U.S. patent application number 16/218576 was filed with the patent office on 2019-09-19 for pharmaceutical combination for the treatment of melanoma.
This patent application is currently assigned to Piramal Enterprises Limited. The applicant listed for this patent is Piramal Enterprises Limited. Invention is credited to Veena AGARWAL, Giridharan PERIYASAMY, Maggie RATHOS, Sreesha SRINIVASA, Ankita SRIVASTAVA.
Application Number | 20190282544 16/218576 |
Document ID | / |
Family ID | 52279411 |
Filed Date | 2019-09-19 |
View All Diagrams
United States Patent
Application |
20190282544 |
Kind Code |
A1 |
AGARWAL; Veena ; et
al. |
September 19, 2019 |
PHARMACEUTICAL COMBINATION FOR THE TREATMENT OF MELANOMA
Abstract
The present invention relates to a pharmaceutical combination
comprising a cyclin dependent kinase (CDK) inhibitor represented by
a compound of formula I (as described herein) or a pharmaceutically
acceptable salt thereof; and at least one anticancer agent selected
from a BRAF inhibitor or a MEK inhibitor, for use in the treatment
of melanoma. The present invention also relates to a method for the
treatment of melanoma comprising administering to a subject in need
thereof, a therapeutically effective amount of a CDK inhibitor and
a therapeutically effective amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor.
Inventors: |
AGARWAL; Veena; (Mumbai,
IN) ; PERIYASAMY; Giridharan; (Bangalore, IN)
; RATHOS; Maggie; (Mumbai, IN) ; SRIVASTAVA;
Ankita; (Mumbai, IN) ; SRINIVASA; Sreesha;
(Mumbai, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Piramal Enterprises Limited |
Mumbai |
|
IN |
|
|
Assignee: |
Piramal Enterprises Limited
Mumbai
IN
|
Family ID: |
52279411 |
Appl. No.: |
16/218576 |
Filed: |
December 13, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14904407 |
Jan 11, 2016 |
|
|
|
PCT/IB2014/063022 |
Jul 11, 2014 |
|
|
|
16218576 |
|
|
|
|
61845749 |
Jul 12, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/437 20130101;
A61P 43/00 20180101; A61K 31/519 20130101; A61P 17/00 20180101;
A61K 31/4025 20130101; A61K 45/06 20130101; A61P 35/00 20180101;
A61K 31/506 20130101; A61P 35/04 20180101; A61K 31/4025 20130101;
A61K 2300/00 20130101; A61K 31/437 20130101; A61K 2300/00 20130101;
A61K 31/506 20130101; A61K 2300/00 20130101; A61K 31/519 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 31/4025 20060101
A61K031/4025; A61K 31/519 20060101 A61K031/519; A61K 45/06 20060101
A61K045/06; A61K 31/437 20060101 A61K031/437; A61K 31/506 20060101
A61K031/506 |
Claims
1. A method of treating melanoma comprising administering to a
subject in need thereof a therapeutically effective amount of a CDK
(cyclin dependent kinase) inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof;
##STR00007## wherein Ar is phenyl, which is substituted by 2
different substituents selected from chlorine and trifluoromethyl;
and a therapeutically effective amount of at least one anticancer
agent selected from a BRAF (serine-threonine protein kinase B-raf)
inhibitor or a MEK (mitogen activated protein kinase)
inhibitor.
2. (canceled)
3. The method according to claim 1, wherein the CDK inhibitor is
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
4. The method according to claim 1, wherein the BRAF inhibitor or
the MEK inhibitor is an inhibitor of V600 mutated form of BRAF.
5. The method according to claim 4, wherein the BRAF inhibitor is
an inhibitor of V600E mutated form of BRAF.
6. The method according to claim 1, wherein the BRAF inhibitor is
selected from sorafenib, vemurafenib, GDC-0879, dabrafenib,
PLX4720, BMS-908662, LGX818, PLX3603, ARQ-736, DP-4978 and
RAF265.
7. The method according to claim 1, wherein the BRAF inhibitor is
vemurafenib.
8. The method according to claim 1, wherein the BRAF inhibitor is
dabrafenib.
9. The method according to claim 1, wherein the MEK inhibitor is an
inhibitor of V600E or V600K mutated form of BRAF.
10. The method according to claim 1, wherein the MEK inhibitor is
selected from selumetinib, binimetinib, PD-0325901, trametinib,
cobimetinib, refametinib, pimasertib, TAK-733 and WX-554.
11. The method according to claim 1, wherein the MEK inhibitor is
trametinib.
12. The method according to claim 1, wherein the said CDK
inhibitor; and the said at least one anticancer agent selected from
a BRAF inhibitor or a MEK inhibitor; are administered
simultaneously to a subject in need thereof.
13. The method according to claim 1, wherein the said CDK inhibitor
and the said at least one anticancer agent selected from a BRAF
inhibitor or a MEK inhibitor; are administered sequentially to a
subject in need thereof.
14. The method according to claim 1, wherein the melanoma is
non-refractory melanoma.
15. The method according to claim 14, wherein the melanoma is
non-refractory BRAF mutant melanoma.
16. The method according to claim 15, wherein the melanoma is
non-refractory BRAFV600 mutant melanoma.
17. The method according to claim 16, wherein the melanoma is
non-refractory BRAFV600E or BRAFV600K mutant melanoma.
18. The method according to claim 1, wherein the melanoma is
recurrent or refractory melanoma.
19. The method according to claim 18, wherein the melanoma is
recurrent or refractory BRAF mutant melanoma.
20. The method according to claim 19, wherein the melanoma is
recurrent or refractory BRAFV600 mutant melanoma.
21. The method according to claim 20, wherein the melanoma is
recurrent or refractory BRAFV600E melanoma or BRAFV600K mutant
melanoma.
22. The method according to claim 1, wherein the melanoma is
metastatic melanoma.
23. The method according to claim 22, wherein the melanoma is
metastatic BRAF mutant melanoma.
24. The method according to claim 23, wherein the melanoma is
metastatic BRAFV600 mutant melanoma.
25. The method according to claim 24, wherein the melanoma is
metastatic BRAFV600E melanoma or BRAFV600K mutant melanoma.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a pharmaceutical
combination comprising a CDK (cyclin dependent kinase) inhibitor
represented by a compound of formula I (as described herein) or a
pharmaceutically acceptable salt thereof and at least one
anticancer agent selected from a BRAF (serine-threonine protein
kinase B-raf) inhibitor or a MEK (mitogen activated protein kinase)
inhibitor, for use in the treatment of melanoma. The invention also
relates to a method of treating melanoma using the pharmaceutical
combination.
BACKGROUND OF THE INVENTION
[0002] Melanoma is the most serious type of skin cancer. Melanoma,
in fact, is a malignant tumor that originates in cells called
melanocytes, which are pigment producing cells. It occurs most
often in the skin but may also develop in the eye or in the lining
of the nose, mouth or genitals. Subsequently, melanoma can spread
to internal organs. When melanoma occurs in the skin it is referred
to as cutaneous melanoma. Melanoma when occurs in the eye, is
called ocular or intraocular melanoma. Incidence of melanoma is
increasing worldwide, it is reported that malignant melanoma is
responsible for 80% of skin cancer deaths (N. Engl. J. Med., 2010,
363, 8, 711-723).
[0003] When melanoma spreads, cancer cells are often found in the
lymph nodes. When the cancer reaches the lymph nodes, it is an
indication that the cancer cells may have spread to other parts of
the body such as the liver, lungs or brain, giving rise to
metastatic melanoma. In fact, melanoma metastases are highly
aggressive and the survival time for patients with metastatic
melanoma averages 3-months only. Unfortunately, no effective
treatment exists for metastatic melanoma. Early diagnosis and
prompt surgical removal are the possible options for patients for a
possible cure. The therapeutic regimen for metastatic melanoma
initially comprised of the drugs such as interleukin-2,
dacarbazine, temozolomide, fotemustine and carboplatin but each is
associated with a poor response rate and poor overall survival. For
instance, dacarbazine was found to be associated with a response
rate of 7-12% and a median overall survival of 5-8 months after
initiation of the treatment (N. Engl. J. Med., 2011, 364, 26,
2507-2516).
[0004] In a study of the mitogen-activated protein (MAP) kinase
pathway in a large panel of common cancers, it has been found that
40-60% of melanomas carry an activating mutation in the gene
encoding the serine-threonine protein kinase B-raf (BRAF). Among
the BRAF mutations observed in melanoma, over 90% are at codon 600,
and among these, over 90% are a single nucleotide mutation
resulting in substitution of glutamic acid for valine (BRAFV600E).
The second most common mutation is BRAFV600K substituting lysine
for valine, which represents 5-6% of melanomas, followed by
BRAFV600R (substituting arginine for valine) and BRAFV600D
(substituting aspartic acid for valine) (Journal of Translational
Medicine, 2012, 10, 85, 1-9).
[0005] BRAF inhibitors have been found to be effective in tumor
shrinkage in the majority of patients with BRAF mutant melanoma at
low nanomolar concentrations. The limiting factors in efficacy are
the drug resistance and the progression-free survival which is
restricted to 5-7 months. A few examples of potent BRAFV600
inhibitors include BAY43-9006 (sorafenib, Bayer), vemurafenib
(PLX4032, Plexxikon; RG7204, RO5185426, Hofmann-LaRoche), GDC-0879
(GlaxoSmithKline), dabrafenib (GSK2118436, GlaxoSmithKline),
PLX4720 (Hofmann-LaRoche), BMS-908662 (XL281, Bristol-Myers
Squibb), LGX818 (Novartis), PLX3603 (RO5212054, Hofmann-LaRoche),
ARQ-736 (ArQule), DP-4978 (Deciphera) and RAF265 (Novartis).
[0006] Vemurafenib is a potent inhibitor of mutant BRAF;
particularly of BRAFV600E mutant. Vemurafenib induced complete or
partial tumor regression in 81% of patients who had melanoma with
the BRAFV600E mutation. Responses were observed at all sites of
disease, including the bone, liver, and small bowel. However, after
a reliable early response to vemurafenib, responsive tumors were
found to develop resistance to treatment. In some patients with
BRAFV600E mutations, the tumors showed resistance without evidence
of an early response (N. Engl. J. Med., 2010, 363, 9, 809-819).
Dabrafenib is a potent and selective RAF kinase inhibitor of human
wild type BRAF and CRAF enzymes as well as the mutant forms
BRAFV600E, BRAFV600K and BRAFV600D.
[0007] MEK1 and MEK2 are dual-specificity kinases that catalyze the
phosphorylation of both tyrosine and threonine in target proteins;
dual-specificity kinases are included within the
protein-serine/threonine kinase family. Protein phosphorylation is
the most widespread class of post-translational modification used
in signal transduction (Biochemical and Biophysical Research
Communications, 2012, 417, 5-10). MEK1 and MEK2 are ubiquitously
expressed hydrophilic non-receptor proteins that participate in the
RAS-RAF-MEK-ERK signal transduction cascade, which is sometimes
denoted as the mitogen activated protein kinase (MAPK) cascade.
Ras-mediated Raf activation triggers activation of MEK1 and MEK2
(MAP/ERK kinases 1 and 2), which in turn phosphorylate ERK1 and
ERK2 (extracellular signal-regulated kinases 1 and 2) on
tyrosine-185 and threonine-183. Activated ERK1 and ERK2 translocate
and accumulate in the nucleus, where they can phosphorylate a
variety of substrates, including transcription factors that control
cellular growth and survival. A controlled regulation of these
cascades is involved in cell proliferation and differentiation,
whereas an unregulated activation of these kinases can result in
oncogenesis. Dysregulation of the RAS/RAF/MEK pathway has been
detected in more than 30% of human tumors, however, mutations in
the MEK1 and MEK2 genes are seldom, so that hyperactivation of
MEK1/2 usually results from gain-of-function mutations in RAS
and/or BRAF. Given the importance of the Ras/Raf/MEK/ERK pathway in
the development of human cancers, the kinase components of the
signal transduction cascade are potentially important targets for
the modulation of disease progression in cancer and other
proliferative diseases.
[0008] A few examples of potent MEK inhibitors include trametinib
(Mekinist.TM.), selumetinib (AstraZeneca), binimetinib (Array
Biopharma), PD-0325901 (Pfizer), cobimetinib (Exelixis),
refametinib (Valeant Pharmaceutical Int.), pimasertib (Santhera
Pharmaceuticals), TAK-733 (Takeda) and WX-554 (UCB Pharma S A).
[0009] Trametinib is a potent selective inhibitor of MEK1 and MEK2.
Significant clinical activity for trametinib is demonstrated in
patients with metastatic malignant melanoma with a BRAFV600E or
BRAFV600K mutation not previously treated with a BRAF inhibitor
when compared to chemotherapy (either dacarbazine or
paclitaxel).
[0010] Thus, it is evident from the above discussion that
metastatic melanoma and resistant BRAF mutant melanoma, continue to
be difficult to treat with existing therapies; and therefore, there
is a continued need for new effective treatments for these
conditions, both, to prevent their progression and to treat the
conditions.
SUMMARY OF THE INVENTION
[0011] In an aspect, the present invention relates to a
pharmaceutical combination comprising a CDK (cyclin dependent
kinase) inhibitor represented by a compound of formula I (as
described herein) or a pharmaceutically acceptable salt thereof and
at least one anticancer agent selected from BRAF (serine-threonine
protein kinase B-raf) inhibitor or a MEK (mitogen activated protein
kinase) inhibitor, for use in the treatment of melanoma.
[0012] In another aspect, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof; a therapeutically effective amount of a CDK inhibitor
represented by a compound of formula I (as described herein) or a
pharmaceutically acceptable salt thereof; in combination with a
therapeutically effective amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor.
[0013] In another aspect, the present invention relates to a
pharmaceutical composition of the present invention comprising a
CDK inhibitor selected from the compounds of formula I or a
pharmaceutically acceptable salt thereof and at least one
anticancer agent selected from a BRAF inhibitor or a MEK inhibitor,
and one or more pharmaceutically acceptable carrier, diluent or
excipient.
[0014] In a further aspect, the present invention relates to use of
a pharmaceutical combination comprising a CDK inhibitor represented
by the compound of formula I or a pharmaceutically acceptable salt
thereof and at least one anticancer agent selected from a BRAF
inhibitor or a MEK inhibitor; for the treatment of melanoma.
[0015] In a further aspect, the present invention relates to use of
a CDK inhibitor represented by the compound of formula I or a
pharmaceutically acceptable salt thereof and at least one
anticancer agent selected from a BRAF inhibitor or a MEK inhibitor;
for the manufacture of a medicament for the treatment of
melanoma.
[0016] In a further aspect, the present invention relates to a
pharmaceutical kit comprising a CDK inhibitor selected from the
compounds of formula I or a pharmaceutically acceptable salt
thereof and at least one anticancer agent selected from a BRAF
inhibitor or a MEK inhibitor.
[0017] Other aspects and further scope of applicability of the
present invention will become apparent from the detailed
description to follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts the effect of compound A (voruciclib) and
vemurafenib alone and in combination, on the cell cycle and
apoptosis in G361 melanoma cells after 5 days using flow
cytometry.
[0019] FIG. 2 depicts the effect of compound A (voruciclib) and
vemurafenib alone and in combination, on the early apoptosis in
G361 melanoma cells treated for 24 h using Annexin staining
method.
[0020] FIG. 3 depicts the effect of compound A (voruciclib) and
vemurafenib alone and in combination, on the cell cycle and
apoptosis in SK-MEL3 melanoma cells after 5 days using flow
cytometry.
[0021] FIG. 4 depicts the effect of compound A (voruciclib) and
vemurafenib alone and in combination (48 h), in A375 cell line.
[0022] FIG. 5 depicts the effect of compound A (voruciclib) and
vemurafenib alone and in combination (48 h), in G361 cell line.
[0023] FIG. 6 depicts the effect of compound A (voruciclib) and
vemurafenib alone and in combination (48 h), in MDA-MB435S cell
line.
[0024] FIGS. 7a and 7b depict the dose response curve of compound A
(voruciclib) and vemurafenib alone (48 h) on A375 parental and
A375R resistant cell line.
[0025] FIGS. 8a and 8b depict the effect of compound A (voruciclib)
and vemurafenib alone and in combination (48 h), in A375 resistant
cell line.
[0026] FIGS. 9a and 9b depict the effect of compound A (voruciclib)
and vemurafenib alone and in combination (48 h), in A375R resistant
cell line.
[0027] FIGS. 10a and 10b depict the effect of compound A
(voruciclib) and trametinib alone and in combination (48 h), in
A375 resistant cell line.
[0028] FIGS. 11a and 11b depict the effect of compound A
(voruciclib) and trametinib alone and in combination (48 h), in
A375R resistant cell line.
[0029] FIGS. 12a and 12b depict the effect of compound A
(voruciclib) and dabrafenib alone and in combination (48 h), in
A375 resistant cell line.
[0030] FIGS. 13a and 13b depict the effect of compound A
(voruciclib) and dabrafenib alone and in combination (48 h), in
A375R resistant cell line.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The general terms used hereinbefore and hereinafter
preferably have the following meanings within the context of this
disclosure, unless otherwise indicated. Thus, the definitions of
the general terms as used in the context of the present invention
are provided herein below:
[0032] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0033] Use of "(s)" as part of a term, includes reference to the
term singly or in plurality, e.g. the term agent(s) may indicate a
single agent or more agents.
[0034] As used herein, the term "at least one" is refers to one or
more. For instance, the term "at least one anticancer agent" means
that the combination comprises a single anticancer agent or more
anticancer agents.
[0035] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, as well as represents a stable compound, which does
not readily undergo transformation such as rearrangement,
cyclization, elimination, etc.
[0036] As used herein, the term "pharmaceutically acceptable" means
that the carrier, diluent, excipients, and/or salt must be
compatible with the other ingredients of the formulation, and not
deleterious to the recipient thereof. "Pharmaceutically acceptable"
also means that the compositions or dosage forms are within the
scope of sound medical judgment, suitable for use for an animal or
human without excessive toxicity, irritation, allergic response, or
other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0037] As used herein, the term "combination" or "pharmaceutical
combination" means the combined administration of the anticancer
agents, in the context of the present invention; a CDK inhibitor (a
compound of formula I) and at least one anticancer agent selected
from a BRAF inhibitor or a MEK inhibitor; which anticancer agents
can be administered independently at the same time or separately
within time intervals that especially allow that the combination
partners show a synergistic effect.
[0038] Cyclin-dependent kinases (CDKs) are a family of enzymes
which become activated in specific phases of the cell cycle. CDKs
consist of a catalytic subunit (the actual cyclin-dependent kinase
or CDK) and a regulatory subunit (cyclin). There are at least nine
CDKs (CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, etc.)
and at least 15 different types of cyclins (cyclin A, B1, B2, D1,
D2, D3, E, H etc.). Each step of the cell cycle is regulated by
such CDK complexes: G1/S transition (CDK2/cyclin A, CDK4/cyclin
D1-D3, CDK6/cyclin D3), S phase (CDK2/cyclin A), G2 phase 30
(CDK1/cyclin A), G2/M transition phase (CDK1/cyclin B).
[0039] As used herein, the term "CDK inhibitor" refers to an agent
that is capable of inhibiting one or more cyclin dependent
kinase(s) (CDK). Aberrant expression and overexpression of these
kinases are evidenced in many disease conditions such as cancer. In
the context of the present invention, the CDK inhibitor contained
in the pharmaceutical combination of the present invention refers
to a compound of formula I or a pharmaceutically acceptable salt
thereof. The compounds of the present invention inhibit CDK1/cyclin
B, CDK2/cyclin E, CDK4/cyclin D, CDK4/cyclin D1 and/or CDK9/cyclin
T1 with specificity.
[0040] As used herein, the term "BRAF inhibitor" refers to an agent
that is capable of inhibiting BRAF kinase or mutated BRAF kinase
activity (one or more mutated forms of serine-threonine protein
kinase B-RAF (BRAF)). Ninety percent of reported BRAF mutations
result in a substitution of glutamic acid for valine at amino acid
600 (the V600E mutation). Accordingly, the term "BRAF inhibitors"
encompasses within its scope a compound that is capable of
inhibiting BRAF or its mutated form; or a compound that is capable
of inhibiting V600 mutated form of BRAF; or a compound which is
capable of inhibiting V600E mutated form of BRAF in both
non-refractory and refractory melanoma.
[0041] As used herein, the term "MEK inhibitor" refers to an agent
that is capable of interacting with a mitogen activated protein
kinase (MEK) and inhibiting its enzymatic activity. Inhibiting MEK
enzymatic activity in turn reduces the ability of MEK to
phosphorylate a substrate peptide or protein. MEK1 and MEK2 are
protein kinases that participate in the RAS-RAF-MEK-ERK signal
transduction cascade. This cascade participates in the regulation
of a large variety of processes including apoptosis, cell cycle
progression, cell migration, differentiation, metabolism, and
proliferation. Accordingly, the term "MEK inhibitors" encompasses
within its scope a compound that is capable of inhibiting MEK.
[0042] As used herein, the term "synergistic" or "synergistic
effect" or "synergism" as used herein refers to the therapeutic
effect of the combination of the compounds (a BRAF inhibitor and a
CDK inhibitor, i.e. the compound of formula I), which is greater
than the additive effect of the compounds used in the
pharmaceutical combination. Advantageously, such synergy between
the active ingredients (the therapeutically active compounds) when
combined, allows for the use of smaller doses of one or both active
ingredients, provides greater efficacy at the same doses, and/or
prevents or delays the build-up of multi-drug resistance. The
combination index (CI) method of Chou and Talalay can be used to
determine the synergy, additive or antagonism effect of the
compounds used in combination. When the CI value is less than 1,
there is synergy between the compounds used in the combination;
when the CI value is equal to 1, there is an additive effect
between the compounds used in the combination and when CI value is
more than 1, there is an antagonistic effect. The synergistic
effect can be attained either by co-formulating the compounds
contained in the pharmaceutical combination or the composition of
the present invention and administering the said compounds
simultaneously through a unit dosage form or as separate
formulations administered simultaneously or sequentially.
[0043] As used herein, "melanoma" refers to a condition
characterized by the growth of a tumor arising from the melanocytic
system of the skin and other organs. Most melanocytes occur in the
skin, but are also found in the meninges, digestive tract, lymph
nodes and eyes. When melanoma occurs in the skin, it is referred to
as cutaneous melanoma. Melanoma can also occur in the eyes and is
called ocular or intraocular melanoma. Melanoma occurs rarely in
the meninges, the digestive tract, lymph nodes or other areas where
melanocytes are found.
[0044] The terms, "mutant melanoma" or "malignant melanoma" used
interchangeably, refers to a melanocytic neoplasm comprising
melanoma cells that have a defect (also referred to as a
"mutation"). Malignant melanoma usually develops from or near a
nevus, consisting of a mass of cells having a marked tendency to
metastasis. 40-60% of melanomas carry an activating mutation in the
gene encoding the serine-threonine protein kinase B-RAF (BRAF).
Among the BRAF mutations observed in melanoma, over 90% are at
codon 600, and among these, over 90% are a single nucleotide
mutation resulting in substitution of glutamic acid for valine
(BRAFV600E). The second most common mutation is BRAFV600K
substituting lysine for valine, which represents 5-6% of melanomas,
followed by BRAFV600R and BRAFV600D. (Journal of Translational
Medicine, 2012, 10, 85, 1-9).
[0045] The term "metastatic melanoma" refers to melanoma that has
spread through the lymphatic system and/or the blood vessels to
other sites of the body including the subcutaneous tissue which
lies underneath the skin, the lymph nodes, and to other organs such
as the lungs, liver, to bone or to the brain. Stage III melanoma is
characterized by the level of lymph node metastasis. There is no
evidence of distant metastasis. Stage IV melanoma is characterized
by the location of distant metastases and the level of serum
lactate dehydrogenase (LDH). This stage is also called as
metastatic melanoma.
[0046] Unless otherwise indicated, the term "melanoma" can also
include recurrent or resistant melanoma. The term "recurrent" or
"resistant" refers to the repeated outbreak of melanoma, or a
progression of the melanoma independently of whether the disease
was cured before said outbreak or progression.
[0047] Thus, the treatment of melanoma for which the pharmaceutical
combination (as described herein) is provided, refers to treatment
of non-refractory, metastatic or refractory (resistant) BRAF mutant
melanoma, particularly BRAFV600 mutant melanoma, and more
particularly BRAFV600E mutant melanoma.
[0048] The term "non-responsive/refractory" as used in reference to
melanoma, is used herein to refer to the subjects or patients
having melanoma who have been treated with the currently available
cancer therapies such as chemotherapy, radiation therapy, surgery,
hormonal therapy and/or biological therapy/immunotherapy; for the
treatment of melanoma; wherein the therapy is not clinically
adequate to treat the patients, such that these patients need
additional effective therapy, i.e, remain unsusceptible to therapy.
The phrase can also describe subjects or patients who respond to
therapy yet suffer from side effects, relapse, develop resistance
or do not experience any relief from one or more symptoms of
melanoma. In various embodiments, "non-responsive/refractory" means
that at least some significant portions of the cancer cells are not
killed or their cell division arrested. The determination of
whether the cancer cells are "non-responsive/refractory" can be
made either in vivo or in vitro by any method known in the art for
assaying the effectiveness of treatment on cancer cells, using the
art-accepted meanings of "refractory" in such a context.
[0049] As used herein the term "treatment cycle" refers to a time
period during which a recurring sequence of administration of a CDK
inhibitor i.e. the compound of formula I or a pharmaceutically
acceptable salt thereof; and at least one anticancer agent selected
from a BRAF inhibitor or a MEK inhibitor, is carried out.
[0050] The term "apoptosis" refers to the natural process of
programmed cell death. It is a process of self-destruction, in
which the cell uses specialized cellular machinery to kill itself.
The cells disintegrate into membrane-bound particles that are then
eliminated by phagocytosis. Apoptosis is a mechanism that enables
metazoans to control cell number and eliminate cells that threaten
the animal's survival.
[0051] The term "subject" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment, more particularly a
human suffering from melanoma. The term "subject" may be used
interchangeably with the term patient. In the context of the
present invention the phrase "a subject in need thereof" means a
subject in need of the treatment for mutant or malignant melanoma.
Alternatively, the phrase "a subject in need thereof" means a
subject (patient) diagnosed with mutant or malignant melanoma.
[0052] The term "mammal" as used herein is intended to encompass
humans, as well as non-human mammals. Non-human mammals include but
are not limited to domestic animals, such as cows, pigs, horses,
dogs, cats, rabbits, rats and mice, and non-domestic animals.
[0053] The term "therapeutically effective amount", as used herein
refers to the amount of a CDK inhibitor i.e. the compound of
formula I or a pharmaceutically acceptable salt thereof and at
least one anticancer agent selected from a BRAF inhibitor or a MEK
inhibitor, that, when administered to a subject in need of such
treatment, is sufficient to provide therapeutic benefit, that shall
include: (i) prevent or delay one or more symptom of melanoma; (ii)
ameliorate or eliminate one or more symptom of melanoma; or (iii)
treat melanoma.
[0054] The term "treat" or "treatment" or "treated" with reference
to melanoma in a subject, preferably a mammal, more preferably a
human include: (i) inhibition of melanoma i.e., arresting the
development of the melanoma; (ii) reduction in the regression of
melanoma; (iii) inhibition of tumor cell infiltration into
peripheral organs; (iv) inhibition (i.e., reduction, slowing down
or complete stopping) of metastasis; (v) amelioration of melanoma,
i.e., reducing the severity of the symptoms associated with
melanoma; and (vi) relief, to some extent, of one or more symptoms
associated with melanoma.
[0055] According to one aspect of the present invention, there is
provided a pharmaceutical combination comprising a CDK inhibitor
selected from a compound of formula I;
##STR00001##
wherein Ar is a phenyl group, which is substituted by 1 or 2
different substituents selected from chlorine and trifluoromethyl;
or a pharmaceutically acceptable salt thereof; and at least one
anticancer agent selected from a BRAF inhibitor or a MEK inhibitor;
for use in the treatment of melanoma.
[0056] According to another embodiment, the CDK inhibitor contained
in the pharmaceutical combination of the present invention is
selected from a compound of formula I; wherein Ar is a phenyl group
substituted by 2 different groups selected from chlorine and
trifluoromethyl or a pharmaceutically acceptable salt thereof.
[0057] According to another further embodiment, the CDK inhibitor
contained in the pharmaceutical combination of the present
invention is selected from a compound of formula I wherein Ar is a
phenyl group substituted by chlorine; or a pharmaceutically
acceptable salt thereof.
[0058] The manufacture of the compounds of formula I or the
pharmaceutically acceptable salts thereof, and the manufacture of
pharmaceutical composition containing the compounds are disclosed
in PCT Patent Publication No. WO2004004632 (corresponding to U.S.
Pat. No. 7,271,193) and PCT Patent Publication No. WO2007148158.
These PCT Patent Publications disclose that the compounds
represented by formula I can be used in the treatment of
proliferative disorders. As indicated herein above the compounds of
formula I may be used in the form of their salts. Preferred salt of
compounds of formula I include acetates, alginates, ascorbates,
aspartates, benzoates, benzenesulfonates, bisulfates, borates,
cinnamates, citrates, ethanesulfonates, fumarates, glucuronates,
glutamates, glycolates, hydrochlorides, hydrobromides,
hydrofluorides, ketoglutarates, lactates, maleates, malonates,
mesylate, nitrates, oxalates, palmoates, perchlorates, phosphates,
picrates, salicylates, succinates, sulfamate, sulfates, tartrates,
tosylate, trifluoroacetic acid salt and other acid addition salts
known to the person skilled in the art.
[0059] In one embodiment, the CDK inhibitor (the compound of
formula 1) contained in the pharmaceutical combination is
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one; or its
pharmaceutically acceptable salt.
[0060] In another embodiment, the CDK inhibitor (the compound of
formula 1) contained in the pharmaceutical combination is
(+)-trans-2-(2-chloro-4-trifluoromethyl
phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl
pyrrolidin-3-yl)-chromen-4-one hydrochloride (referred to herein as
"compound A". Compound A is also referred to herein as
"voruciclib").
[0061] In one embodiment, the CDK inhibitor (compound of formula 1)
contained in the pharmaceutical combination is
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-p-
yrrolidin-3-yl)-chromen-4-one or its pharmaceutically acceptable
salt.
[0062] In another embodiment, the CDK inhibitor (compound of
formula 1) contained in the pharmaceutical combination is
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-p-
yrrolidin-3-yl)-chromen-4-one hydrochloride (referred to herein as
"compound B". Compound B is also referred to herein as
"riviciclib").
[0063] In an embodiment, the BRAF inhibitor contained in the
pharmaceutical combination is an inhibitor of V600 mutated form of
BRAF.
[0064] In an embodiment, the BRAF inhibitor contained in the
pharmaceutical combination is an inhibitor of V600E mutated form of
BRAF.
[0065] In an embodiment, the BRAF inhibitor contained in the
pharmaceutical combination is selected from BAY43-9006 (sorafenib,
Bayer), vemurafenib (PLX4032, Plexxikon; RG7204, RO5185426,
Hofmann-LaRoche), GDC-0879 (GlaxoSmithKline), dabrafenib
(GSK2118436, GlaxoSmithKline), PLX4720 (Hofmann-LaRoche),
BMS-908662 (XL281, Bristol-Myers Squibb), LGX818 (Novartis),
PLX3603 (RO5212054, Hofmann-LaRoche), ARQ-736 (ArQule), DP-4978
(Deciphera) or RAF265 (Novartis).
[0066] In an embodiment, the BRAF inhibitor contained in the
pharmaceutical combination is vemurafenib.
[0067] In an embodiment, the BRAF inhibitor contained in the
pharmaceutical combination is dabrafenib.
[0068] In an embodiment, the MEK inhibitor contained in the
pharmaceutical combination is an inhibitor of V600 mutated form of
BRAF.
[0069] In an embodiment, the MEK inhibitor contained in the
pharmaceutical combination is an inhibitor of V600E or V600K
mutated form of BRAF.
[0070] In an embodiment, the MEK inhibitor contained in the
pharmaceutical combination is selected from selumetinib
(AstraZeneca), binimetinib (Array Biopharma), PD-0325901 (Pfizer),
trametinib (Mekinist.TM.), cobimetinib (Exelixis), refametinib
(Valeant), pimasertib (Santhera Pharmaceuticals), TAK-733 (Takeda)
or WX-554 (UCB Pharma S A).
[0071] In an embodiment, the MEK inhibitor contained in the
pharmaceutical combination is trametinib.
[0072] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; and a
therapeutically effective amount of at least one anticancer agent
selected from a BRAF inhibitor and a MEK inhibitor, wherein said
CDK inhibitor and at least one of the said anticancer agents; are
administered simultaneously.
[0073] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof and a
therapeutically effective amount of at least one anticancer agent
selected from a BRAF inhibitor and a MEK inhibitor, wherein said
CDK inhibitor and atleast one of the said anticancer agents; are
administered sequentially.
[0074] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; and a
therapeutically effectively amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor, wherein the said
anticancer agent is administered prior to the administration of the
CDK inhibitor.
[0075] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; and a
therapeutically effectively amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor, wherein the CDK
inhibitor is administered prior to the administration of the said
anticancer agent.
[0076] In one embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; and a
therapeutically effectively amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor, wherein the CDK
inhibitor and the said anticancer agent are both administered once
a day.
[0077] In another embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof and a
therapeutically effectively amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor, wherein the CDK
inhibitor is administered once a day, while the said anticancer
agent is administered twice a day.
[0078] In yet another embodiment, the present invention relates to
a pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; and a
therapeutically effectively amount of at least one anticancer agent
selected from a BRAF inhibitor or a MEK inhibitor, wherein the CDK
inhibitor and the said anticancer agent are both administered twice
a day.
[0079] In one aspect, the present invention relates to a method for
the treatment of melanoma comprising administering to a subject in
need thereof; a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I (as described herein) or a
pharmaceutically acceptable salt thereof; and a therapeutically
effective amount of at least one anticancer agent selected from a
BRAF inhibitor or a MEK inhibitor.
[0080] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof; a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
at least one anticancer agent selected from a BRAF inhibitor or a
MEK inhibitor, wherein the CDK inhibitor and the said anticancer
agent are administered simultaneously.
[0081] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
at least one anticancer agent selected from a BRAF inhibitor or a
MEK inhibitor, wherein the CDK inhibitor and the said anticancer
agent are administered sequentially.
[0082] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
at least one anticancer agent selected from a BRAF inhibitor or a
MEK inhibitor, wherein the said anticancer agent is administered
prior to the administration of the said CDK inhibitor.
[0083] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of the CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
at least one anticancer agent selected from a BRAF inhibitor or a
MEK inhibitor, wherein the said CDK inhibitor is administered prior
to the administration of said anticancer agent.
[0084] In one embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
at least one anticancer agent selected from a BRAF inhibitor or a
MEK inhibitor, wherein the said CDK inhibitor and the said
anticancer agent are both administered once a day.
[0085] In another embodiment, the present invention relates to a
method of treating melanoma comprising administering to a subject
in need thereof a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof and a therapeutically
effective amount of at least one of a BRAF inhibitor or a MEK
inhibitor, wherein the said CDK inhibitor is administered once a
day and the said anticancer agent is administered twice a day.
[0086] In yet another embodiment, the present invention relates to
a method of treating melanoma comprising administering to a subject
in need thereof a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof and a therapeutically
effective amount of at least one of a BRAF inhibitor or a MEK
inhibitor, wherein the said CDK inhibitor and the said anticancer
agent are both administered twice a day.
[0087] In an embodiment, the method of treating melanoma comprises
administering to the subject in need thereof; the CDK inhibitor
selected from the compound of formula I and at least one anticancer
agent selected from a BRAF inhibitor or a MEK inhibitor in the dose
range described herein.
[0088] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effective amount of a BRAF
inhibitor, wherein the said BRAF inhibitor and said CDK inhibitor
are administered simultaneously.
[0089] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effective amount of a BRAF
inhibitor, wherein the said BRAF inhibitor and the said CDK
inhibitor are administered sequentially.
[0090] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a BRAF
inhibitor, wherein the said BRAF inhibitor is administered prior to
the administration of the said CDK inhibitor.
[0091] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a BRAF
inhibitor, wherein the said CDK inhibitor is administered prior to
the administration of the said BRAF inhibitor.
[0092] In one embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a BRAF
inhibitor, wherein the said CDK inhibitor and the said BRAF
inhibitor are both administered once a day.
[0093] In another embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a BRAF
inhibitor, wherein the said CDK inhibitor is administered once a
day and the said BRAF inhibitor is administered twice a day.
[0094] In yet another embodiment, the present invention relates to
a pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a BRAF
inhibitor, wherein the said CDK inhibitor and the said BRAF
inhibitor are both administered twice a day.
[0095] In one aspect, the present invention relates to a method for
the treatment of melanoma comprising administering to a subject in
need thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I (as described herein) or a
pharmaceutically acceptable salt thereof and a BRAF inhibitor.
[0096] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof and a therapeutically effective amount of a
BRAF inhibitor, wherein the said BRAF inhibitor and the said CDK
inhibitor are administered simultaneously.
[0097] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof and a therapeutically effective amount of a
BRAF inhibitor, wherein the said BRAF inhibitor and the said CDK
inhibitor are administered sequentially.
[0098] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a BRAF inhibitor, wherein the said BRAF inhibitor is administered
prior to the administration of the said CDK inhibitor.
[0099] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a BRAF inhibitor, wherein the said CDK inhibitor is administered
prior to the administration of the said BRAF inhibitor.
[0100] In one embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a BRAF inhibitor, wherein the said CDK inhibitor and the said BRAF
inhibitor are both administered once a day.
[0101] In another embodiment, the present invention relates to a
method of treating melanoma comprising administering to a subject
in need thereof a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof; and a therapeutically
effective amount of a BRAF inhibitor, wherein the said CDK
inhibitor is administered once a day, while the said BRAF inhibitor
is administered twice a day.
[0102] In yet another embodiment, the present invention relates to
a method of treating melanoma comprising administering to a subject
in need thereof a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof; and a therapeutically
effective amount of a BRAF inhibitor, wherein the said CDK
inhibitor and the said BRAF inhibitor are both administered twice a
day.
[0103] In an embodiment, the method of treating melanoma comprises
administering to the subject in need thereof; the BRAF inhibitor
and the CDK inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof; in the dose range
described herein.
[0104] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effective amount of a MEK inhibitor,
wherein the said MEK inhibitor and the said CDK inhibitor are
administered simultaneously.
[0105] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effective amount of a MEK inhibitor,
wherein the MEK inhibitor and the CDK inhibitor are administered
sequentially.
[0106] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; and a
therapeutically effectively amount of a MEK inhibitor, wherein the
said MEK inhibitor is administered prior to the administration of
the said CDK inhibitor.
[0107] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a MEK
inhibitor, wherein the said CDK inhibitor is administered prior to
the administration of the said MEK inhibitor.
[0108] In one embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a MEK
inhibitor, wherein the said CDK inhibitor and the said MEK
inhibitor are both administered once a day.
[0109] In another embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof; and a therapeutically effectively amount of a MEK
inhibitor, wherein the said CDK inhibitor is administered once a
day, while the said MEK inhibitor is administered twice a day.
[0110] In yet another embodiment, the present invention relates to
a pharmaceutical combination for use in the treatment of melanoma,
wherein the said pharmaceutical combination comprises a
therapeutically effective amount of a CDK inhibitor selected from
the compound of formula I or a pharmaceutically acceptable salt
thereof and a therapeutically effectively amount of a MEK
inhibitor, wherein the said CDK inhibitor and the said MEK
inhibitor are both administered twice a day.
[0111] In one aspect, the present invention relates to a method for
the treatment of melanoma comprising administering to a subject in
need thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I (as described herein) or a
pharmaceutically acceptable salt thereof and a MEK inhibitor.
[0112] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a MEK inhibitor, wherein the said MEK inhibitor and the said CDK
inhibitor are administered simultaneously.
[0113] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a MEK inhibitor, wherein the said MEK inhibitor and the said CDK
inhibitor are administered sequentially.
[0114] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a MEK inhibitor, wherein the said MEK inhibitor is administered
prior to the administration of the said CDK inhibitor.
[0115] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of the CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a MEK inhibitor, wherein the said CDK inhibitor is administered
prior to the administration of the said MEK inhibitor.
[0116] In one embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; and a therapeutically effective amount of
a MEK inhibitor, wherein the said CDK inhibitor and the said MEK
inhibitor are both administered once a day.
[0117] In another embodiment, the present invention relates to a
method of treating melanoma comprising administering to a subject
in need thereof a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof; and a therapeutically
effective amount of a MEK inhibitor, wherein the said CDK inhibitor
is administered once a day, while the said MEK inhibitor is
administered twice a day.
[0118] In yet another embodiment, the present invention relates to
a method of treating melanoma comprising administering to a subject
in need thereof a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof; and a therapeutically
effective amount of a MEK inhibitor, wherein the said CDK inhibitor
and the said MEK inhibitor are both administered twice a day.
[0119] In an embodiment, the method of treating melanoma comprises
administering to the subject in need thereof; the MEK inhibitor and
the CDK inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof; in the dose range
described herein.
[0120] In an embodiment, the present invention relates to a
pharmaceutical combination for use in the treatment of melanoma,
wherein said pharmaceutical combination comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound of
formula I or a pharmaceutically acceptable salt thereof; a
therapeutically effective amount of a BRAF inhibitor and a
therapeutically effective amount of a MEK inhibitor.
[0121] In an embodiment, the present invention relates to a method
of treating melanoma comprising administering to a subject in need
thereof a therapeutically effective amount of a CDK inhibitor
selected from the compound of formula I or a pharmaceutically
acceptable salt thereof; a therapeutically effective amount of a
BRAF inhibitor and a therapeutically effective amount of a MEK
inhibitor.
[0122] In one embodiment of the present invention, the melanoma
being treated is non-refractory melanoma.
[0123] In another embodiment of the present invention, the melanoma
being treated is non-refractory BRAF mutant melanoma.
[0124] In yet another embodiment of the present invention, the
melanoma being treated is non-refractory BRAF V600 mutant
melanoma.
[0125] In a further embodiment of the present invention, the
melanoma being treated is non-refractory BRAF V600E or BRAF V600K
mutant melanoma.
[0126] In one embodiment of the present invention, the melanoma
being treated is recurrent or refractory melanoma.
[0127] In another embodiment of the present invention, the melanoma
being treated is resistant BRAF mutant melanoma.
[0128] In yet another embodiment of the present invention, the
melanoma being treated is resistant BRAF V600 mutant melanoma.
[0129] In a further embodiment of the present invention, the
melanoma being treated is resistant BRAF V600E or BRAF V600K mutant
melanoma.
[0130] In one embodiment of the present invention, the melanoma
being treated is metastatic melanoma.
[0131] In another embodiment of the present invention, the melanoma
being treated is metastatic BRAF mutant melanoma.
[0132] In yet another embodiment of the present invention, the
melanoma being treated is metastatic BRAFV600 mutant melanoma.
[0133] In a further embodiment of the present invention, the
melanoma being treated is metastatic BRAFV600E or BRAFV600K mutant
melanoma.
[0134] According to the present invention, administration of the
CDK inhibitors (the compound of formula I) and/or anticancer agent
selected from a BRAF inhibitor and/or a MEK inhibitor can be by any
suitable route, including, without limitation, parenteral, oral,
sublingual, transdermal, topical, intranasal, aerosol, intraocular,
intratracheal or intrarectal.
[0135] In one embodiment, the CDK inhibitor can be administered
orally to generate and maintain good blood levels thereof, while
the anticancer agent(s) selected from a BRAF inhibitor and/or a MEK
inhibitor can be administered parenterally, by intravenous,
subcutaneous, intramuscular, intravascular or infusion route.
[0136] In another embodiment, the CDK inhibitor can be administered
parenterally by intravenous, subcutaneous, intramuscular,
intravascular or infusion route, while the anticancer agent(s)
selected from a BRAF inhibitor and/or a MEK inhibitor can be
administered orally.
[0137] In a further embodiment, both, the CDK inhibitor and the
anticancer agent(s) selected from a BRAF inhibitor and/or a MEK
inhibitor can be administered orally to generate and maintain good
blood levels thereof.
[0138] In a still further embodiment, both the CDK inhibitor of
formula I and the anticancer agent(s) selected from a BRAF
inhibitor and/or a MEK inhibitor can be administered parenterally
by intravenous, subcutaneous, intramuscular intravascular or
infusion route, to generate and maintain good blood levels
thereof.
[0139] In an aspect, the present invention relates to a
pharmaceutical composition for use in the treatment of melanoma,
wherein the said composition comprises a CDK inhibitor selected
from the compounds of formula I or pharmaceutically acceptable
salts thereof; and at least one anticancer agent selected from a
BRAF inhibitor or a MEK inhibitor and one or more pharmaceutically
acceptable carrier, diluent, or excipient. For the production of
pills, tablets, coated tablets and hard gelatin capsules, the
pharmaceutically active excipients that can be used include, but
not limited to, lactose, corn starch or derivatives thereof, gum
arabica, magnesia or glucose, etc. For soft gelatin capsules and
suppositories, the carriers that can be used include, but not
limited to, fats, waxes, natural or hardened oils, etc. Suitable
carriers for the production of solutions, are, for example
injection solutions, or for emulsions or syrups are, for example,
water, physiological sodium chloride solution or alcohols, for
example, ethanol, propanol or glycerol, sugar solutions, such as
glucose solutions or mannitol solutions, or a mixture of the
various solvents which have been mentioned.
[0140] The CDK inhibitor (the compound of formula I) and the
anticancer agent(s) selected from a BRAF inhibitor and/or a MEK
inhibitor can be formulated singly or in combination into
pharmaceutical dosage forms using conventional pharmaceutical
techniques familiar to one skilled in the art such as by means of
blending, granulating, dissolving or lyophilizing.
[0141] In general, compositions intended for pharmaceutical use may
be prepared according to any method known in the art for the
manufacture of pharmaceutical compositions, e.g. Remington--The
Science and Practice of Pharmacy (21.sup.st Edition) (2005),
Goodman & Gilman's The Pharmacological Basis of Therapeutics
(11.sup.th Edition) (2006) and Ansel's Pharmaceutical Dosage Forms
and Drug Delivery Systems (9.sup.th Edition), and Solid-State
Chemistry of Drugs (2.sup.nd Edition) (1999).
[0142] The compositions described herein can be in a form suitable
for oral administration, for example, solid dosage forms such as
tablets, capsules, lozenges, or granules; liquid dosage forms such
as, emulsions, solutions, suspensions; for parenteral injection
(including intravenous, subcutaneous, intramuscular, intravascular
or infusion) for example as a sterile solution, suspension or
emulsion; for topical administration for example as an ointment,
cream, gel or lotion.
[0143] Compositions for oral administrations can be in the form of
tablets, lozenges, aqueous or oily suspensions, granules, powders,
cachets, emulsions, capsules, syrups, or elixirs. Orally
administered compositions may contain one or more optional agents,
for example, sweetening agents such as fructose, aspartame or
saccharin; flavoring agents such as peppermint, oil of wintergreen,
or cherry; coloring agents; and preserving agents, to provide a
pharmaceutically palatable preparation. Selectively permeable
membranes surrounding an osmotically active driving compound are
also suitable for oral administration of the compounds (the CDK
inhibitor, and/or the anticancer agent(s) selected from a BRAF
inhibitor and/or a MEK inhibitor) contained in the pharmaceutical
combination according to the present invention. Compositions
suitable for oral administration can include standard vehicles such
as mannitol, lactose, starch, corn starch, magnesium stearate,
talc, sodium saccharine, cellulose, magnesium carbonate, etc. Such
vehicles are preferably of pharmaceutical grade.
[0144] For ointments, creams, the active ingredient (CDK inhibitor
and/or anticancer agent(s) selected from a BRAF inhibitor and/or a
MEK inhibitor) is formulated in oil-in-water or water-in-oil
base.
[0145] For intramuscular, intraperitoneal, subcutaneous and
intravenous use, sterile solutions of the active ingredient (CDK
inhibitor and/or anticancer agent(s) selected from a BRAF inhibitor
and/or a MEK inhibitor) are usually employed, and the pH of the
solutions should be suitably adjusted and buffered.
[0146] Further, the effect of the compounds i.e. the CDK inhibitors
and/or anticancer agent(s) selected from a BRAF inhibitor and/or a
MEK inhibitor contained in the pharmaceutical composition can be
delayed or prolonged through a proper formulation. For example, a
slowly soluble pellet of the compound may be prepared and
incorporated in a tablet or capsule. The technique can be improved
by making pellets of several different dissolution rates and
filling capsules with a mixture of the pellets. Tablets or capsules
can be coated with a film which resists dissolution for a
predictable period of time. Even the parenteral preparations can be
made long-acting, by dissolving or suspending the compound in oily
or emulsified vehicles which allow it to disperse only slowly in
the serum.
[0147] Although the effective doses of the CDK inhibitor selected
from the compounds of formula I and the anticancer agent(s)
selected from a BRAF inhibitor and/or a MEK inhibitor) used for
administration vary depending on the severity of the disease
(melanoma), the severity of symptoms, the age, sex, body weight and
sensitivity difference of the patient, the mode, time, interval and
duration of administration, the nature and type of formulation,
etc. In certain embodiments, the therapeutic agents contained in
the pharmaceutical combination according to the present invention
are administered in a time frame where both the agents are still
active. One skilled in the art would be able to determine such a
time frame by determining the half life of the administered the
said therapeutic agents. As indicated herein before, the anticancer
agents contained in the pharmaceutical composition can be
administered simultaneously or sequentially. Those skilled in the
art will recognize that several variations are possible within the
scope and spirit of this invention.
[0148] The dosage of the therapeutic agents to be administered
should be selected to produce the desired effect. A suitable dosage
of the CDK inhibitor can be from about 5 mg to about 500 mg. The
dose of the CDK inhibitor, which is to be administered, can cover a
wide range depending on the severity of the melanoma to be treated.
The dose to be administered daily can be selected to obtain the
desired effect. A suitable dose can range from about 50 mg/day to
350 mg/day of the CDK inhibitor. If required, higher or lower daily
doses can also be administered.
[0149] In an embodiment, the BRAF inhibitor can be administered
from about 1 mg/day to about 2500 mg/day and this amount can be
given in a single or multiple doses per day or per dose or per
cycle of treatment.
[0150] In an embodiment, the MEK inhibitor can be administered from
about 0.01 mg/day to 2000 mg/day and this amount can be given in a
single or multiple doses per day or per dose or per cycle of
treatment.
[0151] In an embodiment, the CDK inhibitor and the anticancer
agent(s) selected from a BRAF inhibitor and/or a MEK inhibitor are
both administered once a day. In another embodiment, the CDK
inhibitor and the anticancer agent(s) selected from a BRAF
inhibitor and/or a MEK inhibitor are both administered twice a day.
In a further embodiment, the CDK inhibitor is administered once a
day, while the anticancer agent(s) selected from a BRAF inhibitor
and/or a MEK inhibitor is administered twice a day. However, the
amount of each therapeutic agent contained in the pharmaceutical
combination according to the present invention, when used in
combination will typically be less than an amount that would
produce a therapeutic effect if administered alone. For
convenience, the total daily dose can be divided and administered
in portions during the day if desired.
[0152] The combinations provided by this invention have been
evaluated in certain assay systems, and in several different
administrative schedules in vitro. The experimental details are as
provided herein below. The data presented herein clearly indicate
that the BRAF inhibitor or the MEK inhibitor when combined with a
CDK inhibitor selected from the compounds of formula I or a
pharmaceutically acceptable salt thereof exhibit synergistic
effect. It is clearly indicated that the therapeutic agents when
used in combination in the treatment of melanoma increases
apoptosis or cytotoxicity in proliferative cells than when the
cells are treated with only the CDK inhibitor (i.e. the compound of
formula I or its pharmaceutically acceptable salt, alone) or only
the BRAF inhibitor or only the MEK inhibitor.
[0153] In an aspect, the present invention relates to a
pharmaceutical kit comprising a CDK inhibitor (the compound of
formula I or a pharmaceutically acceptable salt thereof) and at
least one anticancer agent selected from a BRAF inhibitor or a MEK
inhibitor. The pharmaceutical kit may comprise a container
containing a compound of formula I or a pharmaceutically acceptable
salt thereof and at least one anticancer agent selected from a BRAF
inhibitor or a MEK inhibitor as a fixed dose formulation; or the
kit may comprise two or more separate containers for the compound
of formula I or a pharmaceutically acceptable salt thereof; and at
least one anticancer agent selected from a BRAF inhibitor or a MEK
inhibitor. The kit may further comprise a package insert, including
information about the indication, usage, doses, direction for
administration, contraindications, precautions and warnings. The
suitable container that can be used includes a bottle, a vial, an
ampoule, a syringe or a blister pack. The pharmaceutical kit may
optionally comprise a further container comprising a
pharmaceutically acceptable buffer, water for injection,
phosphate-buffered saline, Ringer's solution and dextrose
solution.
[0154] The CDK inhibitors i.e. the compounds of formula I contained
in the pharmaceutical combination of the present invention; may be
prepared according to the methods disclosed in PCT Patent
Publication No. WO2004004632 and PCT Patent Publication No.
WO2007148158.
[0155] The general process for the preparation of compounds of
formula I, or a pharmaceutically acceptable salt thereof, comprises
the following steps:
(a) treating the resolved enantiomerically pure (-)-trans
enantiomer of the intermediate compound of formula VIA,
##STR00002##
with acetic anhydride in the presence of a Lewis acid catalyst to
obtain a resolved acetylated compound of formula VIIA,
##STR00003##
(b) reacting the resolved acetylated compound of formula VIIA with
an acid of formula ArCOOH or an acid chloride of formula ArCOCl or
an acid anhydride of formula (ArCO).sub.2O or an ester of formula
ArCOOCH.sub.3, wherein Ar is as defined hereinabove in reference to
the compound of formula I, in the presence of a base and a solvent
to obtain a resolved compound of formula VIIIA;
##STR00004##
(c) treating the resolved compound of formula VIIIA with a base in
a suitable solvent to obtain the corresponding resolved
.beta.-diketone compound of formula IXA;
##STR00005##
wherein Ar is as defined above; (d) treating the resolved
.beta.-diketone compound of formula IXA with an acid such as
hydrochloric acid to obtain the corresponding cyclized compound of
formula XA,
##STR00006##
(e) subjecting the compound of formula XA to dealkylation by
heating it with a dealkylating agent at a temperature ranging from
120-180.degree. C. to obtain the (+)-trans enantiomer of the
compound of formula I and, optionally, converting the subject
compound into its pharmaceutically acceptable salt.
[0156] The Lewis acid catalyst utilized in the step (a) above may
be selected from: BF.sub.3, Et.sub.2O, zinc chloride, aluminium
chloride and titanium chloride.
[0157] The base utilized in the process step (b) may be selected
from triethylamine, pyridine and a DCC-DMAP combination
(combination of N, N'-dicyclohexyl carbodiimide and
4-dimethylaminopyridine).
[0158] It will be apparent to those skilled in the art that the
rearrangement of the compound of formula VIIIA to the corresponding
.beta.-diketone compound of formula IXA is known as a
Baker-Venkataraman rearrangement (J. Chem. Soc., 1933, 1381 and
Curr. Sci., 1933, 4, 214).
[0159] The base used in the process step (c) may be selected from:
lithium hexamethyl disilazide, sodium hexamethyldisilazide,
potassium hexamethyldisilazide, sodium hydride and potassium
hydride. A preferred base is lithium hexamethyl disilazide.
[0160] The dealkylating agent used in process step (e) for the
dealkylation of the compound of formula IXA may be selected from:
pyridine hydrochloride, boron tribromide, boron trifluoride
etherate and aluminium trichloride. A preferred dealkylating agent
is pyridine hydrochloride.
[0161] Preparation of the starting compound of formula VIA involves
reacting 1-methyl-4-piperidone with a solution of
1,3,5-trimethoxybenzene in glacial acetic acid, to yield
1-methyl-4-(2,4,6-trimethoxyphenyl)-1,2,3,6-tetrahydropyridine,
which is reacted with boron trifluoride diethyl etherate, sodium
borohydride and tetrahydrofuran to yield
1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol. Conversion of
1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol to the compound
of formula VIA involves converting the hydroxyl group present on
the piperidine ring of the compound,
1-methyl-4-(2,4,6-trimethoxyphenyl) piperidin-3-ol to a leaving
group such as tosyl, mesyl, triflate or halide by treatment with an
appropriate reagent such as p-toluenesulfonylchloride,
methanesulfonylchloride, triflic anhydride or phosphorous
pentachloride in the presence of oxygen nucleophiles such as
triethylamine, pyridine, potassium carbonate or sodium carbonate,
followed by ring contraction in the presence of oxygen nucleophiles
such as sodium acetate or potassium acetate in an alcoholic solvent
such as isopropanol, ethanol or propanol.
[0162] The representative compound, compound A (also referred to as
voruciclib) used in the pharmacological assays refers to
(+)-trans-2-(2-chloro-4-trifuorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl--
1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride and was one
of the compounds disclosed in the published PCT Publication No.
WO2007148158.
[0163] Another representative compound, the compound B (also
referred to as riviciclib) used in the pharmacological assays
refers to
(+)-trans-2-(2-chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-py-
rrolidin-3-yl)-chromen-4-one hydrochloride and was one of the
compounds disclosed in published PCT Publication No.
WO2004004632.
[0164] The synergistic effect of the combination according to the
present invention comprising the CDK inhibitor and at least one
anticancer agent selected from a BRAF inhibitor or a MEK inhibitor
is now explained in more detail with reference to preferred
embodiments thereof. It is to be noted that these are provided only
as examples and not intended to limit the invention.
[0165] It is to be understood that the invention may assume various
alternative variations and step sequences, except where expressly
specified to the contrary. Moreover, other than any operating
examples, or where otherwise indicated, all numbers expressing, for
example, quantities of ingredients used in the specification and
claims are to be understood as being modified in all instances by
the term "about". Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the following specification
and attached claims may vary depending upon the desired properties
to be obtained by the present invention.
[0166] Those skilled in the art will recognize that several
variations are possible within the scope and spirit of this
invention. The invention will now be described in greater detail by
reference to the following non-limiting examples. The following
examples further illustrate the invention but, of course, should
not be construed as in any way limiting its scope.
Examples
[0167] The following abbreviations or terms are used herein:
[0168] BF.sub.3: Boron trifluoride
[0169] BF.sub.3.Et.sub.2O: Boron trifluoride diethyl etherate
[0170] CaCl.sub.2: Calcium chloride
[0171] CHCl.sub.3: Chloroform
[0172] CDCl.sub.3: Deuteriated chloroform
[0173] CO.sub.2: Carbon dioxide
[0174] DCC: N, N'-dicyclohexyl carbodiimide
[0175] DMAP: 4-Dimethylaminopyridine
[0176] DMF: N, N-dimethylformamide
[0177] DMSO: Dimethylsulfoxide
[0178] Et.sub.2O: Diethyl ether
[0179] EtOAc: Ethyl acetate
[0180] g: Gram
[0181] h: Hour
[0182] HCl: Hydrochloric acid
[0183] IPA: Isopropyl alcohol
[0184] KBr: Potassium bromide
[0185] Kg: Kilogram
[0186] L: Litre
[0187] MeOH: Methanol
[0188] min: Minute(s)
[0189] mg: Milligram
[0190] mL: Millilitre
[0191] .mu.L: Microlitre
[0192] .mu.M: Micromolar
[0193] mmol: Millimolar
[0194] mol: Mole
[0195] NaCl: Sodium chloride
[0196] Na.sub.2CO.sub.3: Sodium carbonate
[0197] NaHCO.sub.3: Sodium bicarbonate
[0198] Na.sub.2SO.sub.4: Sodium sulfate
[0199] n-BuLi: n-Butyl Lithium
[0200] PEL: Piramal Enterprises Limited
[0201] .degree. C.: Degree Centigrade
[0202] THF: Tetrahydrofuran
Examples
[0203] Preparation of the compound A (voruciclib) and the compound
B (riviciclib), the representative compounds of formula 1 are
presented herein as reference examples:
Reference Example 1
(a) Preparation of
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-hydroxy
methyl-1-methyl pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one
[0204] To a solution of n-BuLi (15% solution in hexane, 2.2 mL, 5
mmol) in THF (10 mL), maintained at 0.degree. C. under nitrogen
atmosphere, hexamethyldisilazane (1.08 mL, 5.1 mmol) was added
dropwise and stirred for 15 min. To this, a solution of
(+)-trans-2-chloro-4-trifluoromethylbenzoic acid
2-(2-acetoxymethyl-1-methyl-pyrrolidin-3-yl)-6-acetyl-3,5-dimethoxyphenyl
ester (1.44 g, 2.5 mmol) in THF (10 mL) was added dropwise,
maintaining the temperature at 0.degree. C. After the addition, the
reaction was allowed to warm to room temperature and stirred for
2.5 h. The reaction mixture was acidified with dilute HCl, and
basified with 10% sodium bicarbonate to pH 8 to 9. The aqueous
layer was extracted with chloroform (3.times.25 mL). The organic
layer was washed with water (25 mL), brine (25 mL) and dried over
anhydrous Na.sub.2SO.sub.4. The organic layer was concentrated
under reduced pressure and dried under vacuum to yield acetic acid
3-{3-[3-(2-chloro-4-trifluromethyl-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
-dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil
(1.3 g, 90.2%). This ester was dissolved in concentrated HCl (10
mL) and stirred for 3 h to effect cyclization. At the end of 3 h,
the reaction mixture was basified with solid NaHCO.sub.3 to pH 8 to
9. The aqueous layer was extracted with chloroform (25.times.3 mL)
and washed with water (25 mL) and brine (25 mL). The organic layer
was dried over anhydrous Na.sub.2SO.sub.4, concentrated under
reduced pressure and dried over vacuum. The residue was purified by
column chromatography with 3% methanol in chloroform and 0.1%
ammonia as eluent to yield the compound,
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methy-
lpyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one as a yellow
solid.
[0205] Yield: 0.56 g (48.2%); .sup.1H NMR (CDCl.sub.3, 300 MHz):
.delta. 7.95 (d, 1H), 7.78 (s, 1H), 7.69 (d, 1H), 6.61 (s, 1H),
6.46 (s, 1H), 4.21 (m, 1H), 4.01 (s, 3H), 3.93 (s, 3H), 3.71 (dd,
1H), 3.41 (d, 1H), 3.26 (m, 1H), 2.84 (m, 1H), 2.70 (m, 1H), 2.44
(s, 3H), 2.10 (m, 2H); MS (ES+): m/z 497 (M+1).
(b) Preparation of
(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydrox-
y-methyl-1-methylpyrrolidin-3-yl)-chromen-4-one
[0206] A mixture of the compound as obtained in part (a) (0.25 g,
0.5 mmol), pyridine hydrochloride (0.25 g, 2.16 mmol) and a
catalytic amount of quinoline was heated at 180.degree. C. for a
period of 2.5 h. The reaction mixture was diluted with methanol (25
mL) and basified with solid Na.sub.2CO.sub.3 to pH 10. The reaction
mixture was filtered, and washed with methanol. The organic layer
was concentrated and the residue purified by column chromatography
using 0.1% ammonia and 4.5% methanol in chloroform as eluent to
yield the compound,
(+)-trans-2-(2-chloro-4-trifluoromethylphenyI)-5,7-dihydroxy-8-(2-hydroxy-
-methyl-1-methylpyrrolidin-3-yl)-chromen-4-one, as a yellow
solid.
[0207] Yield: 0.15 g (63.7%); .sup.1H NMR (CDCl.sub.3, 300 MHz):
.delta. 7.99 (m, 2H), 7.83 (d, 1H), 6.65 (s, 1H), 6.41 (s, 1H),
4.24 (m, 1H), 3.90 (m, 2H), 3.70 (m, 1H), 3.60 (m, 1H), 3.41 (m,
1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.28 (m, 1H); MS (ES+): m/z 470
(M+1).
(c) Preparation of
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-
-methyl-1-methylpyrrolidin-3-yl)-chromen-4-one Hydrochloride
(Compound A or Voruciclib)
[0208] The compound as obtained in (b) (0.1 g, 0.2 mmol) was
suspended in methanol (2 mL) and treated with ethereal HCl and the
organic solvent evaporated to yield the compound,
(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydrox-
y methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
[0209] Yield: 0.1 g (92.8%); .sup.1H NMR (CDCl.sub.3, 300 MHz):
.delta. 8.02 (d, 2H), 7.83 (d, 1H), 6.64 (s, 1H), 6.41 (s, 1H),
4.23 (m, 1H), 3.73 (m, 2H), 3.68 (m, 1H), 3.51 (m, 1H), 3.39 (m,
1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.31 (m, 1H).
Reference Example 2
(a) Preparation of
(+)-trans-2-(2-chlorophenyl)-8-(2-hydroxymethyl-1-methyl
pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one
[0210] Sodium hydride (50%, 0.54 g, 11.25 mmol) was added in
portions to a solution of
(-)-trans-1-[2-hydroxy-3-(2-hydroxymethyl-1-methyl
pyrrolidin-3-yl)-4,6-dimethoxyphenyl)-ethanone (0.7 g, 2.2 mmol) in
dry DMF (15 mL) at 0.degree. C., under nitrogen atmosphere and with
stirring. After 10 min., methyl 2-chlorobenzoate (1.15 g., 6.75
mmol) was added. The reaction mixture was stirred at 25.degree. C.
for 2 h. Methanol was added carefully below 20.degree. C. The
reaction mixture was poured over crushed ice (300 g), acidified
with 1:1 HCl (pH 2) and extracted using EtOAc (2.times.100 mL). The
aqueous layer was basified using a saturated Na.sub.2CO.sub.3 (pH
10) and extracted using CHCl.sub.3 (3.times.200 mL). The organic
layer was dried (anhydrous Na.sub.2SO.sub.4) and concentrated. To
the residue, conc. HCl (25 mL) was added and stirred at room
temperature for 2 h. The reaction mixture was poured over crushed
ice (300 g) and made basic using a saturated aqueous
Na.sub.2CO.sub.3 solution. The mixture was extracted using
CHCl.sub.3 (3.times.200 mL). The organic extract was washed with
water, dried (anhydrous Na.sub.2SO.sub.4) and concentrated to
obtain the compound,
(+)-trans-2-(2-chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl-
)-5,7-dimethoxy-chromen-4-one.
[0211] Yield: 0.67 g (64%); mp: 91-93.degree. C.;
[.alpha.].sub.D.sup.25=+5.8.degree. (c=0.7, methanol); IR (KBr):
3431, 1648, 1598, 1571 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300
MHz): .delta. 7.70 (dd, 1H), 7.52 (m, 1H), 7.45 (m, 2H), 6.50 (s,
1H), 6.44 (s, 1H), 4.17 (m, 1H), 4.00 (s, 3H), 3.97 (s, 3H), 3.64
(dd, 1H), 3.40 (d, 1H), 3.15 (m, 1H), 2.74 (d, 1H), 2.52 (m, 1H),
2.32 (s, 3H), 2.00 (m, 2H); MS (ES+): m/z 430 (M+1).
(b) Preparation of
(+)-trans-2-(2-chlorophenyl)-5,7-dihydroxy-8-(2-hydroxy
methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one
[0212] Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added
to the compound as obtained in part (a) (0.4 g, 0.9 mmol) and
heated at 180.degree. C. for 1.5 h. The reaction mixture was cooled
to 25.degree. C., diluted with MeOH (10 mL) and basified using
Na.sub.2CO.sub.3 to pH 10. The mixture was filtered and the organic
layer was concentrated. The residue was suspended in water (5 mL),
stirred for 30 min., filtered and dried to obtain the compound,
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-p-
yrrolidin-3-yl)-chromen-4-one.
[0213] Yield: 0.25 g (70%); IR (KBr): 3422, 3135, 1664, 1623, 1559
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.56 (d, 1H),
7.36 (m, 3H), 6.36 (s, 1H), 6.20 (s, 1H), 4.02 (m, 1H), 3.70 (m,
2H), 3.15 (m, 2H), 2.88 (m, 1H), 2.58 (s, 3H), 2.35 (m, 1H), 1.88
(m, 1H); MS (ES+): m/z 402 (M+1); Analysis:
C.sub.21H.sub.20ClNO.sub.5 C, 62.24 (62.71); H, 5.07 (4.97); N,
3.60 (3.48); Cl, 9.01 (8.83).
(c) Preparation of
(+)-trans-2-(2-chlorophenyl)-5,7-dihydroxy-8-(2-hydroxy
methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(compound B or riviciclib)
[0214] The compound as obtained in part (b) (0.2 g, 0.48 mmol) was
suspended in IPA (5 mL) and 3.5% HCl (25 mL) was added. The
suspension was heated to get a clear solution. The solution was
cooled and solid filtered to obtain the compound,
(+)-trans-2-(2-chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-py-
rrolidin-3-yl)-chromen-4-one hydrochloride.
[0215] Yield: 0.21 g (97%); nip: 188-192.degree. C.;
[.alpha.].sub.D.sup.25=+21.3.degree. (c=0.2, methanol); .sup.1H NMR
(CD.sub.3OD, 300 MHz): .delta. 7.80 (d, 1H), 7.60 (m, 3H), 6.53 (s,
1H), 6.37 (s, 1H), 4.23 (m, 1H), 3.89 (m, 2H), 3.63 (m, 1H), 3.59
(dd, 1H), 3.38 (m, 1H), 2.90 (s, 3H), 2.45 (m, 1H), 2.35 (m, 1H);
MS (ES+): m/z 402 (M+1) (free base).
Biological Data:
Pharmacological Assays:
Example 1
I. In Vitro Study Involving Use of Combination of Compound a (CDK
Inhibitor, Also Referred to as Voruciclib) and Vemurafenib
(BRAFV600E Inhibitor) in BRAFV600E Mutated Melanoma Cell Lines
Objective:
[0216] To study the effect of the combination of compound A (CDK
inhibitor, also referred to as voruciclib) and vemurafenib
(BRAFV600E inhibitor) on the cell cycle and apoptosis in BRAFV600E
mutated melanoma cell lines.
Materials and Methods:
Cell Lines
[0217] G361 and SK-MEL3 melanoma cell lines obtained from ATCC
(American Type Culture Collection), USA, were used in this study.
G361 is vemurafenib sensitive cell line while SK-MEL3 is
vemurafenib resistant cell line. Both the cell lines are BRAFV600E
mutated.
A. Analysis of Cell Cycle Distribution Using Flow Cytometry:
[0218] The G361/SK-MEL3 melanoma cells were seeded in 25 mm.sup.3
tissue culture flasks. After 24 h, G361 cells were treated with: i)
compound A (1 .mu.M); ii) vemurafenib (1 .mu.M); and iii) compound
A (1 .mu.M) and vemurafenib (1 .mu.M) together for 5 days.
[0219] In case of SK-MEL3 melanoma cells, the cells were treated
with: i) compound A (1 .mu.M); ii) vemurafenib (10 .mu.M); and iii)
compound A (1 .mu.M) and vemurafenib (10 .mu.M) together for 5
days.
[0220] The control cells were left untreated for 5 days. Both
detached and adherent cells were harvested at the end of 5 days.
The cells were washed twice with approximately 5 mL of phosphate
buffered saline (PBS) with centrifugation at 1000 rpm for 10 min.
The cells were resuspended in 500 .mu.L of PBS and fixed in 500
.mu.L ice-cold 70% ethanol. The fixed cells were incubated at room
temperature for 30 min, spun at 1000 rpm for 10 min. To the cell
pellet, 1 mL of chilled 70% ethanol was added and stored below
0.degree. C. till further analysis. The cells were washed twice
with PBS to remove fixative and re-suspended in 250 .mu.L PBS. To
this 12.5 .mu.L of propidium iodide (1 mg/mL in PBS) and 12.5 .mu.L
Rnase A (1 mg/mL) was added. After incubation at 37.degree. C. for
30 min, the cells were analyzed using flow cytometry.
[0221] A flow cytometer (Becton Dickinson FACS Calibur, USA) was
used for these studies in accordance with the manufacturer's
recommendations. The argon ion laser set at 488 nm was used as an
excitation source. Cells with DNA content between 2n and 4n were
designated as being in G1, S and G2/M phases of the cell cycle, as
defined by the level of red fluorescence. Cells exhibiting less
than 2n DNA content were designated as sub-G1 cells. The number of
cells in each cell cycle compartment was expressed as a percentage
of the total number of cells present.
B. Annexin V-FITC Staining (for the Detection of Early
Apoptosis)
[0222] Annexin V-FITC is a sensitive probe for identifying
apoptotic cells. During early apoptosis, the membrane phospholipid
phosphotidyl serine (PS) is translocated from the inner to the
outer leaflet of the plasma membrane, thereby exposing PS to the
external cellular environment. Annexin V is a 35-36 kDa Calcium
dependent phospholipid binding protein that has a high affinity for
PS and binds to cells with exposed PS.
[0223] Propidium iodide is a polar dye, which enters cells through
leaky membranes and hence, is used in conjunction with fluorescein
isothiocyanate (FITC) for detection of late apoptosis.
[0224] The melanoma cells G361 was seeded in 25 mm.sup.3 tissue
culture flasks. After 24 h, cells were treated with: i) compound A
(1 .mu.M); ii) vemurafenib (1 .mu.M); and iii) compound A (1 .mu.M)
and vemurafenib (1 .mu.M) together; for 24 h. The control cells
were left untreated for 24 h. Medium containing floating cells were
collected and pooled with the adherent cells after harvesting with
trypsin at the different time points. The cells were washed twice
with cold PBS with centrifugation at 1000 rpm for 10 minutes. The
cell pellet was resuspended in 1.times. binding buffer (10 mm HEPES
pH 7.4, 140 mM NaCl, 2.5 mM CaCl.sub.2) at a concentration of
1.times.10.sup.6 cells/mL. 100 .mu.L of the solution
(1.times.10.sup.5 cells) were stained with Annexin V-FITC and
propidium iodide. The cells were incubated for 15 min at room
temperature (25-30.degree. C.) in the dark and the sample was
analysed by flow cytometry.
Results:
[0225] The results of these studies are depicted in FIGS. 1-3.
Conclusion:
[0226] FIG. 1 indicates that the vemurafenib treated G361 cells
show significant G1 arrest of 91.94% vs. control cells (72.36%). As
seen from the sub G1 phase the cells treated with compound A and
vemurafenib alone showed only 9.48% and 2.3% apoptosis
respectively, whereas the cells when treated with the combination
of compound A and vemurafenib showed 53.67% cells undergoing
apoptosis, indicating that the combination is synergistic.
[0227] In FIG. 2, the top right quadrant indicates G361 cells which
are in early apoptosis. Subsequently these cells undergo apoptosis
leading to cell death. The cells treated with the combination of
compound A and vemurafenib showed 27% of cells in early apoptosis
(annexin stained cells) versus 8% and 20% for cells treated with
only compound A and vemurafenib respectively. This data indicates
that the combination exhibits synergy.
[0228] In FIG. 3, the SK-MEL3 cells treated with only vemurafenib
showed significant G1 arrest of 76.78% versus control cells
(67.38%). Apoptosis is seen in 29.75% of the cells treated with the
combination of compound A and vemurafenib, as seen from the sub G1
phase while the cells treated with compound A and vemurafenib alone
showed only 19.63% and 15.12% apoptosis respectively, indicating
that the combination is synergistic.
[0229] The results as depicted in FIGS. 1-3 and as explained above,
clearly establish that the combination of compound A and
vemurafenib is synergistic in the vemurafenib sensitive and
resistant BRAFV600E mutated melanoma cell lines and induces greater
apoptosis when used in combination as compared to the compound A
and vemurafenib when used alone.
II. In Vitro Dual Combination Studies of Compound a with
Vemurafenib (BRAFV600E Inhibitor) in Human BRAF-Mutated Melanoma
Cells [0230] Objective: The objective of this study was to evaluate
the efficacy of the combination of compound A (CDK inhibitor) and
vemurafenib (BRAFV600E inhibitor) in BRAF mutated melanoma
cells.
A. Materials
[0230] [0231] Test compounds: compound A (prepared in PEL's Lab);
vemurafenib (Nanjing Chemlin Chemical Industry Co., Ltd, China)
[0232] Vehicle: DMSO (Sigma-Aldrich-Chemie Gmbh, Germany) [0233]
Dose Preparation: compound A and vemurafenib were weighed and
dissolved in the required amount of DMSO to give a required stock
solution. [0234] Test System: Test system included the G361, A375
and MDAMB-435S (BRAFV600E mutated) cell lines, which were procured
from ATCC (American Type Culture Collection), USA.
B. Methods
[0235] The cytotoxicity studies using compound A and vemurafenib in
various combinations, were carried out using the CCK8 live cell
dehydrogenase assay.
i) Cell Counting Kit-8 (CCK8) Live Cell Dehydrogenase Assay
[0236] The human BRAFV600E mutated melanoma cancer cell lines,
G361, A375 and MDAMB-435S were seeded at a density of 3000
cells/well in 199 .mu.L of RPMI 1640 medium in 96-well plate and
incubated overnight to allow the cells to adhere. The cells were
then treated with the respective test compounds. Totally there were
ten groups;
i) 1 .mu.M of vemurafenib alone for 48 h; ii) 0.3 .mu.M/0.1 .mu.M
of compound A alone for 48 h; iii) 1 .mu.M/0.3 .mu.M of compound A
alone for 48 h; iv) 3 .mu.M/1 .mu.M of compound A alone for 48 h;
v) 10 M/3 .mu.M of compound A alone for 48 h; vi) combination of
0.3 .mu.M of compound A and 1 .mu.M of vemurafenib for 48 h; vii)
combination of 1 .mu.M/0.3 .mu.M of compound A and 1 .mu.M of
vemurafenib for 48 h; viii) combination of 3 .mu.M/1 .mu.M of
compound A and 1 .mu.M of vemurafenib for 48 h; ix) combination of
10 .mu.M/3 .mu.M of compound A and 1 .mu.M of vemurafenib for 48 h;
x) control wells were treated with DMSO vehicle for 48 h.
[0237] The plates were incubated in humidified 5% CO.sub.2
incubator at 37.degree..+-.1.degree. C. vemurafenib was used at a
concentration of 1.0 .mu.M for all 3 different cell lines, while
compound A was used at concentrations of 0.3 .mu.M, 1 .mu.M, 3
.mu.M and 10 .mu.M in case of A375 melanoma cells and at
concentrations of 0.1 .mu.M, 0.3 .mu.M, 1.0 .mu.M, 3.0 .mu.M in
case of G361 and MDA MB435S melanoma cells. At the end of the
incubation periods, the plates were assayed using the CCK8
cytotoxicity assay protocol. The synergism was determined by the
combination index (CI) calculated using the Compusyn software by
Chou and Talalay (4). CI<1 is synergistic, CI=1 is additive and
CI>1 is antagonistic.
Statistical Analysis:
[0238] Statistical analysis was performed using the Student t test,
and a p value of <0.05 was considered significant. Data are
expressed as the mean.+-.standard error of the mean (SEM). The mean
value was obtained from at least two independent experiments, each
performed in triplicate.
ii) Cytotoxicity Assay Protocol
[0239] Logarithmically growing cells were plated at a density of
3.times.10.sup.3 cells/well and allowed to recover for 16-18 h. The
cells were challenged with varying concentration of both the
compounds, compound A and vemurafenib for 48 h. After 48 h, cell
toxicity was determined by CCK-8 reagent (Dojindo Molecular
Technologies, Inc, Maryland, and Japan). In accordance with the
manufacturer's instructions, 5 .mu.L/well CCK-8 reagent was added
and plates were incubated for 2 h. The toxicity was determined by
measuring the absorbance on Tecan Sapphire multi-fluorescence
micro-plate reader at a wavelength of 450 nm corrected to 650 nm
and normalized to controls. All the experiments were performed
twice in triplicates.
Treatment Schedule for Compound a and Vemurafenib:
[0240] Tables 1 and 2 indicate the treatment schedule of compound A
and vemurafenib in A375 cells and in G361 and MDA-MB435S cells,
respectively.
TABLE-US-00001 TABLE 1 Treatment schedule of compound A and
vemurafenib in A375 cells Treatment schedule S. No. Treatment
groups for A375 cells (in hours (h)) i compound A (1 .mu.M) 48 h ii
compound A (0.3 .mu.M) 48 h iii compound A (1.0 .mu.M) 48 h iv
compound A (3.0 .mu.M) 48 h v compound A (10.0 .mu.M) 48 h vi
compound A (0.3 .mu.M) & vemurafenib (1 .mu.M) 48 h vii
compound A (1.0 .mu.M) & vemurafenib (1 .mu.M) 48 h viii
compound A (3.0 .mu.M) & vemurafenib (1 .mu.M) 48 h ix compound
A (10.0 .mu.M) & vemurafenib (1 .mu.M) 48 h x Control + Vehicle
(DMSO) 48 h
TABLE-US-00002 TABLE 2 Treatment schedule of compound A and
vemurafenib in G361 and MDA- MB435S cells Treatment schedule S. No.
Treatment groups for G361 & MDA-MB435S cells (in hours (h)) i
vemurafenib (1 .mu.M) 48 h ii compound A (0.1 .mu.M) 48 h iii
compound A (0.3 .mu.M) 48 h iv compound A (1.0 .mu.M) 48 h v
compound A (3.0 .mu.M) 48 h vi compound A (0.1 .mu.M) &
vemurafenib (1 .mu.M) 48 h vii compound A (0.3 .mu.M) &
vemurafenib (1 .mu.M) 48 h viii compound A (1.0 .mu.M) &
vemurafenib (1 .mu.M) 48 h ix compound A (3.0 .mu.M) &
vemurafenib (1 .mu.M) 48 h x Control + Vehicle (DMSO) 48 h
Results:
[0241] The IC.sub.50 values of cytotoxicity for compound A and
vemurafenib and combinations thereof in different cell-lines are
presented in Table 3. FIGS. 4-6 and Tables 4-6 depict the effect of
the combination of compound A and vemurafenib in various melanoma
cell-lines.
TABLE-US-00003 TABLE 3 IC.sub.50 determination of compound A and
vemurafenib in G361, A375 and MDAMB-435S (BRAF.sup.V600E mutated)
cell lines Sr. No. Cell line Compounds IC.sub.50 in .mu.M (.+-.SEM)
1 A375 compound A 5.5 .+-. 0.67 compound A 0.8 .+-. 0.07 2 G361
compound A 0.75 .+-. 0.056 compound A 0.2 .+-. 0.032 3 MDAMB435S
compound A 0.85 .+-. 0.064 compound A 0.7 .+-. 0.058
[0242] In FIGS. 4-6, X-axis shows the concentration of compound A
and vemurafenib alone and of the compounds in combination, left
Y-axis shows % cytotoxicity and right Y-axis shows combination
index values.
[0243] In FIG. 4, the cells treated with vemurafenib at 1 .mu.M
showed 23% inhibition while the cells treated with compound A at 3
.mu.M showed 46% inhibition of cells. However, when the cells were
treated with vemurafenib in combination with compound A at this
suboptimal concentration, a synergistic effect of 83% inhibition of
cells with a combination index (CI) of 0.70 was observed. The
exhibited synergistic data represents the mean for two independent
experiments, each performed in triplicate.
TABLE-US-00004 TABLE 4 Mean combination index values for
combination of compound A and vemurafenib in A375 cells Combination
index (95% CI) Concentration in .mu.M in A375 cells compound A (0.3
.mu.M) & 0.66 (0.58 to 0.75) vemurafenib (1 .mu.M) compound A
(1.0 .mu.M) & 0.68 (0.60 to 0.77) vemurafenib (1 .mu.M)
compound A (3.0 .mu.M) & 0.70 (0.62 to 0.79) vemurafenib (1
.mu.M) compound A (10.0 .mu.M) & 0.76 (0.68 to 0.85)
vemurafenib (1 .mu.M) CI = Confidence Intervals
[0244] In FIG. 5, the cells treated with vemurafenib at 1 .mu.M
showed 30% inhibition while the cells treated with compound A at
0.3 .mu.M showed only 2% inhibition of cells. However, when the
cells were treated with vemurafenib in combination with compound A
at this suboptimal concentration, notable synergistic effect of 52%
inhibition of cells with a combination index value of 0.77 was
observed. When the cells were treated with compound A (3 .mu.M) in
combination with vemurafenib (1 .mu.M), it showed 91% inhibition of
cells with a combination index of 0.9. The exhibited synergistic
data represents the mean for two independent experiments, each
performed in triplicate.
TABLE-US-00005 TABLE 5 Mean combination index values for
combination of compound A and vemurafenib in G361 cells Combination
index (95% CI) in Concentration in .mu.M G361 cells compound A (0.1
.mu.M) & vemurafenib (1 .mu.M) 0.79 (0.74 to 0.84) compound A
(0.3 .mu.M) & vemurafenib (1 .mu.M) 0.77 (0.72 to 0.82)
compound A (1.0 .mu.M) & vemurafenib (1 .mu.M) 0.87 (0.82 to
0.92) compound A (3.0 .mu.M) & vemurafenib (1 .mu.M) 0.97 (0.92
to 1.02) CI = Confidence intervals
[0245] In FIG. 6, the cells treated with vemurafenib at 1 .mu.M
showed 31% inhibition of cells while the cells treated with
compound A at 1 .mu.M showed 32% inhibition of cells. When the
cells were treated with vemurafenib in combination with compound A
at this suboptimal concentration, synergistic effect of 90%
inhibition of cells with a combination index of 0.74 was noted. The
exhibited synergistic data represents the mean for two independent
experiments, each performed in triplicate.
TABLE-US-00006 TABLE 6 Mean combination index values for
combination of compound A and vemurafenib in MDA MB-435S cells
Combination index (95% CI) Concentration in .mu.M in MDA-MB435S
cells compound A (0.1 .mu.M) & vemurafenib (1 .mu.M) 0.83 (0.78
to 0.88) compound A (0.3 .mu.M) & vemurafenib (1 .mu.M) 0.78
(0.72 to 0.83) compound A (1.0 .mu.M) & vemurafenib (1 .mu.M)
0.74 (0.68 to 0.79) compound A (3.0 .mu.M) & vemurafenib (1
.mu.M) 0.90 (0.85 to 0.96) CI = Confidence Intervals
Conclusion:
[0246] The combination of vemurafenib and compound A showed a
marked synergistic effect in BRAF mutated melanoma cells.
REFERENCES
[0247] 1. Smalley K S, Lioni M, Palma M D, Xiao M, Desai B, Egyhazi
S, Hansson J, Wu H, King A J, Van Belle P, Elder D E, Flaherty K T,
Herlyn M, Nathanson K L; "Increased cyclin D1 expression can
mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas";
Mol. Cancer Ther., 2008, 7, 2876-2883. [0248] 2. Smalley K S M and
Flaherty K T; "Integrating BRAF/MEK inhibitors into combination
therapy for Melanoma"; British Journal of Cancer, 2009, 100,
431-435. [0249] 3. Dhomen N, Marais R; "BRAF signaling and targeted
therapies in melanoma" Hematol. Oncol. Clin. North Am., 2009, 23,
3, 529-45. [0250] 4. Ting-Chao Chou; "Theoretical basis,
experimental design, and computerized simulation of synergism and
antagonism in drug combination studies" Pharmacol. Rev., 2006, 58,
621-81.
Example 2
[0251] A. In vitro combination studies of compound A (CDK
inhibitor, also referred to as voruciclib) with one anticancer
agent selected from a BRAF inhibitor (vemurafenib or dabrafenib) or
a MEK inhibitor (trametinib) in a human BRAF-V600E mutated melanoma
cell line (A375) and its vemurafenib resistant derivative
(A375R)
Objective:
[0252] The objective of this study was to evaluate the efficacy of
the combination of CDK inhibitor (voruciclib) with a BRAF inhibitor
(vemurafenib or dabrafenib) or a MEK inhibitor (trametinib) in BRAF
mutated melanoma cells.
B. Materials:
[0253] Test Compounds: compound A (prepared in PEL's Lab.);
vemurafenib (Selleckchem USA, S1267); dabrafenib (Selleckchem USA,
S2807) and trametinib (Selleckchem USA, S2673) [0254] Vehicle: DMSO
(Sigma-Aldrich-Chemie Gmbh, Germany) [0255] Dose Preparation: The
test compounds were weighed and dissolved in the required amount of
DMSO to give a required stock solution. [0256] Test System: Test
system included the A375 (BRAFV600E mutated) cell line, which were
procured from ATCC (American Type Culture Collection), USA and
A375R cell line (vemurafenib resistant--developed in PEL's
Lab.)
C. Methods:
[0257] The cytotoxicity studies using all the test compounds as
single agents and in combinations were carried out using the CCK8
live cell dehydrogenase assay.
Cell Counting Kit-8 (CCK8) Live Cell Dehydrogenase Assay:
[0258] Logarithmically growing human BRAFV600E mutated melanoma
cells were seeded at a density of 1500 cells/well in 30 .mu.L of
Dulbecco's Modified Eagle medium medium (DMEM) in 384-well plate
(Corning, USA) using Tecan automated platform (FreedomEvo Liquid
handling system) and incubated for about 12-16 h to allow the cells
to adhere. The cells were then treated for 48 h with different
doses of compound A and anticancer agents (vemurafenib or
dabrafenib or trametinib) as monotherapy (used singly) and in
combinations. Studies using different dosages were carried out for
each combination (e.g. combination of compound A and vemurafenib).
The treatment ratios of the test compounds when used singly and in
combination in A375 and A375R cells are depicted in tables 7A-7C
(i.e. combination of compound A and vemurafenib; combination of
compound A and dabrafenib; and combination of compound A and
trametinib). The following doses of compound A (.mu.M): 1, 0.5,
0.25, 0.125, 0.06, 0.03, 0.015; vemurafenib (.mu.M) 30, 15, 7.5,
3.75, 1.875, 0.9, 0.4, 0.2, 0.1, 0.05; dabrafenib (.mu.M) 3, 1.5,
0.75, 0.37, 0.18, 0.09, 0.04, 0.02, 0.01, 0.005; trametinib (.mu.M)
1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.015, 0.007, 0.003, 0.001,
were used. The above doses of anticancer agents (vemurafenib or
dabrafenib or trametinib) were mixed in multiple ratios with
compound A as shown in tables 7A-7C. The controls used were only
cells or cells along with the vehicle (DMSO).
[0259] The plates were incubated for 48 h in humidified 5% CO.sub.2
incubator at 37.degree..+-.1.degree. C. Post incubation the plates
were assayed using the CCK8 reagent (Dojindo Molecular
Technologies, Inc, Maryland, and Japan). In accordance with the
manufacturer's instructions, 3 .mu.L CCK-8 reagent was added in
each well of the 384 well plate and plates were incubated for 2 h.
The toxicity was determined by measuring the absorbance on Tecan
Sapphire multi-fluorescence micro-plate reader at a wavelength of
450 nm normalized to controls. All the experiments were performed
in quadruplets.
[0260] The potency of the combination was quantified using Calcusyn
software (Biosoft, Ferguson, MO, USA) which is based on the Chou
Talalay method that calculates a combination index (CI) with CI
values less than 1 indicating synergy.
[0261] Table 7A-7C: Treatment ratios of compound A and anticancer
agents (vemurafenib or dabrafenib or trametinib) as monotherapy
(used singly) and in combinations in A375 and A375R cells.
[0262] The treatment ratios of compound A (denoted as A) and
vemurafenib (denoted as V) alone or in combinations are represented
in table 7A.
TABLE-US-00007 TABLE 7A Concentrations in .mu.M A (0) V (30) V (15)
V (7.5) V (3.75) V (1.88) V (0.94) A (1) A (1) + A (1) + A (1) + A
(1) + A (1) + A (1) + V (30) V (15) V (7.5) V (3.75) V (1.88) V
(0.94) A (0.5) A (0.5) + A (0.5) + A (0.5) + A (0.5) + A (0.5) + A
(0.5) + V (30) V (15) V (7.5) V (3.75) V (1.88) V (0.94) A (0.25) A
(0.25) + A (0.25) + A (0.25) + A (0.25) + A (0.25) + A (0.25) + V
(30) V (15) V (7.5) V (3.75) V (1.88) V (0.94) A (0.125) A (0.125)
+ A (0.125) + A (0.125) + A (0.125) + A (0.125) + A (0.125) + V
(30) V (15) V (7.5) V (3.75) V (1.88) V (0.94) A (0.0625) A
(0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + A
(0.0625) + V (30) V (15) V (7.5) V (3.75) V (1.88) V (0.94) A
(0.0312) A (0.0312) + A (0.0312) + A (0.0312) + A (0.0312) + A
(0.0312) + A (0.0312) + V (30) V (15) V (7.5) V (3.75) V (1.88) V
(0.94) A (0.0156) A (0.0156) + A (0.0156) + A (0.0156) + A (0.0156)
+ A (0.0156) + A (0.0156) + V (30) V (15) V (7.5) V (3.75) V (1.88)
V (0.94) A (0) V (0.47) V (0.23) V (0.12) V (0.059) A (1) A (1) + A
(1) + A (1) + A (1) + V (0.47) V (0.23) V (0.12) V (0.059) A (0.5)
A (0.5) + A (0.5) + A (0.5) + A (0.5) + V (0.47) V (0.23) V (0.12)
V (0.059) A (0.25) A (0.25) + A (0.25) + A (0.25) + A (0.25) + V
(0.47) V (0.23) V (0.12) V (0.059) A (0.125) A (0.125) + A (0.125)
+ A (0.125) + A (0.125) + V (0.47) V (0.23) V (0.12) V (0.059) A
(0.0625) A (0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + V
(0.47) V (0.23) V (0.12) V (0.059) A (0.0312) A (0.0312) + A
(0.0312) + A (0.0312) + A (0.0312) + V (0.47) V (0.23) V (0.12) V
(0.059) A (0.0156) A (0.0156) + A (0.0156) + A (0.0156) + A
(0.0156) + V (0.47) V (0.23) V (0.12) V (0.059)
[0263] The treatment ratios of compound A (denoted as A) and
dabrafenib (denoted as D) alone or in combinations are represented
in table 7B.
TABLE-US-00008 TABLE 7B Concentrations in .mu.M A (0) D (3) D (1.5)
D (0.75) D (0.38) D (0.19) D (0.094) A (1) A (1) + A (1) + A (1) +
A (1) + A (1) + A (1) + D (3) D (1.5) D (0.75) D (0.38) D (0.19) D
(0.094) A (0.5) A (0.5) + A (0.5) + A (0.5) + A (0.5) + A (0.5) + A
(0.5) + D (3) D (1.5) D (0.75) D (0.38) D (0.19) D (0.094) A (0.25)
A (0.25) + A (0.25) + A (0.25) + A (0.25) + A (0.25) + A (0.25) + D
(3) D (1.5) D (0.75) D (0.38) D (0.19) D (0.094) A (0.125) A
(0.125) + A (0.125) + A (0.125) + A (0.125) + A (0.125) + A (0.125)
+ D (3) D (1.5) D (0.75) D (0.38) D (0.19) D (0.094) A (0.0625) A
(0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + A
(0.0625) + D (3) D (1.5) D (0.75) D (0.38) D (0.19) D (0.094) A
(0.0312) A (0.0312) + A (0.0312) + A (0.0312) + A (0.0312) + A
(0.0312) + A (0.0312) + D (3) D (1.5) D (0.75) D (0.38) D (0.19) D
(0.094) A (0.0156) A (0.0156) + A (0.0156) + A (0.0156) + A
(0.0156) + A (0.0156) + A (0.0156) + D (3) D (1.5) D (0.75) D
(0.38) D (0.19) D (0.094) A (0) D (0.047) D (0.023) D (0.012) D
(0.006) A (1) A (1) + A (1) + A (1) + A (1) + D (0.047) D (0.023) D
(0.012) D (0.006) A (0.5) A (0.5) + A (0.5) + A (0.5) + A (0.5) + D
(0.047) D (0.023) D (0.012) D (0.006) A (0.25) A (0.25) + A (0.25)
+ A (0.25) + A (0.25) + D (0.047) D (0.023) D (0.012) D (0.006) A
(0.125) A (0.125) + A (0.125) + A (0.125) + A (0.125) + D (0.047) D
(0.023) D (0.012) D (0.006) A (0.0625) A (0.0625) + A (0.0625) + A
(0.0625) + A (0.0625) + D (0.047) D (0.023) D (0.012) D (0.006) A
(0.0312) A (0.0312) + A (0.0312) + A (0.0312) + A (0.0312) + D
(0.047) D (0.023) D (0.012) D (0.006) A (0.0156) A (0.0156) + A
(0.0156) + A (0.0156) + A (0.0156) + D (0.047) D (0.023) D (0.012)
D (0.006)
[0264] The treatment ratios of compound A (denoted as A) and
trametinib (denoted as T) alone or in combinations are represented
in table 7C.
TABLE-US-00009 TABLE 7C Concentrations in .mu.M A (0) T (1) T (0.5)
T (0.25) T (0.13) T (0.06) T (0.031) A (1) A (1) + A (1) + A (1) +
A (1) + A (1) + A (1) + T (1) T (0.5) T (0.25) T (0.13) T (0.06) T
(0.031) A (0.5) A (0.5) + A (0.5) + A (0.5) + A (0.5) + A (0.5) + A
(0.5) + T (1) T (0.5) T (0.25) T (0.13) T (0.06) T (0.031) A (0.25)
A (0.25) + A (0.25) + A (0.25) + A (0.25) + A (0.25) + A (0.25) + T
(1) T (0.5) T (0.25) T (0.13) T (0.06) T (0.031) A (0.125) A
(0.125) + A (0.125) + A (0.125) + A (0.125) + A (0.125) + A (0.125)
+ T (1) T (0.5) T (0.25) T (0.13) T (0.06) T (0.031) A (0.0625) A
(0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + A (0.0625) + A
(0.0625) + T (1) T (0.5) T (0.25) T (0.13) T (0.06) T (0.031) A
(0.0312) A (0.0312) + A (0.0312) + A (0.0312) + A (0.0312) + A
(0.0312) + A (0.0312) + T (1) T (0.5) T (0.25) T (0.13) T (0.06) T
(0.031) A (0.0156) A (0.0156) + A (0.0156) + A (0.0156) + A
(0.0156) + A (0.0156) + A (0.0156) + T (1) T (0.5) T (0.25) T
(0.13) T (0.06) T (0.031) A (0) T (0.016) T (0.008) T (0.004) T
(0.002) A (1) A (1) + A (1) + A (1) + A (1) + T (0.016) T (0.008) T
(0.004) T (0.002) A (0.5) A (0.5) + A (0.5) + A (0.5) + A (0.5) + T
(0.016) T (0.008) T (0.004) T (0.002) A (0.25) A (0.25) + A (0.25)
+ A (0.25) + A (0.25) + T (0.016) T (0.008) T (0.004) T (0.002) A
(0.125) A (0.125) + A (0.125) + A (0.125) + A (0.125) + T (0.016) T
(0.008) T (0.004) T (0.002) A (0.0625) A (0.0625) + A (0.0625) + A
(0.0625) + A (0.0625) + T (0.016) T (0.008) T (0.004) T (0.002) A
(0.0312) A (0.0312) + A (0.0312) + A (0.0312) + A (0.0312) + T
(0.016) T (0.008) T (0.004) T (0.002) A (0.0156) A (0.0156) + A
(0.0156) + A (0.0156) + A (0.0156) + T (0.016) T (0.008) T (0.004)
T (0.002)
Results:
[0265] The results of these studies are depicted in FIGS. 7-13.
Conclusion:
[0266] 1. From FIGS. 7a and 7b, it can be concluded that A375 cell
line is very sensitive to vemurafenib whereas A375R is resistant.
Further, both A375 and A375R are equally sensitive to compound A
(voruciclib).
[0267] 2. From FIGS. 8a and 8b, it can be concluded that the
combination of compound A (voruciclib) and vemurafenib shows a
strong synergy (CI<0.5) in A375 cell line.
[0268] 3. From FIGS. 9a and 9b, it can be concluded that the
combination of compound A (voruciclib) and vemurafenib shows a
strong synergy (CI<0.5) in A375R cell line.
[0269] 4. From FIGS. 10a and 10b, it can be concluded that the
combination of compound A (voruciclib) and dabrafenib shows a
strong synergy (CI<0.5) in A375 cell line.
[0270] 5. From FIGS. 11a and 11b, it can be concluded that the
combination of compound A (voruciclib) and dabrafenib shows a
strong synergy (CI<0.5) in A375R cell line.
[0271] 6. From FIGS. 12a and 12b, it can be concluded that the
combination of compound A (voruciclib) and trametinib shows a
strong synergy (CI<0.5) in A375 cell line.
[0272] 7. From FIGS. 13a and 13b, it can be concluded that the
combination of compound A (voruciclib) and trametinib shows a
strong synergy (CI<0.5) in A375R cell line.
* * * * *