) was used to assess the pharmacokinetic parameters. The areas
under the concentration time curve (AUC) were calculated by linear
trapezoidal rule. The ratio (AUC.sub.combo/AUC.sub.single) of AUC
for combination therapy of Compound 44 with each component of CHOP
(AUC.sub.combo) to AUC of each CHOP component alone
(AUC.sub.single) and indicated similar bioavailability when CHOP
components were administered alone or with Compound 44.
Example 6: Analysis of Compound 44 and CHOP Combination Therapy in
Mouse Xenograft Models
Mice
[0361] Female Fox Chase SCID.RTM. Mice
(CB17/Icr-Prkdc.sub.scid/IcrIcoCrl, Charles River Laboratories) or
athymic nude mice (Crl:NU(Ncr)-Fox1.sub.nu, Charles River
Laboratories) were 8 weeks old and had a body-weight (BW) range of
16.0-21.1 g on day 1 of the study. The animals were fed ad libitum
water (reverse osmosis 1 ppm Cl) and NIH 31 Modified and Irradiated
Lab Diet.RTM. consisting of 18.0% crude protein, 5.0% crude fat,
and 5.0% crude fiber. The mice were housed on irradiated
Enrich-o'Cobs.TM. bedding in static microisolators on a 12-hour
light cycle at 20-22.degree. C. (68-72.degree. F.) and 40-60%
humidity. All procedures comply with the recommendations of the
Guide for Care and Use of Laboratory Animals with respect to
restraint, husbandry, surgical procedures, feed and fluid
regulation, and veterinary care.
Tumor Cell Culture
[0362] Human lymphoma cell lines line were obtained from different
sources (ATCC, DSMZ) and maintained at Piedmont as suspension
cultures in RPMI-1640 medium containing 100 units/mL penicillin G
sodium salt, 100 g/mL streptomycin, and 25 g/mL gentamicin. The
medium was supplemented with 10% fetal bovine serum and 2 mM
glutamine. The cells were cultured in tissue culture flasks in a
humidified incubator at 37.degree. C., in an atmosphere of 5%
CO.sub.2 and 95% air.
In Vivo Tumor Implantation
[0363] Human lymphoma cell lines were harvested during mid-log
phase growth, and resuspended in PBS with 50% Matrigel.TM. (BD
Biosciences). Each mouse received 1.times.10.sup.7 cells (0.2 mL
cell suspension) subcutaneously in the right flank. Tumors were
calipered in two dimensions to monitor growth as the mean volume
approached the desired 80-120 mm.sup.3 range. Tumor size, in
mm.sup.3, was calculated from:
Tumor .times. volume .times. = w 2 .times. l 2 ##EQU00001##
where w=width and l=length, in mm, of the tumor. Tumor weight can
be estimated with the assumption that 1 mg is equivalent to 1
mm.sup.3 of tumor volume. After 10-30 days (depending on the cell
line used) mice with 108-126 mm.sup.3 tumors were sorted into
treatment groups with mean tumor volumes of 117-119 mm.sup.3.
Test Articles
[0364] Compounds of Formula (IIa) were stored at room temperature
and protected from light. On each treatment day, a fresh compound
formulations were prepared by suspending the powders in 0.5% sodium
carboxymethylcellulose (NaCMC) and 0.1% Tween.RTM. 80 in deionized
water. The Compound 44 vehicle, 0.5% NaCMC and 0.1% Tween.RTM. 80
in deionized water, was used to treat the control groups at the
same schedules. Formulations were stored away from light at
4.degree. C. prior to administration.
[0365] Several chemotherapeutica were used in parallel to Epizyme
compounds. Cyclophosphamide (Baxter, Lot #016591), was
reconstituted to 20 mg/mL with sterile saline and stored at
4.degree. C. A fresh dosing solution was prepared for each dose by
dilution with saline. Doxorubicin (Doxorubicin Meiji.RTM., Meiji
Pharmaceutical Co. Ltd., 1 mg/mL) was stored at 4.degree. C. and
diluted with saline on each treatment day. Vincristine (Hospira,
Inc., 1 mg/mL) was diluted with saline on each treatment day.
Prednisone (Boehringer Ingelheim GmbH, 1 mg/mL) was diluted with
PBS at the beginning of each 5-day dosing cycle.
Treatment Plan
[0366] Mice were treated at compound doses ranging from 75-600
mg/kg and at TID (3 times a day every 8 h), BID (twice a day every
12 h) or QD (once a day) schedules for various amount of days by
oral gavage or injections via the intravenous, intraperitoneal or
subcutaneous routes. Each dose was delivered in a volume of 0.2
mL/20 g mouse (10 mL/kg), and adjusted for the last recorded weight
of individual animals. The maximal treatment length was 28
days.
Median Tumor Volume (MTV) and Tumor Growth Inhibition (TGI)
Analysis
[0367] Treatment efficacy was determined on the last treatment day.
MTV(n), the median tumor volume for the number of animals, n,
evaluable on the last day, was determined for each group. Percent
tumor growth inhibition (% TGI) can be defined several ways. First,
the difference between the MTV(n) of the designated control group
and the MTV(n) of the drug-treated group is expressed as a
percentage of the MTV(n) of the control group:
% .times. TGI = ( M .times. T .times. V .function. ( n ) control -
M .times. T .times. V .function. ( n ) treated M .times. T .times.
V .function. ( n ) control ) .times. 1 .times. 0 .times. 0
##EQU00002##
[0368] Another way of calculating % TGI is taking the change of the
tumor size from day 1 to day n into account with n being the last
treatment day.
% .times. TGI = ( .DELTA. .times. M .times. T .times. V control -
.DELTA. .times. M .times. T .times. V treated .DELTA. .times. M
.times. T .times. V control ) .times. 100 ##EQU00003## .DELTA.MT
.times. V control = M .times. T .times. V .function. ( n ) control
- M .times. T .times. V .function. ( 1 ) control ##EQU00003.2##
.DELTA.MT .times. V treated = M .times. T .times. V .function. ( n
) treated - M .times. T .times. V .function. ( 1 ) treated
##EQU00003.3##
Tumor Growth Delay Analysis
[0369] Alternatively, mice were kept alive after the last treatment
day for tumor growth delay analysis. Tumors were callipered
twice-weekly and each test animal was euthanized when its neoplasm
reached the endpoint volume of 2000 mm.sup.3 or on the
pre-specified last day of the study, whichever came first. The
time-to-endpoint (TTE) for each mouse was calculated from the
following equation:
TTE .function. ( days ) = log 10 ( endpoint .times. volume , mm 3 )
- b m ##EQU00004##
where b is the intercept and m is the slope of the line obtained by
linear regression of a log-transformed tumor growth data set. The
data sets were composed of the first observation that exceeded the
study endpoint volume and the three consecutive observations that
immediately preceded the attainment of the endpoint volume. Animals
that did not reach the volume endpoint were assigned a TTE value
equal to the last day of the study (prespecified). Any animal
classified as a treatment-related (TR) death was to be assigned a
TTE value equal to the day of death. Any animal classified as a
nontreatment-related (NTR) death was excluded from TTE calculations
and all further analyses.
[0370] Treatment outcome was determined from tumor growth delay
(TGD), defined as the increase in the median TTE in a treatment
group compared to the control group:
TGD=T-C
expressed in days, or as a percentage of the median TTE of the
control group:
% .times. TGD = T - C C .times. 1 .times. 0 .times. 0 ##EQU00005##
[0371] where: [0372] T=median TTE for a treatment group [0373]
C=median TTE for the control group
Toxicity
[0374] Animals were weighed daily on Days 1-5, and then twice
weekly until the completion of the study. The mice were examined
frequently for overt signs of any adverse, treatment related side
effects, which were documented. Acceptable toxicity for the maximum
tolerated dose (MTD) was defined as a group mean BW loss of less
than 20% during the test, and not more than 10% mortality due to TR
deaths. A death was to be classified as TR if it was attributable
to treatment side effects as evidenced by clinical signs and/or
necropsy, or due to unknown causes during the dosing period. A
death was to be classified as NTR if there was evidence that the
death was unrelated to treatment side effects. NTR deaths during
the dosing interval would typically be categorized as NTRa (due to
an accident or human error) or NTRm (due to necropsy-confirmed
tumor dissemination by invasion and/or metastasis). Orally treated
animals that die from unknown causes during the dosing period may
be classified as NTRu when group performance does not support a TR
classification and necropsy, to rule out a dosing error, is not
feasible.
Sampling
[0375] On several days during the study mice were sampled in a
pre-specified fashion. Sampling included non-terminal bleeds (0.25
mL) from the mandibular vein without anesthesia and full volume
blood collection via terminal cardiac puncture under CO2
anesthesia. Blood samples were processed for plasma, with K2-EDTA
as anti-coagulant. The plasma samples were frozen at -80.degree. C.
and stored prior to bioanalysis of compound levels.
[0376] Tumors were harvested from specified mice under RNAse free
conditions and bisected. A 2 mm thick slice from one half of each
tumor was formalin-fixed for 24 h and transferred to 70% ethanol.
The fixed tumor tissues were paraffin embedded. The remaining tumor
tissue from each animal was snap frozen in liquid N2 and pulverized
with a mortar and pestle.
[0377] Specified mice were sampled for the surrogate tissues
including spleen, skin, bone marrow, and whiskers. Each tissue was
isolated and fixed and/or snap frozen.
Statistical and Graphical Analyses
[0378] All statistical and graphical analyses were performed with
Prism 3.03 (GraphPad) for Windows. Several analyses methods were
applied. Median D29 tumor volumes were compared with the
Kruskal-Wallis test, and a post hoc Dunn's multiple comparison
test. These tests were performed three times.
[0379] The two-tailed statistical analyses were conducted at
P=0.05. Prism reports results as non-significant (ns) at P>0.05,
significant (symbolized by "*") at 0.01<P<0.05, very
significant ("**") at 0.001<P<0.01 and extremely significant
("***") at P>0.001.
[0380] To test statistical significance between the control and
treated groups over the whole treatment time course either a
Repeated measures ANOVA test followed by Dunnets multiple
comparison post test or a 2 way ANOVA test were employed.
[0381] For graphical representations s "box and whiskers" diagram
was constructed to show the distribution of individual tumor
volumes for each group. The box represents the 25.sub.th to
75.sub.th percentile of observations, the horizontal line
corresponds to the median value, and the "whiskers" indicate the
maximum and minimum values. Median or mean (.+-.SEM) tumor volumes
were graphed on a semilog or linear plot as functions of time.
Group mean BW changes during the study were plotted as percent
change, .+-.SEM, from Dl.
[0382] A scatter plot was constructed to show TTE values, by group.
The TTE plot includes NTR deaths, which are excluded from all other
graphical analyses. When an animal exited the study because of
tumor size, the final tumor volume recorded for the animal was
included with the data used to calculate the median volume at
subsequent time points. The percentage of animals in each group
remaining in the study versus time was presented in a Kaplan-Meier
survival plot.
Histone Extraction
[0383] For isolation of histones, 60-90 mg tumor tissue was
homogenized in 1.5 ml nuclear extraction buffer (10 mM Tris-HCl, 10
mM MgCl2, 25 mM KCl, 1% Triton X-100, 8.6% Sucrose, plus a Roche
protease inhibitor tablet 1836145) and incubated on ice for 5
minutes. Nuclei were collected by centrifugation at 600 g for 5
minutes at 4.degree. C. and washed once in PBS. Supernatant was
removed and histones extracted for one hour, with vortexing every
15 minutes, with 0.4 N cold sulfuric acid. Extracts were clarified
by centrifugation at 10000 g for 10 minutes at 4.degree. C. and
transferred to a fresh microcentrifuge tube containing 10.times.
volume of ice cold acetone. Histones were precipitated at
-20.degree. C. for 2 hours-overnight, pelleted by centrifugation at
10000 g for 10 minutes and resuspended in water.
ELISA
[0384] Histones were extracted from tumor samples as described
above. Histones were prepared in equivalent concentrations in
coating buffer (PBS+0.05% BSA) yielding 0.5 ng/ul of sample, and
100 ul of sample or standard was added in duplicate to 2 96-well
ELISA plates (Thermo Labsystems, Immulon 4HBX #3885). The plates
were sealed and incubated overnight at 4.degree. C. The following
day, plates were washed 3.times. with 300 ul/well PBST (PBS+0.05%
Tween 20; 10.times.PBST, KPL #51-14-02) on a Bio Tek plate washer.
Plates were blocked with 300 ul/well of diluent (PBS+2% BSA+0.05%
Tween 20), incubated at RT for 2 hours, and washed 3.times. with
PBST. All antibodies were diluted in diluent. 100 ul/well of
anti-H3K27me3 (CST #9733, 50% glycerol stock 1:1,000) or anti-total
H3 (Abcam ab1791, 50% glycerol 1:10,000) was added to each plate.
Plates were incubated for 90 min at RT and washed 3.times. with
PBST. 100 ul/well of anti-Rb-IgG-HRP (Cell Signaling Technology,
7074) was added 1:2,000 to the H3K27Me3 plate and 1:6,000 to the H3
plate and incubated for 90 min at RT. Plates were washed 4.times.
with PB ST. For detection, 100 ul/well of TMB substrate (BioFx
Laboratories, #TMBS) was added and plates incubated in the dark at
RT for 5 min. Reaction was stopped with 100 ul/well 1N H2504.
Absorbance at 450 nm was read on SpectaMax M5 Microplate
reader.
Efficacy Study in SUDHL6 Xenograft Model
[0385] The efficacy of treatment with Compound 44 and in
combination with CHOP on tumor growth inhibition in vivo was
determined in the SUDHL6 xenograft model. Comparison of tumor
growth and survival rate established that the administration of
Compound 44 and CHOP inhibited or delayed tumor growth and
increased survival of the tumor-bearing mice. Athymic nude mice
were subcutaneously injected with 1.times.10.sup.7 SUDHL6 human
lymphoma cells. Compound 44 was administered once a day (QD), twice
a day (BID), or three times a day (TID) at the indicated
concentrations (75 mg/kg, 150 mg/kg, or 225 mg/kg). Mice received
CHOP on day 1 and 8. Tumor volume was measured twice a week until
the endpoint of 60 days or when tumor volume reached 2000 mm.sup.3,
whichever occurred first.
[0386] The combination therapy of Compound 44 and CHOP showed
inhibition of tumor growth over the course of treatment (28 days)
in comparison to mice treated with CHOP or Compound 44 alone (FIG.
6A). Significantly, mice receiving combination therapy of Compound
44 and CHOP exhibited a durable regression of tumor size; 7 out of
12 mice demonstrated complete responses on day 60 in the 225 mg/kg
BID and CHOP combination group (FIG. 6A). A Kaplan-Meier curve was
determined to show the survival of the mice in the study. 57% of
mice that received the combination therapy of Compound 44 and CHOP
survived 60 days after injection (FIG. 6B). Single agent efficacy
with Compound 44 was not observed over the 28 day treatment,
however high intra-group variability may have masked the treatment
effect of Compound 44 as a single agent.
Efficacy Study in WSU-DLCL2 Xenograft Model
[0387] The efficacy of treatment with Compound 44 and in
combination with CHOP on tumor growth inhibition in vivo was
determined in the WSU-DLCL2 xenograft model. Comparison of tumor
growth established that the administration of Compound 44 and CHOP
inhibited or delayed tumor growth of the tumor-bearing mice. SCID
mice were subcutaneously injected with 1.times.10.sup.7 WSU-DLCL2
human lymphoma cells. Compound 44 was administered once a day (QD),
twice a day (BID), or three times a day (TID) at the indicated
concentrations (150 mg/kg, 225 mg/kg, 300 mg/kg, or 600 mg/kg).
Mice received CHOP on day 1 and 22 (CHOP on day 1 and 8 were not
tolerated in SCID mice). Tumor volume was measured twice a week
until the endpoint of 28 days.
[0388] Compound 44 alone and in combination with CHOP therapy
resulted in tumor growth inhibition (FIG. 7A). Administration at
150 mg/kg three times a day (TID) and 225 mg/kg twice a day (BID)
of Compound 44 as a single agent resulted in significantly smaller
tumors by volume when compared to control vehicle treated mice
(p<0.05). Moreover, the combination therapy of Compound 44 and
CHOP showed statistically significant inhibition of tumor growth in
comparison to control vehicle treated mice (p<0.001).
Statistical analyses were calculated using Repeated measure ANOVA
followed by Dunnett post test. Tumor growth inhibition was also
calculated, revealing that treatment of tumors with Compound 44
resulted in greater tumor growth inhibition at all dosages (Table
2). Importantly, the combination treatment of Compound 44 with CHOP
resulted in the greatest tumor growth inhibition.
TABLE-US-00007 TABLE 2 Tumor Growth Inhibition (TGI) of WSU-DLCL2
xenograft model. Group % TGI from day 1 % TGI from day 7 150 mg/kg
TID 73 86 225 mg/kg BID 71 80 300 mg/kg BID 57 67 600 mg/kg QD 58
70 CHOP combo 93 100 CHOP 45 51
[0389] To examine the absorption and distribution of the
administered agents, pharmacokinetic analysis was performed to
determine Compound 44 concentration (ng/mL) in the plasma of
tumor-bearing mice (FIG. 7B). Plasma samples were obtained on day
28 at 5 minutes prior to the last dose ("trough") and 3 hours after
the last dose ("post"). Plasma samples were analyzed by LC-MS/MS to
determine concentration Compound 44 (ng/mL). Compound 44 levels
were not significantly different between mice that received 225
mg/kg of Compound 44 twice a day (BID) as a single agent and 225
mg/kg of Compound 44 (BID) with CHOP at the trough timepoint.
However, 3 hours post-administration of the last dose, Compound 44
levels were significantly increased in mice that received 225 mg/kg
of Compound 44 twice a day (BID) with CHOP compared to single agent
Compound 44.
[0390] Pharmacokinetic analysis was also performed to determine
Compound 44 concentration (ng/g) in tumor tissue harvested from
mice on day 28 at 3 hours after the last administration of
treatment (FIG. 7C).
[0391] Efficacy of target inhibition in vivo was determined by
analysis of histone methylation status in tumors after 28 days of
treatment. Western Blot analysis (FIG. 8A) demonstrated that
methylation of H3K27 was inhibited by Compound 44 at all dosages
and dosing schedules in WSU-DLCL2 tumors. Histones were extracted
as described above. Protein concentrations for acid extracted
histones were determined by BCA assay (Pierce). 400-800 ng of each
lysate was fractionated on 10-20% Tris-Glycine gel (Biorad),
transferred using iBlot (7 minutes on program 3, using
Nitrocellulose transfer stacks), and probed with the following
antibodies in Odyssey blocking buffer: rabbit anti-H3K27me3 (CST
9733; 1:20000 dilution) and mouse anti-Total H3 (CST 3638; 1:20000
dilution). Following primary Ab incubation, membranes were probed
with IRDye 800CW Donkey-anti-mouse IgG (LiCOR #926-32212) and Alexa
Fluor 680 goat-anti-rabbit IgG (Invitrogen #A-21076) secondary Ab
and imaged using the LiCOR Odyssey system. Signal from total
histone H3 protein was used as control.
[0392] Analysis of histone methylation in WSU-DLCL2 tumors by ELISA
confirmed that methylation of histones was inhibited by Compound 44
at all dosages and dosing schedules (FIG. 8B). Tumors from mice
that received vehicle or CHOP did not exhibit any inhibition of
methylation at H3K27. Histones were extracted from tumors after 28
days of treatment and analyzed by ELISA as described above.
Efficacy Study in SUDHL10 Xenograft Model
[0393] The efficacy of treatment with Compound 44 and in
combination with COP (Cyclosphosphamide, Oncovin [vincristine], and
Prednisone) on tumor growth inhibition in vivo was determined in
the SUDHL10 xenograft model. Comparison of tumor growth established
that the administration of Compound 44 and COP inhibited or delayed
tumor growth of the tumor-bearing mice. SCID mice were
subcutaneously injected with 1.times.10.sup.7 SUDHL10 human
lymphoma cells. Compound 44 was administered twice a day (BID), or
three times a day (TID) at the indicated concentrations (125 mg/kg,
250 mg/kg, or 500 mg/kg). Mice received COP on day 1 and 22 (CHOP
on day 1 and 8 were not tolerated in SCID mice). Tumor volume was
measured twice a week until the endpoint of 28 days. Half the
cohort were euthanized after 28 days of treatment, while the other
half were analyzed until a 60 day endpoint for analysis of tumor
growth delay.
[0394] FIG. 9A shows that administration of Compound 44 alone
showed statistically significant tumor growth inhibition as a
single agent at 250 mg/kg and 500 mg/kg when delivered twice a day
(BID), when compared to control vehicle treated mice (p<0.001).
Moreover, the combination therapy of Compound 44 at 250 mg/kg twice
a day (BID) and COP showed statistically significant inhibition of
tumor growth in comparison to control vehicle treated mice
(p<0.001). Statistical analyses were calculated using Repeated
measure ANOVA followed by Dunnett post test. One mouse in the 500
mg/kg BID group was euthanized on day 15 due to poor body
condition. Mice receiving Compound 44 and COP combination therapy
exhibited 8% body weight loss on day 25, in which dosing was
stopped but resumed at day 27. On day 29, 1/16 mice and 2/15 mice
were without tumor in 250 mg/kg and 500 mg/kg group. Strikingly,
10/14 mice receiving Compound 44 and COP combination therapy were
without tumor on day 29.
[0395] Tumor growth inhibition was also calculated, revealing that
treatment of SUDHL10 xenograft tumors with Compound 44, with or
without COP resulted in effective tumor growth inhibition at all
dosages (Table 3).
TABLE-US-00008 TABLE 3 Tumor Growth Inhibition (TGI) in SUDHL10
xenograft model. Group % TGI from day 1 % TGI from day 7 125 mg/kg
BID 54 57 250 mg/kg BID 101 113 500 mg/kg BID 104 115 COP 42 43 COP
combo 107 108
[0396] Tumors were weighed after mice were euthanized on day 28
(FIG. 9B). Tumors from mice receiving single agent administration
of Compound 44 at 125 mg/kg or COP were significantly smaller
compared to control mice (vehicle). Tumors from mice receiving
combination therapy of Compound 44 and COP were significantly
smaller than tumors from mice receiving Compound 44 as a single
agent at both 250 mg/kg and 500 mg/kg dosages.
[0397] Half the cohort was maintained to day 60, to determine
efficacy treatment by determining tumor growth delay (FIG. 9C).
Tumor growth in mice treated with 250 mg/kg and 500 mg/kg doses of
Compound 44 as a single agent exhibited small tumors at day 28,
while control and COP treated mice exhibited large tumors where the
tumors continued to grow after day 28. Strikingly, mice receiving
the combination therapy not only had smaller tumors than all other
treatment groups, but also displayed significant and durable tumor
growth delay.
[0398] FIG. 9D shows a Kaplan-Meier curve depicting the survival
rate of mice treated with Compound 44 alone or in combination in
COP. Administration of Compound 44 as a single agent at 125 mg/kg,
250 mg/kg, and 500 mg/kg increased the survival of mice compared to
control mice or mice treated with COP only. Significantly, combined
administration of Compound 44 with COP improved the survival rate
of mice compared to single agent administration.
[0399] Pharmacokinetic and pharmacodynamic studies were performed
on the SUDHL10 xenograft model. FIG. 10A shows pharmacokinetic
analysis of Compound 44 concentrations in plasma levels (ng/mL).
Plasma samples were obtained from tumor-bearing mice on day 28 at
either 5 minutes prior to the last dose ("trough") or 3 hours after
the last dose ("post"). Levels of Compound 44 was determined by
LC-MS. Pharmacodynamic analysis was performed by ELISA and
determining the ratio of tri-methylated H3K27 in tumor samples.
Pharmacodynamic analysis was also performed in other tissues,
specifically the spleen and bone marrow, of the tumor-bearing mice
to show the efficacy of inhibition of histone methylation by
Compound 44 in normal tissues of the surrogate mouse (FIGS. 10B and
10C).
Example 7: Synergistic Effects of Compound 44 and Anti-Cancer
Agents Combination Therapy
[0400] Methods
[0401] The human lymphoma cell lines WSU-DLCL2 (DSMZ; ACC 575),
SU-DHL-10 (DSMZ; ACC 576), and Toledo (ATCC; CRL-2631) were
obtained from the indicated sources and maintained in RPMI-1640
media supplemented with 10%-20% heat inactivated fetal bovine serum
and 2 mM glutamine. The cells were cultured in tissue culture
flasks in a humidified incubator at 37.degree. C., in an atmosphere
of 5% CO2 and 95% air. The WSU-DLCL2 and SU-DHL-10 contain the Y641
EZH2 mutation, and the Toledo cell line contains WT EZH2.
[0402] The in vitro anti-proliferative effects of the combination
of Compound 44 with the following drugs: AraC, Cisplatin,
Decitabine, Dexamethasone, Everolimus, Prednisolone, and
Doxorubicin were investigated. WSU-DLCL2, SU-DHL-10, or Toledo
human lymphoma cells were treated with increasing concentrations of
Compound 44 in flasks. After 4 days of treatment, the WSU-DLCL2 or
SU-DHL-10 cells were split to initial seeding density and plated
into a 96-well tissue culture plate with each concentration of
Compound 44 in one row of the plate. After 6 days the Toledo cells
were split to initial seeding density and plated into a 96-well
tissue culture plate with each concentration of Compound 44 in one
row of the plate. Increasing doses of drugs were then added to the
plates. One dose per column forming a matrix of Compound 44 and
drug doses. After incubation for an additional 3 days cell
viability of WSU-DLCL2 or SU-DHL-10 was measured using Promega Cell
Titer-Glo reagent followed by luminescence detection. After 5 days
viability of Toledo cells was measured using Promega Cell Titer-Glo
reagent followed by luminescence detection.
[0403] Data Analysis
[0404] Synergy was determined using the software package Calcusyn
by Biosoft based on the Chou-Talalay method for drug combination
which uses the median-effect equation (Chou 2006). First, raw
luminescence values were converted to percent inhibition or
fraction affected (Fa) calculated using controls for maximum
inhibition and DMSO treated cells for minimum inhibition control
located on each plate. Percent inhibition values for each constant
ratio of compound combinations were entered into Calcusyn to
determine the combination index values. Combination index values
less than 1 indicated synergy.
[0405] For test compounds that did not inhibit cells viability by
50%, synergy cannot be determined using Calcusyn. Instead, the data
was reported as fold IC50 shift of the IC50 for Compound 44.
Alternatively, if Compound 44 did not have an effect, as with the
Toledo cells, the fold IC50 shift was reported for the drug instead
of Compound 44.
[0406] Results
[0407] Treatment of WSU-DLCL2 and SU-DHL-10 cells with Compound 44
and either AraC, Cisplatin, Doxorubicin, Decitabine, or Everolimus
demonstrated synergistic reduction in cell viability. Combination
index values for combinations with Compound 44 and Decitabine in
WSU-DLCL2 and SU-DHL-10 cells were below 0.1 which is denoted by
very strong synergism according to the Chou-Talalay
characterization (Chou 2006). Combination index values for
combinations with Compound 44 and Everolimus in WSU-DLCL2 cells
were below 0.1 and SU-DHL-10 cells between 0.1-0.3 which is denoted
by very strong synergism and strong synergism respectively.
Combinations with Compound 44 and either AraC, Cisplatin, or
Doxorubicin in WSU-DLCL2 and SU-DHL-10 cells had combination index
values between 0.3-0.7 which denotes synergism (Chou 2006).
Combination of Compound 44 with Prednisolone enhanced the Compound
44 potency by 7-fold for WSU-DLCL2 and 3-fold for SU-DHL-10 cells
at the top dose of Prednisolone. Additionally, combination with
Compound 44 and Dexamethasone enhanced the Compound 44 potency by
17-fold for WSU-DLCL2 cells and 3-fold for SU-DHL-10 cells at the
top does of Dexamethasone.
[0408] The Toledo cell line showed no sensitivity to Compound 44
and no combination benefit was seen when Compound 44 was combined
with either AraC, Cisplatin, Doxorubicin, Decitabine, Everolimus,
Prednisolone, or Dexamethasone. Thus the combination benefit seen
in these experiments appears to be dependent on EZH2 mutation.
TABLE-US-00009 TABLE 4 List of compounds and cell lines WSU SUDHL10
Toledo Prednisolone 7-fold 3-fold enhancement No enhancement
enhancement Doxorubicin Synergism Synergism No enhancement CI
0.3-0.7 CI 0.3-0.7 Cisplatin Synergism n/a No enhancement CI
0.3-0.7 AraC Synergism Synergism No enhancement CI 0.3-0.7 CI
0.3-0.7 Everolimus Very strong Strong synergism No enhancement
synergism CI <0.1 CI 0.1-0.3 Decitabine Very strong Very strong
No enhancement synergism synergism CI <0.1 CI <0.1
Dexamethasone 17-fold 3-fold enhancement No enhancement
enhancement
INCORPORATION BY REFERENCE
[0409] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an admission that any is
pertinent prior art, nor does it constitute any admission as to the
contents or date of the same. The invention having now been
described by way of written description, those of skill in the art
will recognize that the invention can be practiced in a variety of
embodiments and that the foregoing description and examples below
are for purposes of illustration and not limitation of the claims
that follow.
EQUIVALENTS
[0410] The invention can be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
Sequence CWU 1
1
121746PRTHomo sapiens 1Met Gly Gln Thr Gly Lys Lys Ser Glu Lys Gly
Pro Val Cys Trp Arg1 5 10 15Lys Arg Val Lys Ser Glu Tyr Met Arg Leu
Arg Gln Leu Lys Arg Phe 20 25 30Arg Arg Ala Asp Glu Val Lys Ser Met
Phe Ser Ser Asn Arg Gln Lys 35 40 45Ile Leu Glu Arg Thr Glu Ile Leu
Asn Gln Glu Trp Lys Gln Arg Arg 50 55 60Ile Gln Pro Val His Ile Leu
Thr Ser Val Ser Ser Leu Arg Gly Thr65 70 75 80Arg Glu Cys Ser Val
Thr Ser Asp Leu Asp Phe Pro Thr Gln Val Ile 85 90 95Pro Leu Lys Thr
Leu Asn Ala Val Ala Ser Val Pro Ile Met Tyr Ser 100 105 110Trp Ser
Pro Leu Gln Gln Asn Phe Met Val Glu Asp Glu Thr Val Leu 115 120
125His Asn Ile Pro Tyr Met Gly Asp Glu Val Leu Asp Gln Asp Gly Thr
130 135 140Phe Ile Glu Glu Leu Ile Lys Asn Tyr Asp Gly Lys Val His
Gly Asp145 150 155 160Arg Glu Cys Gly Phe Ile Asn Asp Glu Ile Phe
Val Glu Leu Val Asn 165 170 175Ala Leu Gly Gln Tyr Asn Asp Asp Asp
Asp Asp Asp Asp Gly Asp Asp 180 185 190Pro Glu Glu Arg Glu Glu Lys
Gln Lys Asp Leu Glu Asp His Arg Asp 195 200 205Asp Lys Glu Ser Arg
Pro Pro Arg Lys Phe Pro Ser Asp Lys Ile Phe 210 215 220Glu Ala Ile
Ser Ser Met Phe Pro Asp Lys Gly Thr Ala Glu Glu Leu225 230 235
240Lys Glu Lys Tyr Lys Glu Leu Thr Glu Gln Gln Leu Pro Gly Ala Leu
245 250 255Pro Pro Glu Cys Thr Pro Asn Ile Asp Gly Pro Asn Ala Lys
Ser Val 260 265 270Gln Arg Glu Gln Ser Leu His Ser Phe His Thr Leu
Phe Cys Arg Arg 275 280 285Cys Phe Lys Tyr Asp Cys Phe Leu His Pro
Phe His Ala Thr Pro Asn 290 295 300Thr Tyr Lys Arg Lys Asn Thr Glu
Thr Ala Leu Asp Asn Lys Pro Cys305 310 315 320Gly Pro Gln Cys Tyr
Gln His Leu Glu Gly Ala Lys Glu Phe Ala Ala 325 330 335Ala Leu Thr
Ala Glu Arg Ile Lys Thr Pro Pro Lys Arg Pro Gly Gly 340 345 350Arg
Arg Arg Gly Arg Leu Pro Asn Asn Ser Ser Arg Pro Ser Thr Pro 355 360
365Thr Ile Asn Val Leu Glu Ser Lys Asp Thr Asp Ser Asp Arg Glu Ala
370 375 380Gly Thr Glu Thr Gly Gly Glu Asn Asn Asp Lys Glu Glu Glu
Glu Lys385 390 395 400Lys Asp Glu Thr Ser Ser Ser Ser Glu Ala Asn
Ser Arg Cys Gln Thr 405 410 415Pro Ile Lys Met Lys Pro Asn Ile Glu
Pro Pro Glu Asn Val Glu Trp 420 425 430Ser Gly Ala Glu Ala Ser Met
Phe Arg Val Leu Ile Gly Thr Tyr Tyr 435 440 445Asp Asn Phe Cys Ala
Ile Ala Arg Leu Ile Gly Thr Lys Thr Cys Arg 450 455 460Gln Val Tyr
Glu Phe Arg Val Lys Glu Ser Ser Ile Ile Ala Pro Ala465 470 475
480Pro Ala Glu Asp Val Asp Thr Pro Pro Arg Lys Lys Lys Arg Lys His
485 490 495Arg Leu Trp Ala Ala His Cys Arg Lys Ile Gln Leu Lys Lys
Asp Gly 500 505 510Ser Ser Asn His Val Tyr Asn Tyr Gln Pro Cys Asp
His Pro Arg Gln 515 520 525Pro Cys Asp Ser Ser Cys Pro Cys Val Ile
Ala Gln Asn Phe Cys Glu 530 535 540Lys Phe Cys Gln Cys Ser Ser Glu
Cys Gln Asn Arg Phe Pro Gly Cys545 550 555 560Arg Cys Lys Ala Gln
Cys Asn Thr Lys Gln Cys Pro Cys Tyr Leu Ala 565 570 575Val Arg Glu
Cys Asp Pro Asp Leu Cys Leu Thr Cys Gly Ala Ala Asp 580 585 590His
Trp Asp Ser Lys Asn Val Ser Cys Lys Asn Cys Ser Ile Gln Arg 595 600
605Gly Ser Lys Lys His Leu Leu Leu Ala Pro Ser Asp Val Ala Gly Trp
610 615 620Gly Ile Phe Ile Lys Asp Pro Val Gln Lys Asn Glu Phe Ile
Ser Glu625 630 635 640Tyr Cys Gly Glu Ile Ile Ser Gln Asp Glu Ala
Asp Arg Arg Gly Lys 645 650 655Val Tyr Asp Lys Tyr Met Cys Ser Phe
Leu Phe Asn Leu Asn Asn Asp 660 665 670Phe Val Val Asp Ala Thr Arg
Lys Gly Asn Lys Ile Arg Phe Ala Asn 675 680 685His Ser Val Asn Pro
Asn Cys Tyr Ala Lys Val Met Met Val Asn Gly 690 695 700Asp His Arg
Ile Gly Ile Phe Ala Lys Arg Ala Ile Gln Thr Gly Glu705 710 715
720Glu Leu Phe Phe Asp Tyr Arg Tyr Ser Gln Ala Asp Ala Leu Lys Tyr
725 730 735Val Gly Ile Glu Arg Glu Met Glu Ile Pro 740
74522723DNAHomo sapiens 2ggcggcgctt gattgggctg ggggggccaa
ataaaagcga tggcgattgg gctgccgcgt 60ttggcgctcg gtccggtcgc gtccgacacc
cggtgggact cagaaggcag tggagccccg 120gcggcggcgg cggcggcgcg
cgggggcgac gcgcgggaac aacgcgagtc ggcgcgcggg 180acgaagaata
atcatgggcc agactgggaa gaaatctgag aagggaccag tttgttggcg
240gaagcgtgta aaatcagagt acatgcgact gagacagctc aagaggttca
gacgagctga 300tgaagtaaag agtatgttta gttccaatcg tcagaaaatt
ttggaaagaa cggaaatctt 360aaaccaagaa tggaaacagc gaaggataca
gcctgtgcac atcctgactt ctgtgagctc 420attgcgcggg actagggagt
gttcggtgac cagtgacttg gattttccaa cacaagtcat 480cccattaaag
actctgaatg cagttgcttc agtacccata atgtattctt ggtctcccct
540acagcagaat tttatggtgg aagatgaaac tgttttacat aacattcctt
atatgggaga 600tgaagtttta gatcaggatg gtactttcat tgaagaacta
ataaaaaatt atgatgggaa 660agtacacggg gatagagaat gtgggtttat
aaatgatgaa atttttgtgg agttggtgaa 720tgcccttggt caatataatg
atgatgacga tgatgatgat ggagacgatc ctgaagaaag 780agaagaaaag
cagaaagatc tggaggatca ccgagatgat aaagaaagcc gcccacctcg
840gaaatttcct tctgataaaa tttttgaagc catttcctca atgtttccag
ataagggcac 900agcagaagaa ctaaaggaaa aatataaaga actcaccgaa
cagcagctcc caggcgcact 960tcctcctgaa tgtaccccca acatagatgg
accaaatgct aaatctgttc agagagagca 1020aagcttacac tcctttcata
cgcttttctg taggcgatgt tttaaatatg actgcttcct 1080acatcgtaag
tgcaattatt cttttcatgc aacacccaac acttataagc ggaagaacac
1140agaaacagct ctagacaaca aaccttgtgg accacagtgt taccagcatt
tggagggagc 1200aaaggagttt gctgctgctc tcaccgctga gcggataaag
accccaccaa aacgtccagg 1260aggccgcaga agaggacggc ttcccaataa
cagtagcagg cccagcaccc ccaccattaa 1320tgtgctggaa tcaaaggata
cagacagtga tagggaagca gggactgaaa cggggggaga 1380gaacaatgat
aaagaagaag aagagaagaa agatgaaact tcgagctcct ctgaagcaaa
1440ttctcggtgt caaacaccaa taaagatgaa gccaaatatt gaacctcctg
agaatgtgga 1500gtggagtggt gctgaagcct caatgtttag agtcctcatt
ggcacttact atgacaattt 1560ctgtgccatt gctaggttaa ttgggaccaa
aacatgtaga caggtgtatg agtttagagt 1620caaagaatct agcatcatag
ctccagctcc cgctgaggat gtggatactc ctccaaggaa 1680aaagaagagg
aaacaccggt tgtgggctgc acactgcaga aagatacagc tgaaaaagga
1740cggctcctct aaccatgttt acaactatca accctgtgat catccacggc
agccttgtga 1800cagttcgtgc ccttgtgtga tagcacaaaa tttttgtgaa
aagttttgtc aatgtagttc 1860agagtgtcaa aaccgctttc cgggatgccg
ctgcaaagca cagtgcaaca ccaagcagtg 1920cccgtgctac ctggctgtcc
gagagtgtga ccctgacctc tgtcttactt gtggagccgc 1980tgaccattgg
gacagtaaaa atgtgtcctg caagaactgc agtattcagc ggggctccaa
2040aaagcatcta ttgctggcac catctgacgt ggcaggctgg gggattttta
tcaaagatcc 2100tgtgcagaaa aatgaattca tctcagaata ctgtggagag
attatttctc aagatgaagc 2160tgacagaaga gggaaagtgt atgataaata
catgtgcagc tttctgttca acttgaacaa 2220tgattttgtg gtggatgcaa
cccgcaaggg taacaaaatt cgttttgcaa atcattcggt 2280aaatccaaac
tgctatgcaa aagttatgat ggttaacggt gatcacagga taggtatttt
2340tgccaagaga gccatccaga ctggcgaaga gctgtttttt gattacagat
acagccaggc 2400tgatgccctg aagtatgtcg gcatcgaaag agaaatggaa
atcccttgac atctgctacc 2460tcctcccccc tcctctgaaa cagctgcctt
agcttcagga acctcgagta ctgtgggcaa 2520tttagaaaaa gaacatgcag
tttgaaattc tgaatttgca aagtactgta agaataattt 2580atagtaatga
gtttaaaaat caacttttta ttgccttctc accagctgca aagtgttttg
2640taccagtgaa tttttgcaat aatgcagtat ggtacatttt tcaactttga
ataaagaata 2700cttgaacttg tccttgttga atc 27233751PRTHomo sapiens
3Met Gly Gln Thr Gly Lys Lys Ser Glu Lys Gly Pro Val Cys Trp Arg1 5
10 15Lys Arg Val Lys Ser Glu Tyr Met Arg Leu Arg Gln Leu Lys Arg
Phe 20 25 30Arg Arg Ala Asp Glu Val Lys Ser Met Phe Ser Ser Asn Arg
Gln Lys 35 40 45Ile Leu Glu Arg Thr Glu Ile Leu Asn Gln Glu Trp Lys
Gln Arg Arg 50 55 60Ile Gln Pro Val His Ile Leu Thr Ser Val Ser Ser
Leu Arg Gly Thr65 70 75 80Arg Glu Cys Ser Val Thr Ser Asp Leu Asp
Phe Pro Thr Gln Val Ile 85 90 95Pro Leu Lys Thr Leu Asn Ala Val Ala
Ser Val Pro Ile Met Tyr Ser 100 105 110Trp Ser Pro Leu Gln Gln Asn
Phe Met Val Glu Asp Glu Thr Val Leu 115 120 125His Asn Ile Pro Tyr
Met Gly Asp Glu Val Leu Asp Gln Asp Gly Thr 130 135 140Phe Ile Glu
Glu Leu Ile Lys Asn Tyr Asp Gly Lys Val His Gly Asp145 150 155
160Arg Glu Cys Gly Phe Ile Asn Asp Glu Ile Phe Val Glu Leu Val Asn
165 170 175Ala Leu Gly Gln Tyr Asn Asp Asp Asp Asp Asp Asp Asp Gly
Asp Asp 180 185 190Pro Glu Glu Arg Glu Glu Lys Gln Lys Asp Leu Glu
Asp His Arg Asp 195 200 205Asp Lys Glu Ser Arg Pro Pro Arg Lys Phe
Pro Ser Asp Lys Ile Phe 210 215 220Glu Ala Ile Ser Ser Met Phe Pro
Asp Lys Gly Thr Ala Glu Glu Leu225 230 235 240Lys Glu Lys Tyr Lys
Glu Leu Thr Glu Gln Gln Leu Pro Gly Ala Leu 245 250 255Pro Pro Glu
Cys Thr Pro Asn Ile Asp Gly Pro Asn Ala Lys Ser Val 260 265 270Gln
Arg Glu Gln Ser Leu His Ser Phe His Thr Leu Phe Cys Arg Arg 275 280
285Cys Phe Lys Tyr Asp Cys Phe Leu His Arg Lys Cys Asn Tyr Ser Phe
290 295 300His Ala Thr Pro Asn Thr Tyr Lys Arg Lys Asn Thr Glu Thr
Ala Leu305 310 315 320Asp Asn Lys Pro Cys Gly Pro Gln Cys Tyr Gln
His Leu Glu Gly Ala 325 330 335Lys Glu Phe Ala Ala Ala Leu Thr Ala
Glu Arg Ile Lys Thr Pro Pro 340 345 350Lys Arg Pro Gly Gly Arg Arg
Arg Gly Arg Leu Pro Asn Asn Ser Ser 355 360 365Arg Pro Ser Thr Pro
Thr Ile Asn Val Leu Glu Ser Lys Asp Thr Asp 370 375 380Ser Asp Arg
Glu Ala Gly Thr Glu Thr Gly Gly Glu Asn Asn Asp Lys385 390 395
400Glu Glu Glu Glu Lys Lys Asp Glu Thr Ser Ser Ser Ser Glu Ala Asn
405 410 415Ser Arg Cys Gln Thr Pro Ile Lys Met Lys Pro Asn Ile Glu
Pro Pro 420 425 430Glu Asn Val Glu Trp Ser Gly Ala Glu Ala Ser Met
Phe Arg Val Leu 435 440 445Ile Gly Thr Tyr Tyr Asp Asn Phe Cys Ala
Ile Ala Arg Leu Ile Gly 450 455 460Thr Lys Thr Cys Arg Gln Val Tyr
Glu Phe Arg Val Lys Glu Ser Ser465 470 475 480Ile Ile Ala Pro Ala
Pro Ala Glu Asp Val Asp Thr Pro Pro Arg Lys 485 490 495Lys Lys Arg
Lys His Arg Leu Trp Ala Ala His Cys Arg Lys Ile Gln 500 505 510Leu
Lys Lys Asp Gly Ser Ser Asn His Val Tyr Asn Tyr Gln Pro Cys 515 520
525Asp His Pro Arg Gln Pro Cys Asp Ser Ser Cys Pro Cys Val Ile Ala
530 535 540Gln Asn Phe Cys Glu Lys Phe Cys Gln Cys Ser Ser Glu Cys
Gln Asn545 550 555 560Arg Phe Pro Gly Cys Arg Cys Lys Ala Gln Cys
Asn Thr Lys Gln Cys 565 570 575Pro Cys Tyr Leu Ala Val Arg Glu Cys
Asp Pro Asp Leu Cys Leu Thr 580 585 590Cys Gly Ala Ala Asp His Trp
Asp Ser Lys Asn Val Ser Cys Lys Asn 595 600 605Cys Ser Ile Gln Arg
Gly Ser Lys Lys His Leu Leu Leu Ala Pro Ser 610 615 620Asp Val Ala
Gly Trp Gly Ile Phe Ile Lys Asp Pro Val Gln Lys Asn625 630 635
640Glu Phe Ile Ser Glu Tyr Cys Gly Glu Ile Ile Ser Gln Asp Glu Ala
645 650 655Asp Arg Arg Gly Lys Val Tyr Asp Lys Tyr Met Cys Ser Phe
Leu Phe 660 665 670Asn Leu Asn Asn Asp Phe Val Val Asp Ala Thr Arg
Lys Gly Asn Lys 675 680 685Ile Arg Phe Ala Asn His Ser Val Asn Pro
Asn Cys Tyr Ala Lys Val 690 695 700Met Met Val Asn Gly Asp His Arg
Ile Gly Ile Phe Ala Lys Arg Ala705 710 715 720Ile Gln Thr Gly Glu
Glu Leu Phe Phe Asp Tyr Arg Tyr Ser Gln Ala 725 730 735Asp Ala Leu
Lys Tyr Val Gly Ile Glu Arg Glu Met Glu Ile Pro 740 745
75042591DNAHomo sapiens 4ggcggcgctt gattgggctg ggggggccaa
ataaaagcga tggcgattgg gctgccgcgt 60ttggcgctcg gtccggtcgc gtccgacacc
cggtgggact cagaaggcag tggagccccg 120gcggcggcgg cggcggcgcg
cgggggcgac gcgcgggaac aacgcgagtc ggcgcgcggg 180acgaagaata
atcatgggcc agactgggaa gaaatctgag aagggaccag tttgttggcg
240gaagcgtgta aaatcagagt acatgcgact gagacagctc aagaggttca
gacgagctga 300tgaagtaaag agtatgttta gttccaatcg tcagaaaatt
ttggaaagaa cggaaatctt 360aaaccaagaa tggaaacagc gaaggataca
gcctgtgcac atcctgactt ctgtgagctc 420attgcgcggg actagggagg
tggaagatga aactgtttta cataacattc cttatatggg 480agatgaagtt
ttagatcagg atggtacttt cattgaagaa ctaataaaaa attatgatgg
540gaaagtacac ggggatagag aatgtgggtt tataaatgat gaaatttttg
tggagttggt 600gaatgccctt ggtcaatata atgatgatga cgatgatgat
gatggagacg atcctgaaga 660aagagaagaa aagcagaaag atctggagga
tcaccgagat gataaagaaa gccgcccacc 720tcggaaattt ccttctgata
aaatttttga agccatttcc tcaatgtttc cagataaggg 780cacagcagaa
gaactaaagg aaaaatataa agaactcacc gaacagcagc tcccaggcgc
840acttcctcct gaatgtaccc ccaacataga tggaccaaat gctaaatctg
ttcagagaga 900gcaaagctta cactcctttc atacgctttt ctgtaggcga
tgttttaaat atgactgctt 960cctacatcct tttcatgcaa cacccaacac
ttataagcgg aagaacacag aaacagctct 1020agacaacaaa ccttgtggac
cacagtgtta ccagcatttg gagggagcaa aggagtttgc 1080tgctgctctc
accgctgagc ggataaagac cccaccaaaa cgtccaggag gccgcagaag
1140aggacggctt cccaataaca gtagcaggcc cagcaccccc accattaatg
tgctggaatc 1200aaaggataca gacagtgata gggaagcagg gactgaaacg
gggggagaga acaatgataa 1260agaagaagaa gagaagaaag atgaaacttc
gagctcctct gaagcaaatt ctcggtgtca 1320aacaccaata aagatgaagc
caaatattga acctcctgag aatgtggagt ggagtggtgc 1380tgaagcctca
atgtttagag tcctcattgg cacttactat gacaatttct gtgccattgc
1440taggttaatt gggaccaaaa catgtagaca ggtgtatgag tttagagtca
aagaatctag 1500catcatagct ccagctcccg ctgaggatgt ggatactcct
ccaaggaaaa agaagaggaa 1560acaccggttg tgggctgcac actgcagaaa
gatacagctg aaaaaggacg gctcctctaa 1620ccatgtttac aactatcaac
cctgtgatca tccacggcag ccttgtgaca gttcgtgccc 1680ttgtgtgata
gcacaaaatt tttgtgaaaa gttttgtcaa tgtagttcag agtgtcaaaa
1740ccgctttccg ggatgccgct gcaaagcaca gtgcaacacc aagcagtgcc
cgtgctacct 1800ggctgtccga gagtgtgacc ctgacctctg tcttacttgt
ggagccgctg accattggga 1860cagtaaaaat gtgtcctgca agaactgcag
tattcagcgg ggctccaaaa agcatctatt 1920gctggcacca tctgacgtgg
caggctgggg gatttttatc aaagatcctg tgcagaaaaa 1980tgaattcatc
tcagaatact gtggagagat tatttctcaa gatgaagctg acagaagagg
2040gaaagtgtat gataaataca tgtgcagctt tctgttcaac ttgaacaatg
attttgtggt 2100ggatgcaacc cgcaagggta acaaaattcg ttttgcaaat
cattcggtaa atccaaactg 2160ctatgcaaaa gttatgatgg ttaacggtga
tcacaggata ggtatttttg ccaagagagc 2220catccagact ggcgaagagc
tgttttttga ttacagatac agccaggctg atgccctgaa 2280gtatgtcggc
atcgaaagag aaatggaaat cccttgacat ctgctacctc ctcccccctc
2340ctctgaaaca gctgccttag cttcaggaac ctcgagtact gtgggcaatt
tagaaaaaga 2400acatgcagtt tgaaattctg aatttgcaaa gtactgtaag
aataatttat agtaatgagt 2460ttaaaaatca actttttatt gccttctcac
cagctgcaaa gtgttttgta ccagtgaatt 2520tttgcaataa tgcagtatgg
tacatttttc aactttgaat aaagaatact tgaacttgtc 2580cttgttgaat c
25915707PRTHomo sapiens 5Met Gly Gln Thr Gly Lys Lys Ser Glu Lys
Gly Pro Val Cys Trp Arg1 5 10 15Lys Arg Val Lys Ser Glu Tyr Met Arg
Leu Arg Gln Leu Lys Arg Phe 20 25 30Arg Arg Ala Asp Glu Val Lys Ser
Met Phe Ser Ser Asn Arg Gln Lys 35 40 45Ile Leu Glu Arg Thr Glu Ile
Leu Asn Gln Glu Trp Lys Gln Arg Arg 50 55 60Ile Gln Pro Val His Ile
Leu Thr Ser Val Ser Ser Leu Arg Gly Thr65
70 75 80Arg Glu Val Glu Asp Glu Thr Val Leu His Asn Ile Pro Tyr Met
Gly 85 90 95Asp Glu Val Leu Asp Gln Asp Gly Thr Phe Ile Glu Glu Leu
Ile Lys 100 105 110Asn Tyr Asp Gly Lys Val His Gly Asp Arg Glu Cys
Gly Phe Ile Asn 115 120 125Asp Glu Ile Phe Val Glu Leu Val Asn Ala
Leu Gly Gln Tyr Asn Asp 130 135 140Asp Asp Asp Asp Asp Asp Gly Asp
Asp Pro Glu Glu Arg Glu Glu Lys145 150 155 160Gln Lys Asp Leu Glu
Asp His Arg Asp Asp Lys Glu Ser Arg Pro Pro 165 170 175Arg Lys Phe
Pro Ser Asp Lys Ile Phe Glu Ala Ile Ser Ser Met Phe 180 185 190Pro
Asp Lys Gly Thr Ala Glu Glu Leu Lys Glu Lys Tyr Lys Glu Leu 195 200
205Thr Glu Gln Gln Leu Pro Gly Ala Leu Pro Pro Glu Cys Thr Pro Asn
210 215 220Ile Asp Gly Pro Asn Ala Lys Ser Val Gln Arg Glu Gln Ser
Leu His225 230 235 240Ser Phe His Thr Leu Phe Cys Arg Arg Cys Phe
Lys Tyr Asp Cys Phe 245 250 255Leu His Pro Phe His Ala Thr Pro Asn
Thr Tyr Lys Arg Lys Asn Thr 260 265 270Glu Thr Ala Leu Asp Asn Lys
Pro Cys Gly Pro Gln Cys Tyr Gln His 275 280 285Leu Glu Gly Ala Lys
Glu Phe Ala Ala Ala Leu Thr Ala Glu Arg Ile 290 295 300Lys Thr Pro
Pro Lys Arg Pro Gly Gly Arg Arg Arg Gly Arg Leu Pro305 310 315
320Asn Asn Ser Ser Arg Pro Ser Thr Pro Thr Ile Asn Val Leu Glu Ser
325 330 335Lys Asp Thr Asp Ser Asp Arg Glu Ala Gly Thr Glu Thr Gly
Gly Glu 340 345 350Asn Asn Asp Lys Glu Glu Glu Glu Lys Lys Asp Glu
Thr Ser Ser Ser 355 360 365Ser Glu Ala Asn Ser Arg Cys Gln Thr Pro
Ile Lys Met Lys Pro Asn 370 375 380Ile Glu Pro Pro Glu Asn Val Glu
Trp Ser Gly Ala Glu Ala Ser Met385 390 395 400Phe Arg Val Leu Ile
Gly Thr Tyr Tyr Asp Asn Phe Cys Ala Ile Ala 405 410 415Arg Leu Ile
Gly Thr Lys Thr Cys Arg Gln Val Tyr Glu Phe Arg Val 420 425 430Lys
Glu Ser Ser Ile Ile Ala Pro Ala Pro Ala Glu Asp Val Asp Thr 435 440
445Pro Pro Arg Lys Lys Lys Arg Lys His Arg Leu Trp Ala Ala His Cys
450 455 460Arg Lys Ile Gln Leu Lys Lys Asp Gly Ser Ser Asn His Val
Tyr Asn465 470 475 480Tyr Gln Pro Cys Asp His Pro Arg Gln Pro Cys
Asp Ser Ser Cys Pro 485 490 495Cys Val Ile Ala Gln Asn Phe Cys Glu
Lys Phe Cys Gln Cys Ser Ser 500 505 510Glu Cys Gln Asn Arg Phe Pro
Gly Cys Arg Cys Lys Ala Gln Cys Asn 515 520 525Thr Lys Gln Cys Pro
Cys Tyr Leu Ala Val Arg Glu Cys Asp Pro Asp 530 535 540Leu Cys Leu
Thr Cys Gly Ala Ala Asp His Trp Asp Ser Lys Asn Val545 550 555
560Ser Cys Lys Asn Cys Ser Ile Gln Arg Gly Ser Lys Lys His Leu Leu
565 570 575Leu Ala Pro Ser Asp Val Ala Gly Trp Gly Ile Phe Ile Lys
Asp Pro 580 585 590Val Gln Lys Asn Glu Phe Ile Ser Glu Tyr Cys Gly
Glu Ile Ile Ser 595 600 605Gln Asp Glu Ala Asp Arg Arg Gly Lys Val
Tyr Asp Lys Tyr Met Cys 610 615 620Ser Phe Leu Phe Asn Leu Asn Asn
Asp Phe Val Val Asp Ala Thr Arg625 630 635 640Lys Gly Asn Lys Ile
Arg Phe Ala Asn His Ser Val Asn Pro Asn Cys 645 650 655Tyr Ala Lys
Val Met Met Val Asn Gly Asp His Arg Ile Gly Ile Phe 660 665 670Ala
Lys Arg Ala Ile Gln Thr Gly Glu Glu Leu Phe Phe Asp Tyr Arg 675 680
685Tyr Ser Gln Ala Asp Ala Leu Lys Tyr Val Gly Ile Glu Arg Glu Met
690 695 700Glu Ile Pro7056200PRTHomo sapiens 6Ser Cys Pro Cys Val
Ile Ala Gln Asn Phe Cys Glu Lys Phe Cys Gln1 5 10 15Cys Ser Ser Glu
Cys Gln Asn Arg Phe Pro Gly Cys Arg Cys Lys Ala 20 25 30Gln Cys Asn
Thr Lys Gln Cys Pro Cys Tyr Leu Ala Val Arg Glu Cys 35 40 45Asp Pro
Asp Leu Cys Leu Thr Cys Gly Ala Ala Asp His Trp Asp Ser 50 55 60Lys
Asn Val Ser Cys Lys Asn Cys Ser Ile Gln Arg Gly Ser Lys Lys65 70 75
80His Leu Leu Leu Ala Pro Ser Asp Val Ala Gly Trp Gly Ile Phe Ile
85 90 95Lys Asp Pro Val Gln Lys Asn Glu Phe Ile Ser Glu Tyr Cys Gly
Glu 100 105 110Ile Ile Ser Gln Asp Glu Ala Asp Arg Arg Gly Lys Val
Tyr Asp Lys 115 120 125Tyr Met Cys Ser Phe Leu Phe Asn Leu Asn Asn
Asp Phe Val Val Asp 130 135 140Ala Thr Arg Lys Gly Asn Lys Ile Arg
Phe Ala Asn His Ser Val Asn145 150 155 160Pro Asn Cys Tyr Ala Lys
Val Met Met Val Asn Gly Asp His Arg Ile 165 170 175Gly Ile Phe Ala
Lys Arg Ala Ile Gln Thr Gly Glu Glu Leu Phe Phe 180 185 190Asp Tyr
Arg Tyr Ser Gln Ala Asp 195 2007114PRTHomo sapiens 7His Leu Leu Leu
Ala Pro Ser Asp Val Ala Gly Trp Gly Ile Phe Ile1 5 10 15Lys Asp Pro
Val Gln Lys Asn Glu Phe Ile Ser Glu Tyr Cys Gly Glu 20 25 30Ile Ile
Ser Gln Asp Glu Ala Asp Arg Arg Gly Lys Val Tyr Asp Lys 35 40 45Tyr
Met Cys Ser Phe Leu Phe Asn Leu Asn Asn Asp Phe Val Val Asp 50 55
60Ala Thr Arg Lys Gly Asn Lys Ile Arg Phe Ala Asn His Ser Val Asn65
70 75 80Pro Asn Cys Tyr Ala Lys Val Met Met Val Asn Gly Asp His Arg
Ile 85 90 95Gly Ile Phe Ala Lys Arg Ala Ile Gln Thr Gly Glu Glu Leu
Phe Phe 100 105 110Asp Tyr8342DNAHomo sapiens 8catctattgc
tggcaccatc tgacgtggca ggctggggga tttttatcaa agatcctgtg 60cagaaaaatg
aattcatctc agaatactgt ggagagatta tttctcaaga tgaagctgac
120agaagaggga aagtgtatga taaatacatg tgcagctttc tgttcaactt
gaacaatgat 180tttgtggtgg atgcaacccg caagggtaac aaaattcgtt
ttgcaaatca ttcggtaaat 240ccaaactgct atgcaaaagt tatgatggtt
aacggtgatc acaggatagg tatttttgcc 300aagagagcca tccagactgg
cgaagagctg ttttttgatt ac 3429746PRTHomo
sapiensMISC_FEATURE(641)..(641)X is any amino acid other than
tyrosine (Y). 9Met Gly Gln Thr Gly Lys Lys Ser Glu Lys Gly Pro Val
Cys Trp Arg1 5 10 15Lys Arg Val Lys Ser Glu Tyr Met Arg Leu Arg Gln
Leu Lys Arg Phe 20 25 30Arg Arg Ala Asp Glu Val Lys Ser Met Phe Ser
Ser Asn Arg Gln Lys 35 40 45Ile Leu Glu Arg Thr Glu Ile Leu Asn Gln
Glu Trp Lys Gln Arg Arg 50 55 60Ile Gln Pro Val His Ile Leu Thr Ser
Val Ser Ser Leu Arg Gly Thr65 70 75 80Arg Glu Cys Ser Val Thr Ser
Asp Leu Asp Phe Pro Thr Gln Val Ile 85 90 95Pro Leu Lys Thr Leu Asn
Ala Val Ala Ser Val Pro Ile Met Tyr Ser 100 105 110Trp Ser Pro Leu
Gln Gln Asn Phe Met Val Glu Asp Glu Thr Val Leu 115 120 125His Asn
Ile Pro Tyr Met Gly Asp Glu Val Leu Asp Gln Asp Gly Thr 130 135
140Phe Ile Glu Glu Leu Ile Lys Asn Tyr Asp Gly Lys Val His Gly
Asp145 150 155 160Arg Glu Cys Gly Phe Ile Asn Asp Glu Ile Phe Val
Glu Leu Val Asn 165 170 175Ala Leu Gly Gln Tyr Asn Asp Asp Asp Asp
Asp Asp Asp Gly Asp Asp 180 185 190Pro Glu Glu Arg Glu Glu Lys Gln
Lys Asp Leu Glu Asp His Arg Asp 195 200 205Asp Lys Glu Ser Arg Pro
Pro Arg Lys Phe Pro Ser Asp Lys Ile Phe 210 215 220Glu Ala Ile Ser
Ser Met Phe Pro Asp Lys Gly Thr Ala Glu Glu Leu225 230 235 240Lys
Glu Lys Tyr Lys Glu Leu Thr Glu Gln Gln Leu Pro Gly Ala Leu 245 250
255Pro Pro Glu Cys Thr Pro Asn Ile Asp Gly Pro Asn Ala Lys Ser Val
260 265 270Gln Arg Glu Gln Ser Leu His Ser Phe His Thr Leu Phe Cys
Arg Arg 275 280 285Cys Phe Lys Tyr Asp Cys Phe Leu His Pro Phe His
Ala Thr Pro Asn 290 295 300Thr Tyr Lys Arg Lys Asn Thr Glu Thr Ala
Leu Asp Asn Lys Pro Cys305 310 315 320Gly Pro Gln Cys Tyr Gln His
Leu Glu Gly Ala Lys Glu Phe Ala Ala 325 330 335Ala Leu Thr Ala Glu
Arg Ile Lys Thr Pro Pro Lys Arg Pro Gly Gly 340 345 350Arg Arg Arg
Gly Arg Leu Pro Asn Asn Ser Ser Arg Pro Ser Thr Pro 355 360 365Thr
Ile Asn Val Leu Glu Ser Lys Asp Thr Asp Ser Asp Arg Glu Ala 370 375
380Gly Thr Glu Thr Gly Gly Glu Asn Asn Asp Lys Glu Glu Glu Glu
Lys385 390 395 400Lys Asp Glu Thr Ser Ser Ser Ser Glu Ala Asn Ser
Arg Cys Gln Thr 405 410 415Pro Ile Lys Met Lys Pro Asn Ile Glu Pro
Pro Glu Asn Val Glu Trp 420 425 430Ser Gly Ala Glu Ala Ser Met Phe
Arg Val Leu Ile Gly Thr Tyr Tyr 435 440 445Asp Asn Phe Cys Ala Ile
Ala Arg Leu Ile Gly Thr Lys Thr Cys Arg 450 455 460Gln Val Tyr Glu
Phe Arg Val Lys Glu Ser Ser Ile Ile Ala Pro Ala465 470 475 480Pro
Ala Glu Asp Val Asp Thr Pro Pro Arg Lys Lys Lys Arg Lys His 485 490
495Arg Leu Trp Ala Ala His Cys Arg Lys Ile Gln Leu Lys Lys Asp Gly
500 505 510Ser Ser Asn His Val Tyr Asn Tyr Gln Pro Cys Asp His Pro
Arg Gln 515 520 525Pro Cys Asp Ser Ser Cys Pro Cys Val Ile Ala Gln
Asn Phe Cys Glu 530 535 540Lys Phe Cys Gln Cys Ser Ser Glu Cys Gln
Asn Arg Phe Pro Gly Cys545 550 555 560Arg Cys Lys Ala Gln Cys Asn
Thr Lys Gln Cys Pro Cys Tyr Leu Ala 565 570 575Val Arg Glu Cys Asp
Pro Asp Leu Cys Leu Thr Cys Gly Ala Ala Asp 580 585 590His Trp Asp
Ser Lys Asn Val Ser Cys Lys Asn Cys Ser Ile Gln Arg 595 600 605Gly
Ser Lys Lys His Leu Leu Leu Ala Pro Ser Asp Val Ala Gly Trp 610 615
620Gly Ile Phe Ile Lys Asp Pro Val Gln Lys Asn Glu Phe Ile Ser
Glu625 630 635 640Xaa Cys Gly Glu Ile Ile Ser Gln Asp Glu Ala Asp
Arg Arg Gly Lys 645 650 655Val Tyr Asp Lys Tyr Met Cys Ser Phe Leu
Phe Asn Leu Asn Asn Asp 660 665 670Phe Val Val Asp Ala Thr Arg Lys
Gly Asn Lys Ile Arg Phe Ala Asn 675 680 685His Ser Val Asn Pro Asn
Cys Tyr Ala Lys Val Met Met Val Asn Gly 690 695 700Asp His Arg Ile
Gly Ile Phe Ala Lys Arg Ala Ile Gln Thr Gly Glu705 710 715 720Glu
Leu Phe Phe Asp Tyr Arg Tyr Ser Gln Ala Asp Ala Leu Lys Tyr 725 730
735Val Gly Ile Glu Arg Glu Met Glu Ile Pro 740 74510695PRTHomo
sapiens 10Met Gly Gln Thr Gly Lys Lys Ser Glu Lys Gly Pro Val Cys
Trp Arg1 5 10 15Lys Arg Val Lys Ser Glu Tyr Met Arg Leu Arg Gln Leu
Lys Arg Phe 20 25 30Arg Arg Ala Asp Glu Val Lys Ser Met Phe Ser Ser
Asn Arg Gln Lys 35 40 45Ile Leu Glu Arg Thr Glu Ile Leu Asn Gln Glu
Trp Lys Gln Arg Arg 50 55 60Ile Gln Pro Val His Ile Leu Thr Ser Cys
Ser Val Thr Ser Asp Leu65 70 75 80Asp Phe Pro Thr Gln Val Ile Pro
Leu Lys Thr Leu Asn Ala Val Ala 85 90 95Ser Val Pro Ile Met Tyr Ser
Trp Ser Pro Leu Gln Gln Asn Phe Met 100 105 110Val Glu Asp Glu Thr
Val Leu His Asn Ile Pro Tyr Met Gly Asp Glu 115 120 125Val Leu Asp
Gln Asp Gly Thr Phe Ile Glu Glu Leu Ile Lys Asn Tyr 130 135 140Asp
Gly Lys Val His Gly Asp Arg Glu Cys Gly Phe Ile Asn Asp Glu145 150
155 160Ile Phe Val Glu Leu Val Asn Ala Leu Gly Gln Tyr Asn Asp Asp
Asp 165 170 175Asp Asp Asp Asp Gly Asp Asp Pro Glu Glu Arg Glu Glu
Lys Gln Lys 180 185 190Asp Leu Glu Asp His Arg Asp Asp Lys Glu Ser
Arg Pro Pro Arg Lys 195 200 205Phe Pro Ser Asp Lys Ile Phe Glu Ala
Ile Ser Ser Met Phe Pro Asp 210 215 220Lys Gly Thr Ala Glu Glu Leu
Lys Glu Lys Tyr Lys Glu Leu Thr Glu225 230 235 240Gln Gln Leu Pro
Gly Ala Leu Pro Pro Glu Cys Thr Pro Asn Ile Asp 245 250 255Gly Pro
Asn Ala Lys Ser Val Gln Arg Glu Gln Ser Leu His Ser Phe 260 265
270His Thr Leu Phe Cys Arg Arg Cys Phe Lys Tyr Asp Cys Phe Leu His
275 280 285Pro Phe His Ala Thr Pro Asn Thr Tyr Lys Arg Lys Asn Thr
Glu Thr 290 295 300Ala Leu Asp Asn Lys Pro Cys Gly Pro Gln Cys Tyr
Gln His Leu Glu305 310 315 320Gly Ala Lys Glu Phe Ala Ala Ala Leu
Thr Ala Glu Arg Ile Lys Thr 325 330 335Pro Pro Lys Arg Pro Gly Gly
Arg Arg Arg Gly Arg Leu Pro Asn Asn 340 345 350Ser Ser Arg Pro Ser
Thr Pro Thr Ile Asn Val Leu Glu Ser Lys Asp 355 360 365Thr Asp Ser
Asp Arg Glu Ala Gly Thr Glu Thr Gly Gly Glu Asn Asn 370 375 380Asp
Lys Glu Glu Glu Glu Lys Lys Asp Glu Thr Ser Ser Ser Ser Glu385 390
395 400Ala Asn Ser Arg Cys Gln Thr Pro Ile Lys Met Lys Pro Asn Ile
Glu 405 410 415Pro Pro Glu Asn Val Glu Trp Ser Gly Ala Glu Ala Ser
Met Phe Arg 420 425 430Val Leu Ile Gly Thr Tyr Tyr Asp Asn Phe Cys
Ala Ile Ala Arg Leu 435 440 445Ile Gly Thr Lys Thr Cys Arg Gln Val
Tyr Glu Phe Arg Val Lys Glu 450 455 460Ser Ser Ile Ile Ala Pro Ala
Pro Ala Glu Asp Val Asp Thr Pro Pro465 470 475 480Arg Lys Lys Lys
Arg Lys His Arg Leu Trp Ala Ala His Cys Arg Lys 485 490 495Ile Gln
Leu Lys Lys Gly Gln Asn Arg Phe Pro Gly Cys Arg Cys Lys 500 505
510Ala Gln Cys Asn Thr Lys Gln Cys Pro Cys Tyr Leu Ala Val Arg Glu
515 520 525Cys Asp Pro Asp Leu Cys Leu Thr Cys Gly Ala Ala Asp His
Trp Asp 530 535 540Ser Lys Asn Val Ser Cys Lys Asn Cys Ser Ile Gln
Arg Gly Ser Lys545 550 555 560Lys His Leu Leu Leu Ala Pro Ser Asp
Val Ala Gly Trp Gly Ile Phe 565 570 575Ile Lys Asp Pro Val Gln Lys
Asn Glu Phe Ile Ser Glu Tyr Cys Gly 580 585 590Glu Ile Ile Ser Gln
Asp Glu Ala Asp Arg Arg Gly Lys Val Tyr Asp 595 600 605Lys Tyr Met
Cys Ser Phe Leu Phe Asn Leu Asn Asn Asp Phe Val Val 610 615 620Asp
Ala Thr Arg Lys Gly Asn Lys Ile Arg Phe Ala Asn His Ser Val625 630
635 640Asn Pro Asn Cys Tyr Ala Lys Val Met Met Val Asn Gly Asp His
Arg 645 650 655Ile Gly Ile Phe Ala Lys Arg Ala Ile Gln Thr Gly Glu
Glu Leu Phe 660 665 670Phe Asp Tyr Arg Tyr Ser Gln Ala Asp Ala Leu
Lys Tyr Val Gly Ile 675 680 685Glu Arg Glu Met Glu Ile Pro 690
6951124PRTHomo sapiens
11Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly Val1
5 10 15Lys Lys Pro His Arg Tyr Arg Pro 20122682DNAHomo sapiens
12gacgacgttc gcggcgggga actcggagta gcttcgcctc tgacgtttcc ccacgacgca
60ccccgaaatc cccctgagct ccggcggtcg cgggctgccc tcgccgcctg gtctggcttt
120atgctaagtt tgagggaaga gtcgagctgc tctgctctct attgattgtg
tttctggagg 180gcgtcctgtt gaattcccac ttcattgtgt acatcccctt
ccgttccccc caaaaatctg 240tgccacaggg ttactttttg aaagcgggag
gaatcgagaa gcacgatctt ttggaaaact 300tggtgaacgc ctaaataatc
atgggccaga ctgggaagaa atctgagaag ggaccagttt 360gttggcggaa
gcgtgtaaaa tcagagtaca tgcgactgag acagctcaag aggttcagac
420gagctgatga agtaaagagt atgtttagtt ccaatcgtca gaaaattttg
gaaagaacgg 480aaatcttaaa ccaagaatgg aaacagcgaa ggatacagcc
tgtgcacatc ctgacttctt 540gttcggtgac cagtgacttg gattttccaa
cacaagtcat cccattaaag actctgaatg 600cagttgcttc agtacccata
atgtattctt ggtctcccct acagcagaat tttatggtgg 660aagatgaaac
tgttttacat aacattcctt atatgggaga tgaagtttta gatcaggatg
720gtactttcat tgaagaacta ataaaaaatt atgatgggaa agtacacggg
gatagagaat 780gtgggtttat aaatgatgaa atttttgtgg agttggtgaa
tgcccttggt caatataatg 840atgatgacga tgatgatgat ggagacgatc
ctgaagaaag agaagaaaag cagaaagatc 900tggaggatca ccgagatgat
aaagaaagcc gcccacctcg gaaatttcct tctgataaaa 960tttttgaagc
catttcctca atgtttccag ataagggcac agcagaagaa ctaaaggaaa
1020aatataaaga actcaccgaa cagcagctcc caggcgcact tcctcctgaa
tgtaccccca 1080acatagatgg accaaatgct aaatctgttc agagagagca
aagcttacac tcctttcata 1140cgcttttctg taggcgatgt tttaaatatg
actgcttcct acatcctttt catgcaacac 1200ccaacactta taagcggaag
aacacagaaa cagctctaga caacaaacct tgtggaccac 1260agtgttacca
gcatttggag ggagcaaagg agtttgctgc tgctctcacc gctgagcgga
1320taaagacccc accaaaacgt ccaggaggcc gcagaagagg acggcttccc
aataacagta 1380gcaggcccag cacccccacc attaatgtgc tggaatcaaa
ggatacagac agtgataggg 1440aagcagggac tgaaacgggg ggagagaaca
atgataaaga agaagaagag aagaaagatg 1500aaacttcgag ctcctctgaa
gcaaattctc ggtgtcaaac accaataaag atgaagccaa 1560atattgaacc
tcctgagaat gtggagtgga gtggtgctga agcctcaatg tttagagtcc
1620tcattggcac ttactatgac aatttctgtg ccattgctag gttaattggg
accaaaacat 1680gtagacaggt gtatgagttt agagtcaaag aatctagcat
catagctcca gctcccgctg 1740aggatgtgga tactcctcca aggaaaaaga
agaggaaaca ccggttgtgg gctgcacact 1800gcagaaagat acagctgaaa
aagggtcaaa accgctttcc gggatgccgc tgcaaagcac 1860agtgcaacac
caagcagtgc ccgtgctacc tggctgtccg agagtgtgac cctgacctct
1920gtcttacttg tggagccgct gaccattggg acagtaaaaa tgtgtcctgc
aagaactgca 1980gtattcagcg gggctccaaa aagcatctat tgctggcacc
atctgacgtg gcaggctggg 2040ggatttttat caaagatcct gtgcagaaaa
atgaattcat ctcagaatac tgtggagaga 2100ttatttctca agatgaagct
gacagaagag ggaaagtgta tgataaatac atgtgcagct 2160ttctgttcaa
cttgaacaat gattttgtgg tggatgcaac ccgcaagggt aacaaaattc
2220gttttgcaaa tcattcggta aatccaaact gctatgcaaa agttatgatg
gttaacggtg 2280atcacaggat aggtattttt gccaagagag ccatccagac
tggcgaagag ctgttttttg 2340attacagata cagccaggct gatgccctga
agtatgtcgg catcgaaaga gaaatggaaa 2400tcccttgaca tctgctacct
cctcccccct cctctgaaac agctgcctta gcttcaggaa 2460cctcgagtac
tgtgggcaat ttagaaaaag aacatgcagt ttgaaattct gaatttgcaa
2520agtactgtaa gaataattta tagtaatgag tttaaaaatc aactttttat
tgccttctca 2580ccagctgcaa agtgttttgt accagtgaat ttttgcaata
atgcagtatg gtacattttt 2640caactttgaa taaagaatac ttgaacttgt
ccttgttgaa tc 2682
* * * * *
References