U.S. patent application number 17/289873 was filed with the patent office on 2021-12-30 for methods of treating cancer in biomarker-identified patients with non-covalent inhibitors of cyclin-dependent kinase 7 (cdk7).
The applicant listed for this patent is Syros Pharmaceuticals, Inc.. Invention is credited to John Graeme Hodgson, Liv Helena Johannessen.
Application Number | 20210401859 17/289873 |
Document ID | / |
Family ID | 1000005886525 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210401859 |
Kind Code |
A1 |
Hodgson; John Graeme ; et
al. |
December 30, 2021 |
METHODS OF TREATING CANCER IN BIOMARKER-IDENTIFIED PATIENTS WITH
NON-COVALENT INHIBITORS OF CYCLIN-DEPENDENT KINASE 7 (CDK7)
Abstract
The present invention relates to methods of identifying patients
suffering from various types of cancer who are more likely to
respond to treatment with a CDK7 inhibitor conforming to structural
Formula (I), (Ia), a species thereof, or a specified form thereof
(as described herein), either when administered or used alone or in
combination with a second therapeutic agent (e.g., another
anti-cancer therapy). Patients are identified based on one or more
features (e.g., gene copy number or expression level) of certain
biomarkers (e.g., RB1 or another member of the E2F pathway). In
addition, the present invention relates to methods of treating an
identified patient with a compound conforming to structural Formula
(I), (Ia), a species thereof, or a specified form thereof, either
alone or in combination with a second therapeutic agent. In another
aspect, the present invention features kits including instructions
for treating a patient identified as described herein.
Inventors: |
Hodgson; John Graeme;
(Boxborough, MA) ; Johannessen; Liv Helena;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Syros Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
1000005886525 |
Appl. No.: |
17/289873 |
Filed: |
November 1, 2019 |
PCT Filed: |
November 1, 2019 |
PCT NO: |
PCT/US19/59535 |
371 Date: |
April 29, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62754398 |
Nov 1, 2018 |
|
|
|
62877189 |
Jul 22, 2019 |
|
|
|
62915983 |
Oct 16, 2019 |
|
|
|
62927469 |
Oct 29, 2019 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/675 20130101 |
International
Class: |
A61K 31/675 20060101
A61K031/675; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating a selected patient, the method comprising
administering a therapeutically effective amount of a compound of
structural Formula (I): ##STR00028## or a pharmaceutically
acceptable salt thereof, wherein the compound or the
pharmaceutically acceptable salt thereof is optionally within a
pharmaceutical composition; R.sup.1 is methyl or ethyl; R.sup.2 is
methyl or ethyl; R.sup.3 is 5-methylpiperidin-3-yl,
5,5-dimethylpiperidin-3-yl, 6-methylpiperdin-3-yl, or
6,6-dimethylpiperidin-3-yl, wherein one or more hydrogen atoms in
R.sup.3 is optionally replaced by deuterium; R.sup.4 is --CF.sub.3
or chloro; and the selected patient has been determined to have a
cancer in which (a) a gene selected from RB1, RBL1, RBL2, CDKN2A,
CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C, and FBWX7 is
mutated, is genetically deleted, contains an epigenetic alteration,
is translocated, is transcribed at a level equal to or below a
pre-determined threshold, or encodes a protein that is translated
at a level equal to or below a pre-determined threshold or has
decreased activity relative to a reference standard; (b) a gene
selected from E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CDK1,
CDK2, CDK4, CDK6, CCNA1, CCNB1, CCND1, CCND2, CCND3, CCNE1, CCNE2,
and BRAF is mutated, is genetically gained or amplified, contains
an epigenetic alteration, is translocated, transcribed at a level
equal to or above a pre-determined threshold, or encodes a protein
that is translated at a level equal to or above a pre-determined
threshold or has increased activity relative to a reference
standard; or (c) the gene Bcl2-like 1 is mutated, contains an
epigenetic alteration, is translocated, is transcribed at a level
equal to or below a pre-determined threshold, or encodes a BCL-xL
protein that is translated at a level equal to or below a
pre-determined threshold or has decreased activity relative to a
reference standard.
2. The method of claim 1, wherein (i) R.sup.1 is methyl and R.sup.2
is methyl or (ii) R.sup.1 is methyl and R.sup.2 is ethyl.
3. The method of claim 1, wherein (i) R.sup.1 is ethyl and R.sup.2
is ethyl or (ii) R.sup.4 is --CF.sub.3.
4. The method of claim 1, wherein R.sup.4 is chloro.
5. The method of claim 1, wherein R.sup.3 is
5-methylpiperidin-3-yl.
6. The method of claim 1, wherein R.sup.3 is
5,5-dimethylpiperidin-3-yl.
7. The method of claim 1, wherein R.sup.3 is
6-methylpiperdin-3-yl.
8. The method of claim 1, wherein R.sup.3 is
6,6-dimethylpiperidin-3-yl.
9. The method of claim 1, wherein the compound has structural
Formula (Ia): ##STR00029## is a pharmaceutically acceptable salt
thereof, wherein R.sup.3 is ##STR00030##
10.-12. (canceled)
13. The method of claim 9, wherein the compound is: ##STR00031## or
is a pharmaceutically acceptable salt of any one of the foregoing
compounds.
14. The method of claim 13, wherein the compound is ##STR00032## or
a pharmaceutically acceptable salt thereof.
15.-18. (canceled)
19. The method of claim 1, wherein the cancer is a blood cancer, a
breast cancer, Ewing's sarcoma, fallopian tube cancer, a GI tract
cancer, a glioma, a lung cancer, melanoma, an osteosarcoma, an
ovarian cancer, a pancreatic cancer, a primary peritoneal cancer,
prostate cancer, retinoblastoma, or a squamous cell cancer of the
head or neck.
20. (canceled)
21. The method of claim 19, wherein the patient has undergone, is
presently undergoing, or is prescribed treatment with a Bcl-2
inhibitor.
22. The method of claim 21, wherein the Bcl-2 inhibitor is
venetoclax and/or wherein the patient has a breast cancer, a blood
cancer, an ovarian cancer, or a lung cancer.
23. The method of claim 19, wherein the patient has been determined
to have a cancer in which (a) RB1 or CDKN2A is mutated, contains an
epigenetic alteration, is translocated, is transcribed at a level
equal to or below a pre-determined threshold, or encodes a protein
that is translated at a level equal to or below a pre-determined
threshold or has decreased activity relative to a reference
standard; and/or (b) CDK6, CCND2, or CCNE1 is mutated, has a copy
number alteration, contains an epigenetic alteration, is
translocated, transcribed at a level equal to or above a
pre-determined threshold, or encodes a protein that is translated
at a level equal to or above a pre-determined threshold or has
increased activity relative to a reference standard.
24. The method of claim 19, wherein the patient has undergone, is
presently undergoing, or is prescribed treatment with a selective
estrogen receptor modulator (SERM), a selective estrogen receptor
degrader (SERD), a PARP inhibitor, or a platinum-based therapeutic
agent.
25. The method of claim 24, wherein the patient has undergone, is
presently undergoing, or is prescribed treatment with a SERM or
SERD and has an HR+ breast cancer; the patient has undergone, is
presently undergoing, or is prescribed treatment with a PARP
inhibitor and has breast cancer, fallopian tube cancer, a glioma,
ovarian cancer, or primary peritoneal cancer; or the patient has
undergone, is presently undergoing, or is prescribed treatment with
a platinum-based therapeutic agent and has an ovarian cancer.
26. The method of claim 19, wherein the patient has undergone, is
presently undergoing, or is prescribed treatment with a BET
inhibitor with a CDK4/6 inhibitor; with a FLT3 inhibitor; or with a
MEK inhibitor.
27. The method of claim 26, wherein the patient who has undergone,
is presently undergoing, or is prescribed treatment with the CDK4/6
inhibitor has a breast cancer, a pancreatic cancer, or a squamous
cell cancer of the head or neck; the patient who has undergone, is
presently undergoing, or is prescribed treatment with the FLT3
inhibitor has a blood cancer; or the patient who has undergone, is
presently undergoing, or is prescribed treatment with the BET
inhibitor has a breast cancer, a blood cancer, Ewing's sarcoma, or
an osteosarcoma.
28. The method of claim 1, wherein the patient has undergone, is
presently undergoing, or is prescribed treatment with a second
anti-cancer agent.
29. The method of claim 28, wherein the second anti-cancer agent is
a Bcl-2 inhibitor a CDK9 inhibitor; a hormone receptor degradation
agent; a Flt3 (FMS-like tyrosine kinase 3) inhibitor; a PARP
inhibitor; a BET inhibitor; a platinum-based therapeutic agent; a
CDK4/6 inhibitor; a MEK inhibitor; or a phosphoinositide 3-kinase
(PI3 kinase) inhibitor.
30. The method of claim 29, wherein the Bcl-2 inhibitor is
venetoclax, the PARP inhibitor is olaparib or niraparib, the
platinum-based anti-cancer agent is carboplatin or oxaliplatin, the
CDK4/6 inhibitor is palbociclib, ribociclib, abemaciclib, or
trilaciclib, and the hormone receptor degradation agent is
fulvestrant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. provisional application No. 62/754,398, filed Nov. 1, 2018;
U.S. provisional application No. 62/877,189, filed Jul. 22, 2019;
U.S. provisional application No. 62/915,983, filed Oct. 16, 2019,
and U.S. provisional application No. 62/927,469, filed Oct. 29,
2019. The content of each of these prior applications is hereby
incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] The long evolution of healthcare has reached a point in time
where the promise of biomarker analysis is beginning to be
realized. When physicians can stratify patients, even those who
share many similar physiological traits and exhibit common symptoms
of a given disease, into more specific groups, they can better
tailor treatment and optimize the outcome for each patient.
However, it is challenging to develop molecular diagnostics and few
are commercially available.
SUMMARY OF THE INVENTION
[0003] The present invention features, inter alia, diagnostic
methods for identifying cancer patients for treatment with a
non-covalent CDK7 inhibitor described herein (i.e., diagnostic
methods for selecting a patient for treatment) and methods for
treating identified patients with such an inhibitor, either alone
or in combination with one or more additional therapeutic agents
(e.g., a second anti-cancer agent), as described further below. The
diagnostic methods include a step of identifying a patient
suffering from a cancer that is likely to respond well to treatment
with a non-covalent CDK7 inhibitor represented by structural
Formula (I), (Ia), a species thereof, or a specified form thereof,
as shown and described further below. The treatment methods include
a step of administering such a non-covalent CDK7 inhibitor to an
identified patient, whose response can be, for example, significant
tumor growth inhibition (TGI; e.g., more than about 80-90% TGI
and/or continued tumor suppression for a period of time after
cessation of treatment). Thus, the present invention encompasses
methods in which a patient is only diagnosed as being a good
candidate for treatment (i.e., identified for treatment), methods
in which a patient who has been determined to be a good candidate
for treatment (e.g., previously identified) is treated, and methods
requiring that a patient be both diagnosed and treated as described
herein.
The diagnostic methods that identify a patient for treatment
include a step of analyzing one or more of the biomarkers described
herein in a biological sample obtained from the patient by
determining, having determined, or receiving information concerning
the state of the biomarker. In various embodiments, the biomarker
is analyzed to determine: whether it is present and/or in what
amount (e.g., analyzed for a genetic deletion or amplification
(e.g., copy number variation (CNV)); its location (e.g.,
chromosomal translocation); its sequence (i.e., the analysis can
include determining whether the gene is present in wild type form
or includes a mutation); whether it includes an epigenetic
modification (e.g., histone and/or DNA methylation or histone
acetylation); whether it is associated with a super-enhancer (SE)
or a SE of a certain strength; its level of expression (as
evidenced by, for example, the level of transcribed RNA (e.g.,
primary RNA or mRNA)); and/or whether a protein encoded by the
biomarker gene has an aberrant level of expression or activity (in
case of doubt, a protein encoded by a biomarker gene described
herein can also serve as the biomarker). The state of a biomarker
can be assessed by examining any one or more of the features just
listed, and when we refer to "analyzing a/the biomarker," we mean
analyzing one or more of these features (i.e., sequence, copy
number, association with a SE, a level of RNA expression, and so
forth, as provided above). For example, when we refer to analyzing
the biomarker RB1, we mean analyzing or determining whether an RB1
gene is, for example, absent in a biological sample, contains a
mutation (e.g., a mutation predisposing a patient to cancer), is
translocated, has a CNV (copy number alteration (CNA)), bears an
epigenetic modification, is associated with a super-enhancer (SE),
is overexpressed or under-expressed (as evidenced by, for example,
its level of RNA (e.g., primary RNA or mRNA), and/or encodes a
protein with a level of expression or activity that is above or
below a predetermined threshold level. As this implies, each
feature analyzed can be determined to be equal to or above a
pre-determined threshold level or equal to or below a
pre-determined threshold level, as described further below. More
specifically, in the methods of the present invention, one can
analyze a biomarker selected from the genes BRAF, c-myc (also known
as MYC), CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1,
ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway
member (E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1,
CCND2, CCND3, CCNE1, or CCNE2; see also the Table below), or the
proteins encoded thereby, by determining, having determined, and/or
receiving information that the state of such a biomarker, as
evidenced by a feature just described (e.g., RNA level) is equal to
or above (e.g., above) a pre-determined threshold level.
Alternatively, or in addition, one can analyze a biomarker selected
from the genes BCL2-like 1, CDK7, CDK9, CDKN2A, and RB (also known
as RB1 or another E2F pathway member, such as RBL1, RBL2, CDKN2A,
CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C, and FBXW7), or the
proteins encoded thereby, by determining, having determined, and/or
receiving information that the state of such biomarker is equal to
or below (e.g., below) a pre-determined threshold level. The
proteins encoded by the genes just listed as useful biomarkers in
the present methods are known in the art. For example, BRAF encodes
B-Raf; c-myc encodes MYC, CCNE1 encodes cyclin E1 (see Koff et al.,
Cell 66:1217-1228, 1991); FGFR1 encodes FGFR1, a cell surface
membrane receptor with tyrosine kinase activity; RB encodes pRB,
which binds to the activator domain of activator E2F; BCL2-like 1
encodes BCL-xL, a transmembrane protein in mitochondria; CDK7
encodes CDK7; CDK9 encodes CDK9; PIK3CA encodes the p110.alpha.
protein (a catalytic subunit of the class I PI3-kinases), and
CDKN2A encodes p16 and p14arf. Aliases, chromosomal locations,
splice variants, and homologs of the genes and proteins described
herein as biomarkers, in Homo sapiens and species other than Homo
sapiens, are also known.
[0004] The treatment methods of the invention and corresponding
"uses" include administering, or the use of, a compound of Formula
(I), any of which may be included in a pharmaceutically acceptable
composition and administered, e.g., by a route and regimen
described herein, to a patient identified as described herein.
Compounds useful in the present methods have structural Formula
(I):
##STR00001##
or a pharmaceutically acceptable salt, solvate, stereoisomer or
mixture of stereoisomers, tautomer, or isotopic form thereof,
wherein R.sup.1 is methyl or ethyl; R.sup.2 is methyl or ethyl;
R.sup.3 is 5-methylpiperidin-3-yl, 5,5-dimethylpiperidin-3-yl,
6-methylpiperdin-3-yl, or 6,6-dimethylpiperidin-3-yl, wherein one
or more hydrogen atoms in R.sup.3 is optionally replaced by
deuterium; and R.sup.4 is --CF.sub.3 or chloro. More specifically,
in a compound of Formula (I) or in the pharmaceutically acceptable
salt, solvate, stereoisomer or mixture of stereoisomers, tautomer,
isotopic form, or other specified form thereof (i) IV is methyl and
R.sup.2 is methyl or (ii) R.sup.1 is methyl and R.sup.2 is ethyl.
In other embodiments, R.sup.1 is ethyl and R.sup.2 is ethyl. In
some aspects of any one of these embodiments, R.sup.4 is
--CF.sub.3. In other aspects of any one of these embodiments,
R.sup.4 is chloro. In various aspects of any of the preceding
embodiments, R.sup.3 is 5-methylpiperidin-3-yl, R.sup.3 is
5,5-dimethylpiperidin-3-yl, R.sup.3 is 6-methyl-piperdin-3-yl, or
R.sup.3 is 6,6-dimethylpiperidin-3-yl, wherein one or more hydrogen
atoms in R.sup.3 is optionally replaced by deuterium. A compound of
Formula (I) can have structural Formula (Ia):
##STR00002##
and the invention encompasses pharmaceutically acceptable salts,
solvates (e.g., hydrates), tautomers, isotopic forms, or other
specified forms of a compound of Formula (Ia), wherein R.sup.3
is
##STR00003##
[0005] More specifically, in a compound of Formula (Ia) or a
pharmaceutically acceptable salt, solvate, tautomer, isotopic form,
or other specified form thereof (i) R.sup.1 is methyl and R.sup.2
is methyl or (ii) R.sup.1 is methyl and R.sup.2 is ethyl. In other
embodiments, R.sup.1 is ethyl and R.sup.2 is ethyl. In some
embodiments, in a compound of Formula (Ia) or a specified form
thereof, R.sup.4 is --CF.sub.3. In other embodiments, in a compound
of Formula (Ia) or a specified form thereof, R.sup.4 is chloro. In
some embodiments, a compound of Formula (I) or (Ia) is:
##STR00004##
and the invention encompasses methods and the use of
pharmaceutically acceptable salts, solvates (e.g., hydrates),
tautomers, isotopic forms or other specified forms of any one of
the three foregoing compounds. In one embodiment, the compound
is
##STR00005##
or a pharmaceutically acceptable salt thereof. The invention also
encompasses solvates (e.g., hydrates), tautomers, isotopic forms or
other specified forms of the foregoing compound. In isotopic forms,
one or more hydrogen atoms in R.sup.3 is replaced with deuterium.
In other embodiments, none of the hydrogen atoms of a compound
(e.g., none of the hydrogen atoms in R.sup.3) are replaced with
deuterium. Any compound of Formula (I), (Ia), or a species thereof
can be of a "specified form," by which we mean a salt, solvate
(e.g., hydrate), stereoisomer (or mixture thereof), tautomer, or
isotopic form of a compound of Formula (I), (Ia), or a species
thereof. Also within the meaning of "specified form" are forms of a
compound that manifest a combination of the attributes, features,
or properties of a salt, solvate, stereoisomer, tautomer, or
isotopic form. For example, the methods and uses of the invention
can be carried out with a salt that has been solvated (e.g., a
hydrated) or a salt of a stereoisomer, tautomer, or isotopic form
of a compound of Formula I, I(a), or a species thereof; with a
solvate (e.g., hydrate) containing a salt, stereoisomer, tautomer,
or isotopic form of a compound of Formula I, I(a), or a species
thereof; with a stereoisomer of a compound of Formula I, I(a), or a
species thereof that is in the form of a salt or solvate (e.g.,
hydrate) or is a tautomer or isotopic form of a compound of Formula
I, I(a), or a species thereof, with a tautomer of a compound of
Formula I, I(a), or a species thereof that is in the form of a salt
or solvate (e.g., hydrate) or that is a stereoisomer or isotopic
form of a compound of Formula I, I(a), or a species thereof; or
with an isotopic form of a compound of Formula I, I(a), or a
species thereof that is a salt, solvate (e.g., hydrate),
stereoisomer, or tautomer of a compound of Formula I, I(a), or a
species thereof. Any of these specified forms can be
pharmaceutically acceptable and/or contained within a
pharmaceutically acceptable composition (e.g., formulated for oral
administration) for use in a method described herein.
[0006] Accordingly, the invention features treatment methods
including a step of administering a compound of structural Formula
(I), or a pharmaceutically acceptable salt, solvate, stereoisomer
or mixture of stereoisomers, tautomer, or isotopic form thereof,
optionally within a pharmaceutical composition, wherein R',
R.sup.2, R.sup.3, and R.sup.4 are as defined herein, in treating
cancer in a selected patient, wherein the patient has been
determined to have a cancer in which: (a) a gene selected from RB1,
RBL1, RBL2, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C,
and FBWX7 is mutated, is genetically deleted, contains an
epigenetic alteration, is translocated, is transcribed at a level
equal to or below a pre-determined threshold, or encodes a protein
that is translated at a level equal to or below a pre-determined
threshold or has decreased activity relative to a reference
standard; (b) a gene selected from E2F1, E2F2, E2F3, E2F4, E2F5,
E2F6, E2F7, E2F8, CDK1, CDK2, CDK4, CDK6, CCNA1, CCNB1, CCND1,
CCND2, CCND3, CCNE1, CCNE2, and BRAF is mutated, is genetically
gained or amplified, contains an epigenetic alteration, is
translocated, transcribed at a level equal to or above a
pre-determined threshold, or encodes a protein that is translated
at a level equal to or above a pre-determined threshold or has
increased activity relative to a reference standard; or (c) the
gene Bcl2-like 1 is mutated, contains an epigenetic alteration, is
translocated, is transcribed at a level equal to or below a
pre-determined threshold, or encodes a BCL-xL protein that is
translated at a level equal to or below a pre-determined threshold
or has decreased activity relative to a reference standard. In any
embodiment of this method, the cancer is a blood cancer, preferably
an acute myeloid leukemia (AML), a breast cancer, preferably a
triple negative breast cancer (TNBC) or a hormone receptor positive
(HR+) breast cancer, an osteosarcoma or Ewing's sarcoma, fallopian
tube cancer, a GI tract cancer, preferably colorectal cancer, a
glioma, a lung cancer, preferably small cell or non-small cell lung
cancer, melanoma, an ovarian cancer, preferably a high grade serous
ovarian cancer, epithelial ovarian cancer, or clear cell ovarian
cancer, a pancreatic cancer, a primary peritoneal cancer, prostate
cancer, retinoblastoma, or a squamous cell cancer of the head or
neck. For example, the patient may have such a cancer and can be
treated as described herein when it has been determined that, in a
biological sample obtained from the patient, Bcl2-like 1 is
mutated, contains an epigenetic alteration, is translocated, is
transcribed at a level equal to or below a pre-determined
threshold, or encodes a BCL-xL protein that is translated at a
level equal to or below a pre-determined threshold or has decreased
activity relative to a reference standard, preferably wherein a
level of Bcl2-like 1 mRNA in the cancer is equal to or below the
pre-determined threshold level. Further, such a patient can be one
who has undergone, is presently undergoing, or is prescribed
treatment with a Bcl-2 inhibitor, as known in the art and/or
described herein. In some embodiments, the Bcl-2 inhibitor is
venetoclax and the patient has a breast cancer (e.g., TNBC); a
blood cancer (e.g., AML); an ovarian cancer (e.g., HGSOC); or a
lung cancer (e.g., SCLC or NSCLC). In other embodiments, the
patient may have such a cancer and can be treated as described
herein when it has been determined that, in a biological sample
obtained from the patient: (a) RB1 or CDKN2A is mutated, contains
an epigenetic alteration, is translocated, is transcribed at a
level equal to or below a pre-determined threshold, or encodes a
protein that is translated at a level equal to or below a
pre-determined threshold or has decreased activity relative to a
reference standard, preferably wherein RB1 or CDKN2A mRNA,
preferably RB1 mRNA, is equal to or below the pre-determined
threshold; and/or (b) CDK6, CCND2, or CCNE1 is mutated, has a copy
number alteration, contains an epigenetic alteration, is
translocated, transcribed at a level equal to or above a
pre-determined threshold, or encodes a protein that is translated
at a level equal to or above a pre-determined threshold or has
increased activity relative to a reference standard, preferably
wherein CDK6, CCND2, or CCNE1 mRNA, preferably CCNE1 mRNA, is equal
to or above a pre-determined threshold level. Such a patient can be
one who has undergone, is presently undergoing, or is prescribed
treatment with a selective estrogen receptor modulator (SERM; e.g.,
tamoxifen, raloxifene, or toremifene), a selective estrogen
receptor degrader (SERD; e.g., fulvestrant), a PARP inhibitor
(e.g., olaparib or niraparib); or a platinum-based therapeutic
agent (e.g., cisplatin, oxaliplatin, nedaplatin, carboplatin,
phenanthriplatin, picoplatin, satraplatin (JM216). More
specifically, the patient treated with a SERM or SERD may have an
HR+ breast cancer; the patient treated with a PARP inhibitor may
have a TNBC or a Her2.sup.+/ER.sup.-/PR.sup.- breast cancer,
fallopian tube cancer, glioma, ovarian cancer (e.g., an epithelial
ovarian cancer), or primary peritoneal cancer; and the patient
treated with a platinum-based therapeutic agent may have an ovarian
cancer.
[0007] In any of the present methods where a compound of Formula
(I), (Ia), a species thereof or a specified form thereof is used or
administered, optionally within a pharmaceutical composition, the
patient can be one who has undergone, is presently undergoing, or
is prescribed treatment with a BET inhibitor such as ABBV-075,
BAY-299, BAY-1238097, BMS-986158, CPI-0610, CPI-203, FT-1101,
GS-5829, GSK-2820151, GSK-525762, I-BET151, I-BET762, INCB054329,
JQ1, MS436, OTX015, PFI-1, PLX51107, RVX2135, TEN-010, ZEN-3694, or
a compound disclosed in U.S. application Ser. No. 12/810,564; with
a CDK4/6 inhibitor such as BPI-1178, G1T38, palbociclib,
ribociclib, ON 123300, trilaciclib, or abemaciclib, preferably
palbociclib; with a FLT3 inhibitor such as CDX-301, CG'806,
CT053PTSA, crenolanib (e.g., crenolanib besylate), ENMD-2076,
FF-10101-01, FLYSYN, gilteritinib (ASP2215), HM43239, lestautinib,
ponatinib, NMS-088, sorafenib, sunitinib, pacritinib,
pexidartinib/PLX3397, quizartinib, midostaurin, SEL24, SKI-G-801,
or SKLB1028, preferably crenolanib, gilteritinib, or midostaurin;
or with a MEK inhibitor such as trametinib, cobimetinib, or
binimetinib. More specifically, a patient who has undergone, is
presently undergoing, or is prescribed treatment: with a CDK4/6
inhibitor has a breast cancer, preferably a TNBC or an estrogen
receptor-positive (ER.sup.+) breast cancer, a pancreatic cancer, or
a squamous cell cancer of the head or neck; with a FLT3 inhibitor
has a blood cancer, preferably AML; with a BET inhibitor has a
breast cancer, preferably TNBC, a blood cancer, preferably AML,
Ewing's sarcoma, or an osteosarcoma.
[0008] In any of the present methods where compound of Formula (I),
(Ia), a species thereof or a specified form thereof is used or
administered, optionally within a pharmaceutical composition, the
patient can be one who has undergone, is presently undergoing, or
is prescribed treatment with a Bcl-2 inhibitor such as APG-1252,
APG-2575, BP1002 (prexigebersen), the antisense oligonucleotide
known as oblimersen (G3139), S55746/BCL201, or venetoclax; a CDK9
inhibitor such as alvocidib/DSP-2033/flavopiridol, AT7519, AZD5576,
BAY1251152, BAY1143572, CYC065, nanoflavopiridol, NVP2, seliciclib
(CYC202), TG02, TP-1287, VS2-370 or voruciclib (formerly
P1446A-05); a hormone receptor (e.g., estrogen receptor)
degradation agent, such as fulvestrant; a Flt3 (FMS-like tyrosine
kinase 3) inhibitor such as CDX-301, CG'806, CT053PTSA, crenolanib
(e.g., crenolanib besylate), ENMD-2076, FF-10101-01, FLYSYN,
gilteritinib (ASP2215), HM43239, lestautinib, ponatinib, NMS-088,
sorafenib, sunitinib, pacritinib, pexidartinib/PLX3397,
quizartinib, midostaurin, SEL24, SKI-G-801, or SKLB1028; a PARP
inhibitor such as olaparib, rucaparib, talazoparib, veliparib
(ABT-888), or niraparib; a BET inhibitor such as ABBV-075, BAY-299,
BAY-1238097, BMS-986158, CPI-0610, CPI-203, FT-1101, GS-5829,
GSK-2820151, GSK-525762, I-BET151, I-BET762, INCB054329, JQ1,
MS436, OTX015, PFI-1, PLX51107, RVX2135, TEN-010, ZEN-3694, or a
compound disclosed in U.S. application Ser. No. 12/810,564 (now
U.S. Pat. No. 8,476,260); a platinum-based therapeutic agent such
as cisplatin, oxaliplatin, nedaplatin, carboplatin,
phenanthriplatin, picoplatin, satraplatin (JM216), or triplatin
tetranitrate; a CDK4/6 inhibitor such as BPI-1178, G1T38,
palbociclib, ribociclib, ON 123300, trilaciclib, or abemaciclib; a
MEK inhibitor such as trametinib; or a phosphoinositide 3-kinase
(PI3 kinase) inhibitor, optionally of Class I (e.g., Class IA)
and/or optionally directed against a specific PI3K isoform, such as
idelalisib, copanlisib, duvelisib, or alpelisib; or capecitabine.
More specifically, the second agent is selected from a Bcl-2
inhibitor such as venetoclax, a PARP inhibitor such as olaparib or
niraparib, a platinum-based anti-cancer agent such as carboplatin
or oxaliplatin, a taxane such as paclitaxel, a CDK4/6 inhibitor
such as palbociclib, ribociclib, abemaciclib, or trilaciclib, a
selective estrogen receptor modulator such as tamoxifen, and a
selective estrogen receptor degrader such as fulvestrant.
[0009] The invention also features kits that include a compound of
Formula I, I(a), a species thereof, or a specified form thereof,
and instructional material (e.g., a product insert) that describes
a suitable/identified patient, methods of identifying such a
patient for treatment (e.g., by any one of the diagnostic
stratification methods described herein for analyzing a biomarker),
and/or instructions for administering the compound of Formula I,
I(a), a species thereof, or a specified form thereof, alone or in
combination with at least one other therapeutic agent (e.g., an
additional/second anti-cancer therapeutic including any one or more
of the second agents described herein). The kits of the invention
can also include the second agent (e.g., an anti-cancer agent),
including any one or more of the second agents described herein and
instructions for use in a population of patients identified as
described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a table depicting the inhibitory and dissociation
constants and selectivity of the indicated compounds (three
compounds of the invention and four comparators) against CDK2,
CDK7, CDK9, and CDK12.
[0011] FIG. 2 is a line graph depicting changes in tumor volume
(mm.sup.3) over time (days) in a palbociclib-resistant HR+BC PDX
model (ST1799), as described in the Examples below.
[0012] FIG. 3 is a line graph depicting tumor volume (mm.sup.3)
over time (days) in the palbociclib- and fulvestrant-resistant
HR+BC PDX model ST941, as described in the Examples below.
[0013] FIG. 4 is a panel showing three line graphs that depict
changes in tumor volume (mm.sup.3) over time (days) in PDX models
of TNBC (BR5010; top), small cell lung cancer (LU5178; middle), and
ovarian cancer (OV15398; bottom). The animals were treated with
Compound 101 as described in Example 10. Data obtained from
vehicle-treated (control) animals is represented by filled circles
(upper traces in each graph). Data from animals modeling TNBC and
given 10 mg/kg Compound 101 QD are represented in the top graph by
filled squares; the dose of 5 mg/kg BID is represented by
triangles. Triangles also represent data obtained from the animal
models of SCLC and ovarian cancer treated with Compound 101 in the
middle and bottom graphs.
[0014] FIG. 5 is a panel of line graphs showing tumor growth in the
PDX models indicated and corresponding isobolograms, each generated
as described in Example 11. Compound 101 was applied to cells in
combination with the indicated second agents at the concentrations
shown.
[0015] FIG. 6 is a panel of graphs generated from data collected in
the Compound 101-treated PDX models described in Example 12. Black
lines with squares represent vehicle-treated animals. Gray lines
represent Compound 101-treated animals. Error bars are SEM.
BID=twice daily; CNV=copy number variation; MPK=mg per kg body
weight; PO=oral; QD=once daily; RB=retinoblastoma; SCLC=small cell
lung cancer; TNBC=triple negative breast cancer. Vertical dotted
lines mark the last day of treatment.
[0016] FIG. 7 is a Table summarizing the TGI values and genetic
status of the 12 PDX models studied as described in Example 12.
Models in the table are sorted based on highest to lowest response
at end of study. BID, CNV, RB, SCLC, and TNBC are as defined for
FIG. 6 and elsewhere herein. In case of doubt, CCNE1=the gene
encoding cyclin E1; CDKN2A=cyclin-dependent kinase inhibitor 2A,
EoS=end of study, EoT=end of treatment, HGSOC=high-grade serous
ovarian cancer, OVA=ovarian cancer, and TGI=tumor growth
inhibition. For the LU5210 model, tissue was not available for
confirmation of RB pathway genetics.
DETAILED DESCRIPTION
[0017] Despite the efficacy of compounds of Formula (I), we believe
that such efficacy will be higher in patients that have certain
genetic signatures (i.e., biomarkers in a particular state, which
can be analyzed as described herein). Moreover, we believe the
efficacy of compounds of Formula (I) may be enhanced when combined
with other anti-cancer therapies in newly diagnosed and refractory
cancer patients identified as described herein.
[0018] The following definitions apply to the compositions,
methods, and uses described herein unless the context clearly
indicates otherwise, and it is to be understood that the claims may
be amended to include language within a definition as needed or
desired. Moreover, the definitions apply to linguistic and
grammatical variants of the defined terms (e.g., the singular and
plural forms of a term), and some linguistic variants are
particularly mentioned below (e.g., "administration" and
"administering"). The chemical elements are identified in
accordance with the Periodic Table of the Elements, CAS version,
Handbook of Chemistry and Physics, 75th Ed. Additionally, general
principles of organic chemistry are well established and one of
ordinary skill in the art can consult, if desired, Organic
Chemistry by Thomas Sorrell, University Science Books, Sausalito,
1999; Smith and March, March's Advanced Organic Chemistry, 5.sup.th
Edition, John Wiley & Sons, Inc., New York, 2001; Larock,
Comprehensive Organic Transformations, VCH Publishers, Inc., New
York, 1989; and Carruthers, Some Modern Methods of Organic
Synthesis, 3.sup.rd Edition, Cambridge University Press, Cambridge,
1987.
[0019] The term "about," when used in reference to a value,
signifies any value or range of values that is plus-or-minus 10% of
the stated value (e.g., within plus-or-minus 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9% or 10% of the stated value). For example, a dose of
about 10 mg means any dose as low as 10% less than 10 mg (9 mg),
any dose as high as 10% more than 10 mg (11 mg), and any dose or
dosage range therebetween (e.g., 9-11 mg; 9.1-10.9 mg; 9.2-10.8 mg;
and so on). As another example, a prevalence rank in a population
of about 80% means a prevalence rank of 72-88% (e.g., 79.2-80.8%).
In case of doubt, "about X" can be "X" (e.g., about 80% can be
80%). Where a stated value cannot be exceeded (e.g., 100%), "about"
signifies any value or range of values that is up to and including
10% less than the stated value (e.g., a purity of about 100% means
90%-100% pure (e.g., 95%-100% pure, 96%-100% pure, 97%-100% pure
etc. . . . )). In the event an instrument or technique measuring a
value has a margin of error greater than 10%, a given value will be
about the same as a stated value when they are both within the
margin of error for that instrument or technique.
[0020] The term "administration" and variants thereof, such as
"administering," refer to the administration of a compound
described herein (e.g., a compound of Formula (I), (Ia), a species
thereof or a specified form thereof (e.g., a pharmaceutically
acceptable salt of a compound of Formula (I), (Ia), or a species
thereof), or an additional/second agent), or a composition
containing the compound to a subject (e.g., a human patient) or
system (e.g., a cell- or tissue-based system that is maintained ex
vivo); as a result of the administration, the compound or
composition containing the compound (e.g., a pharmaceutical
composition) is introduced to the subject or system. In addition to
compositions of the invention and second agents useful in
combination therapies, items used as positive controls, negative
controls, and placebos, any of which can also be a compound, can
also be "administered." One of ordinary skill in the art will be
aware of a variety of routes that can, in appropriate
circumstances, be utilized for administration to a subject or
system. For example, the route of administration can be oral (i.e.,
by swallowing a pharmaceutical composition) or may be parenteral.
More specifically, the route of administration can be bronchial
(e.g., by bronchial instillation), by mouth (i.e., oral), dermal
(which may be or comprise topical application to the dermis or
intradermal, interdermal, or transdermal administration),
intragastric or enteral (i.e., directly to the stomach or
intestine, respectively), intramedullary, intramuscular,
intranasal, intraperitoneal, intrathecal, intratumoral, intravenous
(or intra-arterial), intraventricular, by application to or
injection into a specific organ (e.g., intrahepatic), mucosal
(e.g., buccal, rectal, sublingual, or vaginal), subcutaneous,
tracheal (e.g., by intratracheal instillation), or ocular (e.g.,
topical, subconjunctival, or intravitreal). Administration can
involve intermittent dosing (i.e., doses separated by various
times) and/or periodic dosing (i.e., doses separated by a common
period of time (e.g., every so many hours, daily (e.g., once daily
oral dosing), weekly, twice per week, etc.)). In other embodiments,
administration may involve continuous dosing (e.g., perfusion) for
a selected time (e.g., about 1-2 hours).
[0021] Two events, two entities, or an event and an entity are
"associated" with one another if one or more features of the first
(e.g., its presence, level and/or form) are correlated with a
feature of the second. For example, a first entity (e.g., an enzyme
(e.g., CDK7)), gene expression profile, genetic signature (i.e., a
single or combined group of genes in a cell with a uniquely
characteristic pattern of gene expression), metabolite, or event
(e.g., myeloid infiltration)) is associated with an event (e.g.,
the onset or progression of a particular disease), if its presence,
level and/or form correlates with the incidence of, severity of,
and/or susceptibility to the disease (e.g., a cancer disclosed
herein). The biomarkers described herein are associated with an
identified patient in the manner described herein (e.g., by virtue
of their level of expression) and, depending on their status, can
also be associated with a clinical outcome (e.g., a better
prognosis based on an increased likelihood that a treatment regimen
described herein will be more successful as evidenced by, e.g.,
TGI, preferably beyond the cessation of treatment). Associations
are typically assessed across a relevant population. Two or more
entities are physically "associated" with one another if they
interact, directly or indirectly, so that they are and/or remain in
physical proximity with one another in a given circumstance (e.g.,
within a cell maintained under physiological conditions (e.g.,
within cell culture) or within a pharmaceutical composition).
Entities that are physically associated with one another can be
covalently linked to one another or non-covalently associated by,
for example, hydrogen bonds, van der Waals forces, hydrophobic
interactions, magnetism, or combinations thereof. A compound of
Formula (I), (Ia), a species thereof, or a specified form thereof
(e.g., a pharmaceutically acceptable salt) can non-covalently
associate with CDK7.
[0022] The term "biological sample" refers to a sample obtained or
derived from a biological source of interest (e.g., a tissue or
organism (e.g., an animal or human patient) or cell culture). For
example, a biological sample can be a sample obtained from an
individual (e.g., a patient or an animal model) suffering from a
disease (or, in the case of an animal model, a simulation of that
disease in a human patient) to be diagnosed and/or treated by the
methods of this invention or from an individual serving in the
capacity of a reference or control (or whose sample contributes to
a reference standard or control population). The biological sample
can contain a biological cell, tissue or fluid or any combination
thereof. For example, a biological sample can be or can include
ascites; blood; blood cells; a bodily fluid, any of which may
include or exclude cells (e.g., tumor cells (e.g., circulating
tumor cells (CTCs) found in at least blood or lymph vessels)) or
circulating tumor DNA (ctDNA); bone marrow or a component thereof
(e.g., hematopoietic cells, marrow adipose tissue, or stromal
cells); cerebrospinal fluid (CSF); feces; flexural fluid;
free-floating nucleic acids (e.g., circulating tumor DNA);
gynecological fluids; hair; immune infiltrates; lymph; peritoneal
fluid; plasma; saliva; skin or a component part thereof (e.g., a
hair follicle); sputum; surgically-obtained specimens; tissue
scraped or swabbed from the skin or a mucus membrane (e.g., in the
nose, mouth, or vagina); tissue or fine needle biopsy samples;
urine; washings or lavages such as a ductal lavage or
broncheoalveolar lavage; or other body fluids, tissues, secretions,
and/or excretions. Samples of, or samples obtained from, a bodily
fluid (e.g., blood, CSF, lymph, plasma, or urine) may include tumor
cells (e.g., CTCs) and/or free-floating or cell-free nucleic acids
of the tumor. Cells (e.g., cancer cells) within the sample may have
been obtained from an individual patient for whom a treatment is
intended. Samples used in the form in which they were obtained may
be referred to as "primary" samples, and samples that have been
further manipulated (e.g., by removing one or more components of
the sample) may be referred to as "secondary" or "processed"
samples. Such processed samples may contain or be enriched for a
particular cell type (e.g., a CDK7-expressing cell, which may be a
tumor cell), cellular component (e.g., a membrane fraction), or
cellular material (e.g., one or more cellular proteins, including
CDK7, DNA, or RNA (e.g., mRNA), which may encode CDK7 and may be
subjected to amplification). As used herein, the term "biomarker"
refers to an entity whose state correlates with a particular
biological event so that it is considered to be a "marker" for that
event (e.g., the presence of a particular cancer and its
susceptibility to a compound of Formula (I), (Ia), a species
thereof, or a specified form thereof). A biomarker can be analyzed
at the nucleic acid or protein level; at the nucleic acid level,
one can analyze the presence (e.g., copy number alterations
(CNAs)), absence, or chromosomal location of a gene in wild type or
mutant form, epigenetic alterations (in, e.g., methylation), its
association with a super-enhancer, and/or its level of expression
(as evidenced, for example, by primary RNA transcript or mRNA
levels). At the protein level, one can analyze the level of
expression and/or activity of a protein encoded by a biomarker
gene. A biomarker may indicate a therapeutic outcome or likelihood
(e.g., increased likelihood) thereof. Thus, a biomarker can be
predictive or prognostic and is therefore useful in methods of
identifying or treating a patient as described herein.
[0023] The term "cancer" refers to a disease in which biological
cells exhibit an aberrant growth phenotype characterized by loss of
control of cell proliferation to an extent that will be detrimental
to a patient having the disease. A cancer can be classified by the
type of tissue in which it originated (histological type) and/or by
the primary site in the body in which the cancer first developed.
Based on histological type, cancers are generally grouped into six
major categories: carcinomas; sarcomas; myelomas; leukemias;
lymphomas; and mixed types. A cancer treated as described herein
may be of any one of these types and may comprise cells that are
precancerous (e.g., benign), malignant, pre-metastatic, metastatic,
and/or non-metastatic. A patient who has a malignancy or malignant
lesion has a cancer. The present disclosure specifically identifies
certain cancers to which its teachings may be particularly
relevant, and one or more of these cancers may be characterized by
a solid tumor or by a hematologic tumor, which may also be known as
a blood cancer (e.g., of a type described herein). Although not all
cancers manifest as solid tumors, we may use the terms "cancer
cell" and "tumor cell" interchangeably to refer to any malignant
cell.
[0024] The term "combination therapy" refers to those situations in
which a subject is exposed to two or more therapeutic regimens
(e.g., two or more therapeutic agents) to treat a single disease
(e.g., a cancer). The two or more regimens/agents may be
administered simultaneously or sequentially. When administered
simultaneously, a dose of the first agent and a dose of the second
agent are administered at about the same time, such that both
agents exert an effect on the patient at the same time or, if the
first agent is faster- or slower-acting than the second agent,
during an overlapping period of time. When administered
sequentially, the doses of the first and second agents are
separated in time, such that they may or may not exert an effect on
the patient at the same time. For example, the first and second
agents may be given within the same hour or same day, in which case
the first agent would likely still be active when the second is
administered. Alternatively, a much longer period of time may
elapse between administration of the first and second agents, such
that the first agent is no longer active when the second is
administered (e.g., all doses of a first regimen are administered
prior to administration of any dose(s) of a second regimen by the
same or a different route of administration, as may occur in
treating a refractory cancer). For clarity, combination therapy
does not require that two agents be administered together in a
single composition or at the same time, although in some
embodiments, two or more agents, including a compound of Formula
(I), (Ia), a species thereof, or a specified form thereof and a
second agent described herein, may be administered within the same
period of time (e.g., within the same hour, day, week, or
month).
[0025] The terms "cutoff" and "cutoff value" mean a value measured
in an assay that defines the dividing line between two subsets of a
population (e.g., likely responders and non-responders (e.g.,
responders and non-responders to a compound of Formula (I), (Ia), a
species thereof, or a specified form thereof)). In some instances,
values that are equal to or above the cutoff value define one
subset of the population, and values that are lower than the cutoff
value define the other subset of the population. In other
instances, values that are equal to or below the cutoff value
define on subset of the population, and values above the cutoff
value define the other. As described further below, the cutoff or
cutoff value can define the threshold value.
[0026] As used herein, "diagnostic information" is information that
is useful in determining whether a patient has a disease and/or in
classifying (stratifying) the disease into a genotypic or
phenotypic category or any category having significance with regard
to the prognosis of the disease or its likely response to treatment
(either treatment in general or any particular treatment described
herein). Similarly, "diagnosis" refers to obtaining or providing
any type of diagnostic information, including, but not limited to,
whether a patient is likely to have or develop a disease; whether
that disease has or is likely to reach a certain state or stage or
to exhibit a particular characteristic (e.g., resistance to a
therapeutic agent); information related to the nature or
classification of a tumor; information related to prognosis (which
may also concern resistance); and/or information useful in
selecting an appropriate treatment (e.g., selecting a compound of
Formula (I), (Ia), a species thereof, or a specified form thereof
for a patient identified as having a cancer that is likely to
respond to such an inhibitor or other treatment). A patient
classified (stratified) according to a method described herein and
selected for treatment with a compound of Formula (I), (Ia), a
species thereof, or a specified form thereof is likely to respond
well to the treatment, meaning that such a patient is more likely
to be successfully treated than a patient with the same type of
cancer who has not been so identified and is not in the same
strata. Available treatments include therapeutic agents and other
treatment modalities such as surgery, radiation, etc., and
selecting an appropriate treatment encompasses the choice of
withholding a particular therapeutic agent; the choice of a dosing
regimen; and the choice of employing a combination therapy.
Diagnostic information can be used to stratify patients and is thus
useful in identifying and classifying a given patient according to,
for example, biomarker status. Obtaining diagnostic information can
constitute a step in any of the patient stratification methods
described herein.
[0027] One of ordinary skill in the art will appreciate that the
term "dosage form" may be used to refer to a physically discrete
unit of an active agent (e.g., a therapeutic or diagnostic agent)
for administration to a patient. Typically, each such unit contains
a predetermined quantity of active agent. In some embodiments, such
quantity is a unit dosage amount (or a whole fraction thereof)
appropriate for administration in accordance with a dosing regimen
that has been determined to correlate with a desired or beneficial
outcome when administered to a relevant population (i.e., with a
therapeutic dosing regimen). Those of ordinary skill in the art
appreciate that the total amount of a therapeutic composition or
agent administered to a particular patient is determined by one or
more attending physicians and may involve administration of
multiple dosage forms.
[0028] One of ordinary skill in the art will appreciate that the
term "dosing regimen" may be used to refer to a set of unit doses
(typically more than one) that are administered individually to a
patient, separated by equal or unequal periods of time. A given
therapeutic agent typically has a recommended dosing regimen, which
may involve one or more doses, each of which may contain the same
unit dose amount or differing amounts. In some embodiments, a
dosing regimen comprises a first dose in a first dose amount,
followed by one or more additional doses in a second dose amount
that is different from the first dose amount. In some embodiments,
a dosing regimen is correlated with a desired or beneficial outcome
when administered across a relevant population (i.e., the regimen
is a therapeutic dosing regimen).
[0029] As used herein, an "effective amount" of an agent (e.g., a
chemical compound described herein), such as a compound of Formula
(I), refers to an amount that produces or is expected to produce
the desired effect for which it is administered. The effective
amount will vary depending on factors such as the desired
biological endpoint, the pharmacokinetics of the compound
administered, the condition being treated, the mode of
administration, and characteristics of the patient, as discussed
further below and recognized in the art. The term can be applied to
therapeutic and prophylactic methods. For example, a
therapeutically effective amount is one that reduces the incidence
and/or severity of one or more signs or symptoms of the disease.
For example, in treating a cancer, an effective amount may reduce
the tumor burden, stop tumor growth, inhibit metastasis or prolong
patient survival. One of ordinary skill in the art will appreciate
that the term does not in fact require successful treatment be
achieved in any particular individual. Rather, a therapeutically
effective amount is that amount that provides a particular desired
pharmacological response in a significant number of patients when
administered to patients in need of such treatment. In some
embodiments, reference to a therapeutically effective amount may be
a reference to an amount administered or an amount measured in one
or more specific tissues (e.g., a tissue affected by the disease)
or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
Effective amounts may be formulated and/or administered in a single
dose or in a plurality of doses, for example, as part of a dosing
regimen.
[0030] As used herein, an "enhancer" is a region of genomic DNA
that helps regulate the expression of genes; enhancers have been
found up to 1 Mbp away from a gene they regulate. An enhancer may
overlap, but is often not composed of, gene coding regions. An
enhancer is often bound by transcription factors and designated by
specific histone marks.
[0031] The term "patient" refers to any organism that is or may be
subjected to a diagnostic method described herein or to which a
compound described herein, or a specified form thereof, is or may
be administered for, e.g., experimental, diagnostic, prophylactic,
and/or therapeutic purposes. Typical patients include animals
(e.g., mammals such as mice, rats, rabbits, non-human primates, and
humans; domesticated animals, such as dogs and cats; and livestock
or any other animal of agricultural or commercial value). A patient
may be suffering from or susceptible to (i.e., have a higher than
average risk of developing) a disease described herein and may
display one or more signs or symptoms thereof.
[0032] The term "pharmaceutically acceptable," when applied to a
carrier used to formulate a composition disclosed herein (e.g., a
pharmaceutical composition), means a carrier that is compatible
with the other ingredients of the composition and not deleterious
to a patient (e.g., it is non-toxic in the amount required and/or
administered (e.g., in a unit dosage form)).
[0033] The term "pharmaceutically acceptable," when applied to a
salt, solvate, stereoisomer, tautomer, or isotopic form of a
compound described herein, refers to a salt, solvate, stereoisomer,
tautomer, or isotopic form that is, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
humans (e.g., patients) and lower animals (including, but not
limited to, mice and rats used in laboratory studies) without
unacceptable toxicity, irritation, allergic response and the like,
and that can be used in a manner commensurate with a reasonable
benefit/risk ratio. Many pharmaceutically acceptable salts are well
known in the art (see, e.g., Berge et al., J. Pharm. Sci. 66:1-19,
1977). Pharmaceutically acceptable salts of the compounds described
herein include those derived from suitable inorganic and organic
acids and bases. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid, and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid, or malonic acid or by using other
methods known in the art such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, MALAT1e, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate, p
toluenesulfonate, undecanoate, valerate salts, and the like. Salts
derived from appropriate bases include alkali metal, alkaline earth
metal, ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 salts.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
[0034] As used herein, the term "population" means some number of
items (e.g., at least 30, 40, 50, or more) sufficient to reasonably
reflect the distribution, in a larger group, of the value being
measured in the population. Within the context of the present
invention, the population can be a discrete group of humans,
laboratory animals, or cells lines (for example) that are
identified by at least one common characteristic for the purposes
of data collection and analysis. For example, a "population of
samples" refers to a plurality of samples that is large enough to
reasonably reflect the distribution of a value (e.g., a value
related to the state of a biomarker) in a larger group of samples.
The items in the population may be biological samples, as described
herein. For example, each sample in a population of samples may be
cells of a cell line or a biological sample obtained from a patient
or a xenograft (e.g., a tumor grown in a mouse by implanting a
tumorigenic cell line or a patient sample into the mouse). As
noted, individuals within a population can be a discrete group
identified by a common characteristic, which can be the same
disease (e.g., the same type of cancer), whether the sample is
obtained from living beings suffering from the same type of cancer
or a cell line or xenograft representing that cancer.
[0035] The term "prevalence cutoff," as used herein in reference to
a specified value (e.g., the strength of a SE associated with a
biomarker gene) means the prevalence rank that defines the dividing
line between two subsets of a population (e.g., a subset of
"responders" and a subset of "non-responders," which, as the names
imply include patients who are likely or unlikely, respectively, to
experience a beneficial response to a therapeutic agent or agents).
Thus, a prevalence rank that is equal to or higher (e.g., a lower
percentage value) than the prevalence cutoff defines one subset of
the population; a prevalence rank that is lower (e.g., a higher
percentage value) than the prevalence cutoff defines the other
subset.
[0036] As used herein, the term "prevalence rank" for a specified
value (e.g., the mRNA level of a specific biomarker) means the
percentage of a population that are equal to or greater than that
specific value. For example, a 35% prevalence rank for the amount
of mRNA of a specific biomarker in a test cell means that 35% of
the population have that level of biomarker mRNA or greater than
the test cell.
[0037] As used herein, the terms "prognostic information" and
"predictive information" are used to refer to any diagnostic
information that may be used to indicate any aspect of the course
of a disease or condition either in the absence or presence of
treatment. Such information may include, but is not limited to, the
average life expectancy of a patient, the likelihood that a patient
will survive for a given amount of time (e.g., 6 months, 1 year, 5
years, etc.), the likelihood that a patient will be cured of a
disease, the likelihood that a patient's disease will respond to a
particular therapy (wherein response may be defined in any of a
variety of ways). Diagnostic information can be prognostic or
predictive.
[0038] As used herein, the term "rank ordering" means the ordering
of values from highest to lowest or from lowest to highest.
[0039] As used herein, the terms "RB-E2F pathway" and "RB-E2F
family" refer to a set of genes and the proteins encoded thereby,
as the context will make clear, whose expression or activity
regulates the activity of the RB gene family and in turn regulates
the activity of the E2F family of transcription factors that are
required for entry into and progression through the cell cycle. The
table below contains a list of genes in the RB-E2F family, an
indication of a currently understood function of the encoded
proteins and the status of these biomarkers in cancer. We use the
shorthand "activated or overexpressed" to indicate that an
attribute of a gene (e.g., its copy number or level of expression)
or the protein it encodes (e.g., its level of expression or
activity) is higher in some patients with certain cancers than it
is in healthy subjects. A pre-determined threshold for such
activated or overexpressed RB-E2F family members can be determined
by comparative analysis and is a level (e.g., mRNA level, protein
level, gene copy number, strength of enhancer associated with the
gene) that, when found or exceeded in a cancer patient, identifies
that patient as a candidate for treatment as described herein. We
use the shorthand "inactivated or underexpressed" to indicate that
an attribute of a gene (e.g., its copy number, or level of
expression) or a protein it encodes (e.g., its level of expression
or activity) is lower in some patients with certain cancers than it
is in healthy subjects. A pre-determined threshold for such
inactivated or underexpressed RB-E2F family members can be
determined by comparative analysis and is a level (e.g., mRNA
level, protein level, CNV, strength of enhancer associated with the
gene) that, when unattained in a cancer patient, identifies that
patient as a candidate for treatment as described herein.
TABLE-US-00001 Gene Function Status in Cancer E2F1 E2F family -
transcriptional control of cell cycle Activated or overexpressed
entry E2F2 E2F family - transcriptional control of cell cycle
Activated or overexpressed entry E2F3 E2F family - transcriptional
control of cell cycle Activated or overexpressed entry E2F4 E2F
family - transcriptional control of cell cycle Activated or
overexpressed entry E2F5 E2F family - transcriptional control of
cell cycle Activated or overexpressed entry E2F6 E2F family -
transcriptional control of cell cycle Activated or overexpressed
entry E2F7 E2F family - transcriptional control of cell cycle
Activated or overexpressed entry E2F8 E2F family - transcriptional
control of cell cycle Activated or overexpressed entry RB1 RB
family - E2F family inhibition Inactivated or underexpressed RBL1
RB family - E2F family inhibition Inactivated or underexpressed
RBL2 RB family - E2F family inhibition Inactivated or
underexpressed CDK4 RB family inhibition Activated or overexpressed
CDK6 RB family inhibition Activated or overexpressed CDK2 RB family
inhibition Activated or overexpressed CCND1 CDK4/6 regulation
Activated or overexpressed CCND2 CDK4/6 regulation Activated or
overexpressed CCND3 CDK4/6 regulation Activated or overexpressed
CDKN2A CDK4/6 regulation Inactivated or underexpressed CDKN2B
CDK4/6 regulation Inactivated or underexpressed CDKN2C CDK4/6
regulation Inactivated or underexpressed CDKN2D CDK4/6 regulation
Inactivated or underexpressed CCNE1 CDK2 regulation Activated or
overexpressed CCNE2 CDK2 regulation Activated or overexpressed
CDKN1A CDK2 regulation Inactivated or underexpressed CDKN1B CDK2
regulation Inactivated or underexpressed CDKN1C CDK2 regulation
Inactivated or underexpressed FBXW7 CCNE regulation Inactivated or
underexpressed
It will be readily apparent to one of ordinary skill in the art
that for those genes in the RB-E2F pathway that are activated or
overexpressed in cancer, one would select those patients that had
(1) an alteration in the DNA encoding such gene that resulted in
increased expression (e.g. elevated gene copy number, mutation that
led to increased activity, change in methylation that led to
increased expression); (2) an epigenetic alteration associated with
that gene that resulted in increased expression (e.g. histone
methylation or histone acetylation pattern that led to increased
expression); or (3) an increase in the level of expression of mRNA
or protein encoded by that gene. For those genes in the RB-E2F
pathway that are inactivated or under-expressed in cancer, one
would select from those patients that had (1) an alteration in the
DNA encoding that gene that resulted in decreased expression or
activity (e.g. reduced gene copy number, mutation that led to
decreased activity or inactivity, change in methylation that led to
decreased expression); (2) an epigenetic alteration associated with
that gene that resulted in decreased expression (e.g. histone
methylation or histone acetylation pattern that led to decreased
expression); or (3) an decrease in the level of expression of mRNA
or protein encoded by that gene.
[0040] As used herein, a "reference" refers to a standard or
control relative to which a comparison is performed. For example,
an agent, patient, population, sample, sequence, or value of
interest is compared with a reference agent, patient, population,
sample, sequence or value. The reference can be analyzed or
determined substantially simultaneously with the analysis or
determination of the item of interest or it may constitute a
historical standard or control, determined at an earlier point in
time and optionally embodied in a tangible medium. One of ordinary
skill in the art is well trained in selecting appropriate
references, which are typically determined or characterized under
conditions that are comparable to those encountered by the item of
interest. One of ordinary skill in the art will appreciate when
sufficient similarities are present to justify reliance on and/or
comparison to a particular possible reference as a standard or
control.
[0041] As used herein, a "response" to treatment is any beneficial
alteration in a patient's condition that results from, or that
correlates with, treatment. The alteration may be stabilization of
the condition (e.g., inhibition of deterioration that would have
taken place in the absence of the treatment), amelioration of,
delay of onset of, and/or reduction in frequency of one or more
signs or symptoms of the condition, improvement in the prospects
for cure of the condition, greater survival time, and etc. A
response may be a patient's response or a tumor's response.
[0042] As used herein, when the term "strength" is used to refer to
a portion of an enhancer or a SE, it means the area under the curve
of the number of H3K27Ac or other genomic marker reads plotted
against the length of the genomic DNA segment analyzed. Thus,
"strength" is an integration of the signal resulting from measuring
the mark at a given base pair over the span of the base pairs
defining the region being chosen to measure.
[0043] As used herein, the term "super-enhancer" (SE) refers to a
subset of enhancers that contain a disproportionate share of
histone marks and/or transcriptional proteins relative to other
enhancers in a particular cell or cell type. Genes regulated by SEs
are predicted to be of high importance to the function of a cell.
SEs are typically determined by rank ordering all of the enhancers
in a cell based on strength and determining, using available
software such as ROSE (bitbucket.org/young_computation/rose), the
subset of enhancers that have significantly higher strength than
the median enhancer in the cell (see, e.g., U.S. Pat. No.
9,181,580, which is hereby incorporated by reference herein in its
entirety).
[0044] The terms "threshold" and "threshold level" mean a level
that defines the dividing line between two subsets of a population
(e.g., responders and non-responders). A threshold or threshold
level can define a prevalence cutoff or a cutoff value.
[0045] As used herein, the terms "treatment," "treat," and
"treating" refer to reversing, alleviating, delaying the onset of,
and/or inhibiting the progress of a "pathological condition" (e.g.,
a disease, such as cancer) described herein. In some embodiments,
"treatment," "treat," and "treating" require that signs or symptoms
of the disease have developed or have been observed. In other
embodiments, treatment may be administered in the absence of signs
or symptoms of the disease or condition (e.g., in light of a
history of symptoms and/or in light of genetic or other
susceptibility factors). Treatment may also be continued after
symptoms have resolved, for example, to delay or inhibit
recurrence.
[0046] As the invention relates to compositions and methods for
diagnosing and treating patients who have cancer, the terms "active
agent," "anti-cancer agent," "pharmaceutical agent," and
"therapeutic agent" are used interchangeably (unless the context
clearly indicates otherwise) and compounds of Formula (I), (Ia), a
species thereof, or a specified form thereof, would be understood
by one of ordinary skill in the art as active, anti-cancer,
pharmaceutical, or therapeutic agents. As noted, the treatment
methods and uses encompass combination therapies/uses in which a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof is administered or used in combination with one or
more additional agents (e.g., an additional anti-cancer
therapeutic), as described herein. In keeping with convention, in
any embodiment requiring two agents, we may refer to one as the
"first" agent and to the other as the "second" agent to underscore
that the first and second agents are distinct from one another.
Where three agents are employed, we refer to the "third agent."
[0047] As indicated, each therapeutic method and any diagnostic
method that employs a compound of Formula (I), (Ia), a species
thereof, or a specified form thereof may also be expressed in terms
of use and vice versa. For example, the invention encompasses the
use of a compound or composition described herein for the treatment
of a disease described herein (e.g., cancer); a compound or
composition for use in diagnosing and/or treating or a disease
(e.g., cancer); and the use of the compound or composition for the
preparation of a medicament for treating a disease described herein
(e.g., cancer).
[0048] A patient subjected to a diagnostic or therapeutic method
described herein may have a blood cancer, which may also be
referred to as a hematopoietic or hematological cancer or
malignancy, and any of the methods described herein can entail
analyzing a biomarker described herein in a biological sample of,
e.g., blood or lymph, obtained from the patient. More specifically
and in various embodiments, the blood cancer can be a leukemia such
as acute lymphocytic leukemia (ALL; e.g., B cell ALL or T cell
ALL), acute myelocytic leukemia (AML; e.g., B cell AML or T cell
AML), chronic myelocytic leukemia (CML; e.g., B cell CML or T cell
CML), chronic lymphocytic leukemia (CLL; e.g., B cell CLL (e.g.,
hairy cell leukemia) or T cell CLL), chronic neutrophilic leukemia
(CNL), or chronic myelomonocytic leukemia (CMML). The blood cancer
can also be a lymphoma such as Hodgkin lymphoma (HL; e.g., B cell
HL or T cell HL), non-Hodgkin lymphoma (NHL, which can be deemed
aggressive; e.g., B cell NHL or T cell NHL), follicular lymphoma
(FL), chronic lymphocytic leukemia/small lymphocytic lymphoma
(CLL/SLL), mantle cell lymphoma (MCL), a marginal zone lymphoma
(MZL), such as a B cell lymphoma (e.g., splenic marginal zone B
cell lymphoma), primary mediastinal B cell lymphoma (e.g., splenic
marginal zone B cell lymphoma), primary mediastinal B cell
lymphoma, Burkitt lymphoma (BL), lymphoplasmacytic lymphoma (i.e.,
Waldenstrom's macroglobulinemia), immunoblastic large cell
lymphoma, precursor B lymphoblastic lymphoma, or primary central
nervous system (CNS) lymphoma. The B cell NHL can be diffuse large
cell lymphoma (DLCL; e.g., diffuse large B cell lymphoma (DLBCL;
e.g., germinal center B cell-like (GCB) DLBCL or activated B-cell
like (ABC) DLBCL)), and the T cell NHL can be precursor T
lymphoblastic lymphoma or a peripheral T cell lymphoma (PTCL). In
turn, the PTCL can be a cutaneous T cell lymphoma (CTCL) such as
mycosis fungoides or Sezary syndrome, angioimmunoblastic T cell
lymphoma, extranodal natural killer T cell lymphoma, enteropathy
type T cell lymphoma, subcutaneous anniculitis-like T cell
lymphoma, or anaplastic large cell lymphoma.
[0049] In other embodiments, the cancer is characterized by a solid
tumor considered to be either of its primary location or
metastatic. For example, in various embodiments, the cancer or
tumor treated or prevented as described herein is an acoustic
neuroma; adenocarcinoma; adrenal gland cancer; anal cancer;
angiosarcoma (e.g., lymphangiosarcoma,
lymphangio-endotheliosarcoma, hemangiosarcoma); appendix cancer;
benign monoclonal gammopathy (also known as monoclonal gammopathy
of unknown significance (MGUS); biliary cancer (e.g.,
cholangiocarcinoma); bladder cancer; breast cancer (e.g.,
adenocarcinoma of the breast, papillary carcinoma of the breast,
mammary cancer, medullary carcinoma of the breast; any of which may
be present in subjects having a particular profile, such as an
HR+(ER+ or PR+), HER2+, HR- (having neither estrogen nor
progesterone receptors), a triple negative breast cancer (TNBC;
ER-/PR-/HER2-), or a triple-positive breast cancer (ER+/PR+/HER2+);
a brain cancer (e.g., meningioma, glioblastoma, glioma (e.g.,
astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer;
carcinoid tumor, which may be benign; cervical cancer (e.g.,
cervical adenocarcinoma); choriocarcinoma; chordoma;
craniopharyngioma; a cancer present in the large intestine, such as
colorectal cancer (CRC, e.g., colon cancer, rectal cancer, or
colorectal adenocarcinoma); connective tissue cancer; epithelial
carcinoma; ependymoma; endothelio-sarcoma (e.g., Kaposi's sarcoma
or multiple idiopathic hemorrhagic sarcoma); endometrial cancer
(e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g.,
adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's
sarcoma (or other pediatric sarcoma, such as embryonal
rhabdomyosarcoma or alveolar rhabdomyosarcoma); eye cancer (e.g.,
intraocular melanoma, retinoblastoma); familiar hypereosinophilia;
gallbladder cancer; gastric cancer (e.g., stomach adenocarcinoma);
gastrointestinal stromal tumor (GIST); germ cell cancer; head and
neck cancer (e.g., head and neck squamous cell carcinoma, oral
cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g.,
laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,
oropharyngeal cancer)); hypopharynx cancer; inflammatory
myofibroblastic tumors; immunocytic amyloidosis; kidney cancer
(e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma);
liver cancer (e.g., hepatocellular cancer (HCC), malignant
hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell
lung cancer (SCLC), non-small cell lung cancer (NSCLC),
adenocarcinoma, squamous cell carcinoma, or large cell carcinoma of
the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic
mastocytosis); mouth cancer; muscle cancer; myelodys-plastic
syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD)
(e.g., polycythemia vera (PV), essential thrombocytosis (ET),
agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF),
chronic idiopathic myelofibrosis, hypereosinophilic syndrome
(HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF)
type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g.,
gastroentero-pancreatic neuroendocrine tumor (GEP-NET), carcinoid
tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g.,
cystadenocarcinoma, ovarian embryonal carcinoma, ovarian
adenocarcinoma, HGSOC, LGSOC, epithelial ovarian cancer (e.g.,
ovarian clear cell carcinoma or mucinous carcinoa), sex cord
stromal tumors (granulosa cell), and endometroid tumors); papillary
adenocarcinoma; pancreatic cancer (whether an exocrine tumor (e.g.,
pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma
(PDAC)), intraductal papillary mucinous neoplasm (IPMN), or a
neuroendocrine tumor (e.g., PNETs or islet cell tumors); penile
cancer (e.g., Paget's disease of the penis and scrotum); pinealoma;
primary peritoneal cancer, primitive neuroectodermal tumor (PNT);
plasma cell neoplasia; paraneoplastic syndromes; prostate cancer,
which may be castration-resistant (e.g., prostate adenocarcinoma);
rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g.,
squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma,
basal cell carcinoma (BCC)); small bowel or small intestine cancer;
soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH),
liposarcoma, malignant peripheral nerve sheath tumor (MPNST),
chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland
carcinoma; sweat gland carcinoma; synovioma; testicular cancer
(e.g., seminoma, testicular embryonal carcinoma); thyroid cancer
(e.g., papillary carcinoma of the thyroid, papillary thyroid
carcinoma (PTC), medullary thyroid cancer); urethral cancer;
vaginal cancer; and vulvar cancer (e.g., Paget's disease of the
vulva). We use the term "gastrointestinal (GI) tract cancer" to
refer to a cancer present anywhere in the GI tract, including
cancers of the mouth, throat, esophagus, stomach, large or small
intestine, rectum, and anus. As noted above, the cancer can be a
neuroendocrine cancer, and such tumors can be treated as described
herein regardless of the organ in which they present. A biomarker
described herein can be analyzed in a biological sample containing
tumor cells or ctDNA of any of the cancer types just listed.
Further, a patient identified by analyzing a biomarker as described
herein can be "newly diagnosed" and therefor previously unexposed
to a compound of Formula (I), (Ia), a species thereof, or a
specified form thereof and, similarly, previously unexposed to a
second agent as described herein. We may refer to such a patient as
treatment naive.
[0050] The methods of the invention that concern diagnosing and/or
treating a cancer described herein (or use of a compound or
compounds for such purposes) may specifically exclude any one or
more of the types of cancers described herein. For example, the
invention features methods of treating cancer by administering a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof, with the proviso that the cancer is not a breast
cancer; with the proviso that the cancer is not a breast cancer or
a leukemia; with the proviso that the cancer is not a breast
cancer, a leukemia, or an ovarian cancer; and so forth, with
exclusions selected from any of the cancers listed herein and with
the same notion of variable exclusion from lists of elements
relevant to other aspects of the invention (e.g., chemical
substituents of a compound described herein or components of kits
and pharmaceutical compositions). Thus, where elements are
presented as lists (e.g., in Markush group format), every possible
subgroup of the elements is also disclosed, and any element(s) can
be removed from the group.
[0051] In one aspect, the invention features the use of a compound
of Formula (I), (Ia), a species thereof, or a specified form
thereof in treating cancer in a patient who has been identified by
analyzing the biomarker BCL2, RB1, RBL1, RBL2, CDKN2A, CDKN2B,
CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C, or FBXW7 in a biological
sample containing cancer cells or ctDNA from the patient. Analyzing
the biomarker can include analyzing its sequence to detect a
mutation or determining CNA, association with a SE, RNA expression
level (e.g., mRNA expression) or another feature described above as
indicating the state of the biomarker. A patient identified by
analyzing BCL2, RB1, RBL1, RBL2, CDKN2A, CDKN2B, CDKN2C, CDKN2D,
CDKN1A, CDKN1B, CDKN1C, or FBXW7 can be: treated with a
platinum-based therapeutic agent (e.g., carboplatin, cisplatin, or
oxaliplatin) as a second agent; a patient whose cancer has
developed resistance to a platinum-based therapeutic agent (e.g.,
carboplatin, cisplatin, or oxaliplatin); or a patient undergoing
treatment with a CDK4/6 inhibitor used alone or in combination with
one or more of an aromatase inhibitor, a selective estrogen
receptor modulator (SERM), selective estrogen receptor degrader
(SERD), or estrogen suppressant, any of which may be selected from
the descriptions of such agents provided herein or known in the
art. The patient's cancer may have become resistant to the CDK4/6
inhibitor or be at risk of becoming so. In the context of these
uses (e.g., where the patient has been identified by analyzing the
biomarker BCL2, RB1, RBL1, RBL2, CDKN2A, CDKN2B, CDKN2C, CDKN2D,
CDKN1A, CDKN1B, CDKN1C, or FBXW7), the cancer can be a breast
cancer (e.g., a triple negative breast cancer (TNBC), HR+, or other
type of breast cancer described herein), an ovarian cancer (e.g.,
HGSOC), a lung cancer (e.g., SCLC, NSCLC or other lung cancer
described herein), retinoblastoma, or a blood cancer (e.g., acute
myeloid leukemia (AML)).
[0052] The methods of treating such a patient include a step of
administering an effective amount of a compound of Formula (I),
(Ia), a species thereof or a specified form thereof, optionally
within a pharma-ceutical composition described herein and/or
according to a dosing regimen described herein.
[0053] In another aspect, the invention features the use of a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof in treating cancer in a patient who has been
identified by analyzing the biomarker CCNE1, CCNE2, RB1, CDK6,
CCND1, CCND2, CCND3, or CCKN2A in a biological sample containing
cancer cells or ctDNA from the patient. Analyzing the biomarker can
include analyzing its sequence to detect a mutation or determining
CNA, association with a SE, RNA expression level (e.g., mRNA
expression) or another feature described above as indicating the
state of the biomarker. A patient identified by analyzing CCNE1,
CCNE2, RB1, CDK6, CCND1, CCND2, CCND3, or CCKN2A can be a patient
who has undergone, is presently undergoing, or who will undergo
(e.g., has been prescribed) treatment with a Bcl-2 inhibitor, such
as venetoclax, a SERM, such as tamoxifen, a SERD, such as
fulvestrant, or a PARP inhibitor, such as olaparib or niraparib. In
the context of these methods, the patient may have a breast cancer
(e.g., TNBC or an HR+ breast cancer), lymphoma, melanoma (e.g.,
familial melanoma), ovarian cancer (e.g., HGSOC), or pancreatic
cancer (e.g., PDAC). For example, where the biomarker is CDKN2A,
the patient may have TNBC, PDAC, or HGSOC. For example, where the
biomarker is CCNE1, the patient may have TNBC, HGSOC, melanoma
(e.g., familial melanoma), or lymphoma. As noted above, one of
ordinary skill will recognize, as is well established in the art,
the relationship between a given gene and the protein it encodes.
Thus, it will be clear that our reference to, for example, "the
biomarker BCL2" encompasses analysis of the biomarker gene
BCL2-like 1 and the biomarker protein (BCL2) encoded thereby; "the
biomarker CCNE1" encompasses analysis of the biomarker gene CCNE1
and the biomarker protein (cyclin E1) encoded thereby; and so
forth. The methods of treating such a patient include a step of
administering an effective amount of a compound of Formula (I),
(Ia), a species thereof or a specified form thereof, optionally
within a pharmaceutical composition described herein and/or
according to a dosing regimen described herein.
[0054] In another aspect, the invention features the use of a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof in treating cancer in a patient who has been
identified by analyzing the biomarker MYC (see Kalkat et al., Genes
8(6):151, 2017), CDK1, CDK2, CDK4, CDK17, CDK18, CDK19, CCNA1,
CCNB1, ESR-1 or FGFR1 in a biological sample containing cancer
cells or ctDNA from the patient. Analyzing the biomarker can
include analyzing any mutations within MYC, CDK1, CDK2, CDK4,
CDK17, CDK18, CDK19, CCNA1, CCNB1, ESR-1 or FGFR1 or determining
CNA, association with a SE, RNA expression level (e.g., mRNA
expression) or another feature described above as indicating the
state of the biomarker. The patient may have a breast cancer (e.g.,
TNBC or an ovarian cancer (e.g., HGSOC) and may be resistant to a
platinum-based anti-cancer agent, such as carboplatin, cisplatin,
or oxaliplatin, resistant to gemcitabine, resistant to a PARP
inhibitor, such as olaparib or niraparib, or resistant to a taxane,
such as paclitaxel. The methods of treating such a patient include
a step of administering an effective amount of a compound of
Formula (I), (Ia), a species thereof or a specified form thereof,
optionally within a pharmaceutical composition described herein
and/or according to a dosing regimen described herein. C-myc
encodes at least two phosphoproteins with apparent molecular
weights of 62,000 and 66,000 (see Ramsay et al., Proc. Natl. Acad.
Sci. (USA) 81(24):7742-7746, 1984), and it has been determined
through H3K27Ac ChIP-seq (ChIP-sequencing) methods that there is a
SE locus associated with the MYC gene at chr8:128628088-128778308
(Gencode v19 annotation of the human genome build hg19/GRCh37).
[0055] In another aspect, the invention features the use of a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof in treating cancer in a patient who has been
identified by analyzing the biomarker CDK7 or CDK9. Analyzing the
biomarker can include analyzing any mutations within CDK7 or CDK9
or determining CNA, association with a SE, RNA expression level
(e.g., mRNA expression) or another feature described above as
indicating the state of the biomarker. Where the biomarker is CDK7
or CDK9, the patient may have a lymphoma and the
diagnosing/identifying step may more specifically be based on
analysis of CDK7 (e.g., the level of CDK7 mRNA); the patient may
have a breast cancer (e.g., TNBC), with the diagnosing/identifying
step more specifically based on CDK9 (e.g., the level of CDK9
mRNA); the patient may have a TNBC or a lung cancer (e.g., SCLC),
with the diagnosing step more specifically be based CDK19 (e.g., on
the level of CDK19 mRNA). The methods of treating such a patient
include a step of administering an effective amount of a compound
of Formula (I), (Ia), a species thereof or a specified form
thereof, optionally within a pharmaceutical composition described
herein and/or according to a dosing regimen described herein.
[0056] In another aspect, the invention features the use of a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof in treating cancer in a patient who has been
identified by analyzing the biomarker BRAF, E2F1, E2F2, E2F3, E2F4,
E2F5, E2F6, E2F7, or E2F8 in a biological sample containing cancer
cells or ctDNA from the patient. Analyzing the biomarker can
include analyzing its sequence to detect a mutation or determining
CNA, association with a SE, RNA expression level (e.g., mRNA
expression) or another feature described above as indicating the
state of the biomarker. A patient identified by analyzing BRAF,
E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, or E2F8 (by virtue of
having a feature equal to or above a pre-determined threshold, as
described herein) can be a patient who has undergone, is presently
undergoing, or who will undergo (e.g., has been prescribed)
treatment with a PI3K inhibitor, such as alpelisib or capecitabine,
a platinum-based anti-cancer agent, such as carboplatin, cisplatin,
or oxaliplatin, or vincristine. In the context of these methods,
the patient may have a melanoma, lung cancer (e.g., NSCLC), GI
tract cancer (e.g., CRC), thyroid cancer, retinoblastoma, or
leukemia (e.g., hairy cell leukemia). The methods of treating such
a patient include a step of administering an effective amount of a
compound of Formula (I), (Ia), a species thereof or a specified
form thereof, optionally within a pharmaceutical composition
described herein and/or according to a dosing regimen described
herein.
[0057] A compound or other composition described herein (e.g., a
pharmaceutical composition comprising a compound of Formula (I),
(Ia), a species thereof or specified form thereof) can be
administered in a combination therapy (e.g., as defined and further
described herein) with a second agent described herein or a
plurality thereof (i.e., a patient identified as described herein
may be treated with first, second, and third agents). The
additional/second agent employed in a combination therapy is most
likely to achieve a desired effect for the same disorder (e.g., the
same cancer), however it may achieve different effects that aid the
patient. Accordingly, the invention features pharmaceutical
compositions containing a compound of Formula (I), (Ia), a species
thereof, or a specified form thereof (e.g., a pharmaceutically
acceptable salt), optionally in a therapeutically effect amount,
for use in treating a patient identified as described herein. The
pharmaceutical compositions may optionally include any of the
additional/second agents described herein and will include a
pharmaceutically acceptable carrier. The second/additional agent
can be selected from a Bcl-2 inhibitor such as venetoclax, a PARP
inhibitor such as olaparib or niraparib, a platinum-based
anti-cancer agent such as carboplatin, cisplatin, or oxaliplatin, a
taxane such as docetaxel or paclitaxel (or paclitaxel protein-bound
(available as Abraxane.RTM.)), a CDK4/6 inhibitor such as
palbociclib, ribociclib, abemaciclib, or trilaciclib, a selective
estrogen receptor modulator (SERM) such as tamoxifen (available
under the brand names Nolvadex.TM. and Soltamox.TM.), raloxifene
(available under the brand name Evista.TM.), and toremifene
(available as Fareston.TM.) and a selective estrogen receptor
degrader such as fulvestrant (available as Faslodex.TM.), each in a
therapeutically effective amount.
[0058] Unless otherwise specified, when employing a combination of
a compound of Formula (I), (Ia), a species thereof, or a specified
variant thereof and a second therapeutic agent in a method of the
invention, the second therapeutic agent can be administered
concurrently with, prior to, or subsequent to a compound of Formula
(I), (Ia), a species thereof, or a specified form thereof. The
second therapeutic pharmaceutical agent may be administered at a
dose and/or on a time schedule determined for that pharmaceutical
agent. An additional/second therapeutic agent may also be
administered together with the compound of Formula (I), (Ia), a
species thereof, or a specified form thereof in a single dosage
form or administered separately in different dosage forms. In
general, and without limitation, it is expected that the second
therapeutic agents utilized in combination with a compound of
Formula (I), (Ia), a species thereof, or a specified form thereof
will be utilized at levels that do not exceed the levels at which
they are utilized individually. In some embodiments, the levels of
the second therapeutic agent utilized in combination will be lower
than those utilized in a monotherapy due to synergistic
effects.
[0059] In particular combination therapies for a patient identified
as described herein: (a) the cancer is TNBC, an ER+ breast cancer,
pancreatic cancer (e.g., PDAC), or a squamous cell cancer of the
head or neck and the second agent is a CDK4/6 inhibitor; (b) the
cancer is a breast cancer (e.g., TNBC) or an ovarian cancer and the
second agent is a PARP inhibitor; (c) the cancer is a leukemia
(e.g., AML) and the second agent is a FLT3 inhibitor; (d) the
cancer is an ovarian cancer (e.g., HGSOC) and the second agent is a
platinum-based anti-cancer agent; (e) the cancer is a breast cancer
(e.g., TNBC), a leukemia (e.g., AML), Ewing's sarcoma, or an
osteosarcoma and the second agent is a BET inhibitor; (f) the
cancer is a breast cancer (e.g., TNBC), a leukemia (e.g., AML), an
ovarian cancer (e.g., HGSOC), or a lung cancer (e.g., NSCLC) and
the second agent is a Bcl-2 inhibitor. In particular embodiments,
the cancer is AML and the second agent is a Bcl-2 inhibitor, such
as venetoclax; the cancer is an epithelial ovarian cancer, a
fallopian tube cancer, a primary peritoneal cancer, a triple
negative breast cancer or a Her2.sup.+/ER.sup.-/PR.sup.- breast
cancer and the second agent is a PARP inhibitor, such as olaparib
or niraparib; the cancer is an ovarian cancer and the second agent
is a platinum-based anti-cancer agent, such as carboplatin,
cisplatin, or oxaliplatin. As noted above, and regardless of the
biomarker analyzed or the type of cancer in question, a method of
treatment can either be carried out on an identified patient
without an explicit step of analyzing the biomarker or with an
explicit step in which the biomarker is analyzed (e.g., by
obtaining a biological sample from a patient).
[0060] With regard to combination therapies, a patient identified
as described herein can be treated with a combination of a compound
of Formula (I), (Ia), a species thereof, or a specified form
thereof and one or more of a second agent that can be, but is not
limited to, a Bcl-2 inhibitor such as APG-1252, APG-2575, BP1002
(prexigebersen), the antisense oligonucleotide known as oblimersen
(G3139), S55746/BCL201, or venetoclax (e.g., venetoclax tablets
marketed as Venclexta.RTM.); a CDK9 inhibitor such as
alvocidib/DSP-2033/flavopiridol, AT7519, AZD5576, BAY1251152,
BAY1143572, CYC065, nanoflavopiridol, NVP2, seliciclib (CYC202),
TG02, TP-1287, VS2-370 or voruciclib (formerly P1446A-05); a
hormone receptor (e.g., estrogen receptor) degradation agent, such
as fulvestrant (e.g., marketed as Faslodex.RTM. and others); a Flt3
(FMS-like tyrosine kinase 3) inhibitor such as CDX-301, CG'806,
CT053PTSA, crenolanib (e.g., crenolanib besylate), ENMD-2076,
FF-10101-01, FLYSYN, gilteritinib (ASP2215), HM43239, lestautinib,
ponatinib (e.g., marketed as Iclusig.RTM., previously AP24534),
NMS-088, sorafenib (e.g., marketed as Nexavar.RTM.), sunitinib,
pacritinib, pexidartinib/PLX3397, quizartinib, midostaurin (e.g.,
marketed as Rydapt.RTM.), SEL24, SKI-G-801, or SKLB1028; a PARP
inhibitor such as olaparib (e.g., marketed as Lynparza.RTM.),
rucaparib (e.g., marketed as Rubraca.RTM.), talazoparib (e.g.,
marketed as Talzenna.RTM.), veliparib (ABT-888), or niraparib
(e.g., marketed as Zejula.RTM.); a BET inhibitor such as ABBV-075,
BAY-299, BAY-1238097, BMS-986158, CPI-0610, CPI-203, FT-1101,
GS-5829, GSK-2820151, GSK-525762, I-BET151, I-BET762, INCB054329,
JQ1, MS436, OTX015, PFI-1, PLX51107, RVX2135, TEN-010, ZEN-3694, or
a compound disclosed in U.S. application Ser. No. 12/810,564 (now
U.S. Pat. No. 8,476,260), which is hereby incorporated herein by
reference in its entirety; a platinum-based therapeutic agent such
as cisplatin, oxaliplatin (e.g., marketed as Eloxatin.RTM.),
nedaplatin, carboplatin (e.g., marketed as Paraplatin.RTM.),
phenanthriplatin, picoplatin, satraplatin (JM216), or triplatin
tetranitrate; a CDK4/6 inhibitor such as BPI-1178, G1T38,
palbociclib (e.g., marketed as Ibrance.RTM.), ribociclib (e.g.,
marketed as Kisqali.RTM.), ON 123300, trilaciclib, or abemaciclib
(e.g., marketed as Verzenio.RTM.); a MEK inhibitor such as
trametinib (e.g., marketed as Mekinist.RTM.), cobimetinib
(available as Cotellic.RTM.), or binimetinib (Braftovi.RTM.),
useful in combination with a compound of Formula (I), (Ia), a
species thereof, or specified form thereof, in treating, e.g.,
melanoma); or a phosphoinositide 3-kinase (PI3 kinase) inhibitor,
optionally of Class I (e.g., Class IA) and/or optionally directed
against a specific PI3K isoform. The PI3K inhibitor can be
apitolisib (GDC-0980), idelalisib (e.g., marketed as Zydelig.RTM.),
copanlisib (e.g., marketed as Aliqopa.RTM.), duvelisib (e.g.,
marketed as Copiktra.RTM.), pictilisib (GDC-0941), or alpelisib
(e.g., marketed as Piqray.RTM.). In other embodiments, the
additional/second agent can be capecitabine (e.g., marketed as
Xeloda.RTM.). Such PI3K inhibitors can be combined with a compound
of Formula (I), (Ia), a species thereof or specified form thereof
in treating, e.g., HR+ breast cancer, TNBC, lymphoma (e.g.,
follicular lymphoma or non-Hodgkin lymphoma), or leukemia (e.g.,
CLL). In other embodiments, the additional/second agent can be
gemcitabine (combined with a compound of the invention to treat,
e.g., TNBC, CRC, SCLC, or a pancreatic cancer (e.g., PDAC)). In
other embodiments, the additional/second agent can be an
antimetabolite, such as the pyrimidine analog 5-fluorouracil
(5-FU), which may be used in combination with a compound of Formula
(I), (Ia), a species thereof, or a specified form thereof, and one
or more of leucovorin, methotrexate, or oxaliplatin. In other
embodiments, the additional/second agent can be an aromatase
inhibitor, such as exemestane or anastrasole. In other embodiments,
the additional/second agent is an inhibitor of the PI3K/AKT/mTOR
pathway (e.g., gedatolisib). In one embodiment, the methods
encompass the use of or administration of a compound of Formula
(I), (Ia), a species thereof or a specified form thereof, to a
patient identified as described herein, in combination with a MEK
inhibitor, such as trametinib (available as Mekinist.RTM.),
cobimetinib (available as Cotellic.RTM.), or binimetinib (available
as Braftovi.RTM.)
[0061] APG-1252 is a dual Bcl-2/Bcl-xL inhibitor that has shown
promise in early clinical trials when patients having SCLC or
another solid tumor were dosed between 10-400 mg (e.g., 160 mg)
intravenously twice weekly for three weeks in a 28-day cycle (see
Lakhani et al., J. Clin. Oncol. 36:15_suppl, 2594, and
ClinicalTrials.gov identifier NCT03080311). APG-2575 is a Bcl-2
selective inhibitor that has shown promise in preclinical studies
of FL and DLBCL in combination with ibrutinib (see Fang et al.,
AACR Annual Meeting 2019, Cancer Res. 79(13 Suppl):Abstract No.
2058) and has begun clinical trials as a single-agent treatment for
patients with blood cancers; in a dose escalation study, patients
are given 20 mg, once daily, by mouth, for four consecutive weeks
as one cycle. Escalations to 50, 100, 200, 400, 600 and 800 mg are
planned to identify the MTD (see ClinicalTrials.gov identifier
NCT03537482). BP1002 is an uncharged P-ethoxy antisense
oligodeoxynucleotide targeted against Bcl-2 mRNA that may have
fewer adverse effects than other antisense analogs and has shown
promise in inhibiting the growth of human lymphoma cell lines
inclubated with BP1002 for four days and of CJ cells (transformed
FL cells) implanted into SCID mice (see Ashizawa et al., AACR
Annual Meeting 2017, Cancer Res. 77(13 Suppl):Abstract No. 5091).
BP1002 has also been administered in combination with cytarabine
(LDAC) to patients having AML (see ClinicalTrials.gov identifier
NCT04072458). S55746/BCL201 is an orally available, selective Bcl-2
inhibitor that, in mice, demonstrated anti-tumor efficacy in two
blood cancer xenograft models (Casara et al., Oncotarget
9(28):20075-88, 2018). A phase I dose-escalation study was designed
to administer film-coated tablets containing 50 or 100 mg of
S55746, in doses up to 1500 mg, to patients with CLL or a B cell
NHL including FL, MCL, DLBCL, SLL, MZL, and MM (see
ClinicalTrials.gov identifier NCT02920697). Venetoclax tablets have
been approved for treating adult patients with CLL or SLL and, in
combination with azacytidine, or decitabine, or low-dose
cytarabine, for treating newly-diagnosed AML in patients who are at
least 75 years old or who have comorbidities that preclude the use
of intensive induction chemotherapy. Dosing for CLL/SLL can follow
the five-week ramp-up schedule and dosing for AML can follow the
four-day ramp-up, both described in the product insert, together
with other pertinent information (see also U.S. Pat. Nos.
8,546,399; 9,174,982; and 9,539,251, which are hereby incorporated
by reference in their entireties). Alvocidib was studied in
combination with cytarabine/mitoxantrone or cytarabine/daunorubicin
in patients with AML, with the details of administration being
available at ClinicalTrials.gov with the identifier NCT03563560
(see also Yeh et al., Oncotarget 6(5):2667-2679, 2015, Morales et
al., Cell Cycle 15(4):519-527, 2016, and Zeidner et al.,
Haematologica 100(9):1172-1179, 2015). AT7519 has been administered
in a dose escalation format to eligible patients having refractory
solid tumors. While there was some evidence of clinical activity,
the appearance of QTc prolongation precluded further development at
the dose schedule described by Mahadevan et al. (J. Clin. Oncol.
ASCO Abstract No. 3533; see also Santo et al., Oncogene
29:2325-2336, 2010, describing the preclinical activity of AT7519
in MM). AZD5576 induced apoptosis in breast and lung cancer cell
lines at the nanomolar level (see Li et al., Bioorg. Med. Chem.
Lett. 27(15):3231-3237, 2017) and has been examined alone and in
combination with acalabrutinib for the treatment of NHL (see AACR
2017 Abstract No. 4295). BAY1251152 was the subject of a phase I
clinical trial to characterize the MTD in patients with advanced
blood cancers; the agent was infused weekly in 21-day cycles (see
ClinicalTrials.gov identifier NCT02745743; see also Luecking et
al., AACR 2017 Abstract No. 984). Voruciclib is a clinical stage
oral CDK9 inhibitor that represses MCL-1 and sensitizes high-risk
DLBCL to BCL2 inhibition. Dey et al. (Scientific Reports 7:18007,
2017) suggest that the combination of voruciclib and venetoclax is
promising for a subset of high-risk DLBCL patients (see also
ClinicalTrials.gov identifier NCT03547115). Fulvestrant has been
approved for administration to postmenopausal women with advanced
hormone receptor (HR)-positive, HER2-negative breast cancer, with
HR-positive metastatic breast cancer whose disease progressed after
treatment with other anti-estrogen therapies, and in combination
with palbociclib (Ibrance.RTM.). Fulvestrant is administered by
intramuscular injection at 500 or 250 mg (the lower dose being
recommended for patients with moderate hepatic impairment) on days
1, 15, and 29, and once monthly thereafter (see the product insert
for additional information; see also U.S. Pat. Nos. 6,744,122;
7,456,160; 8,329,680; and 8,466,139, each of which are hereby
incorporated by reference herein in their entireties). Ponatinib
has been administered in clinical trials to patients with CML or
ALL (see ClinicalTrials.gov identifiers NCT0066092072,
NCT012074401973, NCT02467270, NCT03709017, NCT02448095,
NCT03678454, and NCT02398825) as well as solid tumors, such as
biliary cancer and NSCLC (NCT02265341, NCT02272998, NCT01813734,
NCT02265341, NCT02272998, NCT01813734, NCT02265341, NCT02272998,
NCT01813734, NCT01935336, NCT03171389, and NCT03704688; see also
the review article by Tan et al., Onco. Targets Ther. 12:635-645,
2019). Additional information regarding the dosing regimen can be
found in the product insert; see also U.S. Pat. Nos. 8,114,874;
9,029,533; and 9,493,470, each of which is hereby incorporated by
reference herein in its entirety. Sorafenib has been approved for
the treatment of kidney and liver cancers, AML, and radioactive
iodine resistant advanced thyroid cancer, and a clinical trial was
initiated in patients with desmoid-type fibromatosis (see
ClinicalTrials.gov identifier NCT02066181). Information regarding
dosage can be found in the product insert, which advises
administration of two, 400 mg tablets twice daily; see also U.S.
Pat. Nos. 7,235,576; 7,351,834; 7,897,623; 8,124,630; 8,618,141;
8,841,330; 8,877,933; and 9,737,488, each of which is hereby
incorporated by reference herein in its entirety. Midostaurin has
been administered to patients having AML, MDS, or systemic
mastocytosis, and has been found to significantly prolong survival
of FLT3-mutated AML patients when combined with conventional
induction and consolidation therapies (see Stone et al., ASH 57th
Annual Meeting, 2015 and Gallogly et al., Ther. Adv. Hematol.
8(9):245-251, 2017; din see also the product insert,
ClinicalTrials.gov identifier NCT03512197, and U.S. Pat. Nos.
7,973,031; 8,222,244; and 8,575,146, each of which is hereby
incorporated by reference herein in its entirety. Alpelisib is a
kinase inhibitor indicated in combination with fulvestrant for the
treatment of postmenopausal women, and men, with
HR+/HER2-/PIK3CA-mutated, advanced or metastatic breast cancer as
deteted by an FDA-approved test following progression on or after
an endocrine-based regimen. The recommended dose is 300 mg (two 150
mg tablets) taken orally once daily with food, which, as for all
chemotherapeutic agents, may be interrupted, reduced, or
discontinued to manage adverse reactions. Paclitaxel is supplied as
a nonaqueous solution intended for dilution with a suitable
parenteral fluid prior to intravenous infusion. Under the brand
name Taxol.RTM., it is supplied in 30 mg, 100 mg, and 300 mg vials
and can be used in a combination therapy described herein to treat
a variety of cancers, including those of the bladder, breast,
esophagus, fallopian tube or ovary, lung, skin (melanoma), and
prostate. Palbociclib has been approved for use in HR+/HER2-
advanced or metastatic breast cancer at a recommended dose of 125
mg daily, by mouth. It can be used to treat a patient as identified
herein with a compound of Formula (I), (Ia), a species thereof, or
a specified form thereof, either alone or in combination with an
aromatase inhibitor or fulvestrant. The information provided here
and publicly available can be used to practice the methods and uses
of the invention. In case of doubt, the invention encompasses
combination therapies that require a compound of the invention or a
specified form thereof and any one or more additional/second
agents, which may be administered at or below a dosage currently
approved for single use (e.g., as described above), to a patient as
described herein. Triplet combinations include a compound of
Formula (I), (Ia), a species thereof, or a specified form thereof
with: alpelisib and fulvestrant or alpelisib and a taxane (for,
e.g., treating NSCLC).
[0062] Where the combination therapy employs a compound of the
invention and: a CDK4/6 inhibitor, the patient can have a breast
cancer (e.g., TNBC or an ER+ breast cancer), pancreatic cancer,
lung cancer (e.g., SCLC or NSCLC), or squamous cell cancer of the
head and neck; a CDK9 inhibitor, the patient can have a breast
cancer and, more specifically, a Her2.sup.+/ER.sup.-/PR.sup.-
breast cancer; a Flt3 inhibitor (e.g., midostaurin), the patient
can have a hematological cancer (e.g., AML); a BET inhibitor, the
patient can have a hematological cancer (e.g., AML), a breast
cancer (e.g., TNBC), an osteosarcoma or Ewing's Sarcoma; a Bcl-2
inhibitor (e.g., venetoclax), the patient can have a breast cancer
(e.g., TNBC), an ovarian cancer, a lung cancer (e.g., NSCLC) or a
hematological cancer (e.g., AML); or a PARP inhibitor (e.g.,
niraparib or olaparib), the patient can have a breast cancer (e.g.,
TNBC or Her2.sup.+/ER.sup.-/PR.sup.- breast cancer), an ovarian
cancer (e.g., an epithelial ovarian cancer), a fallopian tube
cancer, or a primary peritoneal cancer.
[0063] In some aspects relating to using RB-E2F pathway genes (or
the proteins they encode) as biomarkers, the invention provides a
method of treating a patient having a cancer and identified as
described herein, which comprises administering to a patient
identified as having either (a) a level of CCNE1 mRNA or protein in
the cancer equal to or above a pre-determined threshold; and/or (b)
a level of RB1 mRNA or protein in the cancer equal to or below a
pre-determined threshold, an effective amount of a CDK7 inhibitor
of Formula (I). In some aspects of these embodiments, the method
further comprises determining a level of RB1 and/or CCNE1 mRNA or
protein present in a sample of cancer cells from the patient. In
various embodiments, the human patient is diagnosed as having a
cancer sensitive to a CDK7 inhibitor responsive to the
determination; is suffering from ovarian cancer; or is suffering
from a breast cancer (e.g., TNBC or an HR.sup.+ breast cancer). In
some embodiments, a compound of Formula (I), (Ia), a species
thereof or a specified form thereof is co-administered with a PARP
inhibitor. In some embodiments, the compound of Formula (I), (Ia),
a species thereof or a specified form thereof is co-administered
with a SERM (e.g., tamoxifen, raloxifene, or toremifene), a SERD
such fulvestrant, or an agent that inhibits the production of
estrogen (e.g., an aromatase inhibitor such as anastrozole
(available as Arimidex.RTM.), exemestane (available as
Aromasin.RTM.), and letrozole (available as Femara.RTM.),
optionally to treat a cancer that is refractory to palbociclib. Our
data indicate that a compound of Formula (I), (Ia), a species
thereof, or a specified form thereof (e.g., Compound 101) can
induce deep and sustained TGI in combination with fulvestrant in
palbociclib-resistant (PBR) ER+ breast cancer PDX models. Further,
based on data with Compound 101, we believe compounds of the
invention can resensitize palbociclib- and fulvestrant-resistant
(PBR/FSR) ER+ breast cancer PDX tumors to fulvestrant treatment. In
other embodiments, the invention provides methods of treating
cancer in a patient identified as described herein by administering
to the patient a combination of a compound of Formula (I), (Ia), a
species thereof, or a specified form thereof and a platinum-based
standard of care (SOC) anti-cancer agent for such cancer or a
taxane. The cancer can be an ovarian cancer and the SOC anti-cancer
agent can be a platinum-based anti-cancer agent (e.g., carboplatin,
cisplatin, or oxaliplatin). In some embodiments, the human patient
is, has been determined to be, or has become resistant (after some
initial responsiveness) to the platinum-based anti-cancer agent
when administered as either a monotherapy or in combination with an
anti-cancer agent other than a CDK7 inhibitor. In some aspects of
this embodiment, the human patient is determined to have become
resistant to the platinum-based anti-cancer agent when administered
as a monotherapy or in combination with an anti-cancer agent other
than a CDK7 inhibitor after some initial efficacy of that prior
treatment. In some aspects of this embodiment, the SOC anti-cancer
agent is a taxane (e.g., paclitaxel).
[0064] The invention also provides methods of treating a
biomarker-identified HR.sup.+ breast cancer in a human patient
selected on the basis of being resistant to treatment with a CDK4/6
inhibitor comprising the step of administering to the patient a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof. In some embodiments, prior to administration of the
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof, the patient is, has been determined to be, or has
become resistant (after some initial responsiveness) to a prior
treatment with a CDK4/6 inhibitor alone or in combination with
another SOC agent for breast cancer other than a CDK7 inhibitor,
such as an aromatase inhibitor (e.g., letrozole, anastrozole) or a
SERM (e.g., tamoxifen, raloxifene, or toremifene), SERD (e.g.,
fulvestrant), or estrogen suppressant such as anastrozole
(available as Arimidex.RTM.), exemestane (available as
Aromasin.RTM.), or letrozole (available as Femara.RTM.). In other
words, the identified patient is selected for treatment with a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof on the basis of being resistant to prior treatment
with a CDK4/6 inhibitor alone or in combination with another SOC
agent for breast cancer other than a CDK7 inhibitor. In some
embodiments, the compound of Formula (I), (Ia), a species thereof,
or a specified form thereof is co-administered with another SOC
agent, such as an aromatase inhibitor (e.g. anastrozole,
exemestane, or letrozole) and/or a SERM or SERD, e.g., as described
herein, or a second line treatment after failure on an aromatase
inhibitor or fulvestrant. In some embodiments, prior to
administration of the compound of Formula (I), (Ia), a species
thereof, or a specified form thereof, the patient is, has been
determined to be, or has become resistant (after some initial
responsiveness) to treatment with a CDK4/6 inhibitor alone or in
combination with another SOC agent for breast cancer other than a
CDK7 inhibitor, such as an aromatase inhibitor (e.g., anastrozole,
exemestane, or letrozole), or a SERM or SERD such as tamoxifen or
fulvestrant; and the compound of Formula (I), (Ia), a species
thereof, or a specified form thereof is co-administered with a SOC
agent for breast cancer (e.g., a second line treatment after
failure of an aromatase inhibitor or a SERM or SERD such as
tamoxifen or fulvestrant.
[0065] An enhancer or SE can be identified by various methods known
in the art (see Hinsz et al., Cell, 155:934-947, 2013; McKeown et
al., Cancer Discov., 7(10):1136-53, 2017; and PCT/US2013/066957,
each of which are hereby incorporated herein by reference in their
entireties). Identifying a SE can be achieved by obtaining a
biological sample from a patient (e.g., from a biopsy or other
source, as described herein). The important metrics for enhancer
measurement occur in two dimensions: along the length of the DNA
over which genomic markers (e.g., H3K27Ac) are contiguously
detected and the compiled incidence of genomic marker at each base
pair along that span of DNA, the compiled incidence constituting
the magnitude. The measurement of the area under the curve ("AUC")
resulting from integration of length and magnitude analyses
determines the strength of the enhancer. The strength of the BRAF,
MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1,
CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an
E2F pathway member (E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8,
CCND1, CCND2, CCND3, CCNE1, or CCNE2) SEs relative to an
appropriate reference can be used to diagnose (stratify) a patient
and thereby determine whether a patient is likely to respond well
to a compound of Formula (I), (Ia), a species thereof, or a
specified form thereof. It will be readily apparent to one of
ordinary skill in the art, particularly in view of the instant
specification, that if the length of DNA over which the genomic
markers is detected is the same for each of BRAF, MYC, CDK1, CDK2,
CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1,
FGFR1, PIK3CA, or certain genes encoding an E2F pathway member
(E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2,
CCND3, CCNE1, or CCNE2) and the reference/control, then the ratio
of the magnitude of the BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7,
CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or
certain genes encoding an E2F pathway member (E2F1, E2F2, E2F3,
E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3, CCNE1, or CCNE2)
SE relative to the control will be equivalent to the strength and
may also be used to determine whether a patient will be responsive
to a compound of Formula (I), (Ia), a species thereof, or a
specified form thereof. The strength of the BRAF, MYC, CDK1, CDK2,
CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1,
FGFR1, PIK3CA, or certain genes encoding an E2F pathway member
(E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2,
CCND3, CCNE1, or CCNE2) SE in a cell can be normalized before
comparing it to other samples. Normalization is achieved by
comparison to a region in the same cell known to comprise a
ubiquitous SE or enhancer that is present at similar levels in all
cells. One example of such a ubiquitous super-enhancer region is
the MALAT1 super-enhancer locus (chr11:65263724-65266724) (genome
build hg19).
[0066] It has been determined through H3K27Ac ChIP-seq
(ChIP-sequencing) methods that there is a SE locus associated with
the CDK18 gene at chr1:205399084-205515396; a SE locus associated
with the CDK19 gene at chr6:110803523-110896277; a SE locus
associated with the CCNE1 gene at chr19:30418503-30441450; and a SE
locus associated with the FGFR1 gene at chr8:38233326-38595483. All
loci are based on the Gencode v19 annotation of the human genome
build hg19/GRCh37.
[0067] ChIP-seq is used to analyze protein interactions with DNA by
combining chromatin immunoprecipitation (ChIP) with massively
parallel DNA sequencing to identify the binding sites of
DNA-associated proteins. It can be used to map global binding sites
precisely for any protein of interest. Previously, ChIP-on-chip was
the most common technique utilized to study these protein-DNA
relations. Successful ChIP-seq is dependent on many factors
including sonication strength and method, buffer compositions,
antibody quality, and cell number (see, e.g., Furey, Nature Reviews
Genetics 13:840-852, 2012); Metzker, Nature Reviews Genetics
11:31-46, 2010; and Park, Nature Reviews Genetics 10:669-680,
2009). Genomic markers other than H3K27Ac that can be used to
identify SEs using ChIP-seq include P300, CBP, BRD2, BRD3, BRD4,
components of the mediator complex (Loven et al., Cell,
153(2):320-334, 2013), histone 3 lysine 4 monomethylated (H3K4me1),
and other tissue-specific enhancer tied transcription factors
(Smith and Shilatifard, Nature Struct. Mol. Biol., 21(3):210-219,
2014; and Pott and Lieb, Nature Genetics, 47(1):8-12, 2015).
Quantification of enhancer strength and identification of SEs can
be determined using SE scores (McKeown et al., Cancer Discov.
7(10):1136-1153, 2017; DOI: 10.1158/2159-8290.CD-17-0399).
[0068] In some instances, H3K27Ac or other marker ChIP-seq data SE
maps of the entire genome of a cell line or a patient sample
already exist. One would then simply determine whether the strength
or ordinal rank of the enhancer or SE in such maps at the
chr8:128628088-128778308 (genome build hg19) locus was equal to or
above the pre-determined threshold level. In some embodiments, one
would simply determine whether the strength, or ordinal rank of the
enhancer or super-enhancer in such maps at the
chr1:205399084-205515396 (genome build hg19) locus was equal to or
above the pre-determined threshold level.
[0069] It should be understood that the specific chromosomal
location of BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18,
CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes
encoding an E2F pathway member (E2F1, E2F2, E2F3, E2F4, E2F5, E2F6,
E2F7, E2F8, CCND1, CCND2, CCND3, CCNE1, or CCNE2) and MALAT1 may
differ for different genome builds and/or for different cell types.
The same is true for BCL2-like 1, CDK7, CDK9, CDKN2A, and RB1 or
another E2F pathway member that is underexpressed in cancer (E2F1,
E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3,
CCNE1, or CCNE2). However, one skilled in the art, particularly in
view of the instant specification, can determine such different
locations by locating in such other genome builds specific
sequences corresponding to the loci in genome build hg 19.
[0070] Other methods that can be used to identify SEs in the
context of the present methods include chromatin
immunoprecipitation (Delmore et al., Cell, 146(6):904-917, 2011),
chip array (ChIP-chip), and chromatin immunoprecipitation followed
by qPCR (ChIP-qPCR) using the same immunoprecipitated genomic
markers and oligonucleotide sequences that hybridize to the
chr8:128628088-128778308 (genome build hg19) MYC locus or
chr1:205399084-205515396 (genome build hg19) CDK18 locus (for
example). In the case of ChIP-chip, the signal is typically
detected by intensity fluorescence resulting from hybridization of
a probe and input assay sample as with other array-based
technologies. For ChIP-qPCR, a dye that becomes fluorescent after
intercalating the double stranded DNA generated in the PCR reaction
is used to measure amplification of the template.
[0071] In some embodiments, determination of whether a cell has a
BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19,
CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes
encoding an E2F pathway member (see above) SE strength equal to or
above a requisite threshold level is achieved by comparing BRAF,
MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1,
CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an
E2F pathway member (see above) enhancer strength in a test cell to
the corresponding BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17,
CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain
genes encoding an E2F pathway member (see above) strength in a
population of cell samples, wherein each of the cell samples is
obtained from a different source (e.g., a different patient, a
different cell line, a different xenograft) reflecting the same
disease to be treated. In some embodiments, only primary tumor cell
samples from patients are used to determine the threshold level. In
some aspects of these embodiments, at least some of the samples in
the population will have been tested for responsiveness to a
specific CDK7 inhibitor (e.g., a compound of Formula (I), (Ia), a
species thereof or a specified form thereof) to establish: (a) the
lowest M BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18,
CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes
encoding an E2F pathway member (see above) enhancer strength of a
sample in the population that responds to that specific compound
("lowest responder"); and, optionally, (b) the highest BRAF, MYC,
CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1,
CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F
pathway member (see above) enhancer strength of a sample in the
population that does not respond to that specific compound
("highest non-responder"). In these embodiments, a cutoff of BRAF,
MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1,
CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an
E2F pathway member (see above) enhancer strength above which a test
cell would be considered responsive to that specific compound is
set: i) equal to or up to 5% above the BRAF, MYC, CDK1, CDK2, CDK4,
CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
enhancer strength in the lowest responder in the population; or ii)
equal to or up to 5% above the BRAF, MYC, CDK1, CDK2, CDK4, CDK6,
CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
enhancer strength in the highest non-responder in the population;
or iii) a value in between the BRAF, MYC, CDK1, CDK2, CDK4, CDK6,
CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
enhancer strength of the lowest responder and the highest
non-responder in the population.
[0072] In the above embodiments, not all of the samples in a
population necessarily are to be tested for responsiveness to a
specific CDK7 inhibitor (e.g., a compound of Formula (I), (Ia), a
species thereof or a specified form thereof), but all samples are
measured for BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18,
CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PICKCA, or certain genes
encoding an E2F pathway member (see above) enhancer strength. In
some embodiments, the samples are rank ordered based on M BRAF,
MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1,
CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F
pathway member (see above) enhancer strength. The choice of which
of the three methods set forth above to use to establish the cutoff
will depend upon the difference in BRAF, MYC, CDK1, CDK2, CDK4,
CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
enhancer strength between the lowest responder and the highest
non-responder in the population and whether the goal is to minimize
the number of false positives or to minimize the chance of missing
a potentially responsive sample or patient. When the difference
between the lowest responder and highest non-responder is large
(e.g., when there are many samples not tested for responsiveness
that fall between the lowest responder and the highest
non-responder in a rank ordering of BRAF, MYC, CDK1, CDK2, CDK4,
CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
enhancer strength), the cutoff is typically set equal to or is up
to 5% above the BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18,
CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes
encoding an E2F pathway member (see above) enhancer strength in the
lowest responder in the population. This cutoff maximizes the
number of potential responders. When this difference is small
(e.g., when there are few or no samples untested for responsiveness
that fall between the lowest responder and the highest
non-responder in a rank ordering of BRAF, MYC, CDK1, CDK2, CDK4,
CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
enhancer strength), the cutoff is typically set to a value in
between the BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19,
CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes
encoding an E2F pathway member (see above) enhancer strength of the
lowest responder and the highest non-responder. This cutoff
minimizes the number of false positives. When the highest
non-responder has a BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17,
CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain
genes encoding an E2F pathway member (see the Table herein)
enhancer strength that is greater than the lowest responder, the
cutoff is typically set to a value equal to or up to 5% above the
BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1,
CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an
E2F pathway member (see above) enhancer strength in the highest
non-responder in the population. This method also minimizes the
number of false positives.
[0073] In some embodiments, the methods discussed above can be
employed to simply determine if a diseased cell (e.g., a cancer
cell) from a patient has a SE associated with a biomarker gene
described herein (e.g., BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17,
CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain
genes encoding an E2F pathway member (see above) or a protein
encoded thereby). The presence of the SE indicates that the patient
is likely to respond well to a compound of Formula (I), (Ia), a
species thereof, or a specified form thereof. The cell is
determined to have a SE associated with the biomarker (e.g., BRAF,
MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1,
CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F
pathway member (see above) or a protein encoded thereby) when the
enhancer has a strength that is equal to or above the enhancer
associated with MALAT-1. In alternate embodiments, the cell is
determined to have a SE associated with BRAF, MYC, CDK1, CDK2,
CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
when the BRAF-, MYC-, CDK1-, CDK2-, CDK4-, CDK6-, CDK17-, CDK18-,
CDK19-, CCNA1-, CCNB1-, CCNE1-, ESR-1-, FGFR1-, PIK3CA-, or certain
genes encoding an E2F pathway member- (see above) associated
enhancer has a strength that is at least 10-fold greater than the
median strength of all of the enhancers in the cell. In other
embodiments, the cell is determined to have a SE associated with an
aforementioned gene when the gene-associated enhancer has a
strength that is above the point where the slope of the tangent is
1 in a rank-ordered graph of strength of each of the enhancers in
the cell.
[0074] For any biomarker (e.g., in embodiments involving CDK18),
the cutoff value for enhancer strength can be converted to a
prevalence cutoff, which can then be applied to biomarker RNA
levels (e.g., CDK18 mRNA) levels to determine a mRNA cutoff value
in a given mRNA assay.
[0075] In some embodiments, biomarker mRNA levels in a patient (as
assessed, e.g., in a biological sample obtained from the patient)
are compared, using the same assay, to the same gene of
interest/biomarker mRNA levels in a population of patients having
the same disease or condition to identify likely responders to a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof. Analogous comparisons can be made when another
feature of the biomarker is selected for analysis (e.g., its copy
number, chromosomal location, primary RNA level, or expressed
protein level). In embodiments where a biomarker (e.g., CDK18,
CDK19, and CCNE1) correlates with (e.g., is one whose mRNA
expression correlates with) responsiveness to a compound of the
invention, at least some of the samples in the population will have
been tested for responsiveness to the inhibitor (e.g., a compound
of Formula (I), (Ia), a species thereof, or specified form thereof)
to establish: (a) the lowest level (e.g., mRNA level) in a sample
in the population that responds to that specific compound ("lowest
mRNA responder"); and, optionally, (b) the highest level (e.g.,
highest mRNA level) in a sample in the population that does not
respond to that specific compound ("highest mRNA non-responder").
In these embodiments, a cutoff of biomarker mRNA level above which
a test cell would be considered responsive to that specific
compound is set: i) equal to or up to 5% above the level (e.g., the
mRNA level) in the lowest mRNA responder in the population; or ii)
equal to or up to 5% above the level (e.g., the mRNA level) in the
highest mRNA non-responder in the population; or iii) a value in
between the level (e.g., mRNA level) of the lowest responder (e.g.,
lowest mRNA responder) and the highest responder (e.g., highest
mRNA) non-responder in the population.
[0076] In embodiments where mRNA levels positively correlate with
sensitivity to a compound of Formula (I), (Ia), a species thereof
or a specified form thereof, not all of the samples in a population
need to be tested for responsiveness to the compound, but all
samples are measured for the gene of interest mRNA levels. In some
embodiments, the samples are rank ordered based on gene of interest
mRNA levels. The choice of which of the three methods set forth
above to use to establish the cutoff will depend upon the
difference in gene of interest mRNA levels between the lowest mRNA
responder and the highest mRNA non-responder in the population and
whether the cutoff is designed to minimize false positives or
maximize the potential number of responders. When this difference
is large (e.g., when there are many samples not tested for
responsiveness that fall between the lowest mRNA responder and the
highest mRNA non-responder in a rank ordering of mRNA levels), the
cutoff is typically set equal to or up to 5% above the mRNA level
in the lowest mRNA responder. When this difference is small (e.g.,
when there are few or no samples untested for responsiveness that
fall between the lowest mRNA responder and the highest mRNA
non-responder in a rank ordering of mRNA levels), the cutoff is
typically set to a value in between the mRNA levels of the lowest
mRNA responder and the highest mRNA non-responder. When the highest
mRNA non-responder has a mRNA level that is greater than the lowest
mRNA responder, the cutoff is typically set to a value equal to or
up to 5% above the mRNA levels in the highest mRNA non-responder in
the population.
[0077] In embodiments where a biomarker is one whose mRNA
expression inversely correlates with responsiveness to a compound
of Formula (I), (Ia), a species thereof, or a specified form
thereof (i.e., BCL-xL, CDK7, CDK9, or an RB1 family member), at
least some of the samples in the population will have been tested
for responsiveness to the compound in order to establish: (a) the
highest mRNA level of a sample in the population that responds to
that specific compound ("highest mRNA responder"); and, optionally,
(b) the lowest mRNA level of a sample in the population that does
not respond to that specific compound ("lowest mRNA
non-responder"). In these embodiments, a cutoff of mRNA level above
which a test cell would be considered responsive to that specific
compound is set: i) equal to or up to 5% below the mRNA level in
the highest mRNA responder in the population; or ii) equal to or up
to 5% below the mRNA level in the lowest mRNA non-responder in the
population; or iii) a value in between the mRNA level of the lowest
mRNA non-responder and the highest mRNA responder and in the
population.
[0078] In embodiments where mRNA levels inversely correlate with
sensitivity to a compound of the invention, not all of the samples
in a population need to be tested for responsiveness to the
compound, but all samples are measured for the gene of interest
mRNA levels. In some embodiments, the samples are rank ordered
based on gene of interest mRNA levels. The choice of which of the
three methods set forth above to use to establish the cutoff will
depend upon the difference in gene of interest mRNA levels between
the highest mRNA responder and the lowest mRNA non-responder in the
population and whether the cutoff is designed to minimize false
positives or maximize the potential number of responders. When this
difference is large (e.g., when there are many samples not tested
for responsiveness that fall between the highest mRNA responder and
the lowest mRNA non-responder in a rank ordering of mRNA levels),
the cutoff is typically set equal to or up to 5% below the mRNA
level in the highest mRNA responder. When this difference is small
(e.g., when there are few or no samples untested for responsiveness
that fall between the highest mRNA responder and the lowest mRNA
non-responder in a rank ordering of mRNA levels), the cutoff is
typically set to a value in between the mRNA levels of the highest
mRNA responder and the lowest mRNA non-responder. When the highest
mRNA responder has a mRNA level that is lower than the lowest mRNA
responder, the cutoff is typically set to a value equal to or up to
5% below the mRNA levels in the lowest mRNA non-responder in the
population.
[0079] In embodiments involving CDK18, the cutoff for CDK18 mRNA
levels may be determined using the prevalence cutoff established
based on CDK18 enhancer strength, as described above. In some
embodiments, a population is measured for mRNA levels and the prior
determined prevalence cutoff is applied to that population to
determine an mRNA cutoff level. In some aspects of these
embodiments a rank-order standard curve of CDK18 mRNA levels in a
population is created, and the pre-determined prevalence cutoff is
applied to that standard curve to determine the CDK18 mRNA cutoff
level.
[0080] In some aspects of embodiments where a test cell or sample
is compared to a population, the cutoff mRNA level value(s)
obtained for the population is converted to a prevalence rank and
the mRNA level cutoff is expressed as a percent of the population
having the cutoff value or higher, e.g., a prevalence cutoff.
Without being bound by theory, applicants believe that the
prevalence rank of a test sample and the prevalence cutoff in a
population will be similar regardless of the methodology used to
determine mRNA levels.
[0081] A patient can be identified as likely to respond well to a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof if the state of BRAF, MYC, CDK1, CDK2, CDK4, CDK6,
CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,
PIK3CA, or certain genes encoding an E2F pathway member (see above)
as determined by, e.g., RNA (e.g., mRNA levels) in a biological
sample from the patient) corresponds to (e.g., is equal to or
greater than) a prevalence rank in a population of about 80%, 79%,
78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%,
65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%,
42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%,
39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%,
26%, 25%, 24%, 23%, 22%, 21%, or 20% as determined by the state of
BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19,
CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes
encoding an E2F pathway member (see above), respectively,
determined by assessing the same parameter (e.g., mRNA level(s)) in
the population. A patient can be identified as likely to respond
well to a compound of Formula (I), (Ia), a species thereof, or a
specified form thereof if the state of BCL2-like 1, CDK7, CDK9,
CDKN2A, and RB (as determined by, e.g., RNA (e.g., mRNA) levels or
corresponding protein levels in a biological sample from the
patient) is below a prevalence rank in a population of about 80%,
79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%,
66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%,
43%, 42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%,
40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%,
27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20% as determined by the
state of BCL2-like 1, CDK7, CDK9, CDKN2A, and RB, respectively,
determined by assessing the same parameter (e.g., mRNA level(s)) in
the population. In some embodiments, the cutoff value or threshold
is established based on the biomarker (e.g., mRNA) prevalence
value.
[0082] In still other embodiments, a population may be divided into
three groups: responders, partial responders and non-responders,
and two cutoff values (or thresholds) or prevalence cutoffs (or
thresholds) are set or determined. The partial responder group may
include responders and non-responders as well as those patients
whose response to a compound of Formula (I), (Ia), a species
thereof, or a specified form thereof was not as high as the
responder group. This type of stratification may be particularly
useful when, in a population, the highest mRNA non-responder has an
mRNA level that is greater than that of the lowest mRNA responder.
In this scenario, for CDK18 or CDK19, the cutoff level or
prevalence cutoff between responders and partial responders is set
equal to or up to 5% above the CDK18 or CDK19 mRNA level of the
highest CDK18 or CDK19 mRNA non-responder; and the cutoff level or
prevalence cutoff between partial responders and non-responders is
set equal to or up to 5% below the CDK18 or CDK19 mRNA level of the
lowest CDK18 or CDK19 mRNA responder. For BCL-xL, CDK7 or CDK9,
this type of stratification may be useful when the highest mRNA
responder has a mRNA level that is lower than that of the lowest
mRNA non-responder. In this scenario, for BCL-xL, CDK7 or CDK9, the
cutoff level or prevalence cutoff between responders and partial
responders is set equal to or up to 5% below the mRNA level of the
lowest mRNA non-responder; and the cutoff level or prevalence
cutoff between partial responders and non-responders is set equal
to or up to 5% above the mRNA level of the highest mRNA responder.
The determination of whether partial responders should be
administered a compound of Formula (I), (Ia), a species thereof, or
a specified form thereof will depend upon the judgment of the
treating physician and/or approval by a regulatory agency.
[0083] Methods that can be used to quantify specific RNA sequences
in a biological sample are known in the art and include, but are
not limited to, fluorescent hybridization such as utilized in
services and products provided by NanoString Technologies, array
based technology (Affymetrix), reverse transcriptase qPCR as with
SYBR.RTM. Green (Life Technologies) or TaqMan.RTM. technology (Life
Technologies), RNA sequencing (e.g., RNA-seq), RNA hybridization
and signal amplification as utilized with RNAscope.RTM. (Advanced
Cell Diagnostics), or Northern blot. In some cases, mRNA expression
values for various genes in various cell types are publicly
available (see, e.g., broadinstitute.org/ccle; and Barretina et
al., Nature, 483:603-607, 2012).
[0084] In some embodiments, the state of a biomarker (as assessed,
for example, by the level of RNA transcripts) in both the test
biological sample (i.e., from the patient) and the reference
standard or all members of a population is normalized before
comparison. Normalization involves adjusting the determined level
of an RNA transcript by comparison to either another RNA transcript
that is native to and present at equivalent levels in both of the
cells (e.g., GADPH mRNA, 18S RNA), or to a fixed level of exogenous
RNA that is "spiked" into samples of each of the cells prior to
super-enhancer strength determination (Loven et al., Cell,
151(3):476-82, 2012; Kanno et al., BMC Genomics 7:64, 2006; Van de
Peppel et al., EMBO Rep., 4:387-93, 2003).
[0085] A patient (e.g., a human) suffering from a cancer described
herein and identified as described herein based on biomarker status
may have been determined to be resistant (or to be acquiring
resistance after some initial efficacy) to a therapeutic agent that
was administered prior to the compound of Formula (I), (Ia), a
species thereof, or a specified form thereof. For example, the
cancer may be resistant or refractory to a chemotherapeutic agent,
e.g., a Bcl-2 inhibitor such as venetoclax, a BET inhibitor, a
CDK4/6 inhibitor such as palbociclib or ribociclib, a CDK9
inhibitor such as alvocidib, a FLT3 inhibitor, a MEK inhibitor such
a trametinib, a PARP inhibitor, such as olaparib or niraparib, a
PI3K inhibitor, such as alpelisib or capecitabine, a platinum-based
therapeutic agent such as cisplatin, oxaliplatin, nedaplatin,
carboplatin, phenanthriplatin, picoplatin, satraplatin (JM216), or
triplatin tetranitrate, a SERM, such as tamoxifen faloxifene, or
toremifene, or a steroid receptor degrading agent (e.g., a SERD,
such as fulvestrant). Combination therapies including one or more
of these agents are also within the scope of the invention and are
discussed further herein. For example, in one embodiment, the
methods encompass the use of or administration of a compound of
Formula (I), (Ia), a species thereof or a specified form thereof,
in combination with a SERD, such as fulvestrant, to treat a cancer
(e.g., a breast cancer (e.g., an ER+ breast cancer)) resistant to
treatment with a CDK4/6 inhibitor such as palbociclib or
ribociclib.
[0086] In some embodiments, the prior therapeutic agent may be a
platinum-based anti-cancer agent administered as a monotherapy or
in combination with a SOC agent. Most cancer patients eventually
develop resistance to platinum-based therapies by one or more of
the following mechanisms: (i) molecular alterations in cell
membrane transport proteins decrease uptake of the platinum agent;
(ii) molecular alterations in apoptotic signaling pathways that
prevent a cell from inducing cell death; (iii) molecular
alterations of certain genes (e.g. BRCA1/2, CHEK1, CHEK2, RAD51)
that restore the ability of the cell to repair platinum
agent-induced DNA damage. K. N. Yamamoto et al., 2014, PloS ONE
9(8):e105724. The term "molecular alterations" includes increased
or decreased mRNA expression from the genes involved in these
functions; increased or decreased expression of protein from such
genes; and mutations in the mRNA/proteins expressed from those
genes.
[0087] Resistance is typically determined by disease progression
(e.g., an increase in tumor size and/or numbers) during treatment
or a decrease in the rate of shrinkage of a tumor. In some
instances, a patient will be considered to have become resistant to
a platinum-based agent when the patient's cancer responds or
stabilizes while on treatment, but which progresses within 1-6
months following treatment with the agent. Resistance can occur
after any number of treatments with platinum agents. In some
instances, disease progression occurs during, or within 1 month of
completing treatment. In this case, the patient is considered to
have never demonstrated a response to the agent. This is also
referred to a being "refractory" to the treatment. Resistance may
also be determined by a treating physician when the platinum agent
is no longer considered to be an effective treatment for the
cancer.
[0088] In some embodiments, the patient is or has been determined
to be resistant to treatment with a CDK4/6 inhibitor administered
as a monotherapy or in combination with a SOC agent.
[0089] CDK4/6 inhibitors in cancer (e.g., HR.sup.+ breast cancer)
are known to block entry into S phase of the cell cycle by inducing
G1 arrest. Resistance to CDK4/6 inhibitors in cancer (e.g.,
HR.sup.+ metastatic breast cancer) has been shown to be mediated,
in part, by molecular alterations that: 1) enhance CDK4/6 activity,
such as amplifications of CDK6, CCND1, or FGFR1 (Formisano et al.,
SABCS 2017, Publication Number GS6-05; Cruz et al., SABCS 2017
Publication Number PD4-05), or 2) reactivate cell cycle entry
downstream of CDK4/6, such as RB1 loss and CCNE1 amplification
(Condorelli, Ann. Oncol., 2017 PMID: 29236940; Herrera-Abreu,
Cancer Research 2016 PMID: 27020857).
[0090] Unlike platinum-based agents which are typically
administered for a period of time followed by a period without
treatment, CDK4/6 inhibitors, such as palbociclib, ribociclib or
abemaciclib, are administered until disease progression is
observed. In some instances, a patient will be considered to have
become resistant to a CDK4/6 inhibitor when the patient's cancer
initially responds or stabilizes while on treatment, but which
ultimately begins to progress while still on treatment. In some
instances, a patient will be considered to be resistant (or
refractory) to treatment with a CDK4/6 inhibitor if the cancer
progresses during treatment without demonstrating any significant
response or stabilization. Resistance may also be determined by a
treating physician when the CDK4/6 inhibitor is no longer
considered to be an effective treatment for the cancer.
[0091] In case of any doubt, any of the specified forms of a
compound of Formula (I), (Ia), or a species thereof can be included
in a pharmaceutical composition used or administered (e.g., in an
effective amount (e.g., a therapeutically effective amount)
according to a method of the invention. Pharmaceutical compositions
useful in the methods of the invention can be prepared by relevant
methods known in the art of pharmacology. In general, such
preparatory methods include the steps of bringing a compound
described herein, including compounds of Formula (I), (Ia), a
species thereof, or a specified form thereof (e.g., a
pharmaceutically acceptable salt, solvate, stereoisomer, tautomer,
or isotopic form thereof) into association with a carrier and/or
one or more other active ingredients (e.g., a second agent
described herein) and/or accessory ingredients, and then, if
necessary and/or desirable, shaping and/or packaging the product
into a desired single-dose or multi-dose unit (e.g., for oral
dosing). The accessory ingredient may improve the bioavailability
of a compound of Formula (I), (Ia), a species thereof, or a
specified form thereof, may reduce and/or modify its metabolism,
may inhibit its excretion, and/or may modify its distribution
within the body (e.g., by targeting a diseased tissue (e.g., a
tumor). The pharmaceutical compositions can be packaged in various
ways, including in bulk containers and as single unit doses
(containing, e.g., discrete, predetermined amounts of the active
agent) or a plurality thereof, and any such packaged or divided
dosage forms are within the scope of the invention. The amount of
the active ingredient can be equal to the amount constituting a
unit dosage or a convenient fraction of a dosage such as, for
example, one-half or one-third of a dose.
[0092] Relative amounts of the active agent/ingredient, the
pharmaceutically acceptable carrier(s), and/or any additional
ingredients in a pharmaceutical composition of the invention can
vary, depending upon the identity, size, and/or condition of the
subject treated and further depending upon the route by which the
composition is to be administered and the disease to be treated. By
way of example, the composition may comprise between about 0.1% and
99.9% (w/w or w/v) of an active agent/ingredient.
[0093] Pharmaceutically acceptable carriers useful in the
manufacture of the pharmaceutical compositions described herein are
well known in the art of pharmaceutical formulation and include
inert diluents, dispersing and/or granulating agents, surface
active agents and/or emulsifiers, disintegrating agents, binding
agents, preservatives, buffering agents, lubricating agents, and/or
oils. Pharmaceutically acceptable carriers useful in the
manufacture of the pharmaceutical compositions described herein
include, but are not limited to, ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0094] Pharmaceutical compositions used as described herein may be
administered orally. Such orally acceptable dosage forms may be
solid (e.g., a capsule, tablet, sachet, powder, granule, and orally
dispersible film) or liquid (e.g., an ampoule, semi-solid, syrup,
suspension, or solution (e.g., aqueous suspensions or dispersions
and solutions). In the case of tablets, carriers commonly used
include lactose and corn starch. Lubricating agents, such as
magnesium stearate, can also be included. In the case of capsules,
useful diluents include lactose and dried cornstarch. When aqueous
suspensions are formulated, the active agent/ingredient can be
combined with emulsifying and suspending agents. In any oral
formulation, sweetening, flavoring or coloring agents may also be
added. In any of the various embodiments described herein, an oral
formulation can be formulated for immediate release or
sustained/delayed release and may be coated or uncoated. A provided
composition can also be micro-encapsulated.
[0095] Compositions suitable for buccal or sublingual
administration include tablets, lozenges and pastilles.
Formulations can also be prepared for subcutaneous, intravenous,
intramuscular, intraocular, intravitreal, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intraperitoneal intralesional and by intracranial injection or
infusion techniques. Preferably, the compositions are administered
orally, subcutaneously, intraperitoneally or intravenously. Sterile
injectable forms of the compositions of this invention may be
aqueous or oleaginous suspension. These suspensions may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally acceptable diluent or
solvent, for example as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium.
[0096] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by one of ordinary skill in the art that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification.
[0097] Compounds described herein are typically formulated in
dosage unit form, e.g., single unit dosage form, for ease of
administration and uniformity of dosage. The specific
therapeutically or prophylactically effective dose level for any
particular subject or organism will depend upon a variety of
factors including the disease being treated and the severity of the
disorder; the activity of the specific active ingredient employed;
the specific composition employed; the age, body weight, general
health, sex and diet of the subject; the time of administration,
route of administration, and rate of excretion of the specific
active ingredient employed; the duration of the treatment; drugs
used in combination or coincidental with the specific active
ingredient employed; and like factors well known in the medical
arts.
[0098] The amount of a compound required to achieve an optimum
clinical outcome can vary from subject to subject, depending, for
example, on species, age, and general condition of a subject,
severity of the side effects, cancer to be treated, identity of the
particular compound(s) to be administered, and mode of
administration. The desired dosage can be delivered two or three
times a day, once a day, every other day, every third day, every
week, every two weeks, every three weeks, or every four weeks. In
certain embodiments, the desired dosage can be delivered using
multiple administrations (e.g., two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve, thirteen, fourteen, or more
administrations).
[0099] In certain embodiments, an effective amount of a compound
for administration one or more times a day (e.g., once) to a 70 kg
adult human may comprise about 1-100 mg, about 1-50 mg, about 1-35
mg (e.g., about 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 mg), about
2-20 mg, about 3-15 mg or about 10-30 mg (e.g., 10-20 or 10-25 mg).
Here, and wherever ranges are referenced, the end points are
included. The dosages provided in this disclosure can be scaled for
patients of differing weights or body surface and may be expressed
per m.sup.2 of the patient's body surface. In certain embodiments,
compositions of the invention may be administered once per day. The
dosage of a compound of Formula (I), (Ia), a species thereof or a
specified form thereof (e.g., a salt thereof) can be about 1-100
mg, about 1-50 mg, about 1-25 mg, about 2-20 mg, about 5-15 mg,
about 10-15 mg, or about 13-14 mg. In certain embodiments, a
composition of the invention may be administered twice per day. In
some embodiments, the dosage of a compound of Formula I or a
subgenus or species thereof for each administration is about 0.5 mg
to about 50 mg, about 0.5 mg to about 25 mg, about 0.5 mg to about
1 mg, about 1 mg to about 10 mg, about 1 mg to about 5 mg, about 3
mg to about 5 mg, or about 4 mg to about 5 mg.
[0100] As noted, the invention provides pharmaceutical kits
configured for treating cancer that include a compound of Formula
(I), (Ia), a species thereof, or a specified form thereof and,
optionally, an additional/second therapeutic agent (e.g., second
and third agents) selected from the second/additional agents
described herein. For example, the second/additional agent can be:
(a) a Bcl-2 inhibitor or dual Bcl-2/BCL-xL inhibitor, (b) a CDK
inhibitor (e.g., a CDK4/6, CDK7, or CDK9 inhibitor), (c) a Flt3
inhibitor, (d) a PARP inhibitor, (e) a BET inhibitor, (f) an
aromatase inhibitor, (g) a SERM, SERD, or estrogen suppressant, (h)
a MEK inhibitor, or (i) a PI3 kinase inhibitor, which, as noted,
may be selected from those disclosed herein. The kit can include
optional instructions for: (a) reconstituting (if necessary) a
compound of Formula (I), (Ia), a species thereof, or a specified
form thereof and/or the second therapeutic agent; (b) administering
each of the compound of Formula (I), (Ia), a species thereof, or a
specified form thereof and/or the second therapeutic agent; and/or
(c) a list of specific cancers for which the kit is useful or
diagnostic methods by which they may be determined. The kits can
also include any type of paraphernalia useful in administering the
active agent(s) contained therein (e.g., tubing, syringes, needles,
sterile dressings, tape, and the like). Such kits, whether
configured to deliver a monotherapy consisting of a compound of
Formula (I), (Ia), a species thereof, or a specified variant
thereof, or a combination therapy including an additional/second
agent selected from any one of those described herein, find utility
in the diagnostic and treatment methods described herein. In some
instances, the first and second agents will be in separate vessels
(e.g., with the first agent confined to a first container and the
second agent confined to a second container) and/or formulated in a
pharmaceutically acceptable composition, optionally in unit dosage
form, that includes the first agent, the second agent, and a
pharmaceutically acceptable carrier. In some instances, the kits
include a written insert or label with instructions to use the two
(or more) therapeutic agents in a patient suffering from a cancer
(e.g., as described herein) and identified as amenable to treatment
by a method described herein. The instructions may be adhered or
otherwise attached to a vessel or vessels comprising the
therapeutic agents. Alternatively, the instructions and the
vessel(s) can be separate from one another but present together in
a single kit, package, box, bag, or other type of container.
Alternatively, or in addition, the written instructions can specify
and direct the user to a website or other media. The instructions
in the kit will typically be mandated or recommended by a
governmental agency approving the therapeutic use of the
combination (e.g., in a patient population identified as described
herein). The instructions may optionally comprise dosing
information for each therapeutic agent, the types of cancer for
which treatment of the combination was approved or may be
prescribed, physicochemical information about each of the
therapeutics, pharmacokinetic information about each of the
therapeutics, drug-drug interaction information, or diagnostic
information (e.g., based on a biomarker or a method of identifying
a patient for treatment as described herein). The kits of the
invention can also include reagents useful in the diagnostic
methods described herein.
EXAMPLES
[0101] The compounds described herein can be prepared from readily
available starting materials and according to the synthetic
protocols described below. Alternatively, one of ordinary skill in
the art may readily modify the disclosed protocols. For example, it
will be appreciated that where process conditions (e.g., reaction
temperatures, reaction times, mole ratios of reactants, solvents,
pressures, etc.) are given, other process conditions can also be
used. Additionally, and as will be apparent to one of ordinary
skill in the art, protecting groups may be used to prevent certain
functional groups from undergoing undesired reactions. The choice
of a suitable protecting group for a particular functional group as
well as suitable conditions for protection and deprotection are
well known in the art. For example, numerous protecting groups and
guidance for their introduction and removal are disclosed by Greene
et al. (Protecting Groups in Organic Synthesis, Second Edition,
Wiley, New York, 1991, and references cited therein).
[0102] Also included within the Examples below are studies
demonstrating that daily oral dosing of Compound 101 can induce
dose-dependent TGI in ovarian and breast tumor xenografts, with
tumor regression observed at doses as low as one-fifth of MTD. We
also observed Compound 101 plasma exposures that were dose
proportional without accumulation upon repeated dialing dosing at
therapeutic doses in mice (1-6 mg/kg). Compound 101 induced rapid
(4 hours) and sustained (24 hours) dose-dependent pharmacodynamic
responses in xenograft tumor tissue that correlated with TGI,
supporting but not mandating a QD dosing regimen. We also observed
tumor regressions that were sustained after treatment with Compound
101 was discontinued, at well-tolerated doses in multiple PDX
models from SCLC, TNBC, and HGSOC. Sustained regressions were
associated with RB pathway alterations. In a study of combination
therapy, Compound 101 induced robust anti-tumor activity in
combination with fulvestrant in treatment-resistant PDX models of
ER+ breast cancer. Collectively, these studies highlight the broad
potential for compounds of the invention in a variety of solid
tumor types.
Example 1: Synthesis of Benzyl (2R,
5R)-5-amino-2-methyl-piperidine-1-carboxylate and benzyl (2S,
5S)-5-amino-2-methyl-piperidine-1-carboxylate
Step 1: Benzyl
5-(tert-butoxycarbonylamino)-2-methyl-piperidine-1-carboxylate
##STR00006##
[0104] To a solution containing commercially available racemic
trans tert-butyl N-(6-methyl-3-piperidyl)carbamate (5 g, 23.33
mmol, 1 eq,) and NaHCO.sub.3 (13.72 g, 163.32 mmol, 7 eq) in
tetrahydrofuran (THF; 50 mL) and H.sub.2O (50 mL), we added CbzCl
(5.97 g, 35.00 mmol, 4.98 mL, 1.5 eq) dropwise at 0.degree. C. The
mixture was stirred at 15.degree. C. for 2 hours then poured into
water (50 mL) and extracted with ethyl acetate (EtOAc; 50
mL.times.3). The combined organic layer was washed with brine (50
mL.times.3), dried over Na.sub.2SO.sub.4, and filtered. The
filtrate was concentrated under reduced pressure, and the residue
was purified by medium pressure liquid chromatography (MPLC;
SiO.sub.2, PE:EtOAc=5:1 to 1:1) to give the title compound as a
yellow solid (9.7 g, 18.04 mmol, 77.32% yield, 64.8% purity).
Step 2: Benzyl (2R, 5R)-5-amino-2-methyl-piperidine-1-carboxylate
and benzyl (2S, 5S)-5-amino-2-methyl-piperidine-1-carboxylate
##STR00007##
[0106] To a mixture of racemic trans benzyl
5-(tert-butoxycarbonylamino)-2-methyl-piperidine-1-carboxylate (9.7
g, 27.84 mmol, 1 eq) in EtOAc (100 mL) we added HCl/EtOAc (15 mL, 4
M), and the mixture was stirred at 15.degree. C. for 1 hour. We
then filtered the mixture and collected the filter cake. The solid
was dissolved in methanol (MeOH; 15 mL) and the pH was adjusted to
9 using a strongly acidic cation exchange resin (here,
AMBERLYST.RTM. A21) before the mixture was filtered and the
filtrate was concentrated. The residue was separated by
supercritical fluid chromatography (SFC; column: marketed by Daicel
as CHIRALCEL.RTM. (chemicals for use in chromatography) ODH (250
mm.times.30 mm, 5 .mu.m); mobile phase: [0.1% NH.sub.3.H.sub.2O
MeOH]; B %: 28%-28%, 16 min) to afford title compound 1 (1.9 g,
SFC: Rt=2.264 min, 93.2% ee, peak 1) and title compound 2 (1.9 g,
SFC: Rt=2.593 min, 98.6% ee, peak 2), both as light yellow solids.
Peak 1 is structure 3. Peak 2 is structure 4.
Example 2: Synthesis of 7-dimethylphosphoryl-3-[2-[[(3S,
6S)-6-methyl-3-piper-idyl]amino]-5-(trifluoromethyl)pyrimidin-4-yl]-1H-in-
dole-6-carbonitrile (Compound 100)
Step 1: Benzyl (2S,
5S)-5-[[4-(7-chloro-6-cyano-1H-indol-3-yl)-5-(trifluoromethyl)
pyrimidin-2-yl] amino]-2-methyl-piperidine-1-carboxylate
##STR00008##
[0108] We stirred a mixture of
7-chloro-3-[2-chloro-5-(trifluoromethyl)pyrimidin-4-yl]-1H-indole-6-carbo-
nitrile (0.81 g, 2.27 mmol, 1 eq), benzyl
(2S,5S)-5-amino-2-methyl-piperidine-1-carboxylate (732.20 mg, 2.95
mmol, 1.3 eq) and N,N-diisopropylethylamine (DIEA or DIPEA; 879.41
mg, 6.80 mmol, 1.19 mL, 3 eq) in N-methyl-2-pyrrolidone (NMP; 8 mL)
at 140.degree. C. for 1 hour. The reaction mixture was diluted with
H.sub.2O (100 mL) and extracted with EtOAc (50 mL.times.2). The
combined organic layers were washed with brine (100 mL.times.2),
dried over Na.sub.2SO.sub.4, filtered, and concentrated under
reduced pressure to give a residue that was purified by column
chromatography (SiO.sub.2, petroleum ether/ethyl acetate=10:1 to
4:1) to afford title compound as a yellow solid (1.1 g).
Step 2: Benzyl (2S,
5S)-5-[[4-(6-cyano-7-dimethylphosphoryl-1H-indol-3-yl)-5-(trifluoromethyl-
) pyrimidin-2-yl]amino]-2-methyl-piperidine-1-carboxylate
##STR00009##
[0110] We prepared a mixture of benzyl
(2S,5S)-5-[[4-(7-chloro-6-cyano-1H-indol-3-yl)-5-(trifluoromethyl)
pyrimidin-2-yl]amino]-2-methyl-piperidine-1-carboxylate (1.05 g,
1.85 mmol, 1 eq), methylphosphonoylmethane (720.17 mg, 9.23 mmol, 5
eq), K.sub.3PO.sub.4 (783.45 mg, 3.69 mmol, 2 eq), Pd(OAc).sub.2
(41.43 mg, 184.54 .mu.mol, 0.1 eq), xantphos
(C.sub.39H.sub.32OP.sub.2; 106.78 mg, 184.54 .mu.mol, 0.1 eq) and
dimethylformamide (DMF; 10 mL) in a microwave sealed tube, degassed
it, and purged it with N.sub.2 (.times.3). The mixture was then
stirred at 150.degree. C. for 1 hour in microwave. The reaction
mixture was diluted with H.sub.2O (100 mL) and extracted with ethyl
acetate (EtOAc; 50 mL.times.3). The combined organic layers were
washed with brine (150 mL.times.2), dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure to give a residue
that we purified by column chromatography (SiO.sub.2, petroleum
ether/ethyl acetate=10:1 to 1:1) to afford the title compound as a
yellow oil (490 mg).
Step 3: 7-dimethylphosphoryl-3-[2-[[(3S,
6S)-6-methyl-3-piperidyl]amino]-5-(trifluoromethyl)pyrimidin-4-yl]-1H-ind-
ole-6-carbonitrile
##STR00010##
[0112] To a solution of
benzyl(2S,5S)-5-[[4-(6-cyano-7-dimethylphosphoryl-1H-indol-3-yl)-5-(trifl-
uoromethyl)pyrimidin-2-yl]amino]-2-methyl-piperidine-1-carboxylate
(440 mg, 720.64 .mu.mol, 1 eq) in EtOAc (5 mL), we added Pd/C (200
mg, 10% purity) under N.sub.2. We degassed the suspension under
vacuum, purged it with H.sub.2 several times, then stirred the
mixture under H.sub.2 (15 psi) at 20.degree. C. for 3 hours before
filtering it. The filtrate was concentrated to give a residue we
purified by prep-HPLC (high performance liquid chromatography;
neutral condition) to yield the title compound as a white solid
(142.2 mg).
[0113] The reaction was combined with another reaction in 50 mg
scale for purification by liquid chromatography mass spectrometry
(LCMS). LCMS: ET6034-1492-P1C: (M+H.sup.+): 477.1 @2.572 (10-80%
ACN (acetonitrile) in H.sub.2O 4.5 minutes). .sup.1H NMR (400 MHz,
DMSO (dimethylsulfoxide)-d6) .delta. 8.74 (br d, J=7.89 Hz, 1H),
8.65-8.44 (m, 2H), 8.17 (br d, J=15.35 Hz, 1H), 7.84 (brt, J=8.11
Hz, 1H), 7.67 (brt, J=7.02 Hz, 1H), 3.81 (br s, 1H), 3.10 (br d,
J=11.40 Hz, 1H), 2.45-2.38 (m, 1H), 2.02 (d, J=13.59 Hz, 8H), 1.64
(br d, J=11.40 Hz, 1H), 1.49-1.34 (m, 1H), 1.11 (br d, J=10.96 Hz,
1H), 0.97 (br d, J=5.70 Hz, 3H).
Example 3: Synthesis of (S)-6,6-dimethylpiperidin-3-amine
##STR00011##
[0115] We dissolved (S)-tert-butyl (6-oxopiperidin-3-yl)carbamate
(1.00 g, 4.67 mmol) (Tetrahedron Letters, 36:8205, 1995) in THF (47
mL) and cooled the solution to -10.degree. C. Zirconium (IV)
chloride (2.61 g, 11.22 mmol) was added, and the mixture was
stirred for 30 minutes at this temperature. A methylmagnesium
bromide solution (3M in ether, 20.25 mL, 60.75 mmol) was added, and
the reaction mixture was allowed to slowly warm up to room
temperature, at which it was stirred overnight. The solution was
quenched with 30% aqueous NaOH, diluted with EtOAc, filtered, and
then extracted 3 times with EtOAc. The organics were combined,
dried over sodium sulfate, filtered, and concentrated in vacuo to
provide the crude product as a yellow oil that was used without
purification. The oil was dissolved in dichloromethane (DCM; 47 mL)
and trifluoroacetic acid (TFA; 3.58 mL, 46.73 mmol) was added. We
stirred the reaction mixture at room temperature for 16 hours,
concentrated it in vacuo and co-evaporated it a few times with DCM
to provide the crude title compound as a brown oil, which we used
in the next step without further purification.
Example 4: Synthesis of
(S)-7-(dimethylphosphoryl)-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(tr-
ifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (Compound
101)
Step 1: 7-Bromo-1H-indole-6-carboxylic Acid
##STR00012##
[0117] We stirred a solution of vinylmagnesium bromide (1.0 M in
THF (159 mL, 159 mmol) at -78.degree. C. and added to it, dropwise,
over a period of 1 hour, a solution of 2-bromo-3-nitrobenzoic acid
(10.0 g, 39.8 mmol) in THF (159 mL). The reaction mixture was
allowed to reach room temperature and was stirred at that
temperature overnight. The reaction mixture was then poured over
saturated aqueous ammonium chloride (150 mL) and acidified to a pH
2, using aqeous 1M HCl. We extracted the crude product with EtOAc
(3.times.200 mL), dried the extract over sodium sulfate, filtered
it, and concentrated it in vacuo. The residue was then triturated
in DCM (100 mL) and dried overnight with a flow of air to provide
the title compound as a light brown solid (7.58 g, 31.58 mmol, 79%
yield).
Step 2: 7-Bromo-1H-indole-6-carboxamide
##STR00013##
[0119] We stirred a solution of 7-bromo-1H-indole-6-carboxylic acid
(6.58 g, 27.4 mmol) in DMF (54.8 mL) at 0.degree. C. and added
1,1'-carbonyldiimidazole (CDI; 8.89 g. 54.8 mmol) to it portion
wise. The mixture was stirred for 5 minutes, and the intermediate
was observed by LCMS. We then added NH.sub.4OH (39.5 mL, 274 mmol)
at 0.degree. C., and the solution was stirred for 5 minutes. The
reaction was quenched with saturated aqueous ammonium chloride (25
mL) and saturated aqueous sodium chloride (25 mL) then diluted with
2-methyltetrahydroftiran (MeTHF; 50 mL). We separated the phases
and washed the organic layer again with saturated aqueous ammonium
chloride (25 mL) and saturated aqueous sodium chloride (25 mL). The
organic layer was then dried over sodium sulfate, filtered, and
concentrated in vacuo to provide the title compound, which was
carried over to the next step assuming the quantitative yield.
Step 3: 7-Bromo-1H-indole-6-carbonitrile
##STR00014##
[0121] We added Et.sub.3N (triethylamine; 44.1 mL, 315 mmol) to a
suspension of 7-bromo-1H-indole-6-carboxamide (7.53 g, 31.5 mmol)
in DCM (315 mL) at 0.degree. C. and stirred the resulting orange
solution at that temperature until we obtained a homogeneous
solution. MsCl (12.2 mL, 157 mmol) was then added dropwise, and the
solution was stirred at 0.degree. C. for 5 minutes. We diluted the
mixture with ethyl acetate and washed it with saturated aqueous
sodium bicarbonate before extracting the aqueous layer twice more
with ethyl acetate. The organic layers were combined, washed with
brine, dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by filtering it through a pad of
silica (eluting with ethyl acetate) to provide the title compound
as a brown solid (5.80 g, 26.24 mmol, 83% yield).
Step 4:
7-Bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-
-carbonitrile
##STR00015##
[0123] We added AlCl.sub.3 (1.83 g, 13.6 mmol) to a solution of
2,4-dichloro-5-trifluoromethylpyrim-idine (3.66 mL, 27.2 mmol) in
1,2-dichloroethane (DCE; 36.2 mL) and stirred the resulting
suspension at 80.degree. C. for 30 minutes. We added
7-bromo-1H-indole-6-carbonitrile (2.00 g, 9.05 mmol) to the mixture
and stirred the resulting red solution at 80.degree. C. until full
conversion (4 hours). The reaction mixture was then diluted with
MeTHF (100 mL) and washed with water (100 mL). The aqueous layer
was extracted with 2-MeTHF (100 mL), and the organic extracts were
combined, dried over sodium sulfate, filtered, and concentrated in
vacuo. Formation of two possible regioisomers was observed in a
ratio of 3:1 (desired/undesired). We purified the residue by
reverse phase chromatography on C18 (MeCN (acetonitrile) in water,
15 to 80% gradient) to provide the title compound as a beige solid
(1.51 g, 3.76 mmol, 42% yield). .sup.1H NMR (500 MHz, DMSO) .delta.
13.00 (brs, 1H), 9.17 (s, 1H), 8.35 (d, J=8.4 Hz, 1H), 8.16 (d,
J=2.6 Hz, 1H), 7.71 (d, J=8.4 Hz, 1H).
Step 5:
(S)-7-Bromo-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluorom-
ethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile
##STR00016##
[0125] We dissolved
7-bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbon-
itrile (200 mg, 0.498 mmol), (S)-6,6-dimethylpiperidin-3-amine
(95.8 mg, 0.747 mmol), and DIPEA (174 .mu.L, 0.996 mmol) in NMP (4
mL) then stirred the reaction mixture at 130.degree. C. in an oil
bath until full conversion (3 hours). The mixture Was cooled to
room temperature, loaded directly onto a C18 column and purified by
reverse phase chromatography (MeCN with 0.1% FA (formic acid) in
water also containing 0.1% FA, 0 to 100% gradient). The title
compound was obtained as a beige solid (245 mg. 0497 mmol,
quantitative yield).
Step 6:
(S)-7-(dimethylphosphoryl)-3-(2-((6-dimethylpiperidin-3-yl)amino)--
5-(trifluoromethyl)-pyrimidin-4-yl)-1H-indole-6-carbonitrile
##STR00017##
[0127] We combined
(S)-7-bromo-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)--
pyrimidin-4-yl)-1H-indole-6-carbonitrile (180.0 mg, 0.365 mmol),
Xantphos (21.5 trig, 36.5 .mu.mol), palladium (II) acetate (4.14 mg
18.2 .mu.mol), and K.sub.3PO.sub.4 (85.2 mg. 0.401 mmol) in a 2.5
mL microwave vial under nitrogen. Dimethylphosphine oxide (73 mg,
0.912 mmol) was dissolved in anhydrous DMF (1 mL), and the solution
was degassed before combining with the other reactants in a
microwave vial. The sealed vial with the reaction mixture was then
submitted to heat in a microwave reactor at 150.degree. C. for 45
minutes. The reaction mixture was cooled to room temperature,
loaded directly onto a C18 column, and purified by reverse phase
chromatography (MeCN in aqueous 10 mM ammonium formate pH 3.8, 15
to 35% gradient). The title compound was obtained as an off-white
solid (76 mg, 0.155 mmol, 42% yield).
Example 5: Synthesis of (3S)-1-benzyl-5,
5-dimethyl-piperidin-3-amine
Step 1: Methyl (2S)-5-oxopyrrolidine-2-carboxylate
##STR00018##
[0129] We added SOCl.sub.2 (215.62 g, 1.81 mol, 131.47 mL, 2 eq) to
a solution of (2S)-5-oxopyrrolidine-2-carboxylic acid (117 g,
906.18 mmol, 1 eq) in MeOH (500 mL) at 0.degree. C. The mixture was
stirred at 18.degree. C. for 1 hour before the reaction mixture was
concentrated. We diluted the residue with EtOAc (1000 mL) and TEA
(triethylamine; 150 mL) and filtered the solid that was formed. The
filtrate was evaporated to afford the title compound as a light
yellow oil (147 g, crude) to be used directly in the next step
without any further purification.
Step 2: (S)-1-tert-butyl 2-methyl
5-oxopyrrolidine-1,2-dicarboxylate
##STR00019##
[0131] To a solution of methyl (2S)-5-oxopyrrolidine-2-carboxylate
(147 g, 1.03 mol, 1 eq), DMAP (4-dimethylaminopyridine; 15.06 g,
123.24 mmol, 0.12 eq) and TEA (259.80 g, 2.57 mol, 357.35 mL, 2.5
eq) in EtOAc (500 mL) we added tert-butoxycarbonyl tert-butyl
carbonate (291.37 g, 1.34 mol, 306.71 mL, 1.3 eq), dropwise, at
0.degree. C. The mixture was then stirred at 20.degree. C. for 16
hours. We then washed the reaction mixture with HCl (0.5 M, 1000
mL), saturated NaHCO.sub.3 (1000 mL), brine (1500 mL), dried it
over Na.sub.2SO.sub.4, and filtered and concentrated it under
reduced pressure to give a residue that was then purified by
re-crystallization from methyl tert-butyl ether (MTBE; 250 mL). The
reaction mixture was filtered and evaporated to afford the title
compound as a white solid (2 batches obtained; Batch 1: 108 g, 100%
HPLC purity; Batch 2: 53 g, 90% .sup.1H NMR purity).
Step 3: (S)-1-tert-butyl 2-methyl
4,4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate
##STR00020##
[0133] We added LiHMDS (lithium hexamethyldisilazide; 1 M, 172.66
mL, 2.1 eq), dropwise, to a solution of (S)-1-tert-butyl 2-methyl
5-oxopyrrolidine-1, 2-dicarboxylate (20 g, 82.22 mmol, 1 eq) in THF
(500 mL) at -78.degree. C. under N.sub.2 atmosphere. After
addition, the mixture was stirred at that temperature for 30
minutes before we added CH.sub.3I (35.01 g, 246.65 mmol, 15.36 mL,
3 eq), dropwise, at -78.degree. C. under N.sub.2 atmosphere. The
resulting mixture was stirred at 20.degree. C. for 2.5 hours. The
reaction mixture was diluted with saturated aqueous NH.sub.4Cl
(1000 mL) and extracted with EtOAc (300 mL.times.3). The combined
organic layers were washed with brine (500 mL), dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure
to give a residue that was purified by MPLC (SiO.sub.2,
PE:EtOAc=4:1-3:1) to afford the title compound as a light yellow
solid (8 g, 25.95 mmol, 31.56% yield, 88% purity).
Step 4: tert-butyl
N-[(1S)-4-hydroxy-1-(hydroxymethyl)-3,3-dimethyl-butyl]carbamate
##STR00021##
[0135] To a solution of (S)-1-tert-butyl 2-methyl 4,
4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate (4.3 g, 15.85 mmol, 1
eq) in THF (35 mL) we added NaBH.sub.4 (1.80 g, 47.55 mmol, 3 eq),
by portions, at 0.degree. C. under N.sub.2. After addition, EtOH
(ethanol; 8.25 g, 179.09 mmol, 10.47 mL, 11.3 eq) was added
dropwise at 0.degree. C. The resulting mixture was stirred at
20.degree. C. for 16 hours then poured into saturated aqueous
NH.sub.4Cl (250 mL) and extracted with EtOAc (100 mL.times.3). The
combined organic layers were washed with brine (250 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure
to afford the title compound as a colorless oil (3.67 g, crude),
which was used directly in the next step without any further
purification
Step 5:
[(2S)-2-(tert-butoxycarbonylamino)-4,4-dimethyl-5-methylsulfonylox-
y-pentyl]methanesulfonate
##STR00022##
[0137] To a solution of tert-butyl
N-[(1S)-4-hydroxy-1-(hydroxymethyl)-3,3-dimethyl-butyl]carbamate
(3.67 g, 14.84 mmol, 1 eq) and TEA (6.01 g, 59.35 mmol, 8.26 mL, 4
eq) in EtOAc (25 mL) we added methanesulfonyl chloride (5.10 g,
44.52 mmol, 3.45 mL, 3 eq), dropwise, at 0.degree. C. The resulting
mixture was stirred at 20.degree. C. for 12 hours then poured into
H.sub.2O (200 mL). EtOAc (50 mL.times.3) was used to extract the
product. The organic layer was washed with brine (30 mL), dried
over Na.sub.2SO.sub.4, filtered and evaporated to afford the title
compound as a colorless oil (6.06 g crude) that was used directly
in the next step without any further purification.
Step 6: Tert-butyl N-[(3S)-1-benzyl-5,5-dimethyl-3-piperidyl]
carbamate
##STR00023##
[0139] A flask was fitted with
[(2S)-2-(tert-butoxycarbonylamino)-4,
4-dimethyl-5-methyl-sulfonyloxypentyl] methanesulfonate (6.06 g,
15.02 mmol, 1 eq), phenylmethanamine (5.15 g, 48.06 mmol, 5.24 mL,
3.2 eq) and dimethoxyethane (DME; 50 mL). We heated the reaction
mixture to 70.degree. C. for 16 hours then poured it into H.sub.2O
(40 mL). DCM (40 mL.times.3) was used to extract the product. The
organic layer was washed with brine (30 mL), dried over
Na.sub.2SO.sub.4, filtered and evaporated to afford the crude
product, which was purified twice by MPLC (SiO.sub.2,
PE:EtOAc=20:1-10:1) to afford the title compound as a colorless oil
(580 mg, 1.49 mmol, 9.91% yield, 81.7% purity).
Step 7: (3S)-1-benzyl-5, 5-dimethyl-piperidin-3-amine
##STR00024##
[0141] A flask was fitted with tert-butyl N-[(3S)-1-benzyl-5,
5-dimethyl-3-piperidyl]carbamate (300 mg, 942.05 .mu.mol, 1 eq) in
HCl/EtOAc (15 mL). The mixture was stirred at 25.degree. C. for 1
hour, after which some white precipitate formed. We filtered the
mixture, and the cake was washed by EtOAc (5 mL), collected and
dried over vacuum to afford the title compound as a white solid
(220 mg, 738.23 .mu.mol, 78.36% yield, 85.5% purity, HCl) as a
white solid to be used directly in the next step.
Example 6: Synthesis of
(S)-7-(dimethylphosphoryl)-3-(2-((5,5-dimethylpiperidin-3-yl)amino)-5-(tr-
ifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (Compound
102)
Step 1:
(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoro-
methyl)pyrimidin-4-yl)-7-bromo-1H-indole-6-carbonitrile
##STR00025##
[0143] We dissolved
7-bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-H-indole-6-carboni-
trile (168 mg, 0.418 mmol),
(S)-1-benzyl-5,5-dimethylpiperidin-3-amine (128 mg, 0.585 mmol),
and DIPEA (221 .mu.L, 1.26 mmol) in NMP (2 mL). We stirred the
reaction mixture at 130.degree. C. in an oil bath until full
conversion (4 hours). The mixture was cooled to room temperature,
diluted with EtOAc and washed with saturated aqueous LiCl. The
organic layer was separated, dried over sodium sulfate, filtered,
and concentrated in vacuo to provide the crude title compound (240
mg, 0.411 mmol, quant. yield), which was used in the next step
without further purification.
Step 2:
(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoro-
methyl)pyrimidin-4-yl)-7-(dimethylphosphoryl)-1H-indole-6-carbonitrile
##STR00026##
[0145] We combined
(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoro-methyl-
)pyrimidin-4-yl)-7-bromo-1H-indole-6-carbonitrile (240 mg, 0.411
mmol), Xantphos (24.3 mg, 41.1 .mu.mol), palladium (II) acetate
(4.66 mg, 20.6 .mu.mol), and K.sub.3P0.sub.4 (96.0 mg, 0.452 mmol)
in a 2.5 mL microwave vial under nitrogen. Dimethylphosphine oxide
(39.2 mg, 0.494 mmol) was dissolved in anhydrous DMF (1 mL), and
the solution was degassed before combining with the other reactants
in a microwave vial. The sealed vial with the reaction mixture was
then submitted to heat in a microwave reactor at 145.degree. C. for
45 minutes. The reaction mixture was then cooled to room
temperature, diluted with 2-MeTHF and washed with saturated aqueous
NaHCO.sub.3 and brine. The organic layer was separated, dried over
sodium sulfate, filtered, and concentrated in vacuo before the
residue was purified by reverse phase chromatography on C18 (MeCN
in aqueous 10 mM ammonium formate pH 3.8, 0 to 100% (gradient). The
title compound was obtained as a pale brown oil (58.0 mg, 0.10
mmol, 24% yield).
Step 3:
(S)-7-(dimethylphosphoryl)-3-(2-((5,5-dimethylpiperidin-3-yl)amino-
)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile
##STR00027##
[0147] Under a nitrogen atmosphere, to a stirring solution of
(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)-
pyrimidin-4-yl)-7-(dimethylphosphoryl)-1H-indole-6-carbonitrile
(58.0 mg, 0.10 mmol) in EtOH (12.5 mL), we added Pd/C 10% w/w (1.1
mg, 0.01 mmol) and Boc.sub.2O (di-t-butyl decarbonate; 65.5 mg,
0.30 mmol). The reaction mixture was evacuated and back-filled with
nitrogen (.times.3) before being filled with hydrogen. The reaction
mixture was then stirred at room temperature overnight under
hydrogen atmosphere. After 16 hours, we observed an incomplete
conversion and therefore filtered the reaction mixture through a
pad of CELITE.RTM. and concentrated it under reduced pressure. The
reaction was then repeated with the residue as described above.
After almost complete consumption of starting material (48 hours),
the reaction mixture was filtered through a pad of CELITE.RTM. and
concentrated in vacuo to provide the crude product, which was
engaged in the next step. Thus, the obtained oil was re-dissolved
in DCM (5 mL), and TFA (0.23 mL, 3.0 mmol) was added. The reaction
mixture was stirred at room temperature overnight. The mixture was
then concentrated in vacuo, and the residue was purified by reverse
phase chromatography on C18 (MeCN in aqueous 10 mM ammonium formate
pH 3.8, 0 to 100% gradient) to provide the title compound as a
white solid (11.11 mg, 0.023 mmol, 23% yield over two steps).
Example 7: Inhibition of CDK Kinase Activity
[0148] We assayed some compounds for inhibition of CDK7, CDK9,
CDK12, and CDK2 activity at Biortus Biosciences (Jiangyin, Jiangsu
Province, P.R. of China) using kinase assays for each CDK developed
with a Caliper/LabChip EZ Reader (Perkin Elmer, Waltham, Mass.).
These assays measure the amount of phosphorylated peptide substrate
produced as a fraction of the total peptide following an incubation
period at 27.degree. C. with the following components: test
compounds (variable concentrations from 10 .mu.M down to 0.508 nM
in a series of 3-fold serial dilutions), active CDK protein (with
the indicated cyclin, listed below for each CDK), ATP (at either
the K.sub.m concentrations listed below for each CDK/cyclin or 2 mM
ATP), and substrate peptide (listed below) in the following buffer:
2-(N-morpholino)ethanesulfonate (MES buffer, 20 mM), pH 6.75, 0.01%
(v/v) Tween 20 detergent, 0.05 mg/mL bovine serum albumin (BSA),
and 2% DMSO.
[0149] Specifically, the CDK7 inhibition assay used CDK7/Cyclin
H/MAT1 complex (6 nM) and "5-FAM-CDK7tide" peptide substrate (2
.mu.M, synthesized fluorophore-labeled peptide with the sequence
5-FAM-YSPTSPSYSPTSPSYSPTSPSKKKK (SEQ ID NO:1), where "5-FAM" is
5-carboxyfluorescein) with 6 mM MgCl2 in the buffer composition
listed above where the apparent ATP K.sub.m for CDK7/Cyclin H/MAT1
under these conditions is 50 .mu.M. The CDK9 inhibition assay used
CDK9/Cyclin T1 complex (8 nM) and "5-FAM-CDK9tide" peptide
substrate (2 .mu.M, synthesized fluorophore-labeled peptide with
the sequence: 5-FAM-GSRTPMY-NH.sub.2 (SEQ ID NO:2), where 5-FAM is
defined above and NH.sub.2 signifies a C-terminal amide with 10 mM
MgCl.sub.2 in the buffer composition listed above. The CDK12
inhibition assay used CDK12 (aa686-1082)/Cyclin K complex (50 nM)
and "5-FAM-CDK9tide" (2 .mu.M) as defined above, with 2 mM
MgCl.sub.2 in the buffer composition above. The CDK2 inhibition
assay used CDK2/Cyclin E1 complex (0.5 nM) and "5-FAM-CDK7tide" (2
.mu.M) as defined above, with 2 mM MgCl.sub.2 in the buffer
composition listed above.
[0150] The incubation period at 27.degree. C. for each CDK
inhibition assay was chosen such that the fraction of
phosphorylated peptide product produced in each assay, relative to
the total peptide concentration, was approximately 20% (.+-.5%) for
the uninhibited kinase (35 minutes for CDK7, 35 minutes for CDK2, 3
hours for CDK12, and 15 minutes for CDK9). In cases where the
compound titrations were tested and resulted in inhibition of
peptide product formation, these data were fit to produce best-fit
IC.sub.50 values. The best-fit IC.sub.50 values at K.sub.m ATP for
each CDK/Cyclin, except for CDK7/Cyclin H/MAT1, were used to
calculate K.sub.i values, or the apparent affinity of each
inhibitor for each CDK/Cyclin from the kinase activity inhibition
assay, according to the Cheng-Prusoff relationship for ATP
substrate-competitive inhibition (Cheng and Prusoff, Biochem.
Pharmacol., 22(23):3099-3108, 1973), with a correction term for
inhibitor depletion due to the enzyme concentration (Copeland,
"Evaluation of Enzyme Inhibitors in Drug Discover: A Guide for
Medicinal Chemists and Pharmacologists," Second Edition, March,
2013; ISBN: 978-1-118-48813-3):
I .times. C 5 .times. 0 = K i .function. ( 1 + [ Substrate ] K m )
+ [ Enzyme ] 2 ##EQU00001##
[0151] Due to tight-binding inhibition and the limits of the
CDK7/Cyclin H/MAT1 assay, instead of calculating the apparent
K.sub.i values for each inhibitor, the K.sub.d, or direct compound
binding affinity, was measured using surface plasmon resonance
(SPR) as described below.
Example 8: CDK7/Cyclin H Surface Plasmon Resonance (SPR) Assay
Method
[0152] We measured binding kinetics and affinities of selected
compounds to the CDK7/Cyclin H dimer using a Biacore T200 surface
plasmon resonance (SPR) instrument (GE Healthcare). The dimer was
amine-coupled to a CM5 sensor chip at pH 6.5 in 10 mM MES buffer at
a concentration of 12.5 .mu.g/mL with a flow rate of 10 .mu.L/min.
Target protein was immobilized on two flow cells for 12-16 seconds
to achieve immobilized protein levels of 200-400 Response
Units.
[0153] Compounds were titrated from 0.08-20 nM in a 9-step, 2-fold
serial dilution in 10 mM HEPES buffer at pH 7.5 with 150 mM NaCl,
0.05% Surfactant P20, and 0.0002% DMSO. Each compound concentration
cycle was run at 100 .mu.L/min with 70 second contact time, 300
second dissociation time, 60 second regeneration time with 10 mM
glycine pH 9.5, and 400 second stabilization time. For each
compound, 0 nM compound controls and reference flow-cell binding
were subtracted to remove background and normalize data. Compound
titrations were globally fit by Biacore T200 Evaluation Software
(GE Healthcare) using kinetics mode. Best-fit values for compound
binding on-rate (k.sub.on) and dissociation off-rate (k.sub.off)
for CDK7/Cyclin H were determined and these values were used to
calculate the compound affinity (K.sub.d) for CDK7/Cyclin H using
the following equation:
K d .function. ( M ) = k off .function. ( s - 1 ) k o .times. n
.function. ( M - 1 .times. s - 1 ) . ##EQU00002##
Compound selectivity for CDK7 over CDK2, CDK9, or CDK12 were
determined based on the ratios of K.sub.i values for the off-target
CDKs relative to the direct compound binding K.sub.d for CDK7
measured by SPR according to:
Selectivity = K i , off .times. .times. target K d , CDK .times.
.times. 7 . ##EQU00003##
The inhibitory and dissociation constants and selectivity of the
indicated compounds (three compounds of the invention and four
comparators) against CDK2, CDK7, CDK9, and CDK12 are shown in the
table of FIG. 1. As can be seen, each of the compounds of the
invention is at least 1300-fold and up to 40,000-fold more specific
for CDK7 than for the other CDKs tested.
Example 9: Inhibition of Cell Proliferation (Compounds 100-102)
[0154] The HCC70 cell line was derived from human TNBC, and we
tested representative compounds of the invention, at different
concentrations (from 4 .mu.M to 126.4 .mu.M; 0.5 log serial
dilutions), for their ability to inhibit the proliferation of those
cells. More specifically, we tested the same compounds tested above
for CDK7 selectivity (the structures of which are shown in FIG. 1),
and we used the known CDK inhibitors dinaciclib (or
N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)
pyrimidin-2-yl)amino)cyclohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)pi-
colinamide) and triptolide as positive controls. The cells were
grown in ATCC-formulated RPMI-1640 medium (ATCC 30-2001)
supplemented with 10% fetal bovine serum (FBS), at 37.degree. C. in
a humidified chamber in the presence of 5% CO.sub.2. We conducted
proliferation assays over a 72-hour time period using a
CyQUANT.RTM. Direct Cell Proliferation Assay (Life Technologies,
Chicago, Ill. USA) according to the manufacturer's directions and
utilizing the reagents supplied with the kit. The results of the
assay are shown in the Table below.
TABLE-US-00002 Compound HCC70 EC.sub.50 (nM) Compound 100 0.98
Compound 101 5.6 Compound 102 2.1 Comparator 1 0.53 Comparator 2
260 Comparator 3 24 Comparator 4 110
Example 10: TGI in Patient-Derived Xenograft (PDX) Models
[0155] Tumor growth inhibition was evaluated in estrogen
receptor-positive breast cancer (ER+BC) PDX models selected in vivo
for resistance to the CDK4/6 inhibitor palbociclib (ST1799, n=1) or
resistance to both palbociclib and fulvestrant (ST941, n=1). Dosing
was initiated when tumors were 100-200 mm.sup.3. Mice were treated
with either Compound 101, QD (6 mg/kg, once daily, by mouth);
fulvestrant, SC (2.5 mg/kg, once weekly dosing, by subcutaneous
injection); palbociclib, QD (50 mpk, once daily, by mouth) or in
combination of Compound 101 (6 mg/kg, once daily, by mouth) and
fulvestrant (2.5 mg/kg, once weekly, by subcutaneous injection)
over the course of 28 days, followed by 21 days of observation.
Tumor growth inhibition (TGI) was calculated on the last day of
dosing using the formula:
TGI=(V.sub.c1-V.sub.t1)/(V.sub.c0-V.sub.t0), where V.sub.c1 and
V.sub.t1 are the mean volumes of control and treated groups at the
time of tumor extraction, while V.sub.c0 and V.sub.t0 are the same
groups at the start of dosing.
[0156] In the palbociclib-resistant ER+BC PDX (ST1799) model, the
combination of Compound 101 and fulvestrant induced significant TGI
(89%), with no evident tumor regrowth up to 21 days after dosing
cessation, distinguishing the observed effects from Compound 101
(83%), fulvestrant (60%) or palbociclib (21%) when administered as
single agents. Additionally, the combination of Compound 101 and
fulvestrant was superior to the SOC combination of palbociblib and
fulvestrant (75%). In a palbociclib and fulvestrant
double-resistant ER+BC PDX model (ST941), Compound 101 administered
as a single agent resulted in 33% TGI and fulvestrant and
palbociclib as single agents or fulvestrant and palbociclib in
combination had no activity. In contrast, the combination of
Compound 101 and fulvestrant demonstrated significant TGI (68%;
p<0.0001 vs fulvestrant as a single agent), suggesting
re-sensitization to fulvestrant.
[0157] FIG. 2 illustrates the TGI results from the palbociclib
resistant HR+BC PDX model ST1799, and FIG. 3 illustrates the TGI
results from the palbociclib and fulvestrant resistant HR+BC PDX
model ST941. We also observed TGI in four additional PDX models;
BR5010 (modeling TNBC), LU5178 (modeling small cell lung cancer
(SCLC)), OV15398 (modeling high grade serous ovarian cancer
(HGSOC)), and ST390 (modeling pancreatic ductal adenocarcinoma
(PDAC)). In the TNBC model, Compound 101 was orally administered to
tumor-bearing NOD/SCID mice at 10 mg/kg QD or 5 mg/kg BID for 21
days. In the SCLC and HGSOC models, Compound 101 was orally
administered to tumor-bearing NOD/SCID mice at 3 mg/kg BID for 21
days. In the PDAC model, Compound 101 was orally administered to
tumor-bearing NOD/SCID mice at 6 mg/kg QD. In the TNBC, SCLC, and
HGSOC models, tumor volume was measured during the treatment period
and for an additional 21 days after treatment ceased. The % TGI
observed at the end of treatment (day 21) was calculated as:
1-[(Mean TV Compound 101 @ EOT-Mean TV Compound 101 @ Day 0)/(Mean
TV Veh @ EOT-Mean TV Veh @ Day 0)].times.100. The % regression was
calculated as: (Mean TV Compound 101 @ EOT)/(Mean TV Compound 101 @
Day 0).times.100. The same calculations were used for end of study
(day 42). The results are shown in FIG. 4. These results
demonstrate deep and sustained TGI, including regressions, at well
tolerated doses, in a variety of tumor types. Dose-dependent
transcriptional responses in xenograft tissue were observed within
4 hours of dosing and were sustained for 24 hours. Similar TGI was
seen when the same total dose was administered either QD or BID in
the TNBC PDX model, suggesting that the effect was AUC or C.sub.min
driven. Moreover, the TGI observed in SCLC (in the LU5178 PDX
model) had not been observed in previous studies with a covalent
CDK7 inhibitor (data not shown). Regarding the model of PDAC, we
found Compound 101 induced 100% TGI over the time examined
(.about.28 days) at a dose well below the MTD: at day 21, tumor
volume was .about.1,250 mm.sup.3 in vehicle-treated mice but only
about 250 mm.sup.3 in Compound 1-treated mice (6 mg/kg QD, PO).
While Compound 101 could achieve 100% TGI at sub-MTD doses in the
tested PDAC PDX tumors, a covalent CDK7 inhibitor achieved only
modest TGI at its MTD (40 mg/kg BIW, by IV administration, with
evident body weight loss (8.4%) and necrosis at the injection site;
data not shown).
Example 11. In Vitro Studies of Compound 101 in Combination with
Various Second Agents
[0158] In the studies described here, cancer cell lines from HR+
breast cancers (lines T47D; PIK3CA p.H1047R, MCF7; PIK3CA p.E545K),
SCLC, (NCI-H1048) and CRCs (lines RKO; BRAF p.V600E, SW480; KRAS
p.G12V) were grown to 70% confluency in their media of preferences
based on the manufacturer recommendations. In the SCLC cell line
(NCI-H1048), Compound 101 was tested in combination with SOC
chemotherapy agents gemcitabine (a DNA synthesis inhibitor) and
carboplatin (a DNA damage agent). In a CRC cell line (RKO; BRAF
p.V600E), Compound 101 was tested in combination with SOC
chemotherapy agent oxaliplatin (a DNA damage agent). Additionally,
in CRC, Compound 101 was tested in combination with the selective
MAPK pathway inhibitor trametinib in two CRC cell lines harboring
MAPK pathway alterations; RKO (BRAF p.V600E mutant) and SW480 (KRAS
p.G12V mutant). Compound 101 was tested in combination with the SOC
agent capecitabine (an antimetabolite) in HR+MCF-7 cells. In the
HR+ cell lines MCF7 and T47D, which have activating mutations in
the PIK3CA kinases, Compound 101 was tested in combination with the
PIK3CA selective inhibitor alpelisib.
[0159] On the day of assay, cells were lifted and counted using the
Countess II FL (Life Technologies). Using an automated dispenser
(here, Multidrop.TM. Combi Reagent Dispenser), 50 .mu.L of
preferred cell media containing 20,000-50,000 cells/ml was
distributed into black 384-well Nunc plates (Thermo) and allowed to
adhere overnight prior to compound addition. Compound arrays were
distributed to 384 well assay plates using Synergy Plate Format
with an HP D300e Digital Dispenser (HP). Compound 101 and other
TEST agents were dissolved in DMSO to make a stock solution that
allowed for more accurate dispensing. However, due to solubility
and reactivity, platinum agents were dissolved in water with an
addition of 0.03% Tween-20 to allow for dispensing with digital
printer. Compounds were plated in each quadrant of a 384-well plate
in quadruplicate. Each quadrant contained test wells with
combination of SY-1365 and carboplatin or oxaliplatin (TEST/test
agent) as well as single agent columns, and vehicle wells.
[0160] Compound 101 was plated in across from left to right in a
high to low concentration (8 columns), and the varying
concentrations of carboplatin or oxaliplatin (TEST) plated in
synergy wells from top to bottom (7 rows). Concentrations were
selected to cover the full isobologram of activity based on
activity of single agents. Single agents were plated in dose in two
columns, with a third separate column of just DMSO/vehicle treated
wells. A separate plate for each cell line was seeded to allow for
determination of a "Time Zero"/"Day Zero" number of cells to parse
the differential cytostatic vs cytotoxic effects. On the day
compounds were added, viability of the time zero plate was
determined to identify growth inhibition from cell killing
effects.
[0161] After addition of compound, cell plates were incubated for 5
days in a 37.degree. C. incubator. Cell viability was evaluated
using CellTiter-Glo.RTM. 2.0 (Promega) following manufacturer
protocols. Data was analyzed in CalcuSyn utilizing the median
effect principle of presented by Chou-Talalay and visualized using
GraphPad Prism Software. Key parameters assessed were combination
index and dose reduction index.
[0162] We found the combination of Compound 101 with SOC
chemotherapy (gemcitabine or carboplatin in SCLC, oxaliplatin in
CRC, or capecitabine in HR+ breast cancer) showed synergy and was
superior to either agent alone. The combination of Compound 101
with the targeted agent trametinib, a selective MAPK pathway
inhibitor approved for the treatment of BRAF p.V600E mutant
melanoma and NSCLC, show significant synergy in BRAF p.V600E mutant
CRC as well as in KRAS p.G12V mutant CRC, which harbors a different
mutation within the MAPK pathway. The combination of Compound 101
with the targeted agent alpelisib, a selective PIK3CA inhibitor
approved for the treatment of PIK3CA mutant HR+BC, showed
significant synergy in both HR+ cell lines representing the two
most common activating mutation of PIK3CA (p.E545K and p.H1047R).
All synergy was determined using CalcuSyn utilizing the median
effect principle of presented by Chou-Talalay and visualized using
GraphPad Prism Software. Combination effect is reflected by shift
in IC50 of Compound 101 with addition of carboplatin or oxaliplatin
or increased antiproliferative effect with lower amounts of either
single agent. This is visualized in the isobolograms of FIG. 5,
where points below the diagonal line reflect synergy.
Example 12. Deep and Sustained Responses to Compound 101 in TNBC,
HGSOC, and SCLC PDX Models
[0163] We evaluated TGI in 12 different PDX models (Crown
Biosciences) in various tumor indications with PDXs representing
SCLC (n=5; LU5180, LU5178, LU5192, LU5173, LU5210), TNBC (n=4;
BR5010, BR1458, BR5399, BR10014) and HGSOC (n=3; OV15398, OV5392,
OV15631). Dosing was initiated when tumors were 150-300 mm.sup.3.
Mice were treated with either Compound 101, QD (6 or 10 mg/kg once
daily, by mouth) or BID (3 or 5 mg/kg twice daily, by mouth) over
the course of 21 days, followed by 21 days of observation. TGI was
calculated on the last day of dosing using the formula: TGI
(V.sub.c1-V.sub.t1)/(V.sub.c0-V.sub.t0), where V.sub.c1 and
V.sub.t1 are the mean volumes of control and treated groups at the
time of tumor extraction, while V.sub.c0 and V.sub.t0 are the same
groups at the start of dosing.
[0164] To perform whole exome sequencing (WES), we isolated DNA
from passage matched tumors using DNeasy.RTM. Blood and Tissue Kit
via manufacturer protocol and sent it to Wuxi Aptec for WES using
Agilent's SureSelectXT Human All Exon V6 kit. Samples were
sequenced to a depth of .about.300.times.. Reads were trimmed to
remove adapter sequences via Skewer (v0.2.1). Reads were then
mapped and further processed using Sentieon tools: BWA, DeDup,
Realigner, and QualCal (v201808.03). Variants were called using
Sentieon's Haplotyper tool, and initial annotations were performed
using Ensembl's Variant Effect Predictor (VEP, release_96.2).
FATHMM-MLK was also used to annotate variant effects. Variants that
met the following qualifications were included in sample
characterizations: (1) variant is located in a protein-coding gene;
(2) variant affects protein sequence or results in a frameshift;
(3) missense mutations are classified as damaging by SIFT,
PolyPhen, or FATHMM-MLK (.gtoreq.0.75); (4) variant allele
frequency is .gtoreq.10%. Copy-number (CN) variation across capture
regions were called using CNVkit (v0.9.1), and CNs for individual
genes were calculated by using the mean CN across its capture
regions. For model LU5210 mutation/CNV data was made available from
WES data provided by the PDX vendor (Crown Biosciences Inc.).
[0165] At these doses, Compound 101 induced at least 50% TGI at the
end of the 21-day dosing period in all models. In a subset of
models (58%, 7/12), Compound 101 responses were deep (>95% TGI
or regression) and sustained, with no evidence of tumor regrowth
for 21 days after treatment discontinuation (see FIG. 6). Compound
101 was well tolerated, with no evident body weight loss at all
once-daily doses tested, indicating that the MTD is above 10 mg/kg
once daily in tumor-bearing mice. Deep and sustained responses were
observed in each indication tested and were associated with
alterations in the RB pathway including RB1 deletion or mutation,
CDKN2A deletion, or CCNE1 amplification (FIG. 7). These results
highlight the therapeutic potential of Compound 101 in TNBC, HGSOC,
and SCLC, particularly in tumors with RB pathway alterations and
aberrant cell cycle control.
[0166] It should it be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements and/or features, certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein. Where ranges are given, endpoints are
included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0167] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, that there
are many equivalents to the specific embodiments of the disclosure
described and claimed herein. Such equivalents are intended to be
encompassed by the following claims.
Sequence CWU 1
1
2125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Tyr Ser Pro Thr Ser Pro Ser Tyr Ser Pro Thr Ser
Pro Ser Tyr Ser1 5 10 15Pro Thr Ser Pro Ser Lys Lys Lys Lys 20
2527PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Gly Ser Arg Thr Pro Met Tyr1 5
* * * * *