U.S. patent application number 17/201614 was filed with the patent office on 2021-07-01 for synergistic pharmaceutical combination for the treatment of squamous cell carcinoma of head and neck.
The applicant listed for this patent is Piramal Enterprises Limited. Invention is credited to Veena Agarwal, Arun Balakrishnan, Giridharan Periyasamy.
Application Number | 20210196680 17/201614 |
Document ID | / |
Family ID | 1000005459359 |
Filed Date | 2021-07-01 |
United States Patent
Application |
20210196680 |
Kind Code |
A1 |
Agarwal; Veena ; et
al. |
July 1, 2021 |
SYNERGISTIC PHARMACEUTICAL COMBINATION FOR THE TREATMENT OF
SQUAMOUS CELL CARCINOMA OF HEAD AND NECK
Abstract
The present invention relates to a pharmaceutical combination
for use in the treatment of squamous cell carcinoma, comprising a
CDK inhibitor selected from the compounds of formula (I);
##STR00001## or a pharmaceutically acceptable salt thereof and one
or more antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil, docetaxel or cetuximab or a
pharmaceutically acceptable salt thereof. The said pharmaceutical
combination exhibits synergy when used in the treatment of squamous
cell carcinoma of head and neck (SCCHN). The invention also relates
to a pharmaceutical composition comprising the said combination and
a method for the treatment of squamous cell carcinoma of head and
neck (SCCHN), using a therapeutically effective amount of said
combination.
Inventors: |
Agarwal; Veena; (Mumbai,
IN) ; Balakrishnan; Arun; (Mumbai, IN) ;
Periyasamy; Giridharan; (Mumbai, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Piramal Enterprises Limited |
Mumbai |
|
IN |
|
|
Family ID: |
1000005459359 |
Appl. No.: |
17/201614 |
Filed: |
March 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16270643 |
Feb 8, 2019 |
10980776 |
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17201614 |
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15206185 |
Jul 8, 2016 |
10245251 |
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16270643 |
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14122922 |
Nov 27, 2013 |
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PCT/IB2012/052698 |
May 30, 2012 |
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15206185 |
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61491569 |
May 31, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/282 20130101;
A61K 31/513 20130101; A61K 39/39558 20130101; A61K 31/517 20130101;
A61K 33/24 20130101; A61K 31/4025 20130101; A61K 31/44 20130101;
A61K 31/337 20130101 |
International
Class: |
A61K 31/4025 20060101
A61K031/4025; A61K 31/337 20060101 A61K031/337; A61K 31/513
20060101 A61K031/513; A61K 31/517 20060101 A61K031/517; A61K 31/282
20060101 A61K031/282; A61K 31/44 20060101 A61K031/44; A61K 33/24
20060101 A61K033/24; A61K 39/395 20060101 A61K039/395 |
Claims
1-20. (canceled)
21. A method of treating cancer in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a CDK inhibitor of Formula I, or a
pharmaceutically acceptable salt or solvate thereof, and a
therapeutically effective amount of an antineoplastic agent
selected from sorafenib, lapatinib, erlotinib, cisplatin,
5-fluorouracil, docetaxel, and cetuximab, wherein in Formula I Ar
is 2-chloro-phenyl or 2-chloro-4-trifluoromethyl phenyl:
##STR00009##
22. The method of claim 21, wherein the pharmaceutically acceptable
salt is
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methy-
l-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
23. The method of claim 21, wherein the pharmaceutically acceptable
salt is
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydr-
oxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one
hydrochloride.
24. The method of claim 21, wherein the antineoplastic agent is
sorafenib.
25. The method of claim 21, wherein the antineoplastic agent is
lapatinib.
26. The method of claim 21, wherein the antineoplastic agent is
erlotinib.
27. The method of claim 21, wherein the antineoplastic agent is
cisplatin.
28. The method of claim 21, wherein the antineoplastic agent is
5-fluorouracil.
29. The method of claim 21, wherein the antineoplastic agent is
docetaxel.
30. The method of claim 21, wherein the antineoplastic agent is
cetuximab.
31. The method of claim 21, wherein the cancer is squamous cell
carcinoma.
32. The method of claim 21, wherein the cancer is head and neck
cancer.
33. The method of claim 21, wherein the cancer is tongue
cancer.
34. The method of claim 21, wherein the cancer is pharyngeal
cancer.
35. The method of claim 21, further comprising administration of
radiation.
Description
CROSS REFERENCE
[0001] This application is a continuation application of U.S.
application Ser. No. 14/122,922, filed Nov. 27, 2013, which is the
U.S. National Stage of International Application No.
PCT/IB2012/052698, filed May 30, 2012, published in English, which
application claims the benefit of U.S. Provisional Application No.
61/491,569, filed May 31, 2011, all of which are incorporated
herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to a pharmaceutical
combination comprising a cyclin dependent kinase (CDK) inhibitor
selected from the compounds of formula I (as described herein) or a
pharmaceutically acceptable salt thereof and one or more
antineoplastic agents for use in the treatment of squamous cell
carcinoma of head and neck (SCCHN). The pharmaceutical combination
of the present invention exhibits synergy when used in the
treatment of squamous cell carcinoma of head and neck (SCCHN).
Thus, the present invention relates to a synergistic pharmaceutical
combination. The present invention further relates to a
pharmaceutical composition comprising said combination and a method
of treating squamous cell carcinoma of head and neck (SCCHN) in a
subject by administrating said pharmaceutical combination to said
subject.
BACKGROUND OF INVENTION
[0003] Cancer is a group of diseases characterized by the unusual
control of cell growth. There are over 100 different types of
cancers, which are classified by the type of cells initially
affected such as bladder cancer, breast cancer, colon cancer,
rectal cancer, endometrial cancer, kidney (renal cell) cancer,
leukemia, small cell lung cancer, non-small cell lung cancer,
pancreatic cancer, prostate cancer, thyroid cancer, skin cancer,
non-hodgkin's lymphoma and melanoma and head and neck cancer.
Squamous cell carcinoma represents more than 90% of all head and
neck cancers. Head and neck squamous cell carcinomas make up the
vast majority of head and neck cancers, and arise from mucosal
surfaces throughout the anatomical region. These include tumors of
the nasal cavities, paranasal sinuses, oral cavity, nasopharynx,
oropharynx, hypopharynx, and larynx.
[0004] In fact, head and neck cancer (HNC) is the sixth most common
cancer worldwide, with an annual incidence of >640,000 cases
worldwide. More than 90% of head and neck cancers are of squamous
histology (HNSCC). Thirty-five percent to 45% of head and neck
cancer patients ultimately die from their disease. In the United
States alone, squamous cell carcinoma of the head and neck
comprises about 4% of all malignancies. This corresponds to an
estimated 17 per 100,000 persons with newly diagnosed squamous cell
carcinoma of the head and neck per year (Jemal A, Siegel R, Ward E,
et al. Cancer statistics, 2008, CA Cancer J Clin. 2008 March-April;
58(2):71-96). Squamous cell carcinoma of head and neck (SCCHN)
remains a challenging clinical problem, due to persisting high rate
of local and distant failure, as well as the occurrence of second
primaries. Some molecular targeted therapy used in squamous cell
cancers of the head and neck include cetuximab, bevacizumab,
erlotinib and reovirus. The best quality data are available for
cetuximab, a recombinant monoclonal antibody, since the 2006
publication of a randomized clinical trial comparing radiation
treatment plus cetuximab versus radiation treatment alone
("Radiotherapy plus cetuximab for squamous-cell carcinoma of the
head and neck". N Engl J Med 2006; 354 (6): 567-78). Another study
evaluated the impact of adding cetuximab to conventional
chemotherapy involving use of cisplatin versus cisplatin alone.
This study found no improvement in survival or disease-free
survival with the addition of cetuximab to the conventional
chemotherapy (J Clin Oncol. 2005; 23 (34): 8646-54). However,
another study completed in March 2007 found that there was an
improvement in survival. This study is referred to as EXTREME
(Erbitux in First-Line Treatment of Recurrent or Metastatic Head
and Neck Cancer) study which is a European multicenter phase III
trial.
[0005] Further, it is well established in the art that CDK
(Cyclin-dependent kinase) inhibitors are useful in
anti-proliferative therapies for diseases characterized by
excessive cell growth such as cancers and immunological disorders
involving unwanted proliferation of leukocytes. Flavone derivatives
useful as CDK inhibitors are described in PCT Patent Publication
No. WO2004-004632 (U.S. Pat. No. 7,271,193) which patent
application specifically relates to the compounds for inhibition of
cyclin-dependent kinases, process for their preparation, methods of
inhibiting cyclin-dependent kinases and of inhibiting cell
proliferation, use of such compounds in the treatment of
proliferative disorders including cancers. PCT Published
application No. WO2005-053699 (U.S. Pat. No. 7,772,207) relates to
a pharmaceutical product comprising a CDK inhibitor and
1-(2-C-cyano-2-dioxy-p-D-arabino-pentofuranosyl)-N4-palmitoyl
cytosine or a metabolite thereof, as a combined preparation for
simultaneous, sequential or separate administration. PCT Published
application No. WO2008-122779 (U.S. Patent Appl. Pub. 2010-0143350)
describes combination of CDK inhibitor with a tyrosine kinase
inhibitor and use thereof in the treatment of proliferative
disorders. PCT Published application No. WO2008-139271 (U.S. Patent
Appl. Pub. 2010-0305057) relates to pharmaceutical combination
comprising a cytotoxic antineoplastic agent selected from
paclitaxel, docetaxel, doxorubicin or gemcitabine and at least one
cyclin dependent kinase (CDK) inhibitor for use in the treatment of
cancer. PCT Published application No. WO2010-128443 describes a
combination for the treatment of cancer wherein the combination
comprises radiation and at least one cyclin dependent kinase (CDK)
inhibitor or a pharmaceutically acceptable salt or a solvate
thereof.
[0006] Although combinations of anticancer agents have been proven
to have a significant advance in various cancer treatment protocols
including squamous-cell carcinoma of the head and neck (SCCHN),
there are still several unmet needs and room for improvements in
medications for the treatment of SCCHN, which are difficult to
treat, or which have shown resistance to treatment with the
conventional antineoplastic agents. More particularly, the
development of novel combination approach for delivering known
anticancer agents having different mechanism of action would
represent an important advance in the art. Although the protocol
involving combination of anticancer agents having different
mechanism of action may work in case of some combinations, it may
not work in the same manner for other combination of anticancer
agents and such combination may not always result in a combination
having advantageous therapeutic effects. However, the inventors of
the present invention have found that a pharmaceutical combination
of anticancer agents comprising a cyclin dependant kinase (CDK)
inhibitor and one or more antineoplastic agent provides greater
efficacy than when the CDK inhibitors or the antineoplastic agents
are used alone for the treatment of squamous-cell carcinoma of the
head and neck (SCCHN).
SUMMARY OF THE INVENTION
[0007] According to one aspect of the present invention, there is
provided a pharmaceutical combination for use in the treatment of
squamous cell carcinoma of the head and neck (SCCHN), comprising a
cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula (I) or a pharmaceutically acceptable salt thereof and
one or more antineoplastic agents.
[0008] In another aspect, the present invention provides
pharmaceutical compositions for use the treatment of squamous cell
carcinoma of the head and neck (SCCHN), comprising a combination of
a cyclin dependent kinase (CDK) inhibitor selected from the
compounds of formula (I) or a pharmaceutically acceptable salt or
solvates thereof and one or more antineoplastic agents along with
at least one pharmaceutically acceptable carrier.
[0009] In another aspect, the present invention relates to a method
for the treatment of squamous cell carcinoma of the head and neck
(SCCHN) in a subject, comprising administering to the subject a
therapeutically effective amount of a cyclin dependent kinase (CDK)
inhibitor selected from the compounds of formula (I) or
pharmaceutically acceptable salts thereof in combination with a
therapeutically effective amount of one or more antineoplastic
agents.
[0010] According to another aspect, the present invention provides
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of the head and neck (SCCHN); comprising a
therapeutically effective amount of a cyclin dependent kinase (CDK)
inhibitor selected from the compounds of formula (I) or a
pharmaceutically acceptable salt thereof and a therapeutically
effective amount of one or more antineoplastic agents wherein said
combination exhibits synergistic effect.
[0011] In yet another aspect, the present invention relates to a
kit comprising a cyclin dependent kinase (CDK) inhibitor selected
from the compounds of formula (I) and one or more antineoplastic
agents; wherein said kit may further include a package insert
comprising printed instructions directing the use of the combined
treatment as a method for treating squamous cell carcinoma of the
head and neck.
[0012] 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
[0013] FIG. 1a is a graphical representation of the percentage
inhibition results of dosing of sorafenib and lapatinib in SCC25
cells.
[0014] FIG. 1b is a graphical representation of the percentage
inhibition results of dosing of compound A and compound B in SCC25
cells.
[0015] FIG. 2a is a graphical representation of the percentage
inhibition results of dosing of sorafenib and lapatinib in
Detroit-562 cells
[0016] FIG. 2b is a graphical representation of the percentage
inhibition results of dosing of compound A and compound B in
Detroit-562 cells.
[0017] FIG. 3a is a graphical representation of the percentage
inhibition results of dosing of sorafenib and lapatinib in FADU
cells.
[0018] FIG. 3b is a graphical representation of the percentage
inhibition results of dosing of compound A and compound B in FADU
cells.
[0019] FIG. 4a is a graphical representation of the percentage
inhibition results of dosing of erlotinib in Detroit-562 cells.
[0020] FIG. 4b is a graphical representation of the percentage
inhibition results of dosing of erlotinib in FADU cells.
[0021] FIG. 5a is a graphical representation of the percentage
inhibition results of dosing of cisplatin, 5-fluorouracil and
docetaxel in Detroit-562 cells.
[0022] FIG. 5b is a graphical representation of the percentage
inhibition results of dosing of cisplatin, 5-fluorouracil and
docetaxel in FADU cells.
[0023] FIG. 6a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and sorafenib in SCC-25 cells.
[0024] FIG. 6b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound B
and sorafenib in SCC-25 cells.
[0025] FIG. 7a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and sorafenib in Detroit-562 cells.
[0026] FIG. 7b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound B
and sorafenib in Detroit-562 cells.
[0027] FIG. 8a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and sorafenib in FADU cells.
[0028] FIG. 8b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound B
and sorafenib in FADU cells.
[0029] FIG. 9a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and lapatinib in SCC-25 cells.
[0030] FIG. 9b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound B
and lapatinib in SCC-25 cells.
[0031] FIG. 10a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and lapatinib in Detroit-562 cells.
[0032] FIG. 10b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound B
and lapatinib in Detroit-562 cells.
[0033] FIG. 11a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and lapatinib in FADU cells.
[0034] FIG. 11b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound B
and lapatinib in FADU cells.
[0035] FIG. 12a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and erlotinib in Detroit-562 cells.
[0036] FIG. 12b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound A
and erlotinib in FADU cells.
[0037] FIG. 13a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound
A, cisplatin and 5-FU in Detroit-562 cells.
[0038] FIG. 13b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound
A, cisplatin and 5-fluorouracil in FADU cells.
[0039] FIG. 14a is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound
A, docetaxel, cisplatin and 5-FU in Detroit-562 cells.
[0040] FIG. 14b is graphical representation of the percentage
cytotoxicity results of single and combination dosing of compound
A, docetaxel, cisplatin and 5-FU in FADU cells.
[0041] FIG. 15a is graphical representation of activation of
Caspase 3 in SCC-25 cells with single and combination dosing of
sorafenib and compound A.
[0042] FIG. 15b is graphical representation of activation of
Caspase 3 in SCC-25 cells with single and combination dosing of
sorafenib and compound B.
[0043] FIG. 16a is graphical representation of activation of
Caspase 3 in SCC-25 cells with single and combination dosing of
lapatinib and compound A.
[0044] FIG. 16b is graphical representation of activation of
Caspase 3 in SCC-25 cells with single and combination dosing of
lapatinib and compound B.
[0045] FIG. 17a is graphical representation of body weight profile
in FaDu xenografts treated with single and combination dosing of
compound A, cisplatin and cetuximab.
[0046] FIG. 17b is graphical representation of tumor growth
inhibition in FaDu xenografts treated with single and combination
dosing of compound A, cisplatin and cetuximab.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention encompasses pharmaceutical
combinations for use in the treatment of squamous cell carcinoma of
the head and neck (SCCHN), comprising a cyclin dependent kinase
(CDK) inhibitor selected from the compounds of formula (I) (as
described herein) or a pharmaceutically acceptable salt thereof and
one or more antineoplastic agents, wherein said combination
exhibits synergistic effect.
[0048] According to the present invention there is provided a
pharmaceutical composition for use in the treatment of squamous
cell carcinoma of the head and neck (SCCHN) comprising a
therapeutically effective amount of a cyclin dependent kinase (CDK)
inhibitor selected from the compounds of formula (I) or a
pharmaceutically acceptable salt thereof and one or more
antineoplastic agents and optionally a pharmaceutically acceptable
carrier.
[0049] The present invention further provides a method for the
treatment of squamous cell carcinoma of head and neck in a subject,
which comprises administering to the said subject a therapeutically
effective amount of a CDK inhibitor selected from the compounds of
formula (I) (as described herein) or a pharmaceutically acceptable
salt or solvate thereof; and a therapeutically effective amount of
one or more antineoplastic agents selected from the group
consisting of sorafenib, lapatinib, erlotinib, cisplatin,
5-fluorouracil and docetaxel or a pharmaceutically acceptable salt
thereof; wherein the said CDK inhibitor and the said antineoplastic
agents contained in the combination are administered either
simultaneously or sequentially.
[0050] The general terms used hereinbefore and hereinafter
preferably have within the context of this disclosure the following
meanings, unless otherwise indicated. Thus, the definitions of the
general terms as used in the context of the present invention are
provided herein below:
[0051] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0052] The phrase "a cyclin dependent kinase (CDK) inhibitor" or
"CDK inhibitor" as used herein means a compound that exhibits
activity against one or more known cyclin dependent kinases. In the
context of the present invention the CDK inhibitor is a pyrrolidine
substituted flavone compound disclosed in PCT Published Application
No. WO2004004632, which application is incorporated herein by
reference in its entirety. The CDK inhibitor according to the
present invention is specifically selected from a compound of
Formula I as described herein below or a pharmaceutically
acceptable salt or solvate thereof. Further, the term "CDK
inhibitor" as used herein may either refer to the compound of
formula I and/or a pharmaceutically acceptable salt or solvate of
the compound of formula I.
[0053] The term "antineoplastic agent" is synonymous to "a
chemotherapeutic agent" or "an anticancer agent" and refers to a
therapeutic agent, which acts by inhibiting or preventing the
growth of neoplasms. The term "antineoplastic agent" or
"anti-cancer agent" in general refers to the compounds which
prevent the cancer cells from multiplying (i.e. anti-proliferative
agents). In general, the antineoplastic agent(s) fall into two
classes, anti-proliferative cytotoxic agents and anti-proliferative
cytostatic agents. Cytotoxic agents prevent cancer cells from
multiplying by: (1) interfering with the cell's ability to
replicate DNA; and (2) inducing cell death and/or apoptosis in the
cancer cells. The cytostatic agents act via modulating, interfering
or inhibiting the processes of cellular signal transduction which
regulate cell proliferation.
[0054] The phrase "pharmaceutically acceptable salts" refers to the
acid addition salt of compound of formula I (as described herein)
and of an antineoplastic agent, wherein the acid is selected from
an inorganic acid such as hydrochloric acid, hydrobromic acid; or
an organic acid such as benzene sulfonic acid, maleic acid, oxalic
acid, fumaric acid, succinic acid, p-toluenesulfonic acid and
maleic acid.
[0055] As used herein, the term "combination" or "pharmaceutical
combination", means the combined administration of the anti-cancer
agents namely the CDK inhibitor selected from the compounds
represented by formula I and one or more antineoplastic agents
which acts by inhibiting or preventing the growth of neoplasms or
the administration of the anti-cancer agents namely the CDK
inhibitor selected from the compounds represented by formula I and
the antineoplastic agents selected from cytostatic or cytotoxic
agents; which may be administered independently at the same time or
separately within time intervals that especially allow that the
combination partners to show a synergistic effect.
[0056] As used herein, the term "synergistic" or "synergy" means
that the effect achieved with the combinations of anticancer agents
encompassed in this invention is greater than the sum of the
effects that result from using anti-cancer agents namely the CDK
inhibitor of formula (I) or a pharmaceutically acceptable salt
thereof, antineoplastic agent(s) or a pharmaceutically acceptable
salt thereof, as a monotherapy. Advantageously, such synergy
provides greater efficacy at the same doses, and/or prevents or
delays the build-up of multi-drug resistance.
[0057] As used herein the term "therapeutically effective amount"
in reference to the treatment of squamous cell carcinoma of head
and neck refers to an amount capable of invoking one or more of the
following effects in a subject receiving the combination of the
present invention: (i) inhibition, to some extent, of tumor growth,
including, slowing down and complete growth arrest; (ii) reduction
in the number of tumor cells; (iii) reduction in tumor size; (iv)
inhibition (i.e., reduction, slowing down or complete stopping) of
tumor cell infiltration into peripheral organs; (v) inhibition
(i.e., reduction, slowing down or complete stopping) of metastasis;
(vi) enhancement of anti-tumor immune response, which may, but does
not have to, result in the regression of the tumor; and/or (vii)
relief, to some extent, of one or more symptoms associated with
squamous cell carcinoma of head and neck (SCCHN).
[0058] 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. The term subject
may be interchangeably used with the term "patient" in the context
of the present invention.
[0059] As used herein, the term "simultaneously" means that two or
more therapeutic agents (anticancer agents) are administered
concurrently, "sequentially" means that two or more therapeutic
agents are available to act therapeutically within the same
time-frame and "separately" means that the gap between
administering one agent and the other is significant i.e. the first
administered agent may no longer be present in the bloodstream in a
therapeutically effective amount when the second agent is
administered.
[0060] The term "caspase3 activity" as used herein refers to
increase in apoptosis in cancer cells.
[0061] The term "apoptosis" refers to a type of cell death in which
a series of molecular steps in a cell leads to its death. This is
the body's normal way of getting rid of unneeded or abnormal cells.
The process of apoptosis may be blocked in cancer cells. Also
called programmed cell death. (Dictionary of cancer terms, National
Cancer Institute). The term "increasing apoptosis" is defined as an
increase in the rate of programmed cell death, i.e. more cells are
induced into the death process as compared to exposure (contact)
with either the antineoplastic agent alone or the CDK inhibitor
alone.
[0062] The phrase "pharmaceutically acceptable carrier" refers to
one or more disintegrating agents, binders, excipients, lubricants
and the like which are well known to those skilled in the art.
[0063] In the present invention there is provided a pharmaceutical
combination of anti-cancer agents for use in the treatment of
squamous cell carcinoma of head and neck (SCCHN). The present
inventors have conducted an extensive research for the development
of the pharmaceutical combination of anti-cancer agents and arrived
at the present synergistic pharmaceutical combination. It has been
found that pharmaceutical combination comprising a cyclin dependent
kinase (CDK) inhibitor selected from the compounds of formula I or
a pharmaceutically acceptable salt thereof and one or more
antineoplastic agent exhibits synergistic effect when used in the
treatment of squamous cell carcinoma of the head and neck
(SCCHN).
[0064] The CDK inhibitor is a pyrrolidine substituted flavone
compound that inhibits cyclin dependent kinases. The CDK inhibitor
used in the pharmaceutical combination of the present invention is
selected from the compounds of formula I or pharmaceutically
acceptable salts or solvates thereof as described herein below. The
compounds of formula I are promising CDK inhibitors, which can
inhibit proliferation of many cancer cells. As indicated herein
above the CDK inhibitors of formula (I) may be used in the form of
their pharmaceutically acceptable salts. The salts encompassed
within the term "pharmaceutically acceptable salts" refer to
non-toxic salts of the compounds of this invention. Representative
salts include, but are not limited to acetate, benzoate,
benzenesulfonate, bicarbonate, chloride, citrate, hydrochloride,
mesylate, methylsulfonate, tartrate, tosylate and trifluoroacetate.
Preferred salts of compounds of formula (I) include hydrochloride
salt, methanesulfonic acid and trifluoroacetic acid salt.
[0065] In one embodiment, the CDK inhibitor used in the
pharmaceutical combination of the present invention is selected
from the compounds represented by the following formula I,
##STR00002##
wherein, Ar is a phenyl group, which is unsubstituted or
substituted by 1, 2, or 3 identical or different substituents
selected from: halogen, nitro, cyano, C.sub.1-C.sub.4-alkyl,
trifluoromethyl, hydroxyl or C.sub.1-C.sub.4-alkoxy; or a
pharmaceutically acceptable salt or solvate thereof.
[0066] As indicated herein above the salts of CDK inhibitor refers
to non-toxic salts of the compounds of formula (I) of this
invention. Representative salts include, but are not limited to
acetate, benzoate, benzenesulfonate, bicarbonate, chloride,
citrate, hydrochloride, mesylate, methylsulfonate, tartrate,
tosylate and trifluoroacetate. Preferred salts of compounds of
formula (I) include hydrochloride salt, methanesulfonic acid and
trifluoroacetic acid salt.
[0067] In an embodiment of the invention, the CDK inhibitor is the
(+)-trans isomer of the compound of formula I, as indicated in
Formula IA below,
##STR00003##
[0068] wherein Ar is a phenyl group, which is unsubstituted or
substituted by 1, 2, or 3 identical or different substituents
selected from: halogen, nitro, cyano, C.sub.1-C.sub.4-alkyl,
trifluoromethyl, hydroxyl or C.sub.1-C.sub.4-alkoxy; or a
pharmaceutically acceptable salt or solvate thereof.
[0069] In another embodiment of the present invention, the CDK
inhibitor used in the pharmaceutical combination of the present
invention is a compound of formula I wherein the phenyl group is
substituted by 1, 2 or 3 identical or different substituents
selected from: chlorine, bromine, fluorine or iodine,
C.sub.1-C.sub.4-alkyl or trifluoromethyl; or a pharmaceutically
acceptable salt or solvate thereof.
[0070] In another embodiment of the present invention, the CDK
inhibitor used in the pharmaceutical combination of the present
invention is a compound of formula I wherein the phenyl group is
substituted by chlorine; or a pharmaceutically acceptable salt or
solvate thereof.
[0071] In another embodiment of the present invention, the CDK
inhibitor used in the pharmaceutical combination of present
invention is a compound of formula I wherein the phenyl group is
substituted by two different substituents selected from chlorine
and trifluoromethyl; or a pharmaceutically acceptable salt or
solvate thereof.
[0072] It will be appreciated by those skilled in the art that the
CDK inhibitors represented by the compounds of formula (I) contain
at least two chiral centers and hence, exist in the form of two
different optical isomers (i.e. (+) or (-) enantiomers). All such
enantiomers and mixtures thereof including racemic mixtures are
included within the scope of the invention. The enantiomers of the
compound of formula I can be obtained by methods disclosed in PCT
Application Publication No. WO2004004632 incorporated herein by
reference or the enantiomers of the compound of formula I can also
be obtained by methods well known in the art, such as chiral HPLC
and enzymatic resolution.
[0073] Alternatively, the enantiomers of the compounds of formula I
can be synthesized by using optically active starting materials.
The manufacture of the compounds of formula I, which may be in the
form of pharmaceutically acceptable salts and solvates, and the
manufacture of oral and/or parenteral pharmaceutical composition
containing the above compounds are generally disclosed in PCT
Application Publication No. WO2004004632. This patent application,
which is incorporated herein by reference, discloses that the CDK
inhibitors represented by formula I exhibit significant anticancer
efficacy. As indicated herein above the CDK inhibitors of formula I
may be used in the form of their salts. Preferred salts of
compounds of formula I include hydrochloride, methanesulfonic acid
and trifluoroacetic acid salt.
[0074] According to another embodiment of the present invention,
the CDK inhibitor used in the pharmaceutical combination of the
present invention is selected from
(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl--
pyrrolidin-3-yl)-chromen-4-one hydrochloride (referred to herein as
compound A) or
(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hyd-
roxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(referred to herein as compound B).
[0075] In an embodiment of the present invention, the CDK inhibitor
used in the pharmaceutical combination is
(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl--
pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound A).
[0076] In further embodiment of the present invention, the CDK
inhibitor used in the pharmaceutical combination is
(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hyd-
roxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(compound B).
[0077] The antineoplastic agents are the compounds that prevent
cancer cells from multiplying (i.e. anti-proliferative agents). In
the present invention anti-neoplastic agent included in the
pharmaceutical combination may be selected from either cytostatic
or cytotoxic agents.
[0078] According to an embodiment of the invention, an
anti-neoplastic agent used in the pharmaceutical combination of the
present invention is a cytostatic agent.
[0079] According to another embodiment of the invention, an
anti-neoplastic agent used in the pharmaceutical combination of the
present invention is a cytotoxic agent.
[0080] According to an embodiment of the invention, when the
anti-neoplastic agent used in the pharmaceutical combination of the
present invention is a cytostatic agent, it is selected from small
molecules such as sorafenib, lapatinib or erlotinib or a chimeric
monoclonal antibody such as cetuximab.
[0081] According to another embodiment of the invention, when the
anti-neoplastic agent used in the pharmaceutical combination of the
present invention is a cytotoxic agent, it is selected from
cisplatin, 5-fluorouracil and/or docetaxel or pharmaceutically
acceptable salts thereof.
[0082] According to another embodiment of the invention, the
pharmaceutical combination comprising a CDK inhibitor selected from
the compounds of formula (I) or a pharmaceutically acceptable salt
thereof, and one or more antineoplastic agents, may further include
use of radiation therapy for the treatment of squamous cell
carcinoma of the head and neck (SCCHN).
[0083] The specified anti-neoplastic agents used in the present
invention are commercially readily available.
[0084] Sorafenib is a kinase inhibitor that decreases tumor cells
proliferation in vitro. Sorafenib was shown to inhibit multiple
intracellular (CRAF, BRAF and mutant BRAF) and cell surface kinases
(KIT, FLT-3, RET, VEGFR-1 to 3 and PDGFR-13. Several of these
kinases are thought to be involved in tumor cell signaling,
angiogenesis and apoptosis. Sorafenib inhibited tumor growth and
angiogenesis of human hepatocellular carcinoma and renal cell
carcinoma and several other human tumor xenografts in
immunocompromised mice.
[0085] It is chemically named as
4-(4-{3-[4-chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)N.sup.2-methyl-
pyridine-2-carboxamide-4-methylbenzenesulfonate. Sorafenib is
commercially available and is marketed as Nexavar.RTM. by Bayer in
the United States for the treatment of patients with advanced renal
cell carcinoma (RCC) and those with unresectable hepatocellular
carcinoma (HCC). It is also approved by the European Medicines
Agency for the treatment of patients with HCC and patients with
advanced RCC with whom prior IFN-.alpha. or interleukin-2-based
therapy had failed or considered to be unsuitable for such therapy
("Preclinical overview of sorafenib, a multikinase inhibitor that
targets both Raf and VEGF and PDGF receptor tyrosine kinase
signaling". Molecular Cancer Therapeutics 2008; 7 (10):
3129-40).
[0086] Lapatinib, is a 4-anilinoquinazoline kinase inhibitor of the
intracellular tyrosine kinase domains of both Epidermal Growth
Factor Receptor (EGFR [ErbB1]) and of human Epidermal Receptor Type
2 (HER2[ErbB2]) receptors. Lapatinib inhibits ErbB-driven tumor
cell growth in vitro and in various animal models. It is present as
the monohydrate of the ditosylate salt, with chemical name
N-(3-chloro-4-{[(3-fluorophenyl)
methyl]oxy}phenyl)-6-[5-({[2-(methylsulfonyl) ethyl]
amino}methyl)-2-furanyl]-4-quinazolinaminebis(4-methylbenzenesulfonate)mo-
nohydrate. Lapatinib ditosylate monohydrate is a dual tyrosine
inhibitor which interrupts the HER2 growth receptor and is used in
combination therapy for HER2-positive breast cancer ("Lapatinib in
the treatment of breast cancer" Expert Review of Anticancer Therapy
(Future Drugs) 7 (9): 1183-92). It is marketed under the brand name
TYKERB.RTM. in the United States by GlaxoSmithKline and is
available commercially. Lapatinib inhibits the tyrosine kinase
activity associated with two oncogenes, EGFR (epidermal growth
factor receptor) and HER2/neu (Human EGFR type 2) ("A unique
structure for epidermal growth factor receptor bound to GW572016
(Lapatinib): relationships among protein conformation, inhibitor
off-rate, and receptor activity in tumor cells" Cancer Res. 2004
Sep. 15; 64(18): 6652-9). Lapatinib inhibits receptor signal
processes by binding to the ATP-binding pocket of the EGFR/HER2
protein kinase domain, preventing self-phosphorylation and
subsequent activation of the signal mechanism ("Lapatinib: a novel
dual tyrosine kinase inhibitor with activity in solid tumors".
Annals of Pharmacotherapy: 40 (2); 261-269).
[0087] Erlotinib is an EGFR inhibitor. The drug follows gefitinib
(Iress.RTM.), which was the first drug of this type. Gefitinib and
erlotinib are commercially available epidermal growth factor
receptor tyrosine kinase inhibitors (EGFR-TKIs) that are widely
used for the treatment of non-small-cell lung cancer (NSCLC).
Erlotinib specifically targets the epidermal growth factor receptor
(EGFR) tyrosine kinase, which is highly expressed and occasionally
mutated in various forms of cancer. It binds in a reversible
fashion to the adenosine triphosphate (ATP) binding site of the
receptor (J Clin Oncol, 2007; 25:1960-1966).
[0088] Cisplatin is a platinum compound which acts as a cytotoxic
anticancer agent. This platinum-based chemotherapy drug, which
kills the cancer cells by damaging DNA and inducing apoptosis.
Cisplatin is commercially available for the treatment of various
types of cancers, including sarcomas, some carcinomas (e.g. small
cell lung cancer, and ovarian cancer), lymphomas, and germ cell
tumors. Cisplatin is a non cell cycle specific cytotoxic agent
which is effective against cells that are actively dividing as well
as those that are merely resting before entering the cell cycle and
reacts in vivo, binding to and causing cross linking of DNA which
ultimately triggers apoptosis (programmed cell death).
[0089] Fluorouracil (5-FU) is an antimetabolite and a cytotoxic
anti-cancer agent. 5-FU inhibits DNA synthesis and cell death and
penetrates cerebrospinal fluid well. 5-FU is commercially available
as an antimetabolite that interferes with RNA and DNA synthesis.
5-FU is therapeutically useful for certain types of carcinoma, such
as carcinoma of the colon, rectum, breast, stomach and
pancreas.
[0090] Docetaxel is an antineoplastic agent belonging to the taxoid
family that acts by disrupting the microtubular network in cells
that is essential for mitotic and interphase cellular functions.
Docetaxel binds to free tubulin and promotes the assembly of
tubulin into stable microtubules while simultaneously inhibiting
their disassembly. This leads to the production of microtubule
bundles without normal function and to the stabilization of
microtubules, which results in the inhibition of mitosis in cells.
Docetaxel's binding to microtubules does not alter the number of
protofilaments in the bound microtubules, a feature which differs
from most spindle poisons currently in clinical use. Docetaxel is
marketed worldwide under the name Taxotere.RTM. by Sanofi- and
available commercially.
[0091] Cetuximab is a recombinant, chimeric monoclonal antibody
directed against the epidermal growth factor (EGFR) with
antineoplastic activity. Cetuximab binds to the extracellular
domain of the EGFR, thereby preventing the activation and
subsequent dimerization of the receptor; the decrease in receptor
activation and dimerization may result in an inhibition in signal
transduction and anti-proliferative effects. This agent may inhibit
EGFR-dependent primary tumor growth and metastasis. Cetuximab is
commercially available as Erbitux.RTM. for treatment of metastatic
colorectal cancer and head and neck cancer.
[0092] According to another embodiment, the present invention
relates to a pharmaceutical combination for use in the treatment of
squamous cell carcinoma of head and neck (SCCHN) wherein the
combination comprises a cyclin dependent kinase (CDK) inhibitor
selected from the compounds of formula I or a pharmaceutically
acceptable salt or a solvate thereof and one or more of
antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil or docetaxel or a
pharmaceutically acceptable salt thereof or the monoclonal antibody
cetuximab.
[0093] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck (SCCHN) wherein the combination
comprises a cyclin dependent kinase (CDK) inhibitor selected from
the compounds of formula I or a pharmaceutically acceptable salt or
a solvate thereof and sorafenib.
[0094] Another embodiment of the present invention provides a
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises a
cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula I or a pharmaceutically acceptable salt or a solvate
thereof and lapatinib.
[0095] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises a
cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula I or a pharmaceutically acceptable salt or a solvate
thereof and erlotinib.
[0096] Another embodiment of the present invention provides a
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises a
cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula I or a pharmaceutically acceptable salt or a solvate
thereof; cisplatin and 5-fluorouracil or a pharmaceutically
acceptable salt thereof.
[0097] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises a
cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula I or a pharmaceutically acceptable salt or a solvate
thereof; docetaxel, cisplatin and 5-fluorouracil or a
pharmaceutically acceptable salt thereof.
[0098] Further embodiment of the present invention provides a
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises a
CDK inhibitor selected from compound A or compound B and one or
more anti-neoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil or docetaxel or a
pharmaceutically acceptable salt thereof.
[0099] In another embodiment, the present invention provides a
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
compound A and sorafenib or a pharmaceutically acceptable salt
thereof.
[0100] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
compound A and lapatinib or a pharmaceutically acceptable salt
thereof.
[0101] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
compound A and erlotinib or a pharmaceutically acceptable salt
thereof.
[0102] In another embodiment, the present invention is directed to
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
compound A, cisplatin and 5-fluorouracil or a pharmaceutically
acceptable salt thereof.
[0103] Further embodiment of the present invention is directed to
pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
compound A, docetaxel, cisplatin and 5-fluorouracil or a
pharmaceutically acceptable salt thereof.
[0104] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
compound B and sorafenib or a pharmaceutically acceptable salt
thereof.
[0105] In another embodiment, the present invention is directed to
a pharmaceutical combination for use in the treatment of squamous
cell carcinoma of head and neck wherein the combination comprises
the compound B and lapatinib or a pharmaceutically acceptable salt
thereof.
[0106] According to another embodiment of the present invention,
the pharmaceutical combination comprising the CDK inhibitor
selected from the compounds of formula I and an antineoplastic
agent selected from sorafenib, lapatinib or erlotinib or the
pharmaceutical combination comprising the CDK inhibitor selected
from the compounds of formula I, and an antineoplastic agent
selected from cisplatin and 5-fluorouracil or the pharmaceutical
combination comprising the CDK inhibitor selected from the
compounds of formula I, and an antineoplastic agent selected from
cisplatin, 5-fluorouracil and docetaxel, is not exclusively limited
to those combinations which are obtained by physical association of
said ingredients, but also encompass those which permit a separate
administration, which can be simultaneous, sequential or spaced out
over a period of time so as to obtain maximum efficacy of the
combination. Thus, the pharmaceutical combination may be
administered simultaneously or sequentially for an effective
treatment of squamous cell carcinoma of head and neck.
[0107] According to another embodiment, the present invention is
directed to a pharmaceutical combination for use in the treatment
of squamous cell carcinoma comprising radiation, a CDK inhibitor
selected from the compounds of formula I and one or more
antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil or docetaxel or a
pharmaceutically acceptable salt thereof.
[0108] According to another embodiment, the present invention is
directed to a pharmaceutical combination for use in the treatment
of squamous cell carcinoma comprising radiation, a CDK inhibitor
selected from compound A or compound B and one or more
antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil or docetaxel or a
pharmaceutically acceptable salt thereof.
[0109] According to another embodiment, the present invention
relates to a pharmaceutical combination for use in the treatment of
squamous cell carcinoma of head and neck (SCCHN) wherein the
combination comprises a cyclin dependent kinase (CDK) inhibitor
selected from compound A or compound B, cisplatin and or a
pharmaceutically acceptable salt thereof and the monoclonal
antibody, cetuximab.
[0110] According to another embodiment, the present invention
relates to a pharmaceutical combination for use in the treatment of
squamous cell carcinoma of head and neck (SCCHN) wherein the
combination comprises radiation, a cyclin dependent kinase (CDK)
inhibitor selected from compound A or compound B or a
pharmaceutically acceptable salt thereof and the monoclonal
antibody, cetuximab.
[0111] In further embodiment, the present invention provides a
pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from the compounds of
formula I (as described herein) or a pharmaceutically acceptable
salt or solvate thereof in combination with a therapeutically
effective amount of one or more antineoplastic agents selected from
the group consisting of sorafenib, lapatinib, erlotinib, docetaxel,
cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt
thereof; in association with a pharmaceutically acceptable
carrier.
[0112] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from the compounds of
formula I or a pharmaceutically acceptable salt or solvate thereof
and a therapeutically effective amount of sorafenib in association
with a pharmaceutically acceptable carrier.
[0113] In another further embodiment, the present invention relates
to a pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from the compounds of
formula I or a pharmaceutically acceptable salt or solvate thereof
and a therapeutically effective amount of lapatinib in association
with a pharmaceutically acceptable carrier.
[0114] In another further embodiment, the present invention relates
to a pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from the compounds of
formula I or a pharmaceutically acceptable salt or solvate thereof
and a therapeutically effective amount of erlotinib in association
with a pharmaceutically acceptable carrier.
[0115] In further embodiment, the present invention provides a
pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from the compounds of
formula I or a pharmaceutically acceptable salt or solvate thereof,
a therapeutically effective amount of cisplatin and a
therapeutically effective amount of 5-fluorouracil or a
pharmaceutically acceptable salt thereof; in association with a
pharmaceutically acceptable carrier.
[0116] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from the compound A or
compound B and therapeutically effective amount of one or more
antineoplastic agents or a pharmaceutically acceptable salt
thereof; in association with a pharmaceutically acceptable
carrier.
[0117] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of a CDK inhibitor selected from compound A or
compound B and a therapeutically effective amount of one or more
antineoplastic agents selected from the group consisting of
sorafenib, lapatinib, erlotinib, cisplatin and 5-fluorouracil or
pharmaceutically acceptable salt thereof; in association with a
pharmaceutically acceptable carrier.
[0118] In another further embodiment, the present invention relates
to a pharmaceutical composition which comprises a therapeutically
effective amount of compound A and a therapeutically effective
amount of sorafenibin association with a pharmaceutically
acceptable carrier.
[0119] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of compound A and a therapeutically effective
amount of lapatinib in association with a pharmaceutically
acceptable carrier.
[0120] In further embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of compound B and a therapeutically effective
amount of sorafenib in association with a pharmaceutically
acceptable carrier.
[0121] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of compound B and a therapeutically effective
amount of lapatinib in association with a pharmaceutically
acceptable carrier.
[0122] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of compound A and a therapeutically effective
amount of erlotinib in association with a pharmaceutically
acceptable carrier.
[0123] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of each of the compound A, cisplatin and
5-fluorouracil or a pharmaceutically acceptable salt thereof; in
association with a pharmaceutically acceptable carrier.
[0124] In another embodiment, the present invention relates to a
pharmaceutical composition which comprises a therapeutically
effective amount of each of the compound A, docetaxel, cisplatin
and 5-fluorouracil or a pharmaceutically acceptable salt thereof;
in association with a pharmaceutically acceptable carrier.
[0125] In further embodiment, the present invention is directed to
a method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of a CDK inhibitor selected from
the compounds of formula (I) or a pharmaceutically acceptable salt
or solvate thereof and a therapeutically effective amount of an
anti-neoplastic agent selected from sorafenib, lapatinib or
erlotinib; wherein said CDK inhibitor and said anti-neoplastic
agent or pharmaceutically acceptable salt thereof is administered
simultaneously or sequentially.
[0126] In another embodiment, the present invention is directed to
a method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of a CDK inhibitor selected from
the compounds of formula (I) or a pharmaceutically acceptable salt
or solvate thereof; a therapeutically effective amount of each of
cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt
thereof; wherein said CDK inhibitor, cisplatin and 5-fluorouracil
are administered simultaneously or sequentially.
[0127] In further embodiment, the present invention is directed to
a method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of a CDK inhibitor selected from
the compound A or compound B and a therapeutically effective amount
of antineoplastic agent selected from sorafenib, lapatinib or
erlotinib; wherein said compound A or compound B and antineoplastic
agent is administered simultaneously or sequentially.
[0128] In another embodiment, the present invention is directed to
a method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of a CDK inhibitor selected from
the compound A or compound B and a therapeutically effective amount
of sorafenib; wherein said compound A or compound B and sorafenib
is administered simultaneously or sequentially.
[0129] In another embodiment, the present invention is directed to
a method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of a CDK inhibitor selected from
the compound A or compound B and a therapeutically effective amount
of lapatinib or a pharmaceutically acceptable salt thereof; wherein
said compound A or compound B and lapatinib is administered
simultaneously or sequentially.
[0130] Another embodiment of the present invention provides a
method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of compound A or a
pharmaceutically acceptable salt or solvate thereof and a
therapeutically effective amount of erlotinib; wherein said
compound A and erlotinib is administered simultaneously or
sequentially.
[0131] In another embodiment, the present invention is directed to
a method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of compound A or a
pharmaceutically acceptable salt or solvate thereof; a
therapeutically effective amount of cisplatin and 5-fluorouracil or
a pharmaceutically acceptable salt thereof; wherein said compound
A, cisplatin and 5-fluorouracil are administered simultaneously or
sequentially.
[0132] Another embodiment of the present invention is directed to a
method for the treatment of squamous cell carcinoma of head and
neck in a subject, which comprises administering to said subject a
therapeutically effective amount of CDK inhibitor selected from the
compounds of formula (I) or a pharmaceutically acceptable salt or
solvate thereof and a therapeutically effective amount of an
antineoplastic agent or a pharmaceutically acceptable salt thereof;
wherein said CDK inhibitor and said anti-neoplastic agent or their
pharmaceutically acceptable salts are administered
sequentially.
[0133] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound A or compound B; a
therapeutically effective amount of an antineoplastic agent
selected from sorafenib, lapatinib or erlotinib; wherein said
compound A or compound B and antineoplastic agent selected from
sorafenib, lapatinib or erlotinib is administered sequentially such
that compound A or compound B is administered before or after the
administration of sorafenib or lapatinib or erlotinib.
[0134] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound A and a
therapeutically effective amount of sorafenib; wherein said
compound A and sorafenib is administered sequentially such that
compound A is administered before or after the administration of
sorafenib.
[0135] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound B and a
therapeutically effective amount of sorafenib; wherein said
compound B and sorafenib is administered sequentially such that
compound B is administered before or after the administration of
sorafenib.
[0136] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound A and a
therapeutically effective amount of lapatinib; wherein said
compound A and lapatinib is administered sequentially such that
compound A is administered before or after the administration of
lapatinib.
[0137] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound B and a
therapeutically effective amount of lapatinib; wherein said
compound B and lapatinib is administered sequentially such that
compound B is administered before or after the administration of
lapatinib.
[0138] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound A; a therapeutically
effective amount of erlotinib; wherein said compound A and
erlotinib is administered sequentially such that compound A is
administered before or after the administration of erlotinib.
[0139] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound A; a therapeutically
effective amount of each of cisplatin; and 5-fluorouracil or a
pharmaceutically acceptable salt thereof; wherein said compound A,
cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt
thereof are administered sequentially such that compound A is
administered before or after the administration of cisplatin and/or
5-fluorouracil.
[0140] Accordingly to another embodiment, the present invention
relates to a method for the treatment of squamous cell carcinoma of
head and neck in a subject comprising administering to said subject
a therapeutically effective amount of compound A; a therapeutically
effective amount of each of docetaxel; cisplatin and 5-fluorouracil
or a pharmaceutically acceptable salt thereof; wherein said
compound A, docetaxel, cisplatin and 5-fluorouracil or a
pharmaceutically acceptable salt thereof are administered
sequentially such that compound A is administered before or after
the administration of docetaxel, and/or cisplatin and/or
5-fluorouracil.
[0141] In another embodiment the present invention provides use of
combination of a CDK inhibitor selected from the compound of
formula I or pharmaceutically acceptable salt or solvate thereof
and one or more antineoplastic agents or a pharmaceutically
acceptable salt thereof for the manufacture of a medicament for the
treatment or prevention of squamous cell carcinoma of the head and
neck (SCCHN).
[0142] Another embodiment of the present invention provides use of
pharmaceutical composition comprising a therapeutically effective
amount of CDK inhibitor selected from the compounds of formula (I)
or a pharmaceutically acceptable salt thereof and an antineoplastic
agent for the manufacture of a medicament for the treatment of
squamous cell carcinoma of the head and neck (SCCHN).
[0143] According to the present invention the administration of the
double combination of CDK inhibitor selected from the compound of
formula I and an antineoplastic agent or a pharmaceutically
acceptable salt thereof selected from sorafenib, lapatinib or
erlotinib may produce effects, such as the anti-cancer effects,
greater than those achieved with any of the CDK inhibitor or
sorafenib or lapatinib or erlotinib when used alone.
[0144] It is further provided by the present invention that the
administration of a triple combination of the CDK inhibitor
selected from the compound of formula I as described herein,
cisplatin and 5-fluorouracil may produce effects, such as
anti-cancer effects, greater than those achieved with any of the
CDK inhibitor or cisplatin or 5-fluorouracil used alone, greater
than those achieved with the combination of the CDK inhibitor,
cisplatin and 5-fluorouracil.
[0145] The administration route of the pharmaceutical composition
of the present invention is not particularly limited. In one
embodiment, the active ingredients (the anticancer agents contained
in the combination) comprised in the composition may have to be
administered by different routes either orally or parenterally
depending on the dosage form. The dosage form suitable for oral
administration may be a tablet or capsule, forms of parenteral
administration include intravenous injection, intravenous infusion,
subcutaneous injection, transdermal injection, intraperitoneal
injection and so on. For rectal administration, for example as a
suppository or the route of administration may be by direct
injection into the tumour or by regional delivery or by local
delivery. In the case of tablets for oral use, carriers which are
commonly used include lactose, corn starch, magnesium carbonate,
talc, and sugar, and lubricating agents such as magnesium stearate
are commonly added. For oral administration in capsule form, useful
carriers include lactose, corn starch, magnesium carbonate, talc
and sugar. For, intramuscular, intraperitoneal, subcutaneous and
intravenous use, sterile solutions of the active ingredient are
usually employed, and the pH of the solutions should be suitably
adjusted and buffered.
[0146] In practice of the present invention, CDK inhibitors
selected from the compounds of Formula I may be administered either
orally or parenterally to generate and maintain good blood levels
thereof, while one or more antineoplastic agents may be
administered orally or parenterally, by intravenous, subcutaneous
or intramuscular route or any other suitable route of
administration.
[0147] In one embodiment, the therapeutic agents (the CDK
inhibitors and the antineoplastic agents) contained in the
combination of the invention are formulated in accordance with
routine procedures as a pharmaceutical composition.
[0148] In practice, oral preparations for oral administration may
be produced by adding to the active ingredients fillers, and if
necessary, binders, disintegrants, lubricants, coloring agents,
flavoring agents, etc. and formulating the resultant mixture
according to conventional procedures into tablets, coated tablets,
granules, subtle granules, powders, capsules or the like. Examples
of the filler include but not limited to lactose, corn starch,
white sugar, glucose, sorbitol, crystalline cellulose, silicon
dioxide, etc. Examples of the binder include but not limited to
polyvinyl alcohol, ethylcellulose, methylcellulose, gum arabic,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc.
Examples of the lubricant include but not limited to magnesium
stearate, talc, silica, etc. The coloring agent may be any coloring
agent which is approved to be added to pharmaceutical preparations.
Examples of the flavoring agent include but not limited to cocoa
powder, menthol, aromatic powder, peppermint oil, camphol, cinnamon
powder, etc. Resultant tablets and granules may be appropriately
coated with, for example, sugar or gelatin according to necessity.
When the pharmaceutical composition of the present invention is
administered transdermally in the form of patch, it is preferable
to select the so-called free-form that does not form a salt.
Injection preparations may be produced as intravenous infusion
preparations or intravenous, subcutaneous or intramuscular
injection preparations according to conventional procedures.
Examples of the suspending agent include but not limited to
methylcellulose, polysolvate 80, hydroxyethyl cellulose, gum
arabic, powdered tragacanth, sodium carboxymethylcellulose,
polyoxyethylene sorbitan monolaurate, etc. Examples of the
dissolution aid include but not limited to polyoxyethylene
hydrogenated castor oil, polysolvate 80, nicotinamide,
polyoxyethylene sorbitan monolaurate, macrogol, fatty acid ethyl
ester from castor oil, etc. Examples of the stabilizer include but
not limited to sodium sulfite, sodium metasulfite, etc. Examples of
the preservative include methyl parahydroxybenzoate, ethyl
parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol
etc.
[0149] Although the effective doses of therapeutic agents (the CDK
inhibitors and the antineoplastic or anticancer agents) for
administration vary depending on the severity of symptom, the age,
sex, body weight and sensitivity difference of the patient, the
mode, time, interval and duration of administration, the nature,
formulation and type of the preparation, the type of the active
ingredient, etc. In certain embodiments, the therapeutic agents are
administered in a time frame where both 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 therapeutic
agents. As indicated herein before, the active ingredients
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.
[0150] For effective administration, the therapeutic agents of the
pharmaceutical combination of the present invention are provided in
a particular dose range, for example the CDK inhibitor selected
from compound of formula I such as the compound A may be provided
in a general dose range of 75 mg/m.sup.2/day to 200 mg/m.sup.2/day;
another CDK inhibitor selected from compound of formula I such as
the Compound B may be provided in a general dose range of 50 mg to
350 mg orally. Further, among the antineoplastic agents, cisplatin
may be provided in a dose range of 40 mg/m.sup.2/day to 200
mg/m.sup.2/day, 5-fluourouracil may be provided in dose range of 40
mg/m.sup.2/day to 200 mg/m.sup.2/day, docetaxel may be provided in
a general dose range of 20 mg/m.sup.2/day to 75 mg/m.sup.2/day,
sorafenib may be provided in at least an amount from about 200 mg
to 400 mg (2.times.200 mg tablet) PO bid (orally twice a day),
lapatinib may be provided in a dose ranging from 500 to 1500 mg/d
and erlotinib may be provided in a dose range of about 150 mg/day
to 300 mg/day.
[0151] In a further embodiment, the present invention provides a
kit comprising a therapeutically effective amount of a CDK
inhibitor selected from the compound of formula I or a
pharmaceutically acceptable salt thereof in combination with one or
more antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin and 5-fluorouracil or a pharmaceutically
acceptable salt thereof.
[0152] The combinations provided by this invention have been
evaluated in certain assay systems, the experimental details are as
provided herein below.
[0153] The synergistic efficacy of the combination of present
invention is demonstrated by conducting the in vitro study
involving use of a combination for example a CDK inhibitor of
formula I as described herein as compound A or compound B and one
or more antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil or docetaxel. It is clearly
indicated that the antineoplastic agents when used in combination
with CDK inhibitors in the treatment of squamous cell carcinoma of
head and neck the apoptosis in proliferative cells increases than
when the cells are treated with the CDK inhibitor of formula I
alone or antineoplastic agent alone. For instance, it is clearly
established from the data described herein that the CDK inhibitor
of formula I, compound described herein as the compound A or
compound B in combination with one or more antineoplastic agents
selected from sorafenib, lapatinib, erlotinib, cisplatin,
5-fluorouracil or docetaxel, are synergistically effective in the
treatment of squamous cell carcinoma of the head and neck. The
synergism exhibited by the pharmaceutical combination of the
present invention is also demonstrated through in vivo study data
as indicated herein.
It is understood that modifications that do not substantially
affect the activity of the various embodiments of this invention
are included within the invention disclosed herein. Accordingly,
the following examples are intended to illustrate but not to limit
the present invention.
Example 1
A) General Procedure for the Preparation of the CDK Inhibitors (the
Compounds of Formula I)
[0154] The compounds of formula I may be prepared according to the
methods disclosed in PCT Patent Publication No. WO2004004632 and
PCT Patent Publication No. WO2007148158 which are incorporated
herein by reference.
[0155] The general process for the preparation of the compound of
formula I, or a pharmaceutically acceptable salt thereof, comprises
the following steps: [0156] a) treating the resolved
enantiomerically pure (-)-trans enantiomer of the intermediate
compound of formula VIA,
[0156] ##STR00004## [0157] with acetic anhydride in the presence of
a Lewis acid catalyst to obtain a resolved acetylated compound of
formula VIIA,
[0157] ##STR00005## [0158] 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;
[0158] ##STR00006## [0159] 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;
[0159] ##STR00007## [0160] wherein Ar is as defined above; [0161]
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,
[0161] ##STR00008## [0162] 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.
[0163] 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.
[0164] 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).
[0165] 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., 1381 (1933) and
Curr. Sci., 4,214 (1933)).
[0166] 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.
[0167] 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.
[0168] 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.
B) Preparation of
(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-p-
yrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A)
[0169] Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added
to
(+)-trans-2-(2-chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl-
)-5,7-dimethoxy-chromen-4-one (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.
[0170] Yield: 0.25 g (70%); IR (KBr): 3422, 3135, 1664, 1623, 1559
cm-1; 1H NMR (CDCl3, 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);
[0171] Analysis: C.sub.21H.sub.20ClNO.sub.5C, 62.24 (62.71); H,
5.07 (4.97); N, 3.60 (3.48); Cl, 9.01 (8.83).
[0172] The compound as obtained above (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.
[0173] Yield: 0.21 g (97%); mp: 188-192.degree. C.;
[.alpha.]D25=+21.3.degree. (c=0.2, methanol);
[0174] 1H NMR (CD3OD, 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).
[0175] This compound was subjected to chiral HPLC. Chiral HPLC was
done using column Chiralcel OD-H (250.times.4.6 mm) and solvent
system haxane:ethanol (92:08) with TFA (0.4%). The results are
recorded at 264 nm with solvent flow rate of 1 mL/min. As depicted
in the chiral HPLC showed 100% e.e of the compound,
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl--
pyrrolidin-3-yl)-chromen-4-one hydrochloride.
C) Preparation of
(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydrox-
ymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(Compound B)
[0176] A mixture of the compound,
(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methy-
l pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (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 hrs.
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-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-
-methyl-1-methylpyrrolidin-3-yl)-chromen-4-one, as a yellow
solid.
[0177] Yield: 0.15 g (63.7%);
[0178] 1H NMR (CDCl3, 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).
[0179] The compound (0.1 g, 0.2 mmol) as obtained above 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-
ymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
[0180] Yield: 0.1 g (92.8%);
[0181] 1H NMR (CDCl3, 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).
[0182] In Vitro Studies Involving Use of the Combination Consisting
of A CDK Inhibitor and One or More Antineoplastic Agents
[0183] In vitro studies involving use of a combination comprising a
CDK inhibitor selected from
(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-p-
yrrolidin-3-yl)-chromen-4-one hydrochloride (compound A) and
(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydrox-
ymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(compound B) and one or more anti-neoplastic agents selected from
sorafenib, lapatinib, erlotinib, docetaxel, cisplatin or
5-fluorouracil, exhibiting the synergistic effect of the
combination of the present invention are illustrated in the
following examples.
Example 2
[0184] Materials:
[0185] Sorafenib, lapatinib and erlotinib were obtained from LC
Labs (USA). Cisplatin, 5-fluorouracil and docetaxel were obtained
from Sigma. CK-8 cytotoxicity kit was procured from Dojindo
Molecular Technologies, Japan. Culture media and fetal bovine serum
(FBS) were obtained from Sigma (St. Louis, Mo.) and Gibco (Paisley,
Scotland) respectively. The head and neck cancer cells SCC-25,
Detroit 562, and FADU were obtained from the American Type Culture
Collection (ATCC, Manassas, Va.). Cells were maintained in
Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% FBS,
Penicillin-Streptomycin Solution Stabilized, sterile-filtered, with
100 units penicilin/ml and 100 mg streptomycin/ml. The cells were
grown in 75-cm.sup.2 culture flasks and kept in a humidified
(37.degree. C., 5% CO.sub.2) incubator. Cells were passaged on
reaching 80% confluence.
[0186] Cell Proliferation Assay:
[0187] Logarithmically growing cells were plated at a density of
3.times.10.sup.3 cells/well and allowed to recover overnight. The
cells were challenged with varying concentration of different
anticancer agents (compound A, compound B, sorafenib, lapatinib
erlotinib, cisplatin, docetaxel and 5-fluorouracil) and the control
cells received standard media containing dimethyl sulfoxide (DMSO)
vehicle at a concentration of 0.2%. After 72 hours, cell toxicity
was determined by CCK-8 (Cell Counting Kit-8) reagent (Dojindo
Molecular Technologies, Japan); WST-8
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,
4-disulfophenyl)]-2H-tetrazolium, monosodium salt) assay. In
accordance with the manufacturer's instructions, 5 .mu.l/well CCK-8
reagent was added and plates were incubated for 2 hours. The
toxicity was determined by measuring the absorbance on Tecan
Sapphire multi-fluorescence micro-plate reader (Tecan, Germany,
GmbH) at a wavelength of 450 nm corrected to 650 nm and normalized
to controls.
[0188] A CCK-8 non-radioactive colorimetric assay was carried out
to characterize the in vitro growth of SCC-25, Detroit 562, and
FADU as well as to test the anti-proliferative/cytotoxic activity
of the anticancer agents, compound A, compound B, sorafenib,
lapatinib, erlotinib, cisplatin, docetaxel and 5-fluorouracil when
used in combination. CCK-8 allows convenient assays using Dojindo's
tetrazolium salt, WST
[8[(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2-
H-tetrazolium, monosodium salt], which produces a water-soluble
formazan dye upon bioreduction in the presence of an electron
carrier, 1-Methoxy PMS. CCK-8 solution is added directly to the
cells; no pre-mixing of components is required. CCK-8 is a
sensitive nonradioactive colorimetric assay for determining the
number of viable cells in cell proliferation and cytotoxicity
assays. WST-8 is bio-reduced by cellular dehydrogenases to an
orange formazan product that is soluble in tissue culture medium.
The amount of formazan produced is directly proportional to the
number of living cells. The detection sensitivity of cell
proliferation assays using WST-8 is higher than assays using the
other tetrazolium salts such as MIT, XTT, MTS or WST-1. Optical
Density was determined at measurement wavelength of 450 nm and
reference wave length of 630 nm.
Determination of 50 Percent Inhibitory Concentrations (IC.sub.50)
of the Compound A, Compound B, Sorafenib, Lapatinib, Erlotinib,
Docetaxel, Cisplatin and 5-FU
[0189] In order to determine the IC.sub.50 of compound A, compound
B, sorafenib and lapatinib, in SCC-25, Detroit 562 and FADU cells
and IC.sub.50 of erlotinib, docetaxel, cisplatin and 5-FU in
Detroit 562 and FADU cells, the cells were treated with the
specified anticancer agents ("the test compounds") at the below
mentioned concentrations. All the anticancer agents in the
following doses of final concentration 0.03 .mu.M, 0.1 .mu.M, 0.3
.mu.M, 1 .mu.M, 3 .mu.M, 10 .mu.M, 30 .mu.M and 100 .mu.M were
analyzed for their capacity to exhibit cytotoxicity particularly to
exhibit 50% cytotoxicity. The cells were seeded at a density of
3000 cells/well, in a 200 .mu.L in tissue culture grade 96 well
plate and were allowed to recover for 24 hrs in a humidified
5%.+-.0.2 CO.sub.2 incubator at 37.degree. C..+-.0.5.degree. C.
After 24 hrs, 1 .mu.L of 200.times. (200 times higher than required
concentration is denoted as 200.times.) test compound (compound A,
compound B lapatinib, sorafenib, erlotinib, docetaxel, cisplatin
and 5-fluorouracil) dissolved in neat dimethyl sulfoxide (DMSO) was
added to the cells. The final DMSO concentration was 0.5% in wells.
Plates were incubated for 48 hrs in humidified 5%.+-.0.2 CO.sub.2
incubator at 37.+-.0.5.degree. C. After 48 hrs the plates were
removed from CO.sub.2 incubator and 5 .mu.L of Cell counting Kit
(CCK-8) per well was added. The same plate was kept at 37.degree.
C. for 3 hrs, and allowed to come to room temperature. The
absorbance at a wavelength of 450 nm was read on Tecan safire
reader. The percent cytotoxicity was calculated using the following
formula.
Percent Cytotoxicity = ( OD of Control - OD Treated cells .times.
100 ) OD DMSO control ##EQU00001##
[0190] Dose response studies at 72 hr in SCC-25 cells showed that
the Compound A, Compound B, sorafenib and lapatinib inhibited 50%
growth (IC.sub.50) at 0.4 .mu.M, 1.1 .mu.M, 2.7 .mu.M and 0.5 .mu.M
respectively. The results are presented in Table 1 and are
graphically presented in FIGS. 1a and 1b.
[0191] Dose response studies at 72 hr in Detroit-562 cells showed
that the Compound A, Compound B, sorafenib and lapatinib inhibited
50% growth (IC.sub.50) at 1.3 .mu.M, 14.1 .mu.M, 6.1 .mu.M and 3.9
.mu.M respectively. The results are presented in Table 2 and are
graphically presented in FIGS. 2a and 2b.
[0192] Dose response studies at 72 hr in FADU cells showed that
compound A, compound B, sorafenib and lapatinib inhibited 50%
growth (IC.sub.50) at 1.3 .mu.M, 4.1 .mu.M, 8.4 .mu.M and 2.6 .mu.M
respectively. The results are presented in Table 3 and are
graphically presented in FIGS. 3a and 3b.
[0193] Dose-response studies at 72 hr in Detroit-562 cells showed
that compound A and erlotinib inhibited 50% growth (IC.sub.50) at
1.3 .mu.M, and 2.3 .mu.M respectively. The results are presented in
Table 4 and are graphically presented in FIG. 4a.
[0194] Dose-response studies at 72 hr in FADU cells showed that the
compound A and erlotinib inhibited 50% growth (IC.sub.50) at 1.3
.mu.M, and 10.2 .mu.M respectively. The results are presented in
Table 5 and are graphically presented in FIG. 4b.
[0195] Dose-response studies at 72 hr in Detroit-562 cells showed
that cisplatin, compound A and 5-FU inhibited 50% growth
(IC.sub.50) at 14.6 .mu.M, 1.3 .mu.M, and 6.3 .mu.M respectively.
The results are presented in Table 6 and are graphically
represented in FIG. 5a.
[0196] Dose-response studies at 72 hr in FADU cells showed that the
cisplatin, compound A and 5-FU inhibited 50% growth (IC.sub.50) at
8.3 .mu.M, 1.3 .mu.M, and 11.6 .mu.M respectively. The results are
presented in Table 7 and are graphically represented in FIG.
5b.
[0197] Dose-response studies at 72 hr in Detroit-562 cells showed
that docetaxel, cisplatin, compound A and 5-FU inhibited 10% growth
(IC.sub.10) at 0.009 .mu.M, 0.3 .mu.M, 0.1 .mu.M and 0.31 .mu.M and
50% growth (IC.sub.50) at 0.85 .mu.M, 14.6 .mu.M, 1.3 .mu.M, and
6.3 .mu.M respectively. The results are presented in Table 6 and
are graphically represented in FIG. 5a.
[0198] Dose-response studies at 72 hr in FADU cells showed that
docetaxel, cisplatin, compound A and 5-FU inhibited 10% growth
(IC.sub.10) at 0.003 .mu.M, 0.25 .mu.M, 0.08 .mu.M, and 0.31 .mu.M
and 50% growth (IC.sub.50) at 0.16 .mu.M, 8.3 .mu.M, 1.3 .mu.M, and
11.6 .mu.M respectively.
[0199] The results are presented in Table 7 and are graphically
represented in FIG. 5b.
[0200] Similarly the IC.sub.30, IC.sub.70 and IC.sub.90
concentrations for all the tested compounds (anticancer compounds)
were established from dose in which particular compound shows 30%,
70% and 90% activity respectively in the cytotoxicity assay.
TABLE-US-00001 TABLE 1 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.30, IC.sub.50, IC.sub.70 and IC.sub.90) of
compound A, compound B, sorafenib and lapatinib in SCC-25 cells.
SCC-25 cells (Inhibitory conc. in .mu.M) Anti-cancer agent
IC.sub.30 IC.sub.50 IC.sub.70 IC.sub.90 Compound A 0.1 0.4 4.1 33.3
Compound B 0.2 1.1 4.8 41.8 Sorafenib 0.18 2.7 6.8 11.7 Lapatinib
0.2 0.5 3.3 9.9
TABLE-US-00002 TABLE 2 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.30, IC.sub.50, IC.sub.70 and IC.sub.90) of
compound A, compound B, sorafenib and lapatinib in Detroit-562
cells. Detroit-562 cells (Inhibitory conc. in .mu.M ) Anti-cancer
agent IC.sub.30 IC.sub.50 IC.sub.70 IC.sub.90 Compound A 0.5 1.3
12.1 26.3 Compound B 2.7 14.1 25.2 44.6 Sorafenib 1.8 6.1 11.2 15.3
Lapatinib 1.0 3.9 7.6 12.6
TABLE-US-00003 TABLE 3 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.30, IC.sub.50, IC.sub.70 and IC.sub.90) of
compound A, compound B, sorafenib and lapatinib in FADU cells. FADU
cells (Inhibitory conc. .mu.M) Anti-cancer agent IC.sub.30
IC.sub.50 IC.sub.70 IC.sub.90 Compound A 0.2 1.3 8.3 28.3 Compound
B 2.3 4.1 9.6 31.4 Sorafenib 3.9 8.4 14.8 30.6 Lapatinib 0.8 2.6
8.7 14.3
TABLE-US-00004 TABLE 4 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.30, IC.sub.50, IC.sub.70 and IC.sub.90) of
compound A and erlotinib in Detroit-562 cells. Detroit-562 cells
(Inhibitory conc. In .mu.M) Anti-cancer agent IC.sub.30 IC.sub.50
IC.sub.70 IC.sub.90 Compound A 0.5 1.3 12.1 26.3 Erlotinib 1.4 2.3
6.3 10.7
TABLE-US-00005 TABLE 5 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.30, IC.sub.50, IC.sub.70 and IC.sub.90) of
compound A and erlotinib in FADU cells. FADU cells (Inhibitory
conc. In .mu.M) Anti-cancer agent IC.sub.30 IC.sub.50 IC.sub.70
IC.sub.90 Compound A 0.2 1.3 8.3 28.3 Erlotinib 0.9 10.2 30.7
63
TABLE-US-00006 TABLE 6 10%, 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.10, IC.sub.30, IC.sub.50, IC.sub.70 and
IC.sub.90) of cisplatin, compound A, 5-FU and docetaxel in
Detroit-562 cells. Detroit-562 cells (Inhibitory conc. In .mu.M)
Anti-cancer agent IC.sub.10 IC.sub.30 IC.sub.50 IC.sub.70 IC.sub.90
Cisplatin 0.3 3 14.6 43.2 74.5 Compound A 0.1 0.5 1.3 12.1 26.3
5-FU 0.31 1.1 6.3 17.6 33.4 Docetaxel 0.009 0.3 0.85 8.7 21.8
TABLE-US-00007 TABLE 7 10%, 30%, 50%, 70% and 90% inhibitory
concentrations (IC.sub.10, IC.sub.30, IC.sub.50, IC.sub.70 and
IC.sub.90) of cisplatin, compound A, 5-FU and docetaxel in FADU
cells FADU cells (Inhibitory conc. In .mu.M) Anti-cancer agent
IC.sub.10 IC.sub.30 IC.sub.50 IC.sub.70 IC.sub.90 Cisplatin 0.25 3
8.3 30 74 Compound A 0.08 0.2 1.3 8.3 28.3 5-FU 0.31 2.3 11.6 22.4
38.3 Docetaxel 0.003 0.06 0.16 0.71 35.6
Example 4
Combination Studies of Compound A and Sorafenib in SCC-25,
Detroit-562 and Fadu Cells
[0201] A) SCC-25 Cells
[0202] Sorafenib in the following dose of final concentration 0.18
.mu.M and compound A in the following doses of final concentration
0.1 .mu.M, 0.4 .mu.M and 4.1 .mu.M were analyzed in single dose and
in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; the SCC-25 cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with
plain MEM (minimum essential media) medium. Fresh MEM with 10%
serum (200 .mu.L/well) was added, followed by treatment with
compound A from 24 hrs to 96 hrs. The results are presented in the
following Table 8 and graphically presented in FIG. 6a.
TABLE-US-00008 Sr. Anticancer agent (SCC-25 cells) Combination No.
(Inhibitory conc. in .mu.M) % Cytotoxicity index 1 Sorafenib
IC.sub.30 13 -- 2 Compound A IC.sub.30 13 -- 3 Compound A IC.sub.50
24 -- 4 Compound A IC.sub.70 20 -- 5 Sorafenib IC.sub.30 + Compound
A IC.sub.30 77 0.31 6 Sorafenib IC.sub.30 + Compound A IC.sub.50 85
0.35 7 Sorafenib IC.sub.30 + Compound A IC.sub.70 88 0.41
[0203] B) Detroit-562 Cells.
[0204] Sorafenib in the following dose of final concentration 1.8
.mu.M and compound A in the following doses of final concentration
0.5 .mu.M, 1.3 .mu.M and 12.1 .mu.WI were analyzed in single dose
and in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; Detroit-562 cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with
plain MEM medium. Fresh MEM with 10% serum (2004/well) was added,
followed by treatment with compound A from 24 hrs to 96 hrs. The
results are presented in the following Table 9 and graphically
presented in FIG. 7a.
TABLE-US-00009 Anticancer agent Sr. (Detroit-562 cells) %
Combination No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1
Sorafenib IC.sub.30 14 -- 2 Compound A IC.sub.30 15 -- 3 Compound A
IC.sub.50 25 -- 4 Compound A IC.sub.70 30 -- 5 Sorafenib IC.sub.30
+ Compound A IC.sub.30 78 0.35 6 Sorafenib IC.sub.30 + Compound A
IC.sub.50 84 0.37 7 Sorafenib IC.sub.30 + Compound A IC.sub.70 86
0.39
[0205] C) FADU Cells
[0206] Sorafenib in the following dose of final concentration 3.9
.mu.M and compound A in the following doses of final concentration
0.2 .mu.M, 1.3 .mu.M and 8.3 .mu.L were analyzed in single dose and
in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; the FADU cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with
plain MEM medium. Fresh MEM with 10% serum (200 .mu.L/well) was
added, followed by treatment with compound A from 24 hrs to 96 hrs.
The results are presented in the following Table 10 and graphically
presented in FIG. 8a.
TABLE-US-00010 Sr. Anticancer agent (FADU cells) % Combination No.
(Inhibitory conc. in .mu.M) Cytotoxicity index 1 Sorafenib
IC.sub.30 21 -- 2 Compound A IC.sub.30 12 -- 3 Compound A IC.sub.50
18 -- 4 Compound A IC.sub.70 26 -- 5 Sorafenib IC.sub.30 + Compound
A IC.sub.30 64 0.56 6 Sorafenib IC.sub.30 + Compound A IC.sub.50 86
0.61 7 Sorafenib IC.sub.30 + Compound A IC.sub.70 92 0.63
Example 5
Combination Studies of Compound B and Sorafenib in SCC-25,
Detroit-562 and FADU Cells
[0207] A) SCC-25 Cells
[0208] Sorafenib in the following dose of final concentration 0.18
.mu.M and compound B in the following doses of final concentration
0.2 .mu.M, 1.1 .mu.M and 4.8 .mu.M were analyzed in single dose and
in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; the SCC-25 cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with
plain MEM medium. Fresh MEM with 10% serum (200 .mu.L/well) was
added, followed by treatment with compound B from 24 hrs to 96 hrs.
The results are presented in the following Table 11 and graphically
presented in FIG. 6b.
TABLE-US-00011 Sr. Anticancer agent (SCC-25 cells) % Combination
No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1 Sorafenib
IC.sub.30 13 -- 2 Compound B IC.sub.30 16 -- 3 Compound B IC.sub.50
19 -- 4 Compound B IC.sub.70 26 -- 5 Sorafenib IC.sub.30 + Compound
B IC.sub.30 48 0.68 6 Sorafenib IC.sub.30 + Compound B IC.sub.50 51
0.73 7 Sorafenib IC.sub.30 + Compound B IC.sub.70 56 0.81
[0209] B) Detroit-562 Cells
[0210] Sorafenib in the following dose of final concentration 1.8
.mu.M and compound B in the following doses of final concentration
2.7 .mu.WI, 14.1 .mu.M and 25.2 .mu.M were analyzed in single dose
and in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; the Detroit-562 cells were treated with sorafenib for 0 to
24 hrs. At the end of 24 hrs the cells were washed two times with
plain MEM medium. Fresh MEM with 10% serum (200 .mu.L/well) was
added, followed by treatment with compound B from 24 hrs to 96 hrs.
The results are presented in the following Table 12 and graphically
presented in FIG. 7b.
TABLE-US-00012 Sr. Anticancer agent (Detroit-562 cells) %
Combination No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1
Sorafenib IC.sub.30 13 -- 2 Compound B IC.sub.30 15 -- 3 Compound B
IC.sub.50 19 -- 4 Compound B IC.sub.70 26 -- 5 Sorafenib IC.sub.30
+ Compound B IC.sub.30 65 0.67 6 Sorafenib IC.sub.30 + Compound B
IC.sub.50 74 0.73 7 Sorafenib IC.sub.30 + Compound B IC.sub.70 86
0.76
[0211] C) FADU Cells
[0212] Sorafenib in the following dose of final concentration 3.9
.mu.M and compound B in the following doses of final concentration
2.3 .mu.M, 4.1 .mu.M and 9.6 .mu.M were analyzed in single dose and
in all possible combinations of the dose range for the two drugs
mentioned above. The sequence of treatment is as follows; the FADU
cells were treated with sorafenib for 0 to 24 hrs. At the end of 24
hrs the cells were washed two times with plain MEM medium. Fresh
MEM with 10% serum (200 .mu.L/well) was added, followed by
treatment with compound B from 24 hrs to 96 hrs. The results are
presented in the following Table 13 and graphically presented in
FIG. 8b.
TABLE-US-00013 Sr. Anticancer agent % Combination No. (FADU cells)
(Inhibitory conc. in .mu.M) Cytotoxicity index 1 Sorafenib
IC.sub.30 21 -- 2 Compound B IC.sub.30 14 -- 3 Compound B IC.sub.50
20 -- 4 Compound B IC.sub.70 27 -- 5 Sorafenib IC.sub.30 + Compound
B IC.sub.30 59 0.74 6 Sorafenib IC.sub.30 + Compound B IC.sub.50 77
0.81 7 Sorafenib IC.sub.30 + Compound B IC.sub.70 88 0.84
Example 6
Combination Studies of Compound A and Lapatinib in SCC-25,
Detroit-562 and FADU Cells
[0213] A) SCC-25 Cancer Cells
[0214] Lapatinib in the following dose of final concentration 0.2
.mu.M and compound A in the following doses of final concentration
0.2 .mu.M, 0.5 .mu.M and 3.3 .mu.M were analyzed in single dose and
in all possible combinations of the dose range for the two drugs
mentioned above. The sequence of treatment is as follows; the
SCC-25 cells were treated with lapatinib for 0 to 24 hrs. At the
end of 24 hrs the cells were washed two times with plain MEM
medium. Fresh MEM with 10% serum (200 .mu.L/well) was added,
followed by treatment with compound A from 24 hrs to 96 hrs. The
results are presented in the following Table 14 and graphically
presented in FIG. 9a.
TABLE-US-00014 Sr. Anticancer agent (SCC-25 cells) % Combination
No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1 Lapatinib
IC.sub.30 17 -- 2 Compound A IC.sub.30 12 -- 3 Compound A IC.sub.50
23 -- 4 Compound A IC.sub.70 26 -- 5 Lapatinib IC.sub.30 + Compound
A IC.sub.30 69 0.68 6 Lapatinib IC.sub.30 + Compound A IC.sub.50 83
0.71 7 Lapatinib IC.sub.30 + Compound A IC.sub.70 89 0.75
[0215] B) Detroit-562 Cancer Cells
[0216] Lapatinib in the following dose of final concentration 1
.mu.M and compound A in the following doses of final concentration
0.5 .mu.M, 1.3 .mu.WI and 12.1 .mu.M were analyzed in single dose
and in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; the Detroit-562 cells were treated with lapatinib for 0 to
24 hrs. At the end of 24 hrs the cells were washed two times with
plain MEM medium. Fresh MEM with 10% serum (200 .mu.L/well) was
added, followed by treatment with compound A from 24 hrs to 96 hrs.
The results are presented in the following Table 15 and graphically
presented in FIG. 10a.
TABLE-US-00015 Sr. Anticancer agent (Detroit-562 cells) %
Combination No. (Inhibitory conc. in pM) Cytotoxicity index 1
Lapatinib IC.sub.30 16 -- 2 Compound A IC.sub.30 11 -- 3 Compound A
IC.sub.50 22 -- 4 Compound A IC.sub.70 25 -- 5 Lapatinib IC.sub.30
+ Compound A IC.sub.30 68 0.81 6 Lapatinib IC.sub.30 + Compound A
IC.sub.50 82 0.88 7 Lapatinib IC.sub.30 + Compound A IC.sub.70 88
0.71
[0217] C) FADU Cancer Cells Lapatinib in the following dose of
final concentration 0.8 .mu.M and compound A in the following doses
of final concentration 0.2 .mu.M, 1.3 .mu.M and 8.3 .mu.M were
analyzed in single dose and in all possible combinations of the
dose range for the two drugs mentioned above. The sequence of
treatment is as follows; the FADU cells were treated with lapatinib
for 0 to 24 hrs. At the end of 24 hrs the cells were washed two
times with plain MEM medium. Fresh MEM with 10% serum (200
.mu.L/well) was added, followed by treatment with compound A from
24 hrs to 96 hrs. The results are presented in the following Table
16 and graphically presented in FIG. 11a.
TABLE-US-00016 Sr. Anticancer agent (FADU cells) % Combination No.
(Inhibitory conc. in .mu.M ) Cytotoxicity index 1 Lapatinib
IC.sub.30 19 -- 2 Compound A IC.sub.30 13 -- 3 Compound A IC.sub.50
22 -- 4 Compound A IC.sub.70 24 -- 5 Lapatinib IC.sub.30 + Compound
A IC.sub.30 62 0.64 6 Lapatinib IC.sub.30 + Compound A IC.sub.50 85
0.69 7 Lapatinib IC.sub.30 + Compound A IC.sub.70 89 0.83
Example 7
Combination Studies of Compound B and Lapatinib in SCC-25,
Detroit-562 and FADU Cells
[0218] A) SCC-25 Cancer Cells
[0219] Lapatinib in the following dose of final concentration 0.2
.mu.M and compound B in the following doses of final concentration
0.2 .mu.M, 1.1 .mu.M and 4.8 .mu.M were analyzed in single dose and
in all possible combinations of the dose range for the two drugs
mentioned above. The sequence of treatment is as follows; the
SCC-25 cells were treated with lapatinib for 0 to 24 hrs. At the
end of 24 hrs the cells were washed two times with plain MEM
medium. Fresh MEM with 10% serum (200 .mu.L/well) was added,
followed by treatment with compound A from 24 hrs to 96 hrs. The
results are presented in the following Table 17 and graphically
presented in FIG. 9b.
TABLE-US-00017 Sr. Anticancer agent (SCC-25 cells) % Combination
No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1 Lapatinib
IC.sub.30 17 -- 2 Compound B IC.sub.30 16 -- 3 Compound B IC.sub.50
19 -- 4 Compound B IC.sub.70 31 -- 5 Lapatinib IC.sub.30 + Compound
B IC.sub.30 62 0.61 6 Lapatinib IC.sub.30 + Compound B IC.sub.50 73
0.81 7 Lapatinib IC.sub.30 + Compound B IC.sub.70 77 0.84
[0220] B) Detroit-562 Cancer Cells
[0221] Lapatinib in the following dose of final concentration 1.0
.mu.M and compound B in the following doses of final concentration
2.7 .mu.M, 14.1 .mu.M and 25.2 .mu.M were analyzed in single dose
and in all possible combinations of the dose range for the two
drugs mentioned above. The sequence of treatment is as follows; the
Detroit-562 cells were treated with lapatinib for 0 to 24 hrs. At
the end of 24 hrs the cells were washed two times with plain MEM
medium. Fresh MEM with 10% serum (200 .mu.L/well) was added,
followed by treatment with compound A from 24 hrs to 96 hrs. The
results are presented in the following Table 18 and graphically
presented in FIG. 10b.
TABLE-US-00018 Sr. Anticancer agent (Detroit-562 cells) %
Combination No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1
Lapatinib IC.sub.30 16 -- 2 Compound B IC.sub.30 15 -- 3 Compound B
IC.sub.50 18 -- 4 Compound B IC.sub.70 30 -- 5 Lapatinib IC.sub.30
+ Compound B IC.sub.30 70 0.71 6 Lapatinib IC.sub.30 + Compound B
IC.sub.50 87 0.65 7 Lapatinib IC.sub.30 + Compound B IC.sub.70 90
0.77
[0222] C) FADU Cancer Cells
[0223] Lapatinib in the following dose of final concentration 0.8
.mu.M and compound B in the following doses of final concentration
2.3 .mu.M, 4.1 .mu.M and 9.6 .mu.M were analyzed in single dose and
in all possible combinations of the dose range for the two
anticancer agents mentioned above. The sequence of treatment is as
follows; the FADU cells were treated with lapatinib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with
plain MEM medium. Fresh MEM with 10% serum (200 .mu.L/well) was
added, followed by treatment with compound A from 24 hrs to 96 hrs.
The results are presented in the following Table 19 and graphically
presented in FIG. 11b.
TABLE-US-00019 Sr. Anticancer agent % Combination No. (FADU cells)
(Inhibitory conc. in .mu.M) Cytotoxicity index 1 Lapatinib
IC.sub.30 19 -- 2 Compound B IC.sub.30 21 -- 3 Compound B IC.sub.50
26 -- 4 Compound B IC.sub.70 33 -- 5 Lapatinib IC.sub.30 + Compound
B IC.sub.30 74 0.78 6 Lapatinib IC.sub.30 + Compound B IC.sub.50 89
0.91 7 Lapatinib IC.sub.30 + Compound B IC.sub.70 93 0.71
Example 8
Combination Studies of Compound A and Erlotinib at IC.sub.30
Concentration in Detroit-562 Cells
[0224] The combination of erlotinib and compound A was found to be
synergistic at the IC.sub.30 of both the anticancer agents.
Erlotinib at IC.sub.30 showed cytotoxicity of 20.3% and Compound A
at IC.sub.30, showed cytotoxicity of 8.30%. However, when used as a
combination of erlotinib IC.sub.30 for 24 hrs, followed by compound
A IC.sub.30 for 48 hrs showed an increase in cytotoxicity to the
extent of 60% was noted, which is 32% more cytotoxicity than the
additive effect suggesting a synergistic effect between the two
anticancer agents in Detroit-562 cells with a combination index of
0.35. The results are presented in the following Table 20 and
graphically presented in FIG. 12a.
TABLE-US-00020 Sr. Anticancer agent (Detroit 562 cells) %
Combination No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1
Erlotinib IC.sub.30 20.3 -- 2 Erlotinib IC.sub.50 34.4 -- 3
Erlotinib IC.sub.70 40.0 -- 4 Compound A IC.sub.30 8.30 -- 5
Compound A IC.sub.50 33.80 -- 6 Compound A IC.sub.70 34.32 -- 7
Erlotinib IC.sub.30 + Compound A IC.sub.30 60.64 0.35 8 Erlotinib
IC.sub.30 + Compound A IC.sub.50 77.17 0.36 9 Erlotinib IC.sub.30 +
Compound A IC.sub.70 76.89 0.89
Example 9
Combination Studies of Compound A and Erlotinib in FADU Cells
[0225] The combination of erlotinib and Compound A was found to be
synergistic at the IC.sub.30 of both the anticancer agents.
erlotinib at IC.sub.30 showed cytotoxicity of 16% and Compound A at
IC.sub.30, showed cytotoxicity of 12.3%. However, when used as a
combination of erlotinib at concentration IC.sub.30 for 24 hrs,
followed by compound A at IC.sub.30 concentration for 48 hrs showed
an increase in cytotoxicity to the extent of 77% was noted, which
is 49% more cytotoxicity than the additive effect suggesting a
synergistic effect between the two drugs in FADU cells with a
combination index of 0.23. The results are presented in the
following Table 21 and graphically presented in FIG. 12b.
TABLE-US-00021 Sr. Anticancer agent % Combination No. (FADU cells)
(Inhibitory conc. in .mu.M) Cytotoxicity index 1 Erlotinib
IC.sub.30 16.24 -- 2 Erlotinib IC.sub.50 35.64 -- 3 Erlotinib
IC.sub.70 41.1 -- 4 Compound A IC.sub.30 12.3 -- 5 Compound A
IC.sub.50 26.54 -- 6 Compound A IC.sub.70 31.78 -- 7 Erlotinib
IC.sub.30 + Compound A IC.sub.30 77.191 0.23 8 Erlotinib IC.sub.30
+ Compound A IC.sub.50 80.286 0.31 9 Erlotinib IC.sub.30 + Compound
A IC.sub.70 84.134 0.67
[0226] In Vitro Studies Involving Use of Triple Combination
Consisting of Compound A. Cisplatin and 5-FU
Example 10
Combination Studies of Compound A, Cisplatin and 5-FU at the
IC.sub.30 in Detroit-562 Cells
[0227] The combination of compound A and (cisplatin and 5-FU) was
found to be synergistic at the IC.sub.30 of each anticancer agents.
Compound A at IC.sub.30 showed cytotoxicity of 10.4% and (cisplatin
and 5-FU) at IC.sub.30, showed cytotoxicity of 28.60%. However,
when used as a combination of (cisplatin and 5-FU) IC.sub.30 for 24
hrs, followed by compound A IC.sub.30 for 48 hrs an increase in
cytotoxicity to the extent of 71% was noted, which was 33% more
cytotoxicity than the additive effect suggesting a synergistic
effect between the three anticancer agents in Detroit-562 cells
with a combination index of 0.39. While the double combination
Cisplatin and 5-FU showed a combination index of 0.9. The results
are presented in the following Table 22 and graphically presented
in FIG. 13a.
TABLE-US-00022 Sr. Anticancer agent (Detroit 562 cells) %
Combination No. (Inhibitory conc. in .mu.M) Cytotoxicity index 1
Cisplatin IC.sub.30 4.07 -- 2 Cisplatin IC.sub.50 14.3 -- 3
Cisplatin IC.sub.70 16.2 -- 4 5-FU IC.sub.90 12.59 -- 5 5-FU
IC.sub.50 16.21 -- 6 5-FU IC.sub.70 19.54 -- 7 Compound A IC.sub.30
10.42 -- 8 Compound A IC.sub.50 12.59 -- 9 Compound A IC.sub.70
18.02 -- 10 Cisplatin IC.sub.30 + 5-FU IC.sub.30 28.67 0.9 11
Cisplatin IC.sub.30 + 5-FU IC.sub.50 32.86 1.21 12 Cisplatin
IC.sub.30 + 5-FU IC.sub.70 26.38 1.45 13 Cisplatin IC.sub.30 + 5-FU
IC.sub.30 + 68.38 0.22 Compound A IC.sub.30 14 Cisplatin IC.sub.30
+ 5-FU IC.sub.50 + 81.68 0.31 Compound A IC.sub.30 15 Cisplatin
IC.sub.30 + 5-FU IC.sub.70 + 75.22 0.39 Compound A IC.sub.30 16
Cisplatin IC.sub.30 + 5-FU IC.sub.30 + 77.30 0.56 Compound A
IC.sub.50 17 Cisplatin IC.sub.30 + 5-FU IC.sub.30 + 75.75 0.64
Compound A IC.sub.70
Example 11
Combination Studies of Compound A, Cisplatin and 5-FU at the
IC.sub.30 in FADU Cells
[0228] The combination of compound A and (cisplatin and 5-FU) was
found to be synergistic at the IC.sub.30 of each anticancer agent.
Compound A at IC.sub.30 showed cytotoxicity of 6.1% and (cisplatin
and 5-FU) at IC.sub.30, showed cytotoxicity of 30.1%. However, when
used as a combination of (cisplatin and 5-FU) at IC.sub.30
concentration for 24 hrs, followed by compound A IC.sub.30 for 48
hrs, an increase in cytotoxicity to the extent of 81% was noted,
which was 44% more cytotoxicity than the additive effect suggesting
a synergistic effect between the three drugs in FADU cells with a
combination index of 0.23. While the double combination cisplatin
and 5-FU showed a combination index of 0.89. The results are
presented in the following Table 23 and graphically presented in
FIG. 13b.
TABLE-US-00023 Sr. Anticancer agent (FADU cells) % Combination No.
(Inhibitory conc. in .mu.M) Cytotoxicity index 1 Cisplatin
IC.sub.30 6.2 -- 2 Cisplatin IC.sub.50 11.1 -- 3 Cisplatin
IC.sub.70 17.1 -- 4 5-FU IC.sub.30 17.81 -- 5 5-FU IC.sub.50 21.1
-- 6 5-FU IC.sub.70 28.6 -- 7 Compound A IC.sub.30 6.1 -- 8
Compound A IC.sub.50 21.4 -- 9 Compound A IC.sub.70 27.8 -- 10
Cisplatin IC.sub.30 + 5-FU IC.sub.30 30.8 0.89 11 Cisplatin
IC.sub.30 + 5-FU IC.sub.50 34.53 1.15 12 Cisplatin IC.sub.30 + 5-FU
IC.sub.70 42.82 1.31 13 Cisplatin IC.sub.30 + 5-FU IC.sub.30 +
67.73 0.56 Compound A IC.sub.30 14 Cisplatin IC.sub.30 + 5-FU
IC.sub.50 + 72.43 0.67 Compound A IC.sub.30 15 Cisplatin IC.sub.30
+ 5-FU IC.sub.70 + 72.94 0.78 Compound A IC.sub.30 18 Cisplatin
IC.sub.30 + 5-FU IC.sub.30 + 82.40 0.31 Compound A IC.sub.50 19
Cisplatin IC.sub.30 + 5-FU IC.sub.30 + 83.13 0.41 Compound A
IC.sub.70
Example 12
Combination Studies of Compound A, Cisplatin and 5-FU with
Docetaxel at the IC30 Concentration in Detroit-562 Cells
[0229] The combination of compound A and (cisplatin and 5-FU) with
Docetaxel was found to be synergistic at the IC.sub.30 of each
anticancer agent. Compound A and Docetaxel at IC.sub.30 showed
cytotoxicity of 16.8% and 18.30 respectively (cisplatin and 5-FU)
at IC.sub.30, showed cytotoxicity of 31.3%. However, when used as a
combination of Docetaxel at IC.sub.30 concentration for 12 hrs
followed by (cisplatin and 5-FU) at IC.sub.30 concentration for 12
hrs, followed by compound A at IC.sub.30 concentration for 48 hrs
an increase in cytotoxicity to the extent of 96.38% was noted, with
a combination index of 0.29. The results are presented in the
following Table 24 and graphically presented in FIG. 14a.
TABLE-US-00024 Com- bination Anticancer agent % index Sr. (Detroit
562 cells) Cyto- (C.I. No. (Inhibitory conc. in .mu.M) toxicity
values) 1 Docetaxel IC.sub.10 11.75 -- 2 Docetaxel IC.sub.30 18.30
-- 3 Cisplatin IC.sub.10 6.92 -- 4 Cisplatin IC.sub.30 13.39 -- 5
5-FU IC.sub.10 9.49 -- 6 5-FU IC.sub.30 15.39 -- 7 Compound A
IC.sub.30 16.81 -- 8 Compound A IC.sub.50 21.98 -- 9 Cisplatin
IC.sub.10 + 5FU IC.sub.10 33.09 0.91 10 Cisplatin IC.sub.30 + 5FU
IC.sub.30 31.92 1.1 11 Docetaxel IC.sub.10 + (Cisplatin IC.sub.10 +
56.85 0.85 5FU IC.sub.10) 12 Docetaxel IC.sub.10 + (Cisplatin
IC.sub.30 + 61.77 0.93 5FU IC.sub.30) 13 Docetaxel IC.sub.30 +
(Cisplatin IC.sub.10 + 66.59 0.87 5FU IC.sub.10) 14 Docetaxel
IC.sub.30 + (Cisplatin IC.sub.30 + 71.81 0.81 5FU IC.sub.30) 15
Docetaxel IC.sub.10 + (Cisplatin IC.sub.10 + 81.42 0.67 5FU
IC.sub.10) + Compound A IC.sub.30 18 Docetaxel IC.sub.10 +
(Cisplatin IC.sub.30 + 89.46 0.62 5FU IC.sub.30) + Compound A
IC.sub.30 19 Docetaxel IC.sub.30 + (Cisplatin IC.sub.10 + 91.47
0.37 5FU IC.sub.10) + Compound A IC.sub.30 20 Docetaxel IC.sub.30 +
(Cisplatin IC.sub.30 + 96.38 0.29 5FU IC.sub.30) + Compound A
IC.sub.30
Example 13
Combination Studies of Compound A, Cisplatin and 5-FU with
Docetaxel at the IC.sub.30 Concentration in FADU Cells
[0230] The combination of compound A and (cisplatin and 5-FU) with
docetaxel was found to be synergistic at the IC.sub.30 of each
anticancer agent. Compound A and docetaxel at IC.sub.30
concentration showed cytotoxicity of 11.77% and 20.02 respectively.
(cisplatin and 5-FU) at IC.sub.30 concentration, showed
cytotoxicity of 51.39%. However, when used as a combination of
docetaxel at IC.sub.30 concentration for 12 hrs followed by
(cisplatin and 5-FU) at IC.sub.30 concentration for 12 hrs,
followed by compound A at IC.sub.30 concentration for 48 hrs an
increase in cytotoxicity to the extent of 98.24% was noted, with a
combination index of 0.12. The results are presented in the
following Table 25 and graphically presented in FIG. 14b.
TABLE-US-00025 Anticancer agent (FADU cells) % Combination index
(Inhibitory conc. in .mu.M) Cytotoxicity (C.I. values) Docetaxel
IC.sub.10 14.69 -- Docetaxel IC.sub.30 20.02 -- Cisplatin IC.sub.10
9.16 -- Cisplatin IC.sub.30 9.08 -- 5-FU IC.sub.10 6.81 -- 5-FU
IC.sub.30 19.29 -- Compound A IC.sub.30 11.77 -- Compound A
IC.sub.50 23.86 -- Cisplatin IC.sub.10 + 5FU IC.sub.10 39.03 1.15
Cisplatin IC.sub.30 + 5FU IC.sub.30 51.39 0.91 Docetaxel IC.sub.10
+ (Cisplatin IC.sub.10 + 5FU 59.95 0.94 IC.sub.10) Docetaxel
IC.sub.10 + (Cisplatin IC.sub.30 + 5FU 70.28 0.89 IC.sub.30)
Docetaxel IC.sub.30 + (Cisplatin IC.sub.10 + 5FU 71.16 0.84
IC.sub.10) Docetaxel IC.sub.30 + (Cisplatin IC.sub.30 + 5FU 67.80
0.85 IC.sub.30) Docetaxel IC.sub.10 + (Cisplatin IC.sub.10 + 5FU
74.93 0.63 IC.sub.10) + Compound A IC.sub.30 Docetaxel IC.sub.10 +
(Cisplatin IC.sub.30 + 5FU 84.83 0.67 IC.sub.30) + Compound A
IC.sub.30 Docetaxel IC.sub.30 + (Cisplatin IC.sub.10 + 5FU 92.67
0.31 IC.sub.10) + Compound A IC.sub.30 Docetaxel IC.sub.30 +
(Cisplatin IC.sub.30 + 5FU 98.24 0.12 IC.sub.30) + Compound A
IC.sub.30
Example 14
Analysis of Cleaved Caspase-3 Expression Levels
[0231] This study was conducted to evaluate the mechanisms by which
the combination consisting of sorafenib or lapatinib in combination
with compound A or compound B blocks proliferation and whether it
can induce apoptosis in head and neck cancer cells. The cells were
seeded in 96-well plates at a density of 7.5.times.10.sup.3
cells/well. 24 h post seeding, the minimum essential medium was
replaced with a fresh minimum essential medium with 10% serum. The
anticancer agents (sorafenib or lapatinib in combination with
compound A or compound B) were treated with specific concentration
as mentioned below in SCC-25, Detroit-562 and FADU cells and
incubated for 48 hrs. At the end of 48 hrs, to determine the
protein expression, the cells were in 96 well plate spin down at
800 g for 5 minutes. Culture supernatant was removed and 200 .mu.L
of caspase-3 assay buffer was added and plates were again spin down
at 800 g for 5 minutes. Supernatant were removed and cells were
lysed with 100 .mu.L caspase-3 lysis buffer and incubated for 30
min in orbital shaker at 300 rpm at room temperature. Further
plates were spin down at 800 g for 10 minutes and 90 .mu.L of the
supernatant was transferred into new black well plate. To 90 .mu.L
of lysis solution 100 .mu.L of caspase-3 substrate was added and
incubated for 30 minutes at 37'C. At the end of incubation plates
were read in Tecan Safire multimode reader with an excitation
wavelength of 485 nm and emission wavelength of 535 nm.
[0232] A) Treatment Pattern of Sorafenib and Compound A or Compound
B in SCC-25 Cells for Assessing Caspase-3 Activity
[0233] The treatment with sorafenib for 24 hrs followed by either
compound A or compound B for 48 hrs showed notable elevation of
caspase3 expression than when used alone. It was also observed that
both compound A or compound B were more potent in inducing
caspase-3 activity in combination as graphically represented in
FIG. 15a and FIG. 15b.
[0234] B) Treatment Pattern of Lapatinib and Compound A or Compound
B in SCC-25 Cells for Assessing Caspase-3 Activity
[0235] The treatment with lapatinib for 24 hrs followed by either
compound A or compound B for 48 hrs showed notable elevation of
caspase3 expression than when used alone. It was also observed that
both compound A or compound B were more potent in inducing
caspase-3 activity in combination as graphically represented in
FIG. 16a and FIG. 16b.
Example 15
In Vivo Efficacy Studies in Human Head and Neck Cancer FaDu
(Hypopharyngeal squamous Cell Carcinoma) Xenografts
[0236] In-vivo studies were carried out according to the method
described in Clinical cancer search, 2003, 9, 6052-6061; the
disclosure of which is incorporated herein by reference for the
teaching of the assay.
[0237] Objective
[0238] The objective of this study was to evaluate the antitumor
activity of Compound A in combination with cetuximab or in
combination with both, cisplatin and cetuximab in human head and
neck cancer xenograft model of FaDu.
[0239] The in-vivo studies were carried out using Xenograft models
in Severe combined immune deficiency (SCID) mice strain
-CbySmn.CB17-Prkdcscid/J, by the method described below. The
statistically significant number of mice per group (n=6) was chosen
in order to be able to statistically evaluate the study data.
[0240] Method
[0241] FaDu cells were grown in MEM (minimum essential media)
medium containing non-essential amino acids and 10% fetal calf
serum in 5% CO2 incubator at 37.degree. C. Cells were pelleted by
centrifugation at 1000 rpm for 10 minutes. Cells were resuspended
in pre-chilled mixture of saline to get a count of 6.times.106
cells per mL; 0.2 ml of this cell suspension was kept on ice and
injected by subcutaneous (s.c.) route in SCID mice. Mice were
observed every alternate day for palpable tumor mass. Once the
tumor size reached a size of 3-5 mm in diameter, animals were
randomized into respective groups of treatment and untreated
controls. The treatment groups comprised of 5 groups viz. 1)
Compound A alone (Group 1); 2) cetuximab alone (Group 2); 3)
cisplatin alone (Group 3); 4) Compound A+cetuximab (Group 4); and
5) Compound A+cisplatin+cetuximab (Group 5). The control group
received no treatment. In single drug treatment i.e. in respect of
Groups 1, 2 and 3, the Compound A (35 mpk) was administered by i.p
route once daily for 5 days a week starting from day 1 of the week
for 3 weeks with total of 15 doses; Cisplatin (1 mpk) was
administered i.p. once a week on day 1 of the week with total of 3
doses. Cetuximab (2.5 mpk) was administered twice a week on days 1
and 4 of the week for 3 weeks with total of 6 doses. In the
treatment with combination of drugs namely compound A and
cetuximab, the sequence that was followed included administration
of Compound A for 2 h followed by cetuximab:
[0242] In the treatment with combination of drugs namely compound
A, cisplatin and cetuximab, the sequence that was followed included
administration of cisplatin for 2 h followed by the Compound A for
2 h, followed by cetuximab. Measurement of tumor was done every 2-3
days apart. Growth inhibition percentage (GI %) was calculated at
the end of experiment.
[0243] Terminal Procedures:
[0244] At the end of the experiment, animals were euthanized using
high dose of pentobarbital sodium (100 mg/kg i.p./i.v.) or exposure
to carbon dioxide gas.
[0245] Results
[0246] The results are as presented in Table 26 and graphically
presented in FIG. 17a. The FIG. 17a depicts the average group body
weight over the period of drug (the therapeutic agents)
administration plotted. FIG. 17b depicts the average % tumor weight
of Head and Neck carcinoma (Fadu) xenograft over a period of 18
days.
TABLE-US-00026 TABLE 26 Percent tumor growth inhibition at the end
of treatment i.e. after 18 days. Groups Tumor Growth inhibition (%)
Group 1 (Compound A) 11 Group 2 (cisplatin) 4 Group 3 (cetuximab)
45 Group 4 (combination of 79 Compound A and cetuximab) Group 5
(combination of cisplatin, 77 Compound A and cetuximab)
[0247] The tumor growth inhibition was highly significant with
p<0.001 in the treatment groups namely Group(s) 4 and 5
involving use of combination of antineoplastic agents with tumor
growth (TG) inhibition of 79% and 77% respectively. There was no
significant body weight loss in all the treatment groups.
CONCLUSION
[0248] The combination of Compound A and cetuximab and the
combination of Compound A, cetuximab and cisplatin showed similar
antitumor activity in the human head and neck cancer xenograft
model of FaDu and were significantly higher than either of the
drugs alone.
[0249] Having thus described in detail various embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
* * * * *