PHARMACEUTICAL COMBINATION OF PACLITAXEL AND A CDK INHIBITOR
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical combination comprising paclitaxel,
or its pharmaceutically acceptable salt; and at least one cyclin dependent kinase (CDK)
inhibitor represented by a compound of formula I (as described herein) or a pharmaceutically
acceptable salt thereof, for use in the treatment of triple negative breast cancer (TNBC). The
invention also relates to a method of treating triple negative breast cancer in a subject
comprising administering to the subject a pharmaceutical combination comprising a
therapeutically effective amount of paclitaxel, or its pharmaceutically acceptable salt; and a
therapeutically effective amount of at least one cyclin dependent kinase (CDK) inhibitor
represented by a compound of formula I (as described herein) or a pharmaceutically
acceptable salt thereof.
BACKGROUND OF THE INVENTION
Cancer is a general term used to describe diseases in which abnormal cells divide
without control. Cancer cells can invade adjacent tissues and can spread through the
bloodstream and lymphatic system to other parts of the body. There are different types of
cancers such as the bladder cancer, breast cancer, colon cancer, rectal cancer, head and neck
cancer, endometrial cancer, kidney (renal cell) cancer, leukemia, small cell lung cancer, nonsmall
cell lung cancer, pancreatic cancer, prostate cancer, thyroid cancer, skin cancer, Non-
Hodgkin's Lymphoma and melanoma. Currently there are many treatments available for
cancer than ever before, including chemotherapy, radiation, surgery, hormonal therapy,
immune therapy and gene therapy. Chemotherapy is the most routinely used treatment for
cancer.
The most widely used chemotherapeutic agents (the antineoplastic agents) include
paclitaxel, docetaxel, doxorubicin, etoposide, carboplatin, cisplatin, topotecan and
gemcitabine. These antineoplastic agents have been successfully used for the treatment of
different cancers. However, in due course of time, some cancer patients have been found to
develop resistance to monotherapy involving use of such standard antineoplastic agents.
Tolerance or resistance to a drug represents a major impediment to successful treatment. Such
resistance is often considered as either intrinsic (i.e. present at the onset of treatment) or
acquired (i.e. occurs during the course of chemotherapy). A study involving exposure of
human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of
doxorubicin reported appearance of a new cell line (NCI-H460/R) that was resistant to
doxorubicin and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin (J.
Chemother., 2006, 18, 1, 66-73). Gemcitabine was considered to be the most clinically active
drug for the treatment of pancreatic cancer, however it failed to significantly improve the
condition of pancreatic cancer patients because of the pre-existing or acquired chemo
resistance of the tumor cells to the drug (Oncogene, 2003, 22, 21, 3243-51).
Another problem observed or prevalent in cancer treatments is the severe toxicity
associated with most of the antineoplastic agents. Despite the incidence of resistance and
severe toxicity associated with the conventional antineoplastic agents e.g. gemcitabine and
paclitaxel, these agents still continue to be important in the cancer treatment because they
have the ability to reduce tumor mass. In order to improve the response rate and prevent
toxicity associated with the conventional antineoplastic agents, new therapeutic approaches
are being evaluated.
One such approach is directed to a protocol involving combination of different
anticancer agents. An optimal combination chemotherapy protocol may result in increased
therapeutic efficacy, decreased host toxicity, and minimal or delayed drug resistance. When
drugs with different toxicities are combined, each drug can be used at its optimal dose,
helping to minimise intolerable side effects. Some of the antineoplastic agents have been
found to be synergistically effective when used in combination with other anticancer agents
than when used as a monotherapy.
Cyclophosphamide and 5-fluorouracil act synergistically in ovarian clear cell
adenocarcinoma cells (Cancer Lett., 2001, 162, 1, 39-48). Combination chemotherapy can
also be advantageously used for treating cancers in advanced stages which are difficult to
treat with monotherapy, radiation or surgical treatment, for example, a combination of
paclitaxel and gemcitabine has been reported for the treatment of metastatic non-small cell
lung cancer (Cancer, 2006, 107, 5, 1050-1054). Gemcitabine and carboplatin combination
chemotherapy was relatively safe and effective for treating elderly patients with non-small
cell lung cancer (Cancer Res. Treat., 2008, 40, 116-120). Gemcitabine plus carboplatin
combination is active in advanced TCC (transitional cell carcinoma) with acceptable toxicity
(BMC Cancer, 2007, 7, 98). Treatment with gemcitabine and carboplatin significantly
improves the progression-free survival of patients with platinum-sensitive recurrent ovarian
cancer (Int. J . Gynecol. Cancer, 2005, 15 (Suppl. 1), 36^-1).
Recently, combination of one or more standard antineoplastic agents such as
paclitaxel, cisplatin etc. with a molecularly targeted anticancer agent for the treatment of
cancer has been tried out to improve drug response rates and to address resistance to the
antineoplastic agents. Molecularly targeted agents e.g. imatinib mesylate, flavopiridol etc.
modulate proteins such as kinases whose activities are more specifically associated with
cancerous cells. Researches over a long period of time have proven that the members of the
cyclin-dependent kinase (CDK) family play key roles in various cellular processes. There are
11 members of the CDK family known till now. Among these, CDK1, CDK2, CDK3, CDK4
and CDK6 are known to play important roles in the cell cycle (Adv. Cancer Res., 1995, 66,
181-212). CDKs are activated by forming noncovalent complexes with cyclins such as Atype,
B-type, C-type, D-type (Dl, D2, and D3), and E-type cyclins. Each isozyme of this
family is responsible for particular aspects (cell signaling, transcription, etc.) of the cell cycle,
and some of the CDK isozymes are specific to certain kinds of tissues. Aberrant expression
and overexpression of these kinases are evidenced in many disease conditions. A number of
compounds having potentially useful CDK inhibitory properties have been developed and
reported in the literature.
Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to reach
clinical trial. Flavopiridol has been found to potentiate synergistically the cytotoxic response
of the conventional cytotoxic antineoplastic agents in a variety of cancer cell-lines. For
example, combined docetaxel and flavopiridol treatment for lung cancer cells has been
reported in Radiother. Oncol., 2004, 71, 2, 213-21 and for the treatment of gastric cancer in
Mol. Cancer Ther., 2003, 2, 6, 549-55. PCT publication WO2008139271 discloses the
combinations of a CDK inhibitor, (+)-ira«s-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl -l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride with cytotoxic
neoplastic agents such as doxorubicin, docetaxel, paclitaxel and gemcitabine for the treatment
of non-small cell lung carcinoma and pancreatic cancer.
Although various treatment options are available for the treatment of cancers, this
disease still remains one of the most fatal diseases. Although, all the types of cancers are
fatal, breast cancer still remains a type of fatal cancer. In fact, in women, breast cancer is
among the most common cancers and is the fifth most common cause of cancer deaths.
Different forms of breast cancers can have remarkably different biological characteristics and
clinical behavior. Thus, classification of a patient's breast cancer has become a critical
component for determining a treatment regimen. Breast cancer patients fall into three main
groups:
(i) those with hormone receptor-positive tumors who are managed with a number of
estrogen receptor (ER)- targeted therapy options ± chemotherapy;
(ii) those with HER2+ tumors, who will, in addition, receive HER2-directed therapy with
trastuzumab or, in some situations, lapatinib; and
(iii) those with hormone receptor [ER and progesterone receptor (PR)] -negative and
HER2) breast cancers, for whom chemotherapy is the only modality of systemic
therapy available.
Currently, trastuzumab has been developed as a targeted therapy for breast cancer
patients. Studies have shown that the expression profiles of breast cancer display a systematic
variation and allow classification of breast cancer into five main groups, two of them ER+
(luminal A and B) and three ER- groups [normal breast-like, ERBB2 (also known as HER2)
and 'basal-like']. It has been shown that the basal-like group is enriched for tumors that lack
expression of hormone receptors and of HER2 and has a more aggressive clinical behavior, a
distinctive metastatic pattern and a poor prognosis despite responding to conventional
neoadjuvant and adjuvant chemotherapy regimens. Based on the above it is clear that the
interest in triple-negative breast cancers stems from (i) the lack of tailored therapies for this
group of breast cancer patients and (ii) overlap with the profiles of basal-like cancers
(Histopathology, 2008, 52, 108-118).
Triple-negative breast cancer (TNBC) i.e. tumors that are estrogen receptor (ER)-
negative and progesterone receptor (PR)-negative and do not overexpress human epidermal
growth factor receptor 2 (HER2) account for approximately 15 % of breast cancers, with
approximately 170,000 cases reported worldwide in 2008. Triple-negative breast cancers are
significantly more aggressive (metastatic) than tumors pertaining to other molecular
subgroups. TNBC does not express estrogen (ER), progesterone (PR) and HER2 receptors,
therefore, they are resistant to currently available targeted treatment, including hormonal and
HER2-targeted therapies. Patients with basal-like or triple negative cancers have a
significantly shorter survival following the first metastatic event when compared with those
with non-basal-like/no-triple negative patients. A vast majority of tumors arising in BRCA1
germ-line mutation carriers have morphological features similar to those described in basallike
cancers and they display a triple negative and basal like phenotype.
TNBC constitutes one of the most challenging groups of breast cancers. The only
systemic therapy currently available for patients with such cancers is chemotherapy.
However, the survival of patients with such tumors is still poor and their management may,
therefore, require a more aggressive intervention. As a result the development of targeted
therapies for TNBC is of considerable importance. Recent trials have shown that poly (ADPribosyl)
ation polymerase (PARP) inhibitor, BSI-201 (currently known as Iniparib developed
by Sanofi-Aventis) is highly effective in TNBC (Maturitas, 2009, 63, 269-274). Also TNBC
is characterized by elevated levels of PARP. These characteristics have suggested that PARP
inhibition might be able to potentiate the effects of chemotherapy-induced DNA damage in
TNBC (Community Oncology, 2010, 7, 5, 2, 7-10; Clinical Advances in Hematology and
Oncology, 7, 7, 441-443).
Although triple-negative breast cancers are reported to respond to chemotherapy,
survival of patients with such tumors is still poor and their management may therefore require
a more aggressive alternative intervention. Thus, the development of biologically informed
systemic therapies and targeted therapies for triple-negative breast cancers is of paramount
importance and may prove to be achievable by understanding the complexity of this
heterogeneous group of tumors and using combination therapy (Histopathology, 2008, 52,
108-118).
In view of the above discussion and considering that treatment options for treating
triple negative breast cancer are very limited, a need remains for additional treatment options
and methods for treating TNBC.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a pharmaceutical combination
comprising a therapeutically effective amount of paclitaxel, or its pharmaceutically
acceptable salt; and a therapeutically effective amount of a cyclin dependent kinase (CDK)
inhibitor represented by a compound of formula I (as described herein) or a pharmaceutically
acceptable salt thereof, for use in the treatment of triple negative breast cancer (TNBC).
In one aspect, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel, or a pharmaceutically acceptable salt thereof; in combination with a
therapeutically effective amount of a cyclin dependent kinase (CDK) inhibitor represented by
a compound of formula I (as described herein) or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel, or its pharmaceutically acceptable salt; followed by a therapeutically
effective amount of the CDK inhibitor represented by a compound of formula I or a
pharmaceutically acceptable salt thereof, to the subject.
In a further aspect, the present invention relates to use of a pharmaceutical
combination comprising a therapeutically effective amount of paclitaxel or its
pharmaceutically acceptable salt and a therapeutically effective amount of a CDK inhibitor
represented by the compound of formula I or a pharmaceutically acceptable salt thereof for
the treatment of triple negative breast cancer.
In yet another further aspect, the present invention relates to use of a pharmaceutical
combination comprising paclitaxel or its pharmaceutically acceptable salt and a CDK
inhibitor represented by the compound of formula I or a pharmaceutically acceptable salt
thereof; for the manufacture of a medicament for treating triple negative breast cancer
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
Figure 1: Effect of Compound A on colony formation in breast cancer cell lines (MDAMB-
231, MDA-MB-468 and MCF-7)
Figure 2 : Effect of Compound A on MCTS formation in MCF-7 breast cancer cell line
Figure 3A: Time dependent effect of Compound A on cell cycle progression and
apoptosis in MCF-7 (Her2-, BRCA +/- allelic loss) cell line
Figure 3B: Time dependent effect of Compound A on cell cycle progression and
apoptosis in MDA-MB-231 cell line
Figure 4 : Expression of antiapoptotic protein Bcl-2 in MCF-7 and MDA-MB-231 cell
lines treated with Compound A
Figure 5A: Effect of Compound A on MDA-MB-231 cell line (different phases of the cell
cycle)
Figure 5B: Effect of Compound A on MDA-MB-468 cell line
Figure 5C: Effect of BSI-201 on TNBC MDA-MB-231 and MDA-MB-468 cell lines
Figure 6A: Cyclin Dl level in various breast cancer cell lines
Figure 6B: Effect of Compound A on MCF-7 cell cycle proteins and CDK4 kinase
activity
Figure 7 : Effect of Compound A on PARP enzyme activity in breast cancer cell lines
(MDA-MB-231 and MDA-MB-468) as measured by PAR polymers
Figure 8 : Effect of Compound A (24 h) on PARP and cell cycle proteins in two TNBC
cell lines (MDA-MB-231 and MDA-MB-468)
Figure 9: Effect of Compound A on HIF- inhibition in the U251 HRE and U251
pGL3 cell lines
Figure 10: Effect of Compound A on VEGF inhibition using the VEGF reporter gene
based assay
Figure 11A: Effect of Compound A on the migration of BT-549 breast cancer cell line
Figure 11B: Effect of Compound A on the migration of MDA-MB-231 breast cancer cell
line
Figure 11C: Effect of Compound A on the migration of MCF-7 breast cancer cell line
Figure 12: Effect of Compound A on endothelial tube formation as observed in
Endothelial Cell Tube Formation Assay
Figure 13: Effect of the combination of Paclitaxel for 24 h followed by complete medium
(CM)-Group IA / Compound A (IC50)-Group IVA/ Sunitinib (IC50)-Group
VA for 72 h in MDA-MB-231 cell line
Figure 14: Effect of the combination of Paclitaxel for 24 h followed by complete medium
(CM) - Group IB / Compound A (IC50) - Group IVB / Sunitinib (IC50) -
Group VB for 72 h in BT-549 cell line
Figure 15: Effect of the combination of Paclitaxel for 24 h followed by Complete
medium (CM) - Group IC / Compound A (IC50) - Group IVC / Sunitinib
(IC50) - Group VC for 72 h in MDA-MB-468 cell line
DETAILED DESCRIPTION OF THE INVENTION
It has now been found that the pharmaceutical combination of the present invention,
which comprises paclitaxel, or its pharmaceutically acceptable salt and a CDK inhibitor
selected from the compound of formula I (as described herein) or a pharmaceutically
acceptable salt thereof; exhibits synergistic effect when used in the treatment of triple
negative breast cancer.
In particular, the present invention provides a method of treating, or managing triple
negative breast cancer in a subject comprising administering to the subject a therapeutically
effective amount of paclitaxel in combination with a therapeutically effective amount of a
CDK inhibitor selected from the compounds of formula I .
The CDK inhibitor comprised in the pharmaceutical combination of the present
invention is selected from the compound of formula I as described herein. The CDK
inhibitors represented by the following formula I are disclosed in PCT Patent Publication No.
WO2004004632 (corresponding to U.S. Patent 7,272,193) and PCT Patent Publication No.
WO2007148158, which are incorporated herein by reference. The compounds of formula I
are CDK inhibitors, which inhibit proliferation of different cancer cells. The compounds of
formula I comprised in the pharmaceutical combination of the present invention are effective
against various solid and hematological malignancies. The inventors of the present invention
observed that combining the compounds of formula I with paclitaxel resulted in an increase
in apoptosis, or programmed cell death.
The CDK inhibitors used in the present invention are selected from the compounds
represented by the following formula I,
Formula I
wherein Ar is a phenyl group, which is unsubstituted or substituted by 1, 2, or 3 identical or
different substituents selected from : halogen selected from chloro, bromo, fluoro or iodo;
nitro, cyano, Ci-C4-alkyl, trifluoromethyl, hydroxy, Ci-C4-alkoxy, carboxy, C1-C4-
alkoxycarbonyl, CONH2 or NRiR2;
wherein and R2 are each independently selected from hydrogen or Ci-C4-alkyl.
Compounds of Formula (I) may be prepared according to the methods disclosed in
PCT Publication No. WO2004004632 and PCT Publication No. WO2007148158 which are
incorporated herein by reference.
The general process for the preparation of the compounds of Formula (I), or a
pharmaceutically acceptable salt thereof, comprises the following steps:
(a) treating the resolved enantiomerically pure (-)-trans enantiomer of the intermediate
compound of Formula VIA,
with acetic anhydride in the presence of a Lewis acid catalyst to obtain a resolved acetylated
compound of Formula VIIA,
V I IA
(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^O
or an ester of Formula ArCOOCI¾, 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;
V INA
(c) treating the resolved compound of Formula VIIIA with a base in a suitable solvent to
obtain the corresponding resolved -diketone compound of Formula IXA;
wherein Ar is as defined above;
(d) treating the resolved -diketone compound of Formula IXA with an acid such as
hydrochloric acid to obtain the corresponding cyclized compound of Formula XA,
XA
(e) subjecting the compound of Formula XA to dealkylation by heating it with a dealkylating
agent at a temperature ranging from 120-180 °C to obtain the (+)-trans enantiomer of the
compound of Formula (I) and, optionally, converting the subject compound into its
pharmaceutically acceptable salt.
The Lewis acid catalyst utilized in the step (a) above may be selected from: BF3 Et20 ,
zinc chloride, aluminium chloride and titanium chloride.
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).
It will be apparent to those skilled in the art that the rearrangement of the compound
of Formula VIIIA to the corresponding -diketone compound of Formula IXA is known as a
Baker-Venkataraman rearrangement (J. Chem. Soc, 1933, 1381 and Curr. ScL, 1933, 4,
214).
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.
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.
Preparation of the starting compound of Formula VIA involves reacting l-methyl-4-
piperidone with a solution of 1,3,5-trimethoxybenzene in glacial acetic acid, to yield 1-
methyl-4-(2,4,6-trimethoxyphenyl)-l,2,3,6-tetrahydropyridine, which is reacted with boron
trifluoride diethyl etherate, sodium borohydride and tetrahydrofuran to yield l-methyl-4-
(2,4,6-trimethoxyphenyl)piperidin-3-ol. Conversion of l-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, l-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.
In an embodiment the CDK inhibitor is a compound of formula I wherein the phenyl
group is substituted by 1, 2, or 3 identical or different substituents selected from: halogen
selected from chlorine, bromine, fluorine or iodine; Ci-C4-alkyl and trifluoromethyl.
In another embodiment, the CDK inhibitor is a compound of formula I wherein the
phenyl group is substituted by 1, 2, or 3 halogens selected from chlorine, bromine, fluorine or
iodine.
In another embodiment, the CDK inhibitor is a compound of formula I wherein the
phenyl group is substituted by chlorine.
In a further embodiment, the CDK inhibitor represented by compound of formula I is
(+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-
chromen-4-one or its pharmaceutically acceptable salt.
In a still further embodiment, the CDK inhibitor represented by compound of formula
I is (+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-
yl)-chromen-4-one hydrochloride (designated herein as compound A).
In another embodiment, the CDK inhibitor is a compound of formula I wherein the
phenyl group is disubstituted with a chloro and a trifluoromethyl group.
In a further embodiment, the CDK inhibitor represented by compound of formula I is
(+)-ira«i-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2 -hydroxymethyl-1 -methyl
-pyrrolidin-3-yl)-chromen-4-one; or its pharmaceutically acceptable salt.
In a still further embodiment, the CDK inhibitor represented by compound of formula
I is (+)-ira«i-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-lmethyl-
pyrrolidin-3-yl)-chromen-4-one hydrochloride (designated herein as compound B).
In an embodiment, the CDK inhibitor represented by a compound of formula I is an
antiangiogenic agent.
In an embodiment, the CDK inhibitor represented by a compound of formula I is a
HIF- inhibitor. In an embodiment, the CDK inhibitor represented by a compound of
formula I is a VEG-F inhibitor. In an embodiment, the CDK inhibitor represented by a
compound of formula I is a PARP enzyme inhibitor.
The manufacture of the compounds of formula I, which may be in the form of
pharmaceutically acceptable salts, and the manufacture of oral and/or parenteral
pharmaceutical composition containing the above compounds are disclosed in PCT
Publication No. WO2004004632 (corresponding to U.S. Patent 7,272,193) and PCT
Publication No. WO2007148158. These PCT Publications disclose that the CDK inhibitors
represented by formula I inhibit proliferation of many cancer cells. As indicated herein above
the CDK inhibitors of formula I may be used in the form of their salts. Preferred salts of the
compounds of formula I include hydrochloride salt, methanesulfonic acid salt and
trifluoroacetic acid salt.
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 as described above, by
methods disclosed in PCT Publication Nos. WO2004004632, WO2008007169 and
WO2007148158 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. The term
"enantiomerically pure" describes a compound which is present in an enantiomeric excess
(ee) of greater than 95 . In another embodiment, the enantiomeric excess is greater than
97%. In still another embodiment, the enantiomeric excess is greater than 99 %. The term
"enantiomeric excess" describes the difference between the amount of one enantiomer and
the amount of another enantiomer that is present in the product mixture.
Alternatively, the enantiomers of the compounds of formula I can be synthesized by
using optically active starting materials. Thus, the definition of the compounds of formula I is
inclusive of all possible stereoisomers and their mixtures. The definition of the compounds of
formula I includes the racemic forms and the isolated optical isomers having the specified
activity.
Paclitaxel, a cytotoxic antineoplastic agent comprised in the pharmaceutical
combination of the present invention, is a natural diterpene product isolated from the Pacific
yew tree Taxus brevifolia (Rowinsky et. al., J . Natl. Cancer Inst., 82, 1247-1259 (1990)).
Isolation of paclitaxel and its structure is disclosed in J . Am. Chem. Soc. 93, 2325 (1971). It
is an antimicrotubule agent that promotes the assembly of microtubules from tubulin dimers
and stabilizes microtubules by preventing depolymerization. Paclitaxel is used to treat
patients with lung, ovarian, breast cancer, head and neck cancer, and advanced forms of
Kaposi's sarcoma. Paclitaxel has been approved for clinical use in the treatment of ovarian
cancer (Merkman et al.; Yale Journal Of Biology and Medicine, 64:583, 1991) and for the
treatment of breast cancer (Holmes et al; J . Nat. cancer Inst., 83; 1797, 1991), however, it is
also useful in treating other cancers for example, it has been considered as a potential
candidate for the treatment of head and neck cancer (Forastire et. al., Sem. Oncol., 20: 56,
1990) and lung cancer (M. Ghaemmaghami et al; Chest; 113; 86-91 (1998)). Paclitaxel is
disclosed in U. S. Pat. No. 5,670,537 which is incorporated herein by reference for its
teaching on the use or administration of paclitaxel in the treatment of susceptible cancers.
Paclitaxel is commercially available as an injectable solution, Taxol®. A formulation in
which paclitaxel is bound to albumin is sold under the trademark, Abraxane® (Abraxis
Bioscience, Inc.).
The general terms used hereinbefore and hereinafter preferably have the following
meanings within the context of this disclosure, unless otherwise indicated:
As used herein, the term "combination" or "pharmaceutical combination", means the
combined administration of the anticancer agents namely paclitaxel and the CDK inhibitor
(the compound of formula I) ; which anti-cancer agents may be administered independently
at the same time or separately within time intervals that especially allow that the combination
partners show a synergistic effect.
As used herein, the term "synergistic" means that the effect achieved with the
methods and combinations of this invention is greater than the sum of the effects that result
from using paclitaxel or a pharmaceutically acceptable salt thereof, and a CDK inhibitor, the
compound of formula I or a pharmaceutically acceptable salt thereof, separately.
Advantageously, such synergy provides greater efficacy at the same doses, and/or prevents or
delays the build-up of multi-drug resistance.
A "therapeutically effective amount", in reference to the treatment of triple negative
breast cancer, 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 cancerous 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 or rejection of the tumor; and/or (vii) relief, to some extent, of one or more
symptoms associated with triple negative breast cancer.
As used herein, the terms "manage", "managing" and "management" refer to the
beneficial effects that a subject or a patient derives from the pharmaceutical combination of
the present invention when administered to said patient or subject so as to prevent the
progression or worsening of TNBC.
As used herein the term "triple negative breast cancer(s)" or "TNBC" encompasses
carcinomas of differing histopathological phenotypes. For example, certain TNBC are
classified as "basal-like" ("BL"), in which the neoplastic cells express genes usually found in
normal basal/myoepithelial cells of the breast, such as high molecular weight basal
cytokeratins (CK, CK5/6, CK14, CK17), vimentin, p-cadherin, ccB crystallin, fascin and
caveolins 1 and 2. Certain other TNBC, however, have a different histopathological
phenotype, examples of which include high grade invasive ductal carcinoma of no special
type, metaplastic carcinomas, medullary carcinomas and salivary gland-like tumors of the
breast. The TNBC for the treatment of which the pharmaceutical combination of the present
invention is provided may be non-responsive or refractory TNBC.
The term "non-responsive/refractory" as used herein, is used to describe subjects or
patients having triple negative breast cancer(TNBC) having been treated with currently
available cancer therapies for the treatment of TNBC such as chemotherapy, radiation
therapy, surgery, hormonal therapy and/or biological therapy/immunotherapy wherein the
therapy is not clinically adequate to treat the patients such that these patients need additional
effective therapy, e.g., remain unsusceptible to therapy. The phrase can also describe subjects
or patients who respond to therapy yet suffer from side effects, relapse, develop resistance,
etc. In various embodiments, "non-responsive/refractory" means that at least some significant
portions of the cancer cells are not killed or their cell division arrested. The determination of
whether the cancer cells are "non-responsive/refractory" can be made either in vivo or in vitro
by any method known in the art for assaying the effectiveness of treatment on cancer cells,
using the art-accepted meanings of "refractory" in such a context. A cancer is "nonresponsive/
refractory" where the number of cancer cells has not been significantly reduced,
or has increased.
As used herein the term "treatment cycle" refers to a time period during which a
recurring sequence of administration of paclitaxel or a pharmaceutically acceptable salt
thereof, and a CDK inhibitor of the compound of formula I or a pharmaceutically acceptable
salt thereof, is carried out.
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)
As used herein 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.
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.
In one embodiment, the present invention relates to a method for the treatment of
triple negative breast cancer in a subject comprising administering to the subject a
therapeutically effective amount of paclitaxel or its pharmaceutically acceptable salt and a
cyclin dependent kinase (CDK) inhibitor selected from the compounds of formula I (as
described herein) or a pharmaceutically acceptable salt thereof.
Accordingly, in the method of the present invention, triple negative breast cancer is
treated in a subject by administering to the subject in need thereof, a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt, in combination with a
therapeutically effective amount of a CDK inhibitor selected from the compounds of formula
I or a pharmaceutically acceptable salt thereof, wherein a synergistic effect results.
In an embodiment, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective
amount of a CDK inhibitor selected from the compounds of formula I or a pharmaceutically
acceptable salt thereof, wherein paclitaxel and said CDK inhibitor are administered
sequentially.
In an embodiment, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective
amount of the CDK inhibitor selected from the compounds of formula I or a pharmaceutically
acceptable salt thereof, wherein paclitaxel is administered prior to the administration of said
CDK inhibitor.
In an embodiment, the method of treating triple negative breast cancer of the present
invention comprises administering paclitaxel and the CDK inhibitor in the dose range
described herein.
In an embodiment, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective
amount of the CDK inhibitor selected from the compound A or compound B.
In an embodiment, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective
amount of the CDK inhibitor selected from the compound A or compound B, wherein
paclitaxel and said compound A or compound B are administered sequentially.
In an embodiment, the present invention relates to a method of treating triple negative
breast cancer in a subject comprising administering to the subject a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective
amount of the CDK inhibitor selected from the compound A or compound B, wherein
paclitaxel is administered prior to the administration of the compound A or compound B.
In an embodiment, the present invention relates to a pharmaceutical combination for
use in the treatment of triple negative breast cancer, wherein said pharmaceutical
combination comprises a therapeutically effective amount of paclitaxel or its
pharmaceutically acceptable salt and a therapeutically effective amount of the CDK inhibitor
selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.
In an embodiment, the present invention relates to a pharmaceutical combination for
use in the treatment of triple negative breast cancer, wherein said pharmaceutical
combination comprises a therapeutically effective amount of paclitaxel or its
pharmaceutically acceptable salt and a therapeutically effective amount of the CDK inhibitor
selected from the compounds of formula I or a pharmaceutically acceptable salt thereof,
wherein paclitaxel and said CDK inhibitor are administered sequentially.
In an embodiment, the present invention relates to a pharmaceutical combination for
use in the treatment of triple negative breast cancer, wherein said pharmaceutical
combination comprises a therapeutically effectively amount of paclitaxel or its
pharmaceutically acceptable salt and a therapeutically effective amount of the CDK inhibitor
selected from the compounds of formula I or a pharmaceutically acceptable salt thereof,
wherein paclitaxel is administered prior to the administration of the CDK inhibitor.
In an embodiment, the present invention relates to the use of a pharmaceutical
combination for the manufacture of a medicament for use in the treatment of triple negative
breast cancer, wherein said pharmaceutical combination comprises a therapeutically effective
amount of paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective
amount of the CDK inhibitor represented by a compound of formula I or a pharmaceutically
acceptable salt thereof.
In an embodiment, the CDK inhibitor comprised in the pharmaceutical combination
provided for use in the treatment of triple negative breast cancer, is selected from the
compound A or compound B.
In an embodiment, the CDK inhibitor comprised in the pharmaceutical combination is
the compound A.
In an embodiment, the CDK inhibitor comprised in the pharmaceutical combination is
the compound B.
In an embodiment, the anticancer agents comprised in the pharmaceutical
combination of the present invention may require different routes of administration, because
of their different physical and chemical characteristics. For example, the CDK inhibitors of
Formula I may be administered either orally or parenterally to generate and maintain good
blood levels thereof, while the antineoplastic agent may be administered parenterally, by
intravenous, subcutaneous or intramuscular route.
For oral use, the CDK inhibitors of formula I may be administered, for example, in
the form of tablets or capsules, powders, dispersible granules, or cachets, or as aqueous
solutions or suspensions. 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 (paclitaxel or the CDK inhibitor) are usually employed, and the pH of
the solutions should be suitably adjusted and buffered.
In an embodiment, the sterile solutions of the active ingredient used are prepared in saline or
distilled water.
The actual dosage of the active ingredients i.e. the anticancer agents contained in the
combination may be varied depending upon the requirements of the patient and the severity
of the condition being treated. Generally, treatment is initiated with smaller doses, which are
less than the optimum dose of the compound. Thereafter, the dose of each ingredient is
increased by small amounts until the optimum effect under the circumstances is reached.
However, the amount of each ingredient in the pharmaceutical combination will typically be
less than an amount that would produce a therapeutic effect if administered alone. For
convenience, the total daily dose may be divided and administered in portions during the day
if desired. In an embodiment, paclitaxel or its pharmaceutically acceptable salt, and a CDK
inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt
thereof are administered sequentially in injectable forms, such that paclitaxel is administered
in a synergistically effective dose ranging from 10 mg to 1000 mg each, and the CDK
inhibitor is administered in a synergistically effective dose ranging from 5 mg/m2/day to 1000
mg/m2/day, particularly in a dose ranging from 9 mg/m2/day to about 259 mg/m2/day.
In an embodiment, the pharmaceutical combination provided for use in the treatment
of triple negative breast cancer is administered to a subject in need thereof, for six to eight
treatment cycles, particularly six treatment cycles; two consecutive treatment cycles
comprising the following steps:
i) a single dose administration of the pharmaceutical combination of paclitaxel and
Compound A on day one of the treatment cycle;
ii) from second day, administration of one dose per day of Compound A for four
consecutive days;
iii) a two- day interval wherein no drug (anticancer agent) is administered;
iv) optional administration of Compound A for five consecutive days followed by twoday
interval with no drug (anticancer agent) administration;
v) optionally repeating step iv); and
vi) repeating steps i) to v) as a second treatment cycle, after an interval of three weeks
from the beginning of step i).
In an embodiment, the pharmaceutical combination is administered to a subject in
need thereof, for two to six treatment cycles, before surgery or after surgery or partially
before and partially after surgery.
The combinations provided by this invention have been evaluated in certain assay
systems, and in several different administrative schedules in vitro. The experimental details
are as provided herein below. The data presented herein clearly indicate that paclitaxel when
combined with a CDK inhibitor selected from the compounds of formula I exhibit synergistic
effect. It is clearly indicated that the anticancer agents when used in combination in the
treatment of triple negative breast cancer increases apoptosis or cytotoxicity in proliferative
cells than when the cells are treated with only the CDK inhibitor, the compound of formula I
alone or paclitaxel alone.
The representative compound, the compound A used in the pharmacological assays
refers to (+)-ira«s-2-(2-Chloro-phenyl)-5,7-dmydroxy-8-(2-hydroxymethyl- 1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride and was one of the compounds disclosed in
the published PCT Publication No. WO2004004632, incorporated herein by reference.
The synergistic effect of the combination of the present invention comprising
paclitaxel and a CDK inhibitor is now explained in more detail with reference to preferred
embodiments thereof. It is to be noted that these are provided only as examples and not
intended to limit the invention.
The following abbreviations or terms are used herein:
ATCC : American Type Culture Collection, USA
ATP : Adenosine triphosphate
CHC13 : Chloroform
CDCI3 : Deuteriated chloroform
CO2 : Carbon dioxide
CoA : Coenzyme A (Sigma Aldrich, USA)
DCC : N, N'-dicyclohexyl carbodiimide
DBTA : Dibenzoyl tartaric acid
DMAP : 4-Dimethylaminopyridine
DMF : N, N-dimethylformamide
DMSO : Dimethylsulfoxide
DNA : Deoxyribonucleic acid
DTT : Dithiothreitol (Sigma Aldrich, USA)
EDTA : Ethylene diamine tetra acetic acid
EtOAc : Ethyl acetate
FBS : Fetal bovine serum (Gibco, USA)
FCS : Fetal calf serum (Gibco, USA)
g : Gram
h : Hour
HC1 : Hydrochloric acid
IPA : Isopropyl alcohol
KBr : Potassium bromide
Kg : Kilogram
L : Litre
MgS0 4 : Magnesium sulfate
MeOH : Methanol
Min Minute(s)
mL Millilitre
Microlitre
Micromolar
mmol Millimolar
mol Mole
Na2C0 3 Sodium carbonate
Na2S0 4 Sodium sulfate
NaBH4 Sodium borohydride
NaOH Sodium hydroxide
NCI National Cancer Institute, USA
°C Degree Centigrade
PARP Poly (ADP-ribose) polymerase
PBS Phosphate buffered saline (Sigma Aldrich, USA)
PI Propidium iodide (Sigma Aldrich, USA)
RPMI Roswell Park Memorial Institute, USA
SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electroph
TFA Trifluoroacetic acid
THF Tetrahydrofuran
Cell-lines (Source: ATCC, USA):
TNBC Triple negative breast cancer
MCF-7 (HER low, ER+, PR+, BRCA +/- allelic loss) breast
cancer cell-line
T47-D (HER low, ER +, PR +) breast cancer cell-line
ZR-75-1 (HER low, ER +, PR +) breast cancer cell-line
MDA-MB-468 (HER-, ER-, PR-) triple negative breast cancer cell-line
MDA-MB-231 (HER-, ER-, PR-) triple negative breast cancer cell-line
MDA-MB-435-S (HER-, ER-, PR-) triple negative breast cancer cell-line
MDA-MB-361 (HER-, ER-, PR-) triple negative breast cancer cell-line
HBL-100 (HER-, ER-, PR-) triple negative breast cancer cell-line
BT-549 (HER-, ER-, PR-) triple negative breast cancer cell-line
HUVEC Human umbilical vein endothelial cells
Cell-lines (Source: NCI. USA):
U251 HRE Genetically engineered glioblastoma cells
U25 1 pGL3 : Genetically engineered glioblastoma cells
Antibodies (Source: Cell Signaling Technology, USA):
Cyclin Dl (cell cycle protein)
Bcl-2 (anti-apoptotic protein)
CDK4 (cyclin dependent kinase-4)
Rb (Retinoblastoma)
pRb Ser780 (phospho-retinoblastoma)
PAR (substrate of PARP enzyme)
PARP (Poly (ADP-ribose) polymerase)
-actin (house -keeping protein and used as a loading control for Western blot analysis)
Incubation conditions for cell-lines: 37 °C and 5 % CO2
The invention is further described by the following non-limiting examples which
further illustrate the invention, and are not intended, nor should they be interpreted to, limit
the scope of the invention.
Examples :
Example 1:
Preparation of (+)-t raras-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxy methyl -1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A)
Sodium hydride (50 , 0.54 g, 11.25 mmol) was added in portions to a solution of (-)-transl-[
2-Hydroxy-3-(2 -hydro xymethyl-l -methylpyrrolidin-3-yl)-4,6-dimethoxy phenyl)-
ethanone (0.7 g., 2.2 mmol) in dry DMF (15 mL) at 0 °C, under nitrogen atmosphere and
with stirring. After 10 min., methyl 2-chlorobenzoate (1. 15 g., 6.75 mmol) was added. The
reaction mixture was stirred at 25 °C for 2 h. Methanol was added carefully below 20 °C. The
reaction mixture was poured over crushed ice (300 g), acidified with 1:1 HCl (pH 2) and
extracted using EtOAc (2 x 100 mL). The aqueous layer was basified using a saturated
Na2C03 (pH 10) and extracted using CHCI3 (3 x 200 mL). The organic layer was dried
(anhydrous Na2S0 4) and concentrated. To the residue, cone. HCl (25 mL) was added and
stirred at room temperature for 2 h. The reaction mixture was poured over crushed ice (300 g)
and made basic using a saturated aqueous Na2C03 solution. The mixture was extracted using
CHCI3 (3 x 200 mL). The organic extract was washed with water, dried (anhydrous Na2S0 4)
and concentrated to obtain the compound, (+)-ira«s-2-(2-chloro-phenyl)-8-(2-
hydroxymethyl- l-methyl-pyrrolidin-3-yl)-5,7-di^ [Yield: 0.67 g (64
) ; mp: 91- 93°C; [ ]D
25 = + 5.8° (c = 0.7, methanol)]
Molten pyridine hydrochloride (4. 1 g, 35.6 mmol) was added to (+)-trans-2-(2-
chloro-phenyl)-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one
(0.4 g, 0.9 mmol) and heated at 180 °C for 1.5 h. The reaction mixture was cooled to 25 °C,
diluted with MeOH (10 mL) and basified using Na2C0 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, (+)-ira«s-2-(2-chloro-phenyl)-5,7-
dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one. [Yield: 0.25 g (70
) ]
(+)-ira«i-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-
chromen-4-one (0.2 g, 0.48 mmol) was suspended in IPA (5 mL) and 3.5 % HC1 (25 mL) was
added. The suspension was heated to get a clear solution. The solution was cooled and solid
filtered to obtain the compound, (+)-ira«s-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl- l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride or Compound A.
Yield: 0.21 g (97 ) ; mp: 188 - 192 °C ; [ ]D
25 = +21 .3° (c = 0. 2, methanol);
Example 2 :
Preparation of (+)-t r «s-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-
hydroxy- methyl -l-methylpyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound
B)
A mixture of the compound of ira«s-l-[2-Hydroxy-3-(2-hydroxymethyl- l -
methylpyrrolidin-3-yl)-4,6-dimethoxy phenyl)- ethanone (1. 16 g, 3.2 mmol), 2-chloro-4-
trifluoromethylbenzoic acid (0.88 g, 4 mmol), DCC ( 1.35 g, 6.5 mmol) and DMAP (0.4 g,
3.27 mmol) were dissolved in dichloromethane (50 mL) and stirred at room temperature for
12 h. The reaction mixture is cooled to 0 °C, the precipitated dicyclohexylurea was filtered
and the organic layer concentrated and residue purified by column chromatography with 1 %
methanol in chloroform and 0.01 % ammonia as eluent to obtain the compound, (+)-trans-2-
chloro-4-trifluoromethylbenzoic acid 2-(2-acetoxymethyl- 1-methyl-pyrrolidin-3-yl)-6-acetyl-
3,5-dimethoxyphenyl ester [Yield: 1.44 g (78.8 )].
To a solution of n-BuLi (15% solution in hexane, 2.2 mL, 5 mmol) in THF (10 mL),
maintained at 0 °C under nitrogen atmosphere, hexamethyldisilazane ( 1.08 mL, 5.1 mmol)
was added dropwise and stirred for 15 min. To this, a solution of (+)-ira«s-2-chloro-4-
trifluoromethylbenzoic acid 2-(2-acetoxymethyl-l-methyl-pyrrolidin-3-yl)-6-acetyl-3,5-
dimethoxyphenyl ester (1.44 g, 2.5 mmol) in THF (10 mL) was added dropwise, maintaining
the temperature at 0 °C. After the addition, the reaction was allowed to warm to room
temperature and stirred for 2.5 h. The reaction mixture was acidified with dilute HC1, and
basified with 10 % sodium bicarbonate to pH 8 to 9. The aqueous layer was extracted with
chloroform (3 x 25 mL). The organic layer was washed with water (25 mL), brine (25 mL)
and dried over anhydrous Na2S0 4. The organic layer was concentrated under reduced
pressure and dried under vacuum to yield acetic acid 3-{3-[3-(2-chloro-4-trifluromethylphenyl)-
3-oxo-propionyl] -2-hydroxy-4,6-dimethoxy-phenyl }-l-methyl-pyrrolidin-2-ylmethyl
ester as an oil (1.3 g, 90.2 ). This ester was dissolved in cone. H (10 mL) and stirred for
3 h to effect cyclisation. At the end of 3 h, the reaction mixture was basified with solid
NaHCC>3 to pH 8 to 9. The aqueous layer was extracted with chloroform (25 x 3 mL) and
washed with water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous
Na2S0 4, concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.1 % ammonia as
eluent to yield the compound , (+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-
hydroxymethyl- 1-methyl pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one as a yellow solid.
[Yield: 0.56 g (48.2 )]
A mixture of (+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-lmethyl
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 °C
for a period of 2.5 h. The reaction mixture was diluted with methanol (25 mL) and basified
with solid Na2C0 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,
(+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy- methyl-1-
methylpyrrolidin-3-yl)-chromen-4-one, as a yellow solid. [Yield: 0.15 g (63.7 )]
(+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy- methyll-
methylpyrrolidin-3-yl)-chromen-4-one (0.1 g, 0.2 mmol) was suspended in methanol (2
mL) and treated with ethereal HC1 and the organic solvent evaporated to yield the compound,
(+)-ira«i-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-lmethyl-
pyrrolidin-3-yl)-chromen-4-one hydrochloride. [ Yield: O.lg (92.8 ) ]
Pharmacological Assays:
Example 3 :
Cytotoxicity assay using Propidium Iodide (PI)
The propidium iodide fluorescence assay (PI) was carried out according to the
procedure mentioned in Anticancer Drugs, 1995, 6, 522-32.
The assay was developed to characterize the in vitro growth of human tumor cell lines
as well as to test the cytotoxic activity of the compounds. Propidium iodide (PI) was used as a
dye, which penetrates only damaged cellular membranes. Intercalation complexes are formed
by PI with double-stranded DNA, which effect an amplification of the fluorescence. After
freezing the cells at -20 °C for 24 h, PI had access to total DNA leading to total cell
population counts. Background readings were obtained from cell-free wells containing media
and propidium iodide.
The human breast cancer cell lines (i.e. MCF-7, T47-D, ZR-75-1, MDA-MB-468,
MDA-MB-231, MDA-MB-435-S, MDA-MB-361, HBL-100, BT-549) were seeded at a
density of 1500-3000 cells/well in 180 of DMEM (Dulbecco's Modified Eagle's Medium,
Gibco, USA) or RPMI 1460, along with 10 % FCS in a 96-well plate and incubated for about
16 h to allow the cells to adhere. The cells were then treated with varying concentrations of
Compound A (0.1 to 3 ) . The above procedure was repeated in three TNBC cell-lines
(MDA-MB-231, MDA-MB-468 and BT-549) for varying concentrations of the Compound
A, paclitaxel (Sigma Aldrich, USA) and Sunitinib (Sutent®, LC Laboratories, USA), i.e. the
concentration range for Compound A was 0.1-3 , the concentration range for paclitaxel
was 0.1-10 while for Sunitinib (Sutent®), the concentration range was 1-100 , for a
total period of 48 h. The plates were incubated in humidified 5 % CO2 incubator at 37 °C ± 1
°C. Control wells were treated with vehicle (DMSO). At the end of the incubation periods,
the plates were assayed using PI cytotoxicity assay protocol. Percent cytoxicity was
calculated at various drug concentrations and from the graph plotted the IC50 values were
determined. The results of this study are presented in Tables 1A and IB.
Table 1A:
Antiproliferative activity of the Compound A, Paclitaxel and Sunitinib for TNBC
Cell lines (IC50 )
Compounds
MDA-MB-231 BT-549 MDA-MB-468
Compound A 1.0 0.8 1.0
Paclitaxel 0.9 NT NT
Sunitinib
7.8 18 10
(Sutent®)
NT = Not tested
Table 1A shows the IC50 values in for the Compound A, Paclitaxel and Sunitinib
(Sutent®) in MDA-MB-231, BT-549 and MDA-MB-468 determined by cytotoxicity assay
done after 48 h of the compound treatment.
Table IB:
Antiproliferative potential (IC50 in ) of Compound A in various breast cancer cell
lines as measured by PI assay
Table IB shows that the Compound A was found to be efficaciously antiproliferative
against all the breast cancer cell lines irrespective of the genetic markers with IC50 ranging
from 0.3 to 1.0 .
Example 4 :
Clonogenic assay or colony forming assay
MDA-MB-231, MDA-MB-468 and MCF-7 cell lines were seeded in RPMI 1460 with
10 % FCS, at a density of 1500 cells/well in six well plates. After 24 h incubation, cells were
treated with IC10, IC30 and IC50 concentrations of Compound A (as determined by the
procedure of Example 3) for a period of 48 h and the ICio„ IC30 and IC50 values are presented
in Table 2. The medium was removed at the end of the treatment and incubated in fresh
medium (without drug) for 14 days. After 14 days the medium was aspirated and colonies
were fixed with methanol and acetic acid mixture in the proportion of 2:1, rinsed with water
and the fixation procedure was repeated. The plates were dried and colonies stained with 0.1
% crystal violet for 5 min. The wells were finally rinsed with water and dried.
Table 2 :
The results are depicted in Figure 1, which shows the visual enhancement in the
response by IC10 , IC30 and IC50 doses of Compound A, in MDA-MB-231, MDA-MB-468 and
MCF-7 cell lines (Seeding density: 1500 cells/plate).
Compound A was found to inhibit the colony forming potential in a dose dependent
manner.
Example 5 :
Effect of Compound A on Multicellular Tumor Spheroid (3D) formation
The assay was carried out according to the method disclosed in Methods in Molecular
Medicine, 2007, 140, 141-151.
The multicellular tumor spheroid (MCTS) model is one of the best-described 3D in
vitro tumor model systems, which depicts many of the characteristics of tumor tissue and
allows reproducible experiments, offering an excellent in vitro screening system. MCTS
were propagated using the hanging drop method. Briefly, the cell monolayer was detached
using trypsin-EDTA. Cell count was adjusted and 20 hanging droplets containing 1,000
cells/drop, were made in bacterial grade petridishes. These hanging drops were incubated for
24 h at 37 °C in a humidified atmosphere of 5 % CO2. The MCTS thus generated were
cultured in the presence or absence of varying concentrations (0.3 to 30 ) of
Compound A for 72 h.
The results are presented in Figure 2.
When MCF-7 cell suspension was co-incubated with varying concentrations of
Compound A (0.3 to 30 ) for propagation of MCTS, the spheroid formation was
arrested from 3 concentration of Compound A onwards. The size of MCTS formed at 1
was also smaller as compared to control. This observation is important from the clinical
point of view, as MCTS have been characterized sufficiently well to simulate the
pathophysiological milieu in a patient tumor. Due to the gradient of oxygen in spheroids,
which leads to formation of tumor hypoxia, it mimics the microenvironment prevailing in the
tumor tissue. Effect of Compound A on spheroidal formation indicates that Compound A
may be effective under hypoxia conditions.
Example 6 :
Time dependent effect of Compound A on cell cycle progression and apoptosis in MCF-
7 (Her low, ER+, PR+, BRCA +/- allelic loss) and TNBC cell-line MDA-MB-231
Time dependent effect of Compound A on cell cycle progression and apoptosis was
evaluated in two breast cancer cell lines. The asynchronous human breast cancer cell lines
MCF-7 (Her low, ER+, PR+, BRCA +/- allelic loss) and MDA-MB-231 cells were seeded in
25 mm3 tissue culture flask at a density of 0.5 xlO6 cells per flask in RPMI 1460 with 10 %
FCS. After 24 h, cells were treated with 4.5 of Compound A for 0, 24, 48 and 72 h. Both
detached and adherent cells were harvested (trypsinised) at different time points as mentioned
in Table 3. After washing in phosphate buffered saline (PBS), cells were fixed in ice-cold 70
% ethanol and stored at -20 °C until further analysis.
Before analysis, cells were washed twice with PBS to remove the fixative and resuspended
in PBS containing 50 g/mL propidium iodide and 50 g/mL RNaseA. After
incubation at room temperature (20-35 °C) for 20 min, cells were analyzed using flow
cytometry. A Becton Dickinson FACS Calibur flow cytometer (BD, USA) was used for these
studies. The argon ion laser set at 488 nm was used as an excitation source. Cells with DNA
content between 2n and 4n were designated as being in Gl, S and G2/M phases of the cell
cycle, as defined by the level of red fluorescence. Cells exhibiting less than 2n DNA content
were designated as sub-Gl (apoptotic population) cells. The number of cells in each cell
cycle compartment was expressed as a percentage of the total number of cells present. The
results are shown in Table 3 and graphically presented in Figure 3A (MCF-7 cell lines) and
Figure 3B (MDA-MB-231 cell lines).
Table 3 : Percent apoptosis
It is evident from the results shown in the above Table that compound A induced
apoptosis in MCF-7 (Her low, ER+, PR+, BRCA +/- allelic loss) and TNBC cell-line MDAMB-
231. Maximum apoptosis was seen at 48 h and 72 h.
Example 7 :
Effect of Compound A in MCF-7 and MDA-MB-231 cells using Western blot analysis:
The Western blot assay was carried out according to the procedure disclosed in
Molecular Cancer Therapeutics, 2007, 6, 918-925 with some modifications.
MCF-7 and MDA-MB-231 cells were seeded in RPMI 1460 medium with 10 % FCS
in 25 mm3 tissue culture flask and incubated for 24 h. The cells were treated with Compound
A at 1.5 and 4.5 . At various time points, i.e. 6, 24 and 30 h the cells were harvested or
trypsinized and lysed using lysis buffer (Sigma Aldrich, USA). Protein content was
estimated. Lysate was applied to Sodium Dodecyl Sulphate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) followed by western blotting (Molecular Cancer Therapeutics,
2007, 6, 918-925). Western blotting was done using specific antibodies to Bcl-2 and actin.
The results are depicted in Figure 4.
It can be seen from Figure 4 that compound A down regulates antiapoptotic protein
Bcl-2 in a dose dependent manner in both the cell lines. In MCF-7 cells, Bcl-2 is significantly
down regulated from 24 h onwards, while in MDA-MB-231 significant down regulation was
observed at 30 h.
Example 8 :
Effect of Compound A on cell cycle progression and apoptosis:
Comparison of the effect of Compound A and PARP inhibitor BSI-201 (Iniparib
developed by Sanofi-Aventis. BSI-201 is prepared in-house) on cell cycle progression and
apoptosis was evaluated in two TNBC cell lines. The asynchronous human TNBC cell lines
MDA-MB-231 and MDA-MB-468 were seeded in 25 mm3 tissue culture flask at a density of
0.5 xlO6 cells per flask in RPMI 1460 with 10 % FCS. After 24 h, cells were treated either
with 1.5 and 3.0 of Compound A or 50 of PARP inhibitor BSI-201 for 72 h. After
the incubation cells were harvested (trypsinised) and processed as given in example 6. The
results are shown in Tables 4A and 4B; and graphically presented in Figures 5A, 5B and
5C.
Table 4A: Comparative analysis of percentage distribution of cells in different cell cycle
phases and apoptosis in MDA-MB-231 treated with Compound A (a CDK inhibitor) and
BSI-201 (a PARP inhibitor)
Table 4B: Comparative analysis of percentage distribution of cells in different cell cycle
phases and apoptosis in MDA-MB-468 treated with Compound A (a CDK inhibitor) and
BSI-201 (a PARP inhibitor)
The TNBC cell lines MDA-MB-231 and MDA-MB-468 showed dose dependent
increase in apoptosis when treated with Compound A. BSI-201 (at 50 ) showed no
induction of apoptosis in MDA-MB-231. However, marginal apoptosis (12.67 ) was
observed in MDA-MB-468.
Example 9 :
Effect of Compound A on MCF-7 cell cycle proteins and CDK4 kinase activity
Step 1: Basal level of cyclin-Dl expression
Basal level of cyclin-Dl expression was studied using western blot analysis
(Molecular Cancer Therapeutics, 2007, 6, 918-925) across various breast cancer cell lines, i.e.
MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-435 S, MDA-MB-453, BT-549 and
HBL-100. These cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm3 tissue
culture flask and incubated for 24 h. The cells were harvested (trypsinised) and lysed using
lysis buffer. Protein content was estimated. Lysate was applied to Sodium Dodecyl
Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by Western blotting.
Western blotting was done using cyclin Dl antibody and actin is used as a loading control.
The results are shown in Figure 6A. High cyclin Dl levels were observed in most of the
breast cancer cell lines including triple negative breast cancer cell lines.
Step 2 : Effect of Compound A on MCF-7 cell cycle proteins and CDK4 kinase activity
MCF-7 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm3 tissue
culture flask and incubated for 24 h. These cells were treated with Compound A at 1.5 .
At various time points viz. 3 h, 6 h, 9 h, 12 h and 24 h the cells were harvested (trypsinised)
and lysed using lysis buffer. Protein content was estimated by Bradford method (Anal.
Biochem., 1976, 72, 248-254). Lysate was applied to Sodium Dodecyl Sulphate-
Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by Western blotting. Western
blotting was done using specific antibodies to various cell cycle proteins viz. cyclin Dl,
CDK4, Rb and pRbSer780.
For immunoprecipitation assay, the MCF-7 cells were synchronized by serum
starvation. These cells were treated with Compound A at 1.5 at various time points viz. 3
h, 6 h, 9 h and 12 h. Cells were harvested (trypsinised) and lysed using lysis buffer, and
protein content was estimated. CDK4-D1 (Cyclin Dl and CD 4) was purified from the
lysate by immunoprecipitation using specific antibody to CDK4. Immune complex was
further purified using Protein A sepharose beads (Sigma Aldrich, USA). Immune complex
was used to determine CDK4 activity using pRb as a substrate and 2P labelled ATP (BRIT,
India). Reaction mixed was applied to SDS-PAGE followed by transfer and autoradiography.
The results are shown in Figure 6B.
Compound A down regulates cyclin Dl and pRb in MCF-7 (Her low, ER+, PR+,
BRCA +/- with allelic loss) in time dependent manner. Cyclin Dl and pRb expression show
decrease from 6 h onwards and significantly at 12 h. There is no significant change in total
Rb except at 24 h. Decrease in CDK4 kinase activity in cell-based assay was seen as early as
3 h onwards.
Example 10:
Effect of Compound A on PARP enzyme activity as measured by PAR polymers
Poly(ADP-ribose) polymerase (PARP) is the principle member of a family of enzyme
possessing poly(ADP-ribosylation) (PAR) catalytic capacity. To study PARP enzyme
activity, PAR polymer formation was measured. MDA-MB-231 and MDA-MB-468 cells
were seeded in RPMI 1460 medium with 10 % FCS in 25 mm3 tissue culture flask and
incubated for 24 h. These cells were treated at 1.5 and 5 of Compound A for 24 h.
The cells were harvested (trypsinised) and lysed using lysis buffer. Western blotting
(Molecular Cancer Therapeutics, 2007, 6, 918-925) was done with specific antibody to PAR.
The results are shown in Figure 7.
Compound A inhibits PARP enzyme activity as observed by the inhibition of PAR
polymer formation in MDA-MB-231 cell line. However it was observed that in MDA-MB-
468 the formation of PAR polymers is not inhibited.
Example 11:
Effect of Compound A (24 h) on PARP and cell cycle proteins in TNBC cell lines
Correlation of PARP activity and cell cycle proteins cyclin Dl, total Rb and pRb 780
were studied in two TNBC cell lines viz. MDA-MB-468 and MDA-MB-231. MDA-MB-231
and MDA-MB-468 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm3
tissue culture flask and incubated for 24 h. These cells were treated at 1.5 and 5 
Compound A for 24 h. The cells were harvested (trypsinised) and lysed using lysis buffer.
Western blotting was carried out (Molecular Cancer Therapeutically effectives, 2007, 6, 918-
925) using specific antibody to PAR, PARP, cyclin Dl, CDK4 and pRb Ser 780. The results
are shown in Figure 8.
In MDA-MB-231, Compound A inhibits PARP enzyme activity as seen by the
inhibition of PAR polymer formation. This is accompanied by dose dependent decrease in
pRb, cyclin Dl and CDK4. While in MDA-MB-468 although there was no change in the
PAR polymer formation, PARP cleavage was prominent, which is an indication of apoptosis.
On treatment of TNBC cell line MDA-MB-231 with the compound A and incubation
for 24h, inhibition of PARP activity was observed in the cell line. However, MDA-MB-468
did not show PARP enzyme inhibition and instead showed cleaved PARP. Both of these are
markers of cells undergoing apoptosis. Thus, it is evident that compound A induces
significant apoptosis in both these cell lines.
Example 12:
Effect of Compound A on HIF-inhibition
Test System in HIF-la reporter gene based assay:
1) U251 HRE: The genetically engineered cells U251 HRE which stably express a
recombinant vector in which the Luciferase reporter gene is under control of three copies of a
canonical HRE.
2) U251 pGL3: A control cell line contains the firefly Luciferase reporter gene under control
of the constitutively active SV40 promoter and enhancer that helps to exclude compounds
that inhibit Luciferase expression in a nonspecific and/or HIF-1 -independent fashion. These
cells expressed high basal levels of Luciferase in normoxic conditions and slightly lower
levels in hypoxic conditions.
U251 HRE cells were inoculated into 96 well white flat-bottomed plates at 10000 -
15000 cells/well in a volume of 180 and incubated for 24 h at 37 °C, 5 % C0 2, and
ambient O2. Compound A was tested at various concentrations viz. 0.01, 0.03, 0.1, 0.3, 1.0,
3.0 and 10 and plates were incubated for 20 h in a modular hypoxia chamber (Billups
Rothenberg, MIC 101, USA) at 37 °C, 5 % C0 2, 1 % 0 2 and 94 % N2. After 20 h incubation,
the plates were removed and incubated at room temperature, 5 % C0 2, and ambient O2 for
1.5 h. 40 of Bright Glo Luciferase reagent (Promega , USA) was added and after 3 min,
luminescence was measured using a Polar Star Plate Reader (USA) in luminescence mode.
Appropriate control cells (U25 1 pGL3) were treated identically, except that they were treated
at 37 °C, 5 % CO2 and ambient O2. Compound toxicity was assayed using the MTS assay.
The results are graphically presented in Figure 9.
Treatment with Compound A effectively blocked the expression of HIF- in a dose
dependent manner in the U251 HRE cell line under hypoxia (< 1 % 0 2) . These compounds
did not inhibit the luciferase expression in the control cell line U25 1 pGL3 under normoxia.
This indicates that Compound A inhibits HIF- specifically.
Example 13:
Effect of Compound A on VEGF inhibition:
A cell line M-9 is MDA-MB-231 which is stably co-transfected with the VEGF-Luc
construct (VEGF promoter in pGL2-basic) and a plasmid containing the Geneticin (G418)
resistance gene which forms VEGF promoter reporter gene. The expression of the reporter
gene in the clone cells, as measured by luciferase activity, is stable.
The effect of Compound A on VEGF inhibition was evaluated using the VEGF
reporter gene based assay.
Reagents for VEGF assay:
Lysis Assay Buffer (IX)
Tris-Phosphate (pH 7.8)-125 mM, DTT-10 mM, EDTA-10 mM, Glycerol-50 %
and Triton X-100-5 .
Luciferase Assay Reagent (LAR)
Luciferase Assay Buffer-8 mL, 530 ATP-530 , 270 CoA-1 mL and 170
Luciferin-1 mL.
Luciferase Assay Buffer (LAB) (IX)
Tricin (pH 7.8)-20 mM, Magnesia Alba- 1.07 mM, MgS04-2.67 mM, EDTA-0.1
mM and DTT-33.3 mM.
ATP Stock made in LAB = 5.85 mg/mL
CoA Stock made in LAB = 2.1 mg/mL
Luciferin Stock made in LAB = 1.5 mg/mL
Protocol for VEGF assay:
1. M-9 cells were sub-cultured and maintained in RPMI-1640 Medium with supplement of
10 % FBS, and 4 /ΐ G418 (Stock 100 mg/mL) in a humidified incubator at 37 °C and 5 %
C0 2.
2. Cells were seeded at density of 3 x 104 cells/well in 180 volume in tissue culture grade
96 well white plates as well as transparent plates and allowed to adhere for 16-20 h in
humidified C0 2 incubator (5 % C0 2) at 37 °C. A total of two sets of plates were made, as the
incubation conditions are different.
3. Compound A, Sutent® and BSI-201 were diluted in medium serially so that final desired
concentrations are achieved in the respective wells (not more than 0.5 % concentration of
DMSO in the wells).
4. INCUBATION CONDITIONS: One set of plates are incubated under ambient atmospheric
condition with 5 % C0 2, referred hereafter as NORMOXIC/OXIC PLATE. While the other
set of plate goes in anoxic condition where the Oxygen concentration is less the 1 , and 94
% Nitrogen, 5 % C0 2, and referred hereafter as HYPOXIC PLATE. Temperature of
incubation is 37 °C and humidity greater than 75 .
5. After 20-24 h incubation under Hypoxic and Normoxic conditions, plates are taken out
from the incubators, medium from all the wells are removed from white plate. Cells are given
a rapid wash with 100-150 Phosphate Buffer Saline (PBS). Cells are lysed with 40-
50 Lysis buffer for 20 min.
6. To all the wells, 100 Luciferase Assay Reagent (LAR) are added, plates are
immediately read for Luminescence on TOPCOUNT™ (Packard, USA). The percentage
inhibitions and Inhibitory Concentration (IC50) or Effective Concentration (EC50) are
calculated in comparison with Control (untreated) values.
IC50 values () for VEGF inhibition under hypoxia:
Compound A : 0.31 
Sutent : 15 
BSI-201 : > 100 
The results are graphically presented in Figure 10.
Treatment with Compound A effectively blocked the expression of VEGF in a dose
dependent manner.
Example 14:
Effect of Compound A in wound healing assay
The wound-healing assay is simple, inexpensive, and one of the earliest developed
methods to study directional cell migration in vitro. This method mimics cell migration
during wound healing in vivo.
Protocol:
1. MCF-7 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm3 tissue
culture flask and incubated for 24 h.
2. The cells were trypsinized and seeded at a density of (0.5 - 2.0) x 10 per well in a
sterile 6 well plate.
3. The plate was incubated for about 16 h in humidified CO2 incubator (5 % CO2) at 37
°C under ambient oxygen levels. The cells were observed to form a confluent
uniform monolayer on the complete surface of the well. The required number of
cells for a confluent monolayer depends on both the particular cell type and size of
dishes.
4. The cell monolayer in a straight line was scraped evenly to create a "scratch" with
a pipette tip. The first image of the scratches was captured before addition of the
compound .
5. Compound A was added at concentrations of 1 and 3 .
6. The plates were then kept in the incubator for further incubation. The time frame for
incubation was determined empirically for the particular cell type used.
7. After the incubation, the dish was placed under a phase contrast microscope (Zeiss
Axio Observer, Germany), reference point was matched, the photographed regions of
the first image were aligned and the second image was captured. For each image
distances between one side of the scratch and the other were measured.
Similar protocol was followed for BT-549 and MDA-MB-231 cell lines.
The results are presented in Figures 11A, 11B and 11C.
Compound A showed potent anti migratory effect in all the breast cancer cell lines
including triple negative breast cancer cell line. The control cells showed complete healing
after an incubation of 24 h. The cells treated with Compound A showed very less migration
from both sides, thus indicating potent anti migratory effect.
Example 15:
Angiogenesis of Compound A in Endothelial Tube Formation Assay
The Tube Formation Assay represents a simple but powerful model for studying
inhibition and induction of angiogenesis. The assay relies on the endothelial cells' ability to
form distinct blood-vessel like tubules in an extracellular matrix (BD Matrigel™ Matrix,
USA) where they can subsequently be visualized by microscopy. It enables analysis of
angiogenic tubules in a 3 dimensional matrix that better resembles the native physiological
environment.
Protocol
Endothelial Cell Tube Formation Assay
Confluent HUVEC (Human umbilical vein endothelial cells) were cultured with
above mentioned endothelial medium to desired confluence. For HUVEC 60-80 %
confluence is recommended.
Endothelial cell suspensions were prepared by trypsinizing the cell monolayers and
resuspending the cells in culture medium with 5-10 % serum. (0.5 - l ) x l06 cells per 180 
of cell suspension were added (per well of 24 well plate) to the medium (BD Matrigel
Matrix) which, had been thawed at 4°C. This suspension was then added to the plates and
kept for incubation. The cells were allowed to adhere for 2-3 h and then Compound A (),
Rotenone () (Sigma-Aldrich, USA) and Topotecan (3) (Sigma-Aldrich, USA) (20 
of 10X stocks) were added to the respective wells. DMSO was used as the control. After 24 -
48 h of incubation the cells were observed under a phase contrast microscope (Zeiss Axio
Observer, Germany) for tube formation and angiogenesis.
The results are shown in Figure 12.
Compound A effectively inhibited endothelial tube formation and thus angiogenesis
in the 3D gel HUVEC tube formation assay. Compound A at 1 was comparable to
Rotenone (standard VEGF inhibitor) and better than Topotecan (known HIF- inhibitor in
clinical trials).
Example 16:
In vitro cytotoxicity assay:
Methods
Effect of the combination of Compound A and paclitaxel on triple negative breast
cancer cell line, MDA-MB-231 using Propidium Iodide (PI) assay
Assay protocol:
The propidium iodide fluorescence assay (PI) was carried out according to the
procedure mentioned in Anticancer Drugs, 1995, 6, 522-32.
The assay was developed to characterize the in vitro growth of human tumor cell lines
as well as to test the cytotoxic activity of the test compounds. Propidium iodide (PI) was used
as a dye, which penetrates only, damaged cellular membranes. Intercalation complexes are
formed by PI with double-stranded DNA, which effect an amplification of the fluorescence.
After freezing the cells at -20 °C for 24 h, PI had access to total DNA leading to total cell
population counts. Background readings were obtained from cell-free wells containing media
and propidium iodide.
The human triple negative breast cancer cell line, MDA-MB-231 was seeded at a
density of 1500-3000 cells/well in 180 of RPMI-1640 medium in a 96-well plate and
incubated for about 16 h in humidified 5 % CO2 incubator at 37 ± 1 °C to allow the cells to
adhere. The cells were then treated according to the schedule for drug treatment presented in
Table 5. The schedule consists of six treatment groups. In every treatment group, 20 of
10X compound (dissolved first in DMSO and then diluted in cell medium, final DMSO
concentration not exceeding 0.5 ) was used in the wells and the plate was incubated in
humidified 5 % CO2 incubator at 37 ± 1 °C. The medium was removed from the wells and
washed with PBS. 100 of PI working solution (7 g/mL) per well was added and the
plates were stored at -80 °C for about 16 h. The plates were thawed and the fluorescence was
measured using the POLARstar optima plate reader (USA) at excitation 536 nm and emission
590 nm.
(PI stock solution of 1 mg/mL was prepared by dissolving 1 mg PI in 1 mL of
distilled water. PI working solution was prepared by adding 140 of stock solution to PBS
to make up the volume to 220 mL (7 g/mL)).
Schedule: It consists of six treatment groups.
1) The MDA-MB-231 cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 
concentrations IC ) and incubated for 24 h followed by removal of medium,
addition of complete medium (CM) and incubation for 72 h (Group IA).
2) The cells were treated with complete medium and incubated for 24 h followed by
removal of medium and addition of Compound A (ICso=l ) and incubation for 72
h (Group IIA).
3) The cells were treated with complete medium and incubated for 24 h followed by
removal of medium, addition of Sunitinib (Sutent®, ICso=7.8 ) and incubation for
72 h (Group IIIA).
4) The cells were treated with varying concentrations of paclitaxel (0.03, 0.1, 0.3, 1.0
and 3.0 ) and incubated for 24 h followed by removal of medium, addition of
Compound A (ICso=l ) and incubation for 72 h (Group IVA).
5) The cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 ) and incubated
for 24 h followed by removal of medium, addition of Sunitinib (Sutent®, ICso=7.8
) and incubation for 72 h (Group VA).
6) The cells were treated with DMSO vehicle and incubated for 24 h followed by
removal of medium, addition of complete medium (CM: medium + 10 % FCS) and
incubation for 72 h (Group VIA).
The schedule of drug treatment is shown in Table 5.
Table 5 :
Group Schedule for drug treatment
At Oh At 24 h
IA Paclitaxel (0.03 ) and 24 h incubation CM and 72 h incubation
Paclitaxel (0.1 ) and 24 h incubation
Paclitaxel (0.3 ) and 24 h incubation
Paclitaxel (1.0 ) and 24 h incubation
Paclitaxel (3.0 ) and 24 h incubation
IIA CM and 24 h incubation Compound A (IC50= 1 )
and 72 h incubation
IIIA CM and 24 h incubation Sunitinib (Sutent®, IC50=7.8
) and 72 h incubation
IVA Paclitaxel (0.03 ) and 24 h incubation Compound A (IC50= 1 )
Paclitaxel (0.1 ) and 24 h incubation and 72 h incubation
Paclitaxel (0.3 ) and 24 h incubation
Paclitaxel (1.0 ) and 24 h incubation
Paclitaxel (3.0 ) and 24 h incubation
VA Paclitaxel (0.03 ) and 24 h incubation Sunitinib (Sutent®, IC50=7.8
Paclitaxel (0.1 ) and 24 h incubation ) and 72 h incubation
Paclitaxel (0.3 ) and 24 h incubation
Paclitaxel (1.0 ) and 24 h incubation
Paclitaxel (3.0 ) and 24 h incubation
VIA Vehicle control and 24 h incubation CM and 72 h incubation
CM = Complete medium
At the end of the incubation periods, the plate was assayed using the PI cytotoxicity
assay protocol. The results are shown in Table 6, Table 7 and Figure 13.
Table 6 :
Group IA (CM and 72h incubation)
Group VIA (Compound A and 72h incubation)
Group VA (Sunitinib and 72h incubation)
The synergistic effects in TNBC MDA-MB-231 cell line have been evaluated using
the CompuSyn software by Chou and Talalay, described in Pharmacological Reviews, 2006,
58, 621-681. Combination index (CI) is used to evaluate if a combination is additive,
synergistic or antagonistic. CI<1 is synergistic, CI=1 is additive and CI>1 is antagonistic. The
combination index as evaluated for the combination groups is shown in Table 7.
Table 7 : CI values for combination groups in MDA- MB-231
The combination of paclitaxel and Compound A was comparatively more synergistic
than paclitaxel and Sutent® as is evident from the value of the combination index in the
TNBC cell lines MDA-MB-231.
Cytotoxicity determination:
The IC50 values in for Compound A, paclitaxel and Sunitinib (Sutent®) in MDAMB-
231, BT-549 and MDA-MB-468 determined by cytotoxicity assay done after 48 h of
compound treatment as determined in Table 1A of Example 3 were used in Example 16.
After completion of the compound treatment i.e. at the end of 48 h, the plates were processed
for PI assay and the percent cytotoxicity was calculated as compared to DMSO (vehicle)
control.
The results of the schedule used in the combination experiments indicated that
Compound A is synergistic when used in combination with paclitaxel.
Example 17:
In vitro cytotoxicity assay:
Methods
Effect of the combination of Compound A and paclitaxel on triple negative breast
cancer cell line, BT-549 using Propidium Iodide (PI) assay
Assay protocol:
The propidium iodide fluorescence assay (PI) was carried out according to the
procedure mentioned in Anticancer Drugs, 1995, 6, 522-32.
The assay was developed to characterize the in vitro growth of human tumor cell lines
as well as to test the cytotoxic activity of the compounds. Propidium iodide (PI) was used as a
dye, which penetrates only, damaged cellular membranes. Intercalation complexes are
formed by PI with double-stranded DNA, which effect an amplification of the fluorescence.
After freezing the cells at -20 °C for 24 h, PI had access to total DNA leading to total cell
population counts. Background readings were obtained from cell-free wells containing media
and propidium iodide.
The human triple negative breast cancer cell line, BT-549 was seeded at a density of
1500-3000 cells/well in 180 of RPMI-1640 medium in a 96-well plate and incubated for
about 16 h in humidified 5 % CO2 incubator at 37 ± 1 °C to allow the cells to adhere. The
cells were then treated according to the schedule in Table 8. Each schedule consists of six
treatment groups. In every treatment group, 20 of 10X compound (dissolved first in
DMSO and then diluted in cell medium, final DMSO concentration not exceeding 0.5 ) was
used in the wells and the plate was incubated in humidified 5 % CO2 incubator at 37 ± 1 °C.
The medium was removed from the wells and washed with PBS. 100 of PI working
solution (7 g/mL) per well was added and the plates were stored at -80 °C for about 16 h.
The plates were thawed and the fluorescence was measured using the POLARstar optima
plate reader (USA) at excitation 536 nm and emission 590 nm.
(PI stock solution of lmg/mL was prepared by dissolving 1mg PI in 1mL of distilled
water. PI working solution was prepared by adding 140 of stock solution to PBS to make
up the volume to 220 mL (7 g/mL)).
Schedule : It consists of six treatment groups.
1) The BT-549 cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 
concentrations) and incubated for 24 h followed by removal of medium, addition of
complete medium and incubation for 72 h (Group IB).
2) The cells were treated with complete medium and incubated for 24 h followed by
removal of medium, addition of Compound A (IC 0= 1 ) and incubation for 72 h
(Group IIB).
3) The cells were treated with complete medium and incubated for 24 h followed by
removal of medium, addition of Sunitinib (Sutent®, ICso=7.8 ) and incubation
for 72 h (Group IIIB).
4) The cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 ) and incubated
for 24 h followed by removal of medium, addition of Compound A (ICso=l ) and
incubation for 72 h (Group IVB).
5) The cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 ) and incubated
for 24 h followed by removal of medium, addition of Sunitinib (Sutent®, ICso=7.8
) and incubation for 72 h (Group VB).
6) The cells were treated with DMSO vehicle and incubated for 24 h followed by
removal of medium, addition of complete medium (CM: medium + 10 % FCS) and
incubation for 72 h (Group VIB).
The schedule of drug treatment is shown in Table 8.
Table 8 :
The combination index as evaluated for the combination groups is shown in Table 9.
The results are shown in Figure 14.
Table 9: CI values for combination groups in BT-549
Group CI
IVB 0.4 - 0.8
VB >1.0
The combination of Paclitaxel and Compound A was comparatively more synergistic
than Paclitaxel and Sutent as indicated by the combination index in the TNBC cell line BT-
549.
Example 18:
In vitro cytotoxicity assay:
Methods
Effect of the combination of Compound A and paclitaxel on triple negative breast
cancer cell line, MDA-MB-468 using Propidium Iodide (PI) assay
Assay protocol:
The propidium iodide fluorescence assay (PI) was carried out according to the
procedure mentioned in Anticancer Drugs, 1995, 6, 522-32.
The assay was developed to characterize the in vitro growth of human tumor cell lines
as well as to test the cytotoxic activity of the compounds. Propidium iodide (PI) was used as a
dye, which penetrates only, damaged cellular membranes. Intercalation complexes are
formed by PI with double-stranded DNA, which effect an amplification of the fluorescence.
After freezing the cells at -20 °C for 24 h, PI had access to total DNA leading to total cell
population counts. Background readings were obtained from cell-free wells containing media
and propidium iodide.
The human triple negative breast cancer cell line, MDA-MB-468 was seeded at a
density of 1500-3000 cells/well in 180 of RPMI-1640 medium in a 96-well plate and
incubated for about 16 h in humidified 5 % C0 2 incubator at 37 ± 1 °C to allow the cells to
adhere. The cells were then treated according to the schedule in Table 10. Each schedule
consists of six treatment groups. In every treatment group, 20 of 10X compound
(dissolved first in DMSO and then diluted in cell medium, final DMSO concentration not
exceeding 0.5 ) was used in the wells and the plate was incubated in humidified 5 % CO2
incubator at 37 ± 1 °C. The medium was removed from the wells and washed with PBS. 100
of PI working solution (7 g/mL) per well was added and the plates were stored at -80 °C
for about 16 h. The plates were thawed and the fluorescence was measured using the
POLARstar optima plate reader (USA) at excitation 536 nm and emission 590 nm.
(PI stock solution of lmg/mL was prepared by dissolving 1mg PI in 1mL of distilled
water. PI working solution was prepared by adding 140 of stock solution to PBS to make
up the volume to 220 mL (7 g/mL)).
Schedule: It consists of six treatment groups.
1) The MDA-MB-468 cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 
concentrations) and incubated for 24 h followed by removal of medium, addition of
complete medium and incubation for 72 h (Group IC).
2) The cells were treated with complete medium and incubated for 24 h followed by
removal of medium, addition of Compound A (ICso=l ) and incubation for 72 h
(Group IIC).
3) The cells were treated with complete medium and incubated for 24 h followed by
removal of medium, addition of Sunitinib (Sutent®, ICso=7.8 ) and incubation for
72 h (Group IIIC).
4) The cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 ) and incubated
for 24 h followed by removal of medium, addition of Compound A (ICso=l ) and
incubation for 72 h (Group IVC).
5) The cells were treated with paclitaxel (0.03, 0.1, 0.3, 1.0 and 3.0 ) and incubated
for 24 h followed by removal of medium, addition of Sunitinib (Sutent®, ICso=7.8
) and incubation for 72 h (Group VC).
6) The cells were treated with DMSO vehicle and incubated for 24 h followed by
removal of medium, addition of complete medium (CM: medium + 10 % FCS) and
incubation for 72 h (Group VIC).
The schedule of drug treatment is shown in Table 10.
Table 10:
Group Schedule for drug treatment
At Oh At 24 h
IC Paclitaxel (0.03 ) and 24 h incubation CM and 72 h incubation
Paclitaxel (0.1 ) and 24 h incubation
Paclitaxel (0.3 ) and 24 h incubation
Paclitaxel (1.0 ) and 24 h incubation
Paclitaxel (3.0 ) and 24 h incubation
IIC CM and 24 h incubation Compound A (IC50= 1 )
and 72 h incubation
IIIC CM and 24 h incubation Sunitinib (Sutent®,
IC50=7.8 ) and 72 h
incubation
IVC Paclitaxel (0.03 ) and 24 h incubation Compound A (IC50= 1 )
Paclitaxel (0.1 ) and 24 h incubation and 72 h incubation
Paclitaxel (0.3 ) and 24 h incubation
Paclitaxel (1.0 ) and 24 h incubation
Paclitaxel (3.0 ) and 24 h incubation
vc Paclitaxel (0.03 ) and 24 h incubation Sunitinib (Sutent®,
Paclitaxel (0.1 ) and 24 h incubation IC50=7.8 ) and 72 h
Paclitaxel (0.3 ) and 24 h incubation incubation
Paclitaxel (1.0 ) and 24 h incubation
Paclitaxel (3.0 ) and 24 h incubation
VIC Vehicle control and 24 h incubation CM and 72 h incubation
The combination index as evaluated for the combination groups is shown in Table 11.
The results are shown in Figure 14.
Table 11: CI values for combination groups in MDA-MB-468
The combination of Paclitaxel and Compound A was comparatively more synergistic
than Paclitaxel and Sutent as indicated by the combination index in the TNBC cell line
MDA-MB-468.
The invention has been described It should be noted that, as used in this specification
and the appended claims, the singular forms "a", "an", and "the" include plural referents
unless the content clearly dictates otherwise. Thus, for example, reference to a composition
containing "a compound" includes a mixture of two or more compounds. It should also be
noted that the term "or" is generally employed in its sense including "and/or" unless the
content clearly dictates otherwise.
All publications and patent applications in this specification are indicative of the level
of ordinary skill in the art to which this invention pertains.
The invention has been described with reference to various specific and preferred
embodiments and techniques. However, it should be understood that many variations and
modifications may be made while remaining within the spirit and scope of the invention.
We claim:
1. A pharmaceutical combination for use in the treatment of triple negative breast cancer,
wherein said pharmaceutical combination comprises a therapeutically effective amount of
paclitaxel or its pharmaceutically acceptable salt and a therapeutically effective amount of the
CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable
salt thereof;
Formula I
wherein Ar is phenyl, which is unsubstituted or substituted by 1, 2, or 3 identical or different
substituents selected from: halogen selected from chlorine, bromine, fluorine or iodine; nitro,
cyano, Ci-C 4-alkyl, trifluoromethyl, hydroxyl, Ci-C 4-alkoxy, carboxy, C1-C4-
alkoxycarbonyl, CONH 2 or NR1R2; wherein and R2 are each independently selected from
hydrogen or Ci-C 4-alkyl.
2. The pharmaceutical combination for the use according to claim 1, wherein the CDK
inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein
the phenyl group is substituted by 1, 2, or 3 identical or different substituents selected from:
halogen selected from chlorine, bromine, fluorine or iodine; Ci-C4-alkyl or trifluoromethyl.
3. The pharmaceutical combination for the use according to claim 1 or 2, wherein the CDK
inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein
the phenyl group is substituted by 1, 2, or 3 halogens selected from chlorine, bromine,
fluorine or iodine.
4. The pharmaceutical combination for the use according to any one of the claims 1 to 3,
wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt
thereof; wherein the phenyl group is substituted by chlorine.
5. The pharmaceutical combination for the use according to claim 4, wherein the CDK
inhibitor represented by compound of formula I is (+)-ira«s-2-(2-Chloro-phenyl)-5,7-
dihydroxy-8-(2-hydroxymethyl- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(Compound A).
6. The pharmaceutical combination for the use according to claim 1 or 2, wherein the CDK
inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein
the phenyl group is disubstituted with a chloro and a trifluoromethyl group.
7. The pharmaceutical combination for the use according to claim 6, wherein the CDK
inhibitor represented by compound of formula I is (+)-ira«i-2-(2-Chloro-4-
trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-
chromen-4-one hydrochloride (compound B) .
8. The pharmaceutical combination for the use according to any one of the claims 1 to 7,
wherein a therapeutically effective amount of paclitaxel, or its pharmaceutically acceptable
salt; and a therapeutically effective amount of the CDK inhibitor represented by a compound
of formula I or a pharmaceutically acceptable salt thereof; are administered sequentially to a
subject in need thereof.
9. The pharmaceutical combination for the use according to claim 8, wherein therapeutically
effective amount of paclitaxel, or its pharmaceutically acceptable salt; is administered prior to
a therapeutically effective amount of the CDK inhibitor represented by a compound of
formula I or a pharmaceutically acceptable salt thereof.
10. The pharmaceutical combination for the use according to any one of the claims 1 to 9,
wherein said combination exhibits therapeutic synergy.
11. A method of treating triple negative breast cancer in a subject comprising administering
to the subject a therapeutically effective amount of paclitaxel or its pharmaceutically
acceptable salt and a therapeutically effective amount of the CDK inhibitor selected from the
compounds of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof.
12. The method according to claim 11, wherein the CDK inhibitor is a compound of formula
I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by 1,
2, or 3 identical or different substituents selected from: halogen selected from chlorine,
bromine, fluorine or iodine; Ci-C4-alkyl or trifluoromethyl.
13. The method according to claim 11 or 12, wherein the CDK inhibitor is a compound of
formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is
substituted by 1, 2, or 3 halogens selected from chlorine, bromine, fluorine or iodine.
14. The method according to any one of the claims 11 to 13, wherein the CDK inhibitor is a
compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl
group is substituted by chlorine.
15. The method according to claim 14, wherein the CDK inhibitor represented by compound
of formula I is (+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride (Compound A).
16. The method according to claim 11 or 12, wherein the CDK inhibitor is a compound of
formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is
disubstituted with a chloro and a trifluoromethyl group.
17. The method according to claim 16, wherein the CDK inhibitor represented by compound
of formula I is (+)-ira«i-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound B).
18. The method according to any one of the claims 11 to 17, wherein a therapeutically
effective amount of paclitaxel, or its pharmaceutically acceptable salt; and a therapeutically
effective amount of the CDK inhibitor represented by a compound of formula I or a
pharmaceutically acceptable salt thereof; are administered sequentially to the subject in need
thereof.
19. The method according to claim 18, wherein therapeutically effective amount of
paclitaxel, or its pharmaceutically acceptable salt; is administered prior to a therapeutically
effective amount of the CDK inhibitor represented by a compound of formula I or a
pharmaceutically acceptable salt thereof.
20. The method according to any one of the claims 11 to 19, wherein paclitaxel and the CDK
inhibitor exhibits therapeutic synergy.
2 1. Use of a pharmaceutical combination as defined in claim 1 for the manufacture of a
medicament for use in the treatment of triple negative breast cancer .
22. The use according to claim 2 1, wherein the CDK inhibitor comprised in the
pharmaceutical combination defined in claim 1 is (+)-ira«s-2-(2-Chloro-phenyl)-5,7-
dihydroxy-8-(2-hydroxymethyl- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(Compound A).
23. The use according to claim 22, wherein the CDK inhibitor comprised in the
pharmaceutical combination defined in claim 1 is (+)-ira«i-2-(2-Chloro-4-
trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-
chromen-4-one hydrochloride (Compound B).